CN103083434A - Yang warming and blood activating traditional Chinese compound preparation for preventing and treating chronic kidney diseases and application thereof - Google Patents
Yang warming and blood activating traditional Chinese compound preparation for preventing and treating chronic kidney diseases and application thereof Download PDFInfo
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Abstract
The invention discloses a yang warming and blood activating traditional Chinese compound preparation for preventing and treating chronic kidney diseases and application thereof. Raw materials of the traditional Chinese compound preparation comprise the components: 15-30g of cistanche, 15-30g of herba epimedii, 10-15g of peach kernel, 10-15g of flowers carthami and 10-15g of moutan bark. In use, the traditional Chinese compound preparation is decocted with water. The invention further provides the application of the traditional Chinese compound preparation for preventing and treating chronic kidney diseases, namely, the traditional Chinese compound preparation can be used for researching pathogenesis of aristolochzc acid nephropathy and use for interferring a rat model with IgAN glomerular sclerosis. According to the yang warming and blood activating traditional Chinese compound preparation for preventing and treating chronic kidney diseases provided by the invention, aiming at the deficiencies in treatment of chronic kidney diseases at present, a novel treating and researching method for chronic kidney diseases is provided, so that the current research status for curative effect in treatment of chronic kidney diseases can be better improved.
Description
Technical field
The present invention relates to a kind of Chinese medicinal formulae and uses thereof, particularly, relate to a kind of be used to preventing and treating warming YANG Huoxue Herbs compound preparation of chronic kidney disease and uses thereof.
Background technology
Chronic kidney disease (Chronic kidney disease, CKD) be the common clinical pathology process of multiple kidney disease except acute nephritis and acute urinary tract infection (kidney acute inflammatory disease) clinically, course of disease delay, finally can develop into whole nephropathy in latter stage (End stage renal disease, ESRD).U.S.'s nephropathy data show, the number of the infected of end-stage renal disease rises to 98.3 people/hundred ten thousand from 1998 to 2002 by 39.4 people/1,000,000 in continuous increase.Estimate accordingly, these data of coming 10 years will be with annual 4.1% rising.In China, Adult Groups CKD total prevalence rate has reached 10.8%.Therefore, how CKD being carried out early prevention and treatment, delays disease progression, is the important topic that various countries' nephropathy researcher faces.
Chronic renal failure is the final home to return to of serious kidney disease.It is reported that China approximately has 130,000 people that chronic kidney hypofunction can occur every year.Although hemodialysis, abdomen thoroughly reach carrying out of renal transplantation new way are provided for this reason, effectively extended patient's life, but expense is high owing to existing, the kidney source is not enough and the problem of various complication, and still difficulty is fully universal, seeks the non-dialysis therapy very crucial undoubtedly to the control of this disease.
Meanwhile, Aristolochic acid nephropathy is that the main of discovered in recent years becomes main disease with the renal tubular interstitium sexually transmitted disease (STD), its disease progression is rapid, prognosis is relatively poor, there is no clinically effective Therapeutic Method, be main mainly with glucocorticoid treatment at present, but medication indication and concrete therapeutic regimen also are not finalized.Its unique curative effect is arranged aspect the treatment of some disease, also it can not be limited use fully due to the Chinese herbal medicine that contains the Aristolochic Acid composition and patent medicine thereof.Inquire into the pathogenesis of this disease and seek effectively prevention and the treatment measure is the crucial problem that solves of needing.
Clinical data shows: in China CKD patient, IgA nephropathy (IgA nephropathy, IgAN) patient accounts for the 30%-40% of primary glomerulopathy at present.Actively seek that to delay or block the glomerular sclerosis carcinogenesis of human be the topic of the common concern of current nephropathy scholar for CKD patient's treatment.In addition, although at present with staphylococcal enterotoxin B (Staphylococcal Enterotoxin B, SEB) or take SEB as main model of inducing near human IgA N pathogenic process, reflect its part pathological features, but the weak point of this model is that the glomerular sclerosis formation time is long and is difficult to control.Therefore, seeking a kind of approximate human IgA N occurrence regularity and glomerular sclerosis time of occurrence experimental animal model early is also a problem that need to urgently solve in the IgAN research process.
Summary of the invention
The purpose of this invention is to provide a kind of compound Chinese medicinal preparation be used to preventing and treating chronic kidney disease, deficiency for present chronic kidney disease treatment, the Therapeutic Method of novel chronic kidney disease is provided, and the method can better improve the present Research of present chronic kidney disease treatment curative effect.
In order to achieve the above object, the invention provides a kind of warming YANG Huoxue Herbs compound preparation be used to preventing and treating chronic kidney disease, the raw material of this compound Chinese medicinal preparation comprises following composition: Herba Cistanches 15-30g, Herba Epimedii 15-30g, Semen Persicae 10-15g, Flos Carthami 10-15g, Cortex Moutan 10-15g.
Herba Cistanches: property sweet-salty, warm nature; Return kidney, large intestine channel, reinforcing the kidney and supporting YANG is arranged, functions of loosening bowel relieving constipation.
Herba Epimedii: property acrid-sweet flavor, warm nature; Return kidney, Liver Channel, kidney invigorating and YANG supporting is arranged, the expelling wind and removing dampness effect.
Semen Persicae: property bitter but sweet flavor; GUIXIN, liver, large intestine channel have blood circulation promoting and blood stasis dispelling, loosening bowel to relieve constipation, function of relieving cough and calming asthma.
Flos Carthami: nature and flavor are bitter, warm nature; GUIXIN, Liver Channel have promoting blood circulation to restore menstrual flow, the stasis-dispelling and pain-killing effect.
Cortex Moutan: the nature and flavor toil is slightly cold; GUIXIN, liver, kidney channel have clearing away heat and cooling blood, the blood circulation promoting and blood stasis dispelling effect.
Herba Cistanches, Herba Epimedii are monarch drug, synergism, and Semen Persicae, Flos Carthami are ministerial drug, synergism, Cortex Moutan are adjuvant drug.
This Chinese medicine compound reality also can be added other adjuvants as required.
Above-mentioned being used for prevented and treated the warming YANG Huoxue Herbs compound preparation of chronic kidney disease, and wherein, the raw material of described compound Chinese medicinal preparation consists of the following composition: Herba Cistanches 30g, Herba Epimedii 15g, Semen Persicae 15g, Flos Carthami 15g, Cortex Moutan 15g.
Above-mentioned being used for prevented and treated the warming YANG Huoxue Herbs compound preparation of chronic kidney disease, and wherein, the usage of described compound Chinese medicinal preparation is for being decocted in water for oral dose.
The described use detailed process that is decocted in water for oral dose comprises: after water added, bubble was fried in shallow oil about upper 30 minute again.General first (big fire) with high heat uses slow fire (little fire) again after being fried boiling, the state that maintenance is boiled is avoided overflow of drug fluid and too fast boiling dry.Should not again and again open pot cover when boiling medicine, prevent that as far as possible abnormal smells from the patient wanders away, reduce the excessive of volatile ingredient.General intense fire 5-10 minute, the kinds left and right can be poured out medicinal liquid in 20 minutes at a simmer, then add water (with for the first time quite or few point) method that go up for another example fries in shallow oil once, divides 2-3 times/day after the medicinal liquid of this decoction is mixed and take.
The present invention also provides a kind of above-mentioned being used for to prevent and treat the purposes of the warming YANG Huoxue Herbs compound preparation of chronic kidney disease, and wherein, described purposes comprises for the pathogenetic research of Aristolochic acid nephropathy, and is used for the control to corresponding Aristolochic acid nephropathy.
The above-mentioned purposes for the warming YANG Huoxue Herbs compound preparation of preventing and treating chronic kidney disease, wherein, described purposes also comprises for the IgAN glomerular sclerosis rat model is intervened and studied.
Warming YANG Huoxue Herbs compound preparation be used to preventing and treating chronic kidney disease provided by the invention has the following advantages:
(1) the CKD control is the difficult problem of present nephropathy research field, and the present invention treats on the basis in the modern medicine standard, tries to explore Chinese medicine in the application in this field.According to the traditional Chinese medical science " prolonged illness is how empty ", " the many stasis of bloods of prolonged illness " theory, invigorate blood circulation by warming YANG and prevent and treat CKD, studies confirm that the present invention has remarkable clinical efficacy; And launch on this basis experimentation, further illustrate its mechanism of action.Therefore, possess research feasibility and reasonability.
(2) warming YANG blood circulation promoting recipe provided by the invention be through Long-term clinical treatment through proved recipe, compared to domestic other unit Chinese medicinal formulae used, rationally (temperature is cool and with, reinforcement and elimination in combination to have flavour of a drug few (being comprised of Herba Cistanches, Herba Epimedii, Semen Persicae, Flos Carthami, Cortex Moutan five kinds of Chinese medicine), medication, make " tonify without causing stagnation is evil; rush down and just do not hinder "), the characteristics such as cost is low, effective, therefore be easy to promote the use of.We apply it clinical at present, and the medicine for treatment of domestic and international similar research is confined to clinical or the experimentation stage more.
(3) aspect the Aristolochic acid nephropathy study of incident mechanism, domestic and international similar research focuses mostly in the aspects such as impact of Aristolochic Acid on renal cells, and Aristolochic Acid rarely has the research report to the kidney blood vessel especially impact of kidney microvasculatures.Therefore, we have successfully copied the rat chronic Model of Aristolochic Acid nephropathy with Caulis Aristolochiae Manshuriensis, study rats with chronic aristolochic acid nephropathy renal ischaemia damage mechanism by using the present invention on this model, cytokine Ang-1, Ang-2, Tie-2, VEGF, BMP-7, Caspase-3 and the CD34 that may participate in this process inquired into, tentatively disclosed the effect of renal ischaemia damage mechanism in the Chronic Aristolochic Acid Nephropathy progress.
(4) the present invention also unites by MOI the IgAN glomerular sclerosis animal model that 5/6 nephrectomy method constructs and has verified that the warming YANG blood circulation promoting recipe can delay the progress of IgAN glomerular sclerosis model.
Description of drawings
Fig. 1 is the experiment flow schematic diagram of embodiment two.
Fig. 2 be embodiment two respectively organize 4 all nephridial tissue Electronic Speculum (* 5000) schematic diagrams.
Fig. 3 be embodiment two respectively organize 8 all nephridial tissue Electronic Speculum (* 5000) schematic diagrams.
Fig. 4 be embodiment two respectively organize 12 all nephridial tissue Electronic Speculum (* 5000) schematic diagrams.
Fig. 5 is protein immunoblot (Western blot) the testing result schematic diagram of the HIF-1 alpha expression of embodiment two.
Fig. 6 is the Western blot testing result schematic diagram that the TGF-β 1 of embodiment two expresses.
Fig. 7 is the Western blot testing result schematic diagram that the MMP-9 of embodiment two expresses.
Fig. 8 is the Western blot testing result schematic diagram that the TIMP-1 of embodiment two expresses.
Fig. 9 is the interior experiment flow schematic diagram of the body of embodiment three.
Figure 10-1 and Figure 10-2 are the experiment in vitro schematic flow sheet of embodiment three.
Figure 11 is that the interior experiment of the body of embodiment three is respectively organized Renal Glomeruli In Rats Electronic Speculum result schematic diagram (* 4200), A normal group, B model group, C warming YANG blood circulation promoting recipe group, D losartan group, E warming YANG blood circulation promoting recipe+losartan group.
Figure 12 is that the interior experiment of the body of embodiment three is respectively organized kidney of rats tubule Electronic Speculum result schematic diagram (* 11500), A normal group, B model group, C warming YANG blood circulation promoting recipe group, D losartan group, E warming YANG blood circulation promoting recipe+losartan group.
Figure 13 is that the interior experiment of the body of embodiment three is respectively organized matter Electronic Speculum result schematic diagram (* 11500) between kidney of rats, A normal group, B model group, C warming YANG blood circulation promoting recipe group, D losartan group, E warming YANG blood circulation promoting recipe+losartan group.
Figure 14 is that the interior experiment of the body of embodiment three is respectively organized renal tissues of rats BMP-7 expression immunohistochemical staining schematic diagram (* 250), A normal group, B model group, C warming YANG blood circulation promoting recipe group, D losartan group, E warming YANG blood circulation promoting recipe+losartan group.
Figure 15 is that the interior experiment of the body of embodiment three is respectively organized renal tissues of rats Caspase-3 expression immunohistochemical staining schematic diagram (* 250), A normal group, B model group, C warming YANG blood circulation promoting recipe group, D losartan group, E warming YANG blood circulation promoting recipe+losartan group.
Figure 16 is that the interior experiment of the body of embodiment three is respectively organized renal tissues of rats CD34 expression immunohistochemical staining schematic diagram (* 250), A normal group, B model group, C warming YANG blood circulation promoting recipe group, D losartan group, E warming YANG blood circulation promoting recipe+losartan group.
Figure 17 observes NRK-52E cell normal morphology schematic diagram under the experiment in vitro inverted microscope of embodiment three.
Figure 18 is that the experiment in vitro of embodiment three is respectively organized the transmission electron microscope observing schematic diagram.
Figure 19 is that the experiment in vitro of embodiment three is respectively organized immunocytochemical method and detected α-smooth muscle actin (expression of results schematic diagram of α-SMA).
Figure 20 is the experiment flow schematic diagram of embodiment four.
Figure 21 is the expression of results schematic diagram (* 400) of respectively organizing rat immunity fluorescence of embodiment four, N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
Figure 22 be embodiment four respectively organize rat staining for glycogen (PAS) om observation schematic diagram (* 400), N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
Figure 23 be embodiment four respectively organize rat transmission electron microscope observing schematic diagram (* 5000), N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
Figure 24 be embodiment four respectively organize rat TGF-β 1 ImmunohistochemistryResults Results schematic diagram (* 200), N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
Figure 25 be embodiment four respectively organize rat FN ImmunohistochemistryResults Results schematic diagram (* 200), N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
Figure 26 be embodiment four respectively organize rat α-SMA ImmunohistochemistryResults Results schematic diagram (* 200), N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
Figure 27 be embodiment four respectively organize P of Rats DGF ImmunohistochemistryResults Results schematic diagram (* 200), N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
Figure 28 be embodiment four respectively organize rat FGF-1 ImmunohistochemistryResults Results schematic diagram (* 200), N sham operated rats, M model group, W warming YANG blood circulation promoting recipe group, L losartan group, W+L combination of Chinese and Western medicine group.
The specific embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is further described.
Embodiment one: improve the clinical research of CKD for the warming YANG Huoxue Herbs compound preparation (warming YANG blood circulation promoting recipe) of preventing and treating chronic kidney disease.
by adopting warming YANG blood circulation promoting recipe treatment CKD2-3 phase yang deficiency blood stasis type patient, observe it to renal function: comprise serum creatinine (serum Scr), serum bladder chalone C (Cys-C), blood urea nitrogen (BUN), 24 hours protein quantifications, endogenous creatinine clearance rate (Ccr), glomerular filtration filter (eGFR) etc., routine blood test: comprise hemoglobin (Hb), erythrocyte (RBC), leukocyte (WBC), platelet (PLT) etc., liver function: comprise glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST) etc., the impact of the experimental index such as electrocardiogram (EKG), be intended to further understand the warming YANG blood circulation promoting recipe in treatment CKD2-3 phase yang deficiency blood stasis type patient's clinical efficacy and safety, seek the optimizing therapeutic regimen of chronic kidney disease.
choose 213 routine CKD2-3 phase yang deficiency blood stasis type patients, adopt random contrast method that it is divided into warming YANG blood circulation promoting recipe group (108 example) and doctor trained in Western medicine integrated therapeutic group (105 example), its Chinese and western medicine integrated therapeutic group gives the doctor trained in Western medicine Primary Care, warming YANG blood circulation promoting recipe group adds on the basis of doctor trained in Western medicine Primary Care uses the warming YANG blood circulation promoting recipe, be 12 weeks the course for the treatment of, Continuous Observation detects two groups for the treatment of front and back renal function (serum Scr after a course for the treatment of, BUN, Cys-C), the 24h urine protein quantitation, Ccr, eGFR, routine blood test (Hb, RBC, WBC, PLT), liver function (ALT, AST), electrocardiogram, the situation of change of traditional Chinese medical science disease integration and clinical symptoms integration.Regular follow-up in the observation process is filled in clinical case observation table and symptom integral table strictly according to the facts.Carry out statistical analysis and efficiency evaluation according to above data.
The present embodiment selected 213 routine cases are goes to a doctor in Shanghai Putuo District Central Hospital, Shanghai City profit group hospital, Shanghai City Sixth People's Hospital Urology Department outpatient service and inpatient year July in January, 2010 to 2012.
One. diagnostic criteria.
Western medicine diagnose standard: 2005, the international nephropathy raising whole prognosis working group of nephropathy of association (KDIGO) has clearly proposed the definition of chronic kidney disease (CKD): (1) kidney injury (Renal Structure or dysfunction) 〉=3 months, companion or descend without glomerular filtration rate (GFR), nephropathy is of science to be checked abnormal or kidney injury (blood, urine composition or imaging examination are abnormal); (2) eGFR＜60mlmin-1(1.73m
2)-1 〉=3 month has or without the kidney injury evidence.
The clinical stages of chronic kidney disease, see Table 1.
The standard by stages of table 1 chronic kidney disease.
| By stages | Describe | GFR［ml/1·73m 2］ |
| 1 | Injury of kidney sign (+), GFR normal or ↑ | >90 |
| 2 | Injury of kidney sign (+), GFR is slight ↓ | 60-89 |
| 3 | The GFR moderate ↓ | 30-59 |
| 4 | GFR is serious ↓ | 15-29 |
| 5 | Renal failure | ＜15 or the dialysis |
Tcm diagnosis standard: with reference in May, 2002 " new Chinese medicine guideline of clinical investigations (trying) "
[8]Relevant chronic renal failure Clinical typing standard will meet deficiency of spleen-YANG and kidneyYANG blood stasis type patient:
Primary symptom: deficient syndrome: spiritlessness and weakness, soreness of the waist and knees, aversion to cold and cold limbs; Excess syndrome: dim complexion, waist cold type of pain or twinge, tongue limit petechia or ecchymosis.
Inferior disease: deficiency syndrome: loose stool, frequent micturition, nocturia, indentations at the margin of the tongue, deep-thready pulse is puckery; Excess syndrome: Urethra astringent pain, frequent micturition, few abdomen cold type of pain or twinge, xerostomia is the wish drink not.
Two. the case choice criteria.
Include the case standard in: CKD2-3 phase diagnostic criteria: the chronic kidney disease medical history is arranged, and glomerular filtration rate GFR is at 30～89 ml/min/1.73mm.According to the evaluation of renal glomerular filtration rate that K/DOQI recommends, MDRD formula: GFR(ml/min/1.73m
2)=186 * (serum creatinine)-1.154 * (age)-0.203 * (0.742 women) in this way=exp (5.228-1.154 * ln (serum creatinine)-0.203 * ln (age)-(0.299 women) in this way).
Syndrome Differentiation of Traditional Chinese Medicine belongs to the yang deficiency blood stasis type, must have binomial deficiency syndrome and binomial excess syndrome in primary symptom to demonstrate,prove.
Filled in the mileometer adjustment of Syndrome Differentiation of Traditional Chinese Medicine stream.
Glomerular filtration rate 30-89ml/min/1.73m
2
Age was at 18 ~ 90 years old.
Do not accept dialysis treatment.
Allly meet above-mentioned standard person, can include the observation case in; Can not participate in test take the next item up as "No" person.
Excluded cases standard: once participated in other clinical trials in nearly three months; Gestation or women breast-feeding their children; Be associated with the serious primary disease such as cardiovascular, liver and hemopoietic system; The psychotic; Obstructive nephropathy.Can not participate in test take the next item up as "Yes" person.
Reject the case standard: those who are allergic to this drug; Completed treatment in accordance with regulations not; Patient's compliance is poor; Can't judge that curative effect or data are not congruent affects the treatment or safety judgement person.
Group technology: take random counter point.Produce stratified random number (1-240) with SAS software, produce corresponding random number.Comprise 20% the case that comes off.Enter the sequencing of Clinical observation according to the patient, according to corresponding random number, be divided into warming YANG blood circulation promoting recipe group and doctor trained in Western medicine integrated therapeutic group.The present embodiment has 240 routine patients and enters clinical trial, and wherein 27 examples come off, complete case 213 examples, wherein warming YANG blood circulation promoting recipe group 108 example and doctor trained in Western medicine integrated therapeutic group 105 examples.
Three. Therapeutic Method.
1. warming YANG blood circulation promoting recipe group therapeutic scheme.
Warming YANG blood circulation promoting recipe composition is shown in Table 2.
Table 2: the constituent of warming YANG blood circulation promoting recipe.
| |
1 | 2 | 3 |
| Herba Cistanches | 30g | 15g | 20g |
| Semen Persicae | 15g | 30g | 20g |
| Flos Carthami | 15g | 10g | 12g |
| Cortex Moutan | 15g | 10g | 13g |
| Herba Epimedii | 15g | 10g | 14g |
Adopt any one of above proportioning, potion on the one is decocted in water for oral dose and takes at twice, each 200ml, each 1 time sooner or later.
2. doctor trained in Western medicine integrated therapeutic group therapeutic scheme.
Nutrition treatment: the supply of protein and essential amino acids: the high-quality low protein diet, be generally 0.5-0.6g/kg, appropriate saccharide, fat are to guarantee enough heats, low-phosphorous diet.Suitable vitamin and trace element give.During anasarca, the sodium amount should be limited in 3g every day left and right.
Symptomatic treatment:
Correction water balance imbalance: SHUANGKE, 25mg/ time, 2-3/ days, oral, GFR＜30%ml/min gave furosemide 20 mg/ time, and 2-3/ days, oral, intravenous injection in case of necessity.
Hyperpotassemia: avoid taking in potassium increased diet; Diuresis furosemide 20 mg/ time, 2-3/ days, oral; Fall the potassium blood fat 5-15g time, 2-3/ days, add water 100 ml and take.Severe rising person, (k〉6.5mg/L, available 10% calcium gluconate 10-20 ml, insulin regular adds quiet of Glucose Liquid, (insulin: sugar=1U:5g), and simultaneously in conjunction with blood gas analysis, the SB 100-200ml of quiet 5%.
Metabolic acidosis: slight CO
2CP 20-15.7mmol/L. sodium bicarbonate 1.0 tid; Severe: CO
2CP＜13.5 mmol/L, the sodium bicarbonate of quiet 5%, but need to detect calcium level, anti-low calcium.
When low calcium and hyperphosphatemia: GFR＜40 ml/min, the 1.2-2.0g/d that replenishes the calcium, blood calcium〉2.75 mmol/L drug withdrawals; Phosphorus falls: g/ 3-4/ of calcium carbonate 1-3 days
Secondary Hyperparathyroidism and renal osteodystrophy treatment: sprout the prosperous 0.5 μ g qd po of lattice
Renal anemia: HB＜110g/L, or HCT＜33% use the EPO treatment, initial dose: minute 2-3 medication of subcutaneous medication adult's 80-120u/kg/ week (approximately 6000 u/W), child＜5 years old, 300 u/kg/ are all, more than 5 years old with reference to adult's scheme; Dose titration, begin treatment or increase dosage 2-4 are in week, and HCT rises＜2%, increases by 50% dosage, after treatment beginning or increase EPO dosage, the HCT rising〉8%/month, or Hb/HCT has reached decrement 25% of target (Europe is 110-120g/L).Enclosing in the situations such as operation, infection, can increase as one sees fit EPO dosage.
Chalybeate replenishes: adult's dosage (elemental iron) was 200 mg/ days, child 2-3 every day mg/kg, and minute 2-3 time is oral; Ferrous sulfate 0.3-0.6g/ time, every day 3 times is in meal, one after each meal.
Folic acid: 5-10mg tid is oral, keeps the treatment phase at EPO, and the chalybeate amount should be 25-100mg/W.
Renal hypertension: Angiotensin-Converting or acceptor inhibitor treatment, other available ca channel blockers, beta-blocker, vasodilation.
Heart failure: heart tonifying diuresis: furosemide 20mg-100mg intravenous injection.Blood vessel dilating: sorbitrate 10-20mg/ time, 6-8 hour once.Heart tonifying: digoxin loading 1-1.5mg, maintenance dose be every day or the next day 0.125mg; Cedilanid uses when acute attack, 0.2-0.4mg/ time.
Infect: select antibiotic to use according to drug sensitive test.Via the renal excretion antibiotic, after giving first loading dose, adjust dosage pressing GFR.In the situation that curative effect is close, should select the little medicine of nephrotoxicity.
Regulate blood fat: hypercholesterolemia, high/low density hyperlipoproteinemia, lescol see fluvastatin 40mg QN is oral; Hypertriglyceridemia, lipanthyl 200mg QN is oral.
Reduce High-grade Proteinuria: select angiotensin converting enzyme inhibitor (ACEI) or Angiotensin Ⅱ receptor antagonist (ARB) medicine, two kinds of use in conjunction when needing.
Four. the course for the treatment of.
This project in the end random packet patient selected after, two groups all take 12 weeks as 1 course for the treatment of, carry out statistical analysis after 1 course for the treatment of.
Five. observation index and detection method.
1. clinical symptoms and integration.
Tcm syndrome integration situation by patient's Chinese medical discrimination primary symptom " light, in, heavy " with " 2,4,6 " minute expression; Inferior disease represents with " 1,2,3 ".Clinical symptoms integration situation according to patient's subjective symptoms " light, in, heavy " with " 1,2,3 " minute expression.
2. biochemical indicator.
Renal function index: (eGFR, formula calculates ml/min/1.73m for serum creatinine (Scr, μ mol/L), blood urea nitrogen (BUN, mmol/L), serum bladder chalone C (CysC, mg/L), endogenous creatinine clearance rate (Ccr, ml/min), glomerular filtration rate
2).
Uroscopy: 24h urine protein quantitation (24hUPr, g/24h).
Routine blood test index: hemoglobin (Hb, g/L), erythrocyte (RBC, * 10
12/ L), leukocyte (WBC, * 10
9/ L), platelet (PLT, * 10
12/ L).
Liver function index: glutamate pyruvate transaminase (ALT, U/L), glutamic oxaloacetic transaminase, GOT (AST, U/L).
Electrocardiogram (EKG).
Six. criterion of therapeutical effect.
1. curative effect of disease criterion: formulate with reference to " new Chinese medicine guideline of clinical investigations ".
Produce effects:
The clinical symptoms integration reduces 〉=60%,
Endogenous creatinine clearance rate (Ccr) or glomerular filtration rate (eGFR) increase 〉=20%,
Serum creatinine (Scr) reduces 〉=20%.
Above
Item is indispensable,
,
Possess 1, can judge.
Effectively:
The clinical symptoms integration reduces 〉=30%,
Endogenous creatinine clearance rate (Ccr) or glomerular filtration rate (eGFR) increase 〉=10%,
Serum creatinine (Scr) reduces 〉=10%;
With logarithm or the inverse of serum creatinine, use the linear regression equation analysis before and after treatment, its slope has clear meaning person.
Above
Indispensability, other possess 1, can judge.
Stable:
Clinical symptoms makes moderate progress, and integration reduces＜30%,
Endogenous creatinine clearance rate (Ccr) or glomerular filtration rate (eGFR) are without reduction, or increase＜10%,
Serum creatinine (Scr) is without increase, or reduction＜l0%.
Above
Item is indispensable,
,
Possess 1, can judge.
Invalid:
Clinical symptoms is without improving or increase the weight of,
Endogenous creatinine clearance rate (Ccr) or glomerular filtration rate (eGFR) reduce,
Serum creatinine (Scr) increases.
Above
Item is indispensable,
,
Possess 1, can judge.
2. traditional Chinese medical science disease curative effect determinate standard: formulate with reference to " new Chinese medicine guideline of clinical investigations ".
Clinical recovery: tcm clinical practice symptom, sign disappear or basic the disappearance, and the syndrome integration reduces 〉=95%.
Produce effects: tcm clinical practice symptom, sign disappear or basic the disappearance, and the syndrome integration reduces 〉=70%.
Effectively: tcm clinical practice symptom, sign disappear or basic the disappearance, and the syndrome integration reduces 〉=30%.
Invalid: all without obviously improving, even increase the weight of, the syndrome integration reduces less than 30% for tcm clinical practice symptom, sign.
Annotate: computing formula (nimodipine method) is: ［ integration before (integration after integration before treatment-treatment) ÷ treatment ］ * 100%.
Seven. statistical method.
All the data SAS 9.2 softwares calculate, measurement data with mean ± standard deviation represent (± s), two two groups of differential counting data are relatively with X 2 test or definite probabilistic method, adopt X 2 test statistic be chi-square value; Relatively with the CMH check, statistic is the CMH chi-square value for two groups of many differential countings data.Before treating, two groups of front and back difference or rates of change relatively adopt Wei Shi sum of ranks (wilcoxon rank sum) check, statistic is Z, relatively adopt Wei Shi signed rank (wilcoxon signed rank) check before and after in group, statistic is S, two groups of effective percentage logic (logistic) regression analyses.P≤0.05 will be regarded as checking statistical significance.
Eight. experimental data and conclusion.
1. Clinical efficacy comparison: see Table shown in 3 ~ table 4.2.
Table 3 liang group curative effect of disease situation and comparison, unit: example.
| Grouping | Number of cases | Produce effects | Effectively | Stable | Invalid | Total effective rate (%) |
| Warming YANG blood circulation promoting recipe group | 97 | 34 | 30 | 20 | 13 | 86.60 ▲ |
| Doctor trained in Western medicine integrated therapeutic group | 97 | 26 | 22 | 13 | 36 | 62.89 |
Effective percentage (%)=(produce effects+effectively+stable)/total number of cases * 100.
Epidemiological Analysis by statistics: two groups of patient disease curative effects relatively adopt the CMH check of control centre's effect, and=8.48, ▲ P=0.0036,
P＜0.05, difference has statistical significance; Prompting A group curative effect is organized apparently higher than B.
The table 3.1 liang efficient logistic Regression Analysis Result of group curative effect of disease (not considering center and grouping reciprocal action).
| The Wald chi-square value | The P value | OR value (95%CI) | |
| Treat after 3 months | |||
| Grouping | 14.55 | 0.0001 | (4.35 2.04,9.27) |
| Center | 6.21 | 0.0449 | |
| Clinical symptoms integration before treatment | 12.82 | 0.0003 |
Whether be effectively dependent variable after treatment, index assignment (effectively=1, invalid=2), match probabilistic model effectively=1 take the B group as reference, is considered center effect during analysis simultaneously, and the clinical symptoms integration before treatment.
Conclusion: the logistic Regression Analysis Result shows, two groups of curative effect of disease effective percentage comparison difference have statistical significance (card side=14.55, P=0.0001, OR(95%CI): 4.35 (2.04,9.27)), the effective percentage of A group is higher than the effective percentage of B group, and conclusion is checked with CMH.
The table 3.2 liang efficient logistic Regression Analysis Result of group curative effect of disease (consideration center and grouping reciprocal action).
| The Wald chi-square value | The P value | |
| Treat after 3 months | ||
| Grouping | 12.35 | 0.0004 |
| Center | 6.61 | 0.0367 |
| Center and grouping mutual | 3.01 | 0.2224 |
| Clinical symptoms integration before treatment | 12.71 | 0.0004 |
Whether be effectively dependent variable after treatment, index assignment (effectively=1, invalid=2), match probabilistic model effectively=1 take the B group as reference, is considered center effect, center and grouping reciprocal action during analysis simultaneously, and the clinical symptoms integration before treatment.
Conclusion: the logistic Regression Analysis Result shows, the reciprocal action not statistically significant (P=0.2224) of center and grouping shows that each center test effect is consistent, and quality is better.Therefore can not consider center and grouping reciprocal action when analyzing curative effect, namely take the result of table 3.1 as main.
Table 4 liang group traditional Chinese medical science disease curative effect situation and comparison, unit: example.
| Grouping | Number of cases | Clinical recovery | Produce effects | Effectively | Invalid | Total effective rate (%) |
| Warming YANG blood circulation promoting recipe group | 99 | 0 | 0 | 52 | 47 | 52.53 ▲ |
| Doctor trained in Western medicine integrated therapeutic group | 98 | 0 | 0 | 23 | 75 | 23.47 |
Effective percentage (%)=(clinical recovery+produce effects+effectively)/total number of cases * 100
Statistical analysis: two groups of patient disease curative effects relatively adopt the CMH check of control centre's effect, and=27.70, ▲ P＜0.0001, difference has statistical significance; Prompting A group curative effect is organized apparently higher than B.
The table 4.1 liang efficient logistic Regression Analysis Result of group traditional Chinese medical science disease curative effect (not considering center and grouping reciprocal action).
| The Wald chi-square value | The P value | OR value (95%CI) | |
| Treat after 3 months | |||
| Grouping | 12.47 | 0.0004 | (12.08 3.03,48.15) |
| Center | 2.63 | 0.2683 | |
| Syndrome total mark before treatment | 29.21 | <0.0001 |
Whether be effectively dependent variable after treatment, index assignment (effectively=1, invalid=2), match probabilistic model effectively=1 take the B group as reference, is considered center effect during analysis simultaneously, and the syndrome total mark before treatment.
Conclusion: the logistic Regression Analysis Result shows, two groups of traditional Chinese medical science disease curative effect effective percentage comparison difference have statistical significance (card side=12.47, P=0.0004, OR(95%CI): 12.08 (3.03,48.15)), the effective percentage of A group is higher than the effective percentage of B group, and conclusion is checked with CMH.
The table 4.2 liang efficient logistic Regression Analysis Result of group traditional Chinese medical science disease curative effect (consideration center and grouping reciprocal action).
| The Wald chi-square value | The P value | |
| Treat after 3 months | ||
| Grouping | 12.82 | 0.0003 |
| Center | 1.71 | 0.4261 |
| Center and grouping mutual | 0.08 | 0.9596 |
| Syndrome total mark before treatment | 27.87 | <0.0001 |
Whether be effectively dependent variable after treatment, index assignment (effectively=1, invalid=2), match probabilistic model effectively=1 take the B group as reference, is considered center effect, center and grouping reciprocal action during analysis simultaneously, and the syndrome total mark before treatment.
Conclusion: the logistic Regression Analysis Result shows, the reciprocal action not statistically significant (P=0.9596) of center and grouping shows that each center test effect is consistent, and quality is better.Therefore can not consider center and grouping reciprocal action when analyzing curative effect, namely take the result of table 4.1 as main.
This shows: warming YANG blood circulation promoting recipe group curative effect of disease total effective rate is 86.60%, higher than the 62.89%(of doctor trained in Western medicine integrated therapeutic group
P＜0.05); Warming YANG blood circulation promoting recipe group traditional Chinese medical science disease curative effect total effective rate is 52.53%, higher than the 23.47%(of doctor trained in Western medicine integrated therapeutic group
P＜0.05).The patient that explanation added on doctor trained in Western medicine integrated therapeutic basis with the warming YANG blood circulation promoting recipe treatment CKD2-3 phase can obviously improve curative effect of disease and traditional Chinese medical science disease curative effect.
2. the variation of clinical symptoms integration before and after the treatment: see Table shown in 5 ~ table 6.
Table 5 liang group clinical symptoms integration situation of change and comparison (± s).
Epidemiological Analysis by statistics: clinical symptoms integral contrasts before two groups of patient treatments, the difference not statistically significant (
P0.05), have clinical comparability.Reduce before after treatment, two groups of clinical symptoms are all treated (▲
P＜0.05).Between two groups of clinical symptoms change groups, statistical significance (■ is arranged relatively
P＜0.05), show that the rear warming YANG blood circulation promoting recipe group clinical symptoms integration for the treatment of descends apparently higher than doctor trained in Western medicine integrated therapeutic group.
Table 6 liang group traditional Chinese medical science disease integration situation of change and comparison (± s).
Epidemiological Analysis by statistics: traditional Chinese medical science disease integral contrasts before two groups of patient treatments, the difference not statistically significant (
P0.05), have clinical comparability.Reduce before after treatment, two groups of traditional Chinese medical science diseases are all treated (▲
P＜0.05).Between two groups of traditional Chinese medical science disease set of variations, statistical significance (■ is arranged relatively
P＜0.05), show that the rear warming YANG blood circulation promoting recipe group traditional Chinese medical science disease integration for the treatment of descends apparently higher than doctor trained in Western medicine integrated therapeutic group.
3. the variation of renal function before and after the treatment: see Table shown in 7 ~ table 11.
Scr(μ mol/L before and after table 7 liang group treatment) situation of change and comparison (± s).
| Grouping | Number of cases | Before treatment | After treatment | Difference (after treatment-treatment is front) |
| Warming YANG blood circulation promoting recipe group | 108 | 115.33±36.18 | 105.66±38.53 ▲■ | -11.92±25.43 |
| Doctor trained in Western medicine integrated therapeutic group | 105 | 118.94±38.31 | 115.84±41.23 ▲ | -5.28±22.88 |
Annotate: with comparison before this group treatment, ▲ P temperature＜0.0001, ▲ P west=0.0470 (P＜0.05).
After two groups of treatments, difference compares, ■ P=0.0140 (P＜0.05).
BUN(mmol/L before and after table 8 liang group treatment) situation of change and comparison (± s).
| Grouping | Number of cases | Before treatment | After treatment | Difference (after treatment-treatment is front) |
| Warming YANG blood circulation promoting recipe group | 107 | 8.86±4.06 | 8.26±3.78 ▲■ | -0.61±2.66 |
| Doctor trained in Western medicine integrated therapeutic group | 105 | 8.77±3.49 | 9.85±4.20 ▲ | 1.07±2.84 |
Annotate: with comparison before this group treatment, ▲ P temperature=0.0489, ▲ P west=0.0002 (P＜0.05).
After two groups of treatments, difference compares, ■ P＜0.0001 (P＜0.05).
CysC(mg/L before and after table 9 liang group treatment) situation of change and comparison (± s).
| Grouping | Number of cases | Before treatment | After treatment | Difference (after treatment-treatment is front) |
| Warming YANG blood circulation promoting recipe group | 105 | 1.25±0.62 | 1.16±0.54 ▲ | -0.10±0.46 |
| Doctor trained in Western medicine integrated therapeutic group | 104 | 1.31±0.57 | 1.30±0.59 | -0.02±0.37 |
Annotate: relatively front with the treatment of this group, ▲ P temperature=0.0184 (P＜0.05).
Ccr(ml/min before and after table 10 liang group treatment) situation of change and comparison (± s).
| Grouping | Number of cases | Before treatment | After treatment | Difference (after treatment-treatment is front) |
| Warming YANG blood circulation promoting recipe group | 107 | 55.62±21.55 | 61.69±23.48 ▲■ | 6.99±9.12 |
| Doctor trained in Western medicine integrated therapeutic group | 105 | 49.55±18.22 | 52.27±20.40 ▲ | 3.07±10.57 |
Annotate: with comparison before this group treatment, ▲ P temperature＜0.0001, ▲ P west=0.0112 (P＜0.05).
After two groups of treatments, difference compares, ■ P=0.0048 (P＜0.05).
EGFR(ml/min/1.73m2 before and after table 11 liang group treatment) situation of change and comparison (± s).
| Grouping | Number of cases | Before treatment | After treatment | Difference (after treatment-treatment is front) |
| Warming YANG blood circulation promoting recipe group | 107 | 54.75±18.35 | 62.60±21.60 ▲■ | 8.69±10.51 |
| Doctor trained in Western medicine integrated therapeutic group | 105 | 53.50±18.09 | 55.24±21.80 | 2.49±14.16 |
Annotate: relatively front with the treatment of this group, ▲ P temperature＜0.0001 (P＜0.05); After two groups of treatments, difference compares, ■ P=0.0003 (P＜0.05).
Before treatment: Scr, the BUN of warming YANG blood circulation promoting recipe group and doctor trained in Western medicine integrated therapeutic group, CysC, Ccr, eGFR no significant difference (P〉0.05), two groups of CKD2-3 phase patient treatments are described before renal function have comparability.
After treatment: warming YANG blood circulation promoting recipe group and doctor trained in Western medicine integrated therapeutic group Scr all descend before treatment, and difference has statistical significance (P＜0.05), and before and after two groups of treatments, difference relatively has statistical significance (P＜0.05).Therefore warming YANG blood circulation promoting recipe group is better than doctor trained in Western medicine integrated therapeutic group to reducing the serum creatinine curative effect.
Before warming YANG blood circulation promoting recipe group and doctor trained in Western medicine integrated therapeutic group BUN treat, statistical significance (P＜0.05) is arranged all, after two groups of BUN treatments, the difference comparing difference has statistical significance (P＜0.05).Warming YANG blood circulation promoting recipe group is being better than doctor trained in Western medicine integrated therapeutic group aspect the reduction blood urea nitrogen.
Warming YANG blood circulation promoting recipe group CysC treats front reduction, and difference has statistical significance.After the treatment of doctor trained in Western medicine integrated therapeutic group, reduction trend is arranged, but the obvious statistical significance of nothing (P〉0.05).Difference not statistically significant between organizing before and after two groups of CysC treatments (P〉0.05).
All raise before treatment after warming YANG blood circulation promoting recipe group and doctor trained in Western medicine integrated therapeutic group Ccr treatment, difference has statistical significance (P＜0.05).Compare between the difference group before and after two groups of Ccr treatments, difference has statistical significance (P＜0.05).Warming YANG blood circulation promoting recipe group is better than doctor trained in Western medicine integrated therapeutic group at rising Ccr.
Have before warming YANG blood circulation promoting recipe group eGFR treats and raise obviously, difference has statistical significance (P＜0.05), before doctor trained in Western medicine integrated therapeutic group eGFR treats, rising trend is arranged, but the difference not statistically significant (P〉0.05), between two groups of eGFR therapeutic effect groups, statistical significance is arranged relatively.Therefore warming YANG blood circulation promoting recipe group is better than doctor trained in Western medicine integrated therapeutic group aspect eGFR keeping.
In sum, the warming YANG blood circulation promoting recipe effectively reduces Scr, BUN, keep and raise Ccr, eGFR, and curative effect is better than doctor trained in Western medicine integrated therapeutic group.Certain effect is arranged aspect renal function stablizing and improve.
4. the variation of twenty-four-hour urine protein quantification before and after the treatment is shown in Table 12.
24h-UPr(g/24h before and after table 12 liang group treatment) situation of change and comparison (± s).
| Grouping | Number of cases | Before treatment | After treatment | Difference (after treatment-treatment is front) |
| Warming YANG blood circulation promoting recipe group | 107 | 1.35±1.45 | 0.92±1.04 ▲■ | -0.40±1.00 |
| Doctor trained in Western medicine integrated therapeutic group | 103 | 1.07±1.10 | 1.05±1.05 | -0.03±0.94 |
Annotate: relatively front with the treatment of this group, ▲ P temperature＜0.0001 (P＜0.05); After two groups of treatments, difference compares, ■ P=0.0006 (P＜0.05).
Before treatment: two groups of 24h urine protein quantitation no significant differences (P〉0.05), two groups of patient treatments are described before the 24h urine protein quantitation have comparability.
After treatment: before warming YANG blood circulation promoting recipe group 24h urine protein quantitation is treated, obvious decline is arranged, difference has statistical significance (P＜0.05), before doctor trained in Western medicine integrated therapeutic group 24h urine protein quantitation is treated, downward trend is arranged, but not statistically significant (P〉0.05), before and after two groups of 24h urine protein quantitation treatments, difference relatively has statistical significance (P＜0.05), therefore warming YANG blood circulation promoting recipe group can effectively reduce albuminuria, therapeutic effect is better than doctor trained in Western medicine integrated therapeutic group.
5. before and after the treatment, routine blood test changes, and sees Table shown in 13 ~ table 16.
Hb(g/L before and after table 13 liang group treatment) relatively (± s).
RBC(before and after table 14 liang group treatment * 1012/L) relatively (± s).
Annotate: after two groups of treatments, difference compares, ▲ P＜0.05.
WBC(before and after table 15 liang group treatment * 109/L) relatively (± s).
PLT(before and after table 16 liang group treatment * 1012/L) relatively (± s).
Before treatment: more equal not statistically significant between the group of two groups of Hb, RBC, WBC, PLT (P〉0.05), two groups of treatments are described before the above indexs of routine blood test have comparability.
After treatment: before and after two groups of Hb, RBC, WBC, PLT in group relatively, the equal not statistically significant of difference (P〉0.05), before and after two groups of Hb, WBC, PLT between group relatively, the equal not statistically significant of difference (P〉0.05); Before treatment, downward trend is arranged after doctor trained in Western medicine integrated therapeutic group RBC treatment, warming YANG blood circulation promoting recipe group is without significant change.After two groups of RBC treatment, comparing difference has statistical significance (P〉0.05), illustrate the treatment of doctor trained in Western medicine integrated therapeutic group afterwards RBC descend than warming YANG blood circulation promoting recipe group.
In sum, two groups of patient's routine blood tests are treated front and back without significant change.Slightly descend after the treatment of doctor trained in Western medicine integrated therapeutic group erythrocyte.
6. changes of liver function before and after treating: see Table shown in 17 ~ table 18.
ALT(U/L before and after table 17 liang group treatment) relatively (± s).
AST(U/L before and after table 18 liang group treatment) relatively (± s).
Before treatment: more equal not statistically significant between the group of two groups of ALT, AST (P〉0.05), illustrate that two groups of front above indexs of liver function for the treatment of have comparability.
After treatment: compare in organizing before and after two groups of ALT, AST, the equal not statistically significant of difference (P〉0.05), compare between group before and after two groups of ALT, AST, the equal not statistically significant of difference (P〉0.05).
In sum, two groups of patient treatments are described before and after liver function without significant change.
7. before and after the treatment, electrocardiogram changes: be shown in Table 19.
Before and after the treatment of table 19 liang group electrocardiogram just, ANOMALOUS VARIATIONS and comparison, unit: example (%).
Check through CMH :=100.21, P＜0.0001, point out two groups of electrocardiogram comparing differences that statistical significance is arranged, illustrate that warming YANG blood circulation promoting recipe group transfers to normal many than the rear electrocardiogram of doctor trained in Western medicine integrated therapeutic group treatment.
8. a situation arises for adverse events: be shown in Table 20.
A situation arises and comparison for table 20 a liang group adverse events.
| Grouping | Number of cases does not occur | Number of cases occurs | Add up to | Incidence (%) | The method of inspection | Statistic | The P value |
| The positive blood circulation promoting recipe group of temperature | 105 | 3 | 108 | 2.78 | Proofread and correct Chi-square Test | Card side=0.00 | 1.0000 |
| Doctor trained in Western medicine integrated therapeutic group | 103 | 2 | 105 | 1.90 |
Epidemiological Analysis by statistics: two groups of adverse events incidence rates relatively, difference not statistically significant (P=1.0000) illustrates two groups of adverse events incidence rate indifferences.
9. situation of change by stages: be shown in Table 21.
Situation of change and comparison by stages before and after table 21 liang group treatment, unit: example (%).
After the treatment of 12 weeks, warming YANG blood circulation promoting recipe group renal function improves that (CKD was converted into for 1 phase by 2 phases, and 3 phases were converted into 2 phases, 1 phase) 28 examples, stable renal function (CKD is by stages constant) 34 examples, (CKD was converted into for 3 phases by 2 phases to renal hypofunction, and 3 phases were converted into for 4 phases) 3 examples, without obvious renal hypofunction (CKD was converted into for 4 phases by 2 phases, and 3 phases were converted into for 5 phases) case.Doctor trained in Western medicine integrated therapeutic group renal function improves 18 examples, stable renal function 66 examples, and renal hypofunction 14 examples are improved and the case that goes down without obvious renal function.Check through CMH :=80.49, P＜0.0001, point out two groups of GFR situations of change that statistical significance (P〉0.05) is arranged, illustrate that the A component phase changes to organize than B.
Embodiment two: the impact of warming YANG blood circulation promoting recipe on the renal failure Renal Function in Rats.
Observe the warming YANG blood circulation promoting recipe to the impact of 5/6 nephrectomy chronic renal failure rats renal function, inquire into the possible mechanism of action that it delays disease progression.
120 SD male rats are divided into sham operated rats (A group), model group (B group), warming YANG blood circulation promoting recipe group (C group), losartan group (D group) and warming YANG blood circulation promoting recipe+losartan group (E group) at random.
Model group: make the chronic renal failure rats model by 5/6 nephrectomy.the SD male rat is carried out adaptability raises, carry out left kidney 2/3 excision the 5th week, getting the ventricumbent position after anesthesia is fixed on operating-table, at back surgery district routine disinfection, drape, get back of the body left side straight cut incision skin, separate psoas major, after entering peritoneum, find kidney, and separate perinephric fat capsule and the kidney base of a fruit with mosquito forceps, successively percutaneous incision undertissue and peplos, SC separate the adrenal gland, avoid damage, fully expose left kidney, expose the kidney base of a fruit, clamp the kidney base of a fruit with the fiber mosquito forceps, cut with ophthalmologic operation and wipe out respectively left kidney each 1/3 left and right cortex up and down, use the absorbable gelatin sponge hemostasis by compression, open mosquito forceps, observation has or not hemorrhage, residual kidney resets, Intraperitoneal injection penicillin prevention infection, close the abdominal cavity, suture muscles and skin, the postoperative routine gives intramuscular injection of penicillin 3d, feed water and observe.After one week, i.e. the 6th week row operation for the second time is with Resection of right kidney.After the kidney of same fully exposure right side, press from both sides under the hilus renalis with mosquito forceps and close the kidney base of a fruit, extract kidney, then clear up the abdominal cavity, extract kidney base of a fruit ligation the end of a thread of not wiping out, and the kidney base of a fruit of ligation is put into the abdominal cavity.Determine that the Intraperitoneal injection penicillin closes abdomen, sews up without cutting short ligation the end of a thread after hemorrhage.The postoperative routine gives intramuscular injection of penicillin.Two operations is excised 5/6 kidney altogether, and whole process of operation all has a people to hold cutter to complete.
Sham operated rats: carry out sham-operation, namely after the rats by intraperitoneal injection anesthetics, free bilateral renal, peel off perirenal fat after, kidney is resetted, the operation process operation is identical with above-mentioned nephrectomy method.
A week begins to give pharmaceutical intervention after modeling or sham-operation: (1) sham operated rats: give normal saline 10mlkg
-1D
-1Gavage; (2) model group: give normal saline 10mlkg
-1D
-1Gavage; (3) warming YANG blood circulation promoting recipe group: give warming YANG blood circulation promoting recipe 30gkg
-1D
-1Gavage; (4) losartan group: losartan 33.3mgkg
-1D
-1Gavage; (5) warming YANG blood circulation promoting recipe+losartan group: give warming YANG blood circulation promoting recipe 30gkg
-1D
-1+ losartan 33.3mgkg
-1D
-1Gavage.
Detect and respectively to organize 4,8,12 serum urea nitrogens at weekend (BUN) after the rat administration, serum creatinine (Scr), twenty-four-hour urine albumen and twenty-four-hour urine NAG enzyme, electron microscopic observation, the expression of using Western blot method detection nephridial tissue HIF-1 α at 12 weekend, TGF-β 1, MMP-9, TIMP-1.
Experiment flow is shown in Figure 1, and experimental data and result are as follows.
1. renal tissue morphological observation.
Electron microscopic observation: sham operated rats glomerule clear in structure, filter membrane is complete, and epithelial cell podocytic process marshalling has no fusion, renal tubular cell microvillus clear in structure, complete, endoplasmic reticulum, structure of mitochondria are normal, the nucleus sub-circular, kernel is clear.After 4 weeks, the glomerule structure is still clear in gavage for model group, and glomerular basement membrane slightly bending thickens, and visible proliferation of mesangial cells has inflammatory reaction, and inflammatory cell infiltration is arranged, and Stromal fibroblasts increases; After 8 weeks, the swelling of part podocyte, flattens at the podocytic process partial fusion, the swelling of part renal tubules, visible cavity in renal cells part endochylema, microvillus swelling, interstitial fibers hypertrophy; After 12 weeks, glomerular capillary open still can, the basement membrane bending thickens, podocyte swelling, the part podocytic process merges, flattens, renal tubules swelling, degeneration, interstitial fibers hyperplasia is obvious; Treatment group with compare with time segment model group, pathological change has alleviating in various degree.Referring to Fig. 2 ~ shown in Figure 4.
2. Western blot testing result.
The HIF-1 alpha expression: HIF-1 α the expression of model group apparently higher than sham operated rats (
P﹤ 0.01), warming YANG blood circulation promoting recipe group, losartan group and warming YANG blood circulation promoting recipe+losartan group express all lower than model group (
P﹤ 0.05).Comparing difference not statistically significant between three treatment groups (
P﹥ 0.05).As shown in Figure 5, compare * with sham operated rats
P＜0.05, * *
P＜0.01; Compare # with model group
P＜0.05, ##
P＜0.01.
TGF-β 1 expresses: TGF-β 1 the expression of model group apparently higher than sham operated rats (
P﹤ 0.01), warming YANG blood circulation promoting recipe group, losartan group warming YANG blood circulation promoting recipe+losartan group express all lower than model group (
P﹤ 0.05).Comparing difference not statistically significant between three treatment groups (
P﹥ 0.05).As shown in Figure 6, compare * with sham operated rats
P＜0.05, * *
P＜0.01; Compare # with model group
P＜0.05, ##
P＜0.01.
MMP-9 expresses: MMP-9 the expression of model group higher than sham operated rats (
P﹤ 0.05), warming YANG blood circulation promoting recipe group, losartan group and warming YANG blood circulation promoting recipe+losartan group express all higher than model group (
P﹤ 0.05).Comparing difference not statistically significant between three treatment groups (
P﹥ 0.05).As shown in Figure 7, compare * with sham operated rats
P＜0.05, * *
P＜0.01; Compare # with model group
P＜0.05, ##
P＜0.01.
TIMP-1 expresses: TIMP-1 the expression of model group apparently higher than sham operated rats (
P﹤ 0.01), warming YANG blood circulation promoting recipe group, losartan group and warming YANG blood circulation promoting recipe+losartan group express all lower than model group (
P﹤ 0.05).Comparing difference not statistically significant between three treatment groups (
P﹥ 0.05).As shown in Figure 8, compare * with sham operated rats
P＜0.05, * *
P＜0.01; Compare # with model group
P＜0.05, ##
P＜0.01.
The warming YANG blood circulation promoting recipe has the protecting renal function effect to chronic renal failure rats, can delay renal failure progress, and the Renal Morphology that reduces the excretion of urine protein, urine NAG and alleviate chronic renal failure changes.
The mechanism that the warming YANG blood circulation promoting recipe delays the renal failure progress may and raise MMP-9 and express relevant with its downward nephridial tissue HIF-1 α, TGF-β 1, TIMP-1 expression.
Embodiment three: the research of warming YANG blood circulation promoting recipe control Chronic Aristolochic Acid Nephropathy mechanism.
Lack understanding for present Fructus Aristolochiae nephropathy pathogenesis, simultaneously for the deficiency for the treatment of, the present invention can provide a kind of Therapeutic Method of novel therapeutic Fructus Aristolochiae nephropathy by the warming YANG blood circulation promoting recipe, and the method can better improve the present Research of present Fructus Aristolochiae treatment of kidney disease curative effect.
The present embodiment comprises experiment and experiment in vitro two parts in body.
Wherein, in body, the purpose of experiment is: research rats with chronic aristolochic acid nephropathy renal ischaemia damage mechanism, analyze may concerning of kidney blood capillary change and injury of renal tubular, apoptosis and interstitial fibrosis; Research Ang-1, Ang-2, Tie-2, VEGF, BMP-7, Caspase-3, the expression of CD34 in renal tissues of rats, clear and definite its effect in the damage of rats with chronic aristolochic acid nephropathy renal ischaemia.The mechanism of research warming YANG blood circulation promoting recipe control Chronic Aristolochic Acid Nephropathy is for the clinical practice the party provides theory and experimental basis.The purpose of experiment in vitro is: inquire into Aristollchic acid sodium salt (AA-Na) to the damaging action mechanism of renal cells; Inquire into the intervention effect mechanism of warming YANG blood circulation promoting recipe Contained Serum.
One. experimental technique.
Experiment in 1 body: flow process as shown in Figure 9.
48 of healthy male SD rats, adaptability is divided into 5 groups according to body weight by table of random number, i.e. Normal group after raising a week; Model group; Warming YANG blood circulation promoting recipe group; The losartan group; (warming YANG blood circulation promoting recipe+losartan) group.Wherein Normal group is 8, all the other every group each 10.Model group is carried out modeling, and method is as described in embodiment two.
Be handled as follows again after modeling: (1) Normal group: with normal saline gavage (10ml.kg
-1.d
-1); (2) model group: by Caulis Aristolochiae Manshuriensis decocting liquid 10ml.kg
-1.d
-1(be equivalent to Caulis Aristolochiae Manshuriensis 40g.kg
-1.d
-1, aristolochic acid A 2.6mg.kg
-1.d
-1) to rat oral gavage; (3) warming YANG blood circulation promoting recipe group (Chinese drug-treated group): on the model group basis, then give warming YANG blood circulation promoting recipe 30g.kg
-1.d
-1Gavage; (4) losartan group (Western medicine group): on the model group basis, then give losartan 33.3mg.kg
-1.d
-1Gavage; (5) (warming YANG blood circulation promoting recipe+losartan) group (Chinese medicine and western medicine in conjunction with group): on the model group basis, then give (warming YANG blood circulation promoting recipe 30g.kg
-1.d
-1+ losartan 33.3mg.kg
-1.d
-1) gavage.
At 21 weekends, collect rat urine and measure twenty-four-hour urine albumen, NAG, β 2-MG; Abdominal aortic blood is measured Scr, BUN, RBC, Hb; The electron microscopic observation Pathological; SABC detects the expression of nephridial tissue Caspase-3 and BMP-7; Adopt the CD34 immunohistochemical staining to reflect the microvascular degree of impairment of nephridial tissue; PCR in real time detects the expression of Ang-l, Ang-2, Tie-2 and VEGFmRNA in nephridial tissue.
2 experiment in vitro: flow process is as shown in Figure 10-1 and Figure 10-2.
1) observe variable concentrations AA-Na(0,5,10,20,40 μ gml under Electronic Speculum
-1) lower rat proximal renal tubular epithelial cells (NRK-52E) form of stimulation and Ultrastructural variation.
2) mtt assay detects Aristolochic Acid (AA) to the impact of NRK-52E cell proliferation, and flow cytometer detects the apoptotic index of NRK-52E cell.
3) immunocytochemical method is observed NRK-52E cellular expression α-smooth muscle actin (situation of change of α-SMA).
4) the ELISA method detects the situation of change of NRK-52E emiocytosis NTx (COL-I).
5) experimental technique that adopts Serum Pharmacology of Chinese Herbal Drugs and cell in vitro to cultivate uses Real-Time PCR method to detect Contained Serum to NRK-52E cellular expression transforming growth factor (TGF-β
1), the impact of VEGF (VEGF), endothelin-1 (ET-1), bone morphogenetic protein (BMP-7) and apoptotic proteins (Caspase-3).
6) the ELISA method detects under the Contained Serum intervention, the situation of change of NRK-52E emiocytosis COL-I.
Two. experimental result.
1. experiment in body.
(1) rat biochemical indicator result: see Table 22 and table 23 shown in.
Compare with Normal group, all obviously risings of model group Rat 24 h urine protein, NAG, β 2-MG, blood Scr, BUN (
P＜0.05), and blood Hb, RBC all obviously descend (
P＜0.01); Compare with model group, each treatment group Rat 24 h urine protein, NAG, β 2-MG, blood Scr, BUN all obviously descend (P＜0.05), and all obviously risings of blood RBC, Hb (
P＜0.05).
Table 22 respectively organize rat body weight, 24h urine protein, urine NAG, urine β 2-MG comparison (
± s).
| Grouping | Body weight (g) | Urine protein (mg/24h) | Urine NAG (μ g/24h) | Urine β 2-MG (μ g/24h) |
| Normal group (7) | 546.00±37.26 | 5.23±1.30 | 2.54±2.42 | 0.89±1.46 |
| Model group (8) | 368.5±29.74 ## | 20.90±2.85 ## | 7.91±2.02 ## | 16.31±9.75 # |
| Warming YANG blood circulation promoting recipe group (8) | 426.75±36.92 △△ | 8.53±2.27 △△ | 5.16±2.23 △ | 2.23±2.10 △ |
| Losartan group (6) | 394.33±29.49 | 9.03±2.10 △△ | 3.35±1.58 △△ | 6.95±5.31 |
| Warming YANG blood circulation promoting recipe+losartan group (6) | 419.33±24.02 △△ | 8.33±2.82 △△ | 5.15±2.37 △ | 3.82±1.96 △ |
Annotate: compare with matched group
# P＜0.05,
## P＜0.01; Compare with model group
△ P＜0.05,
△ △ P＜0.01.
Table 23 respectively organize rat serum Scr, BUN, HB, RBC comparison (± s).
| Grouping | Scr(μmol/L) | BUN(mmol/L) | HB(g/L) | RBC(10^12/L) |
| Normal group (7) | 29.43±2.64 | 8.37±0.88 | 129.57±10.88 | 7.07±0.84 |
| Model group (8) | 50.00±7.16 ## | 13.63±2.04 ## | 89.83±10.36 ## | 4.71±0.58 ## |
| Warming YANG blood circulation promoting recipe group (8) | 40.13±5.03 △△ | 9.46±1.94 △△ | 106.38±10.17 △ | 5.80±0.77 △△ |
| Losartan group (6) | 45.50±10.50 | 9.76±0.89 △△ | 95.67±18.36 | 5.08±0.78 |
| Warming YANG blood circulation promoting recipe+losartan group (6) | 39.00±2.68 △△ | 8.37±2.57 △△ | 110.17±8.47 △△ | 5.89±0.55 △△ |
Annotate: compare with matched group
# P＜0.05,
## P＜0.01; Compare with model group
△ P＜0.05,
△ △ P＜0.01.
(2) rat kidney pathological examination: referring to Figure 11 ~ Figure 13.
After 21 weeks of administration, can find out Normal group nephridial tissue structure normal from Electronic Speculum; The model group glomerule is the shrinkage of ischemic basement membrane, the renal cells degeneration, and necrosis comes off, and basement membrane is exposed, the atrophy of part renal tubules or disappearance, the kidney ID becomes the district a large amount of pencil collagen fiber; The visible glomerular basement membrane ischemic of each treatment group shrinkage degree, renal cells degeneration, necrosis and interstitial fibrosis degree all alleviate.
(3) renal tissues of rats immunohistochemical staining result: be shown in Table 24, and referring to Figure 14 ~ Figure 16.
Compare with Normal group, model group nephridial tissue Caspase-3 increasing expression (
P＜0.05), and BMP-7 express to reduce (
P＜0.01); Compare with model group, each treatment group nephridial tissue Caspase-3 expression minimizing (
P＜0.05), and the BMP-7 increasing expression (
P＜0.05).
CD34 is great expression in the Normal group renal tubular interstitium, and model group express to reduce, with normal group relatively have statistical significance (
P＜0.01); After giving warming YANG blood circulation promoting recipe and losartan therapeutic intervention, the expression of CD34 in renal tubular interstitium than model group obviously increase (
P＜0.05), do not have between each treatment group statistical significance (
P0.05).Prompting: with Normal group relatively, model group kidney microvasculatures obviously reduces, it is lighter that each treatment group and model group are compared microvascular lesions.
Table 24 respectively organize the positive relative area of renal tissues of rats BMP-7, Caspase-3 comparison (
± s).
| Grouping | BMP-7 | Caspase-3 | CD34 |
| Normal group (7) | 6.75±1.83 | 3.18±2.57 | 9.14±3.09 |
| Model group (8) | 2.57±0.89 ## | 9.17±2.67 # | 3.05±0.88 ## |
| Warming YANG blood circulation promoting recipe group (8) | 4.74±0.67 △△ | 3.56±1.30 △ | 5.82±0.83 △△ |
| Losartan group (6) | 4.28±0.78 △ | 3.22±0.83 △ | 5.48±0.94 △ |
| Warming YANG blood circulation promoting recipe+losartan group (6) | 4.53±0.79 △△ | 4.61±1.97 △ | 5.56±1.38 △ |
Annotate: compare with matched group
# P＜0.05,
## P＜0.01; Compare with model group
△ P＜0.05,
△ △ P＜0.01.
(4) renal cortex of rats Real-time PCR testing result: be shown in Table 25.
Model group and normal group matched group compare, the down-regulated expression of nephridial tissue Ang-1, Tie-2 and VEGFmRNA (
P＜0.05), and the up-regulated of Ang-2mRNA (
P＜0.01); Compare with the model group rat, the up-regulated of each treatment group renal tissues of rats Ang-1, Tie-2 and VEGFmRNA (
P＜0.05), the down-regulated expression of Ang-2mRNA (warming YANG blood circulation promoting recipe and losartan associating) (
P＜0.01).
Table 25 respectively organize rat Ang-l, Ang-2, Tie-2, VEGFmRNA comparison (
± s).
| Grouping | Ang-l | Ang-2 | Tie-2 | VEGF |
| Normal group (7) | 0.0089±0.0037 | 0.0025±0.0019 | 0.0015±0.0004 | 0.0058±0.0029 |
| Model group (8) | 0.0014±0.0004 ## | 0.0076±0.0031 ## | 0.0005±0.0002 # | 0.0007±0.0003 # |
| Warming YANG blood circulation promoting recipe group (8) | 0.0041±0.0011 △△ | 0.0065±0.0026 | 0.0014±0.0011 △ | 0.0052±0.0016 △△ |
| Losartan group (6) | 0.0077±0.0022 △△ | 0.0073±0.0032 | 0.0015±0.0008 △△ | 0.0046±0.0014 △△ |
| Warming YANG blood circulation promoting recipe+losartan group (6) | 0.0085±0.0048 △ | 0.0035±0.0014 △△ | 0.0025±0.0008 △△ | 0.0060±0.0019 △△ |
Annotate: compare with matched group
# P＜0.05,
## P＜0.01; Compare with model group
△ P＜0.05,
△ △ P＜0.01.
(5) the correlation analysis result between CD34 and Ang-l, Ang-2, Tie-2 and VEGFmRNA: the expression of the expression of nephridial tissue CD34 and Ang-l, Tie-2 and VEGFmRNA is remarkable positive correlation (r=0.796 in the Chronic Aristolochic Acid Nephropathy model, r=0.8, r=0.817
P＜0.01), with the Ang-2mRNA expression be remarkable negative correlation (r=-0.653,
P＜0.01).Therefore, point out the microvascular damage loss of our Chronic Aristolochic Acid Nephropathy nephridial tissue closely related with Ang-l, Ang-2, Tie-2 and VEGF.
2 experiment in vitro.
(1) NRK-52E cell normal morphology: referring to Figure 17.
Normal rat renal cells (NRK-52E) is oval or nearly cube, is paving stone sample adherent growth, and iuntercellular connects closely, in flakes growth.
(2) transmission electron microscope observing renal cells ultrastructure is referring to Figure 18.
With 0,5,10,20,40 μ gml
-1After AA-Na stimulates NRK-52E cell 24h, the change of cell ultrastructure: 5 μ gml
-1The AA-Na group is basic identical with matched group, 10 μ gml
-1AA-Na and 20 μ gml
-1The AA-Na group mitochondrial swelling occurs and mitochondrial crista gradually disappears, cell membrane foamed phenomenon, 40 μ gml
-1Nuclear membrane is curling thickens for AA-Na group, the karyopycnosis phenomenon.
(3) proliferation function of mtt assay detection AA to renal cells: be shown in Table 26.
AA-Na concentration is at 5 μ gml
-1, 10 μ gml
-1The time, to the propagation of NRK-52E have no significant effect (
P0.05), when AA-Na concentration at 20 and 40 μ gml
-1The time, with 0 μ gml
-1AA-Na organizes relatively, all significantly reductions of absorbance (
P＜0.01), cell proliferation all has been subject to inhibition in various degree, and its depression effect is dose dependent, may be relevant with the cytotoxicity of Aristolochic Acid, and as seen, AA-Na concentration is at 10 μ gml
-1The time, on cell proliferation have no significant effect (
P0.05).
The impact that table 26. AA breeds NRK-52E (n=6,
± s).
Annotate: with matched group (0 μ gml
-1) relatively, * *
P＜0.01.
(4) flow cytometer detects apoptotic ratio: be shown in Table 27.
After the AA-Na irritation cell 24h of variable concentrations, compare 5,10 μ gml with Normal group
-1AA-Na is without obviously short cells apoptosis, 20 μ gml
-1AA-Na have significantly short cells apoptosis (
P＜0.05), especially with 40 μ gml
-1AA-Na group the most obviously (
P＜0.01), 40 μ gml
-1AA-Na has had very strong short cells apoptosis, and apoptosis rate is greater than 80%.AA-Na concentration is at 10 μ gml
-1The time, to cell without obvious apoptosis-promoting effect (
P0.05).MTT experiment by the front draws AA-Na concentration at 10 μ gml
-1The time, to cell without obvious proliferation (
P0.05).Therefore, we select 10 μ gml
-1The AA-Na of concentration carries out following intervention test.
Table 27 AA-NA apoptosis-promoting effect (n=3,
± s).
Annotate: with matched group (0 μ gml
-1) relatively, *
P＜0.05, * *
P＜0.01.
(5) immunocytochemical method detects α-smooth muscle actin (expression of α-SMA) is referring to Figure 19.
Result shows that α-SMA mainly is expressed in endochylema.Matched group minute quantity express alpha-SMA, the AA-Na of each concentration all can stimulate NRK-52E cellular expression α-SMA, and along with the rising of Aristolochic Acid concentration, the expression of α-SMA strengthens gradually.
After the AA-Na of variable concentrations acted on cell, the AA-Na of high concentration caused a large amount of cellular swellings, necrosis, and each organizes the cell number obvious difference, used Image analysis system also can't add up accurately, and still, what Figure 19 can be clear and definite describes the problem.
(6) RT-PCR detects AA-Na stimulates NRK-52E cell different time (12,24,48h) correlation factor m rna expression situation: be shown in Table 28.
Result shows, TGF-β
1MRNA reach when 24h the peak (
P＜0.01), during 48h, expression descend during than 24h (
P＜0.05), ET-1 mRNA 24h express the highest (
P＜0.05), during 48h, the ET-1 mrna expression has downward trend, may be relevant with the AA toxicity of high concentration.Therefore in the test of Contained Serum intervention below, detect TGF-β
1When mRNA and ET-1 mRNA, with AA-Na(10 μ gml
-1) adding the Contained Serum intervention after irritation cell 24h, intervention time also is made as 24h.
Table 28 AA-Na(10 μ gml
-1) on the impact of NRK-52E cell correlation factor mrna expression (n=6,
± s).
Compare with 12h,
* p0.05,
* p0.01, compare with 24h
p＜0.05.
(7) the ELISA method detects COL-I(0,12,24,48h) expression: be shown in Table 29.
AA-Na(10 μ gml
-1) result shows after the irritation cell different time: with Normal group (0 μ gml
-1) relatively, 12,24, three time point extracellular matrix COL-I of place of 48h express all and raise (
P＜0.01), with the 12h time point relatively, 24,48h group COL-I express significantly and raise (
P＜0.01), with 24h group relatively, during the 48h group COL-I expression significantly raise (
P＜0.01).Therefore in the intervention test of back, AA-Na adds the Contained Serum intervention after stimulating NRK-52E cell 48h, intervention time also is made as 48h.
Table 29 COL-I(0,12,24,48h) expression (n=6,
± s).
Compare with normal group
* p0.01, compare with the 12h group
p0.01, compare with the 24h group
## p＜0.01.
(8) mtt assay detects Contained Serum to the impact of rat renal tubular epithelial cells propagation: see Table 30 and table 31 shown in.
According to pertinent literature report and previous experiences; 10% rat blood serum on cell proliferation has no significant effect; therefore at first we select 10% Contained Serum to carry out intervention experiment; use mtt assay and detect its on cell proliferation impact; then; we are set as Contained Serum again 5%, 10%, 20% 3 concentration, and whether 10% Contained Serum can play protective effect in checking.
Result shows, compares with Normal group, and AA-Na group cell proliferation is suppressed, absorbance decline (
P ﹤ 0.01), with AA-Na group relatively, 10% warming YANG blood circulation promoting recipe and losartan Contained Serum can alleviate the inhibitory action of AA-Na on cell proliferation, 5% Chinese medicine Contained Serum to cell have no significant effect (
P〉0.05), 10% Chinese medicine Contained Serum group can alleviate the toxic action of Aristolochic Acid, the promotion cell proliferation (
P ﹤ 0.01), therefore select 10% Contained Serum to intervene test.
Table 30 respectively organize Contained Serum to the inhibitory action of NRK-52E propagation (n=6,
± s).
Compare with Normal group, △ △ P＜0.01 is compared in * * P＜0.01 with the AA-Na group.W warming YANG blood circulation promoting recipe; The L losartan.
The inhibitory action that table 31 variable concentrations Contained Serum is bred NRK-52E (n=6,
± s).
Compare with Normal group,
* P＜0.01,Compare with the AA-Na group,
△ △ P＜0.01W warming YANG blood circulation promoting recipe.Compare with Normal group,
* P＜0.01Compare with the AA-Na group,
△ △ P＜0.01W warming YANG blood circulation promoting recipe.
(9) Real-Time PCR detects the correlation factor expression.
AA-Na(10 μ gml
-1) and after Contained Serum intervened, each organized rat renal tubular epithelial cells TGF β
1The expression of mRNA: be shown in Table 32.
After Contained Serum is intervened 24h, compare AA-Na group rat renal tubular epithelial cells TGF-β with Normal group
1The mrna expression rise (
P ﹤ 0.05), compare warming YANG blood circulation promoting recipe group TGF β with the AA-Na group
1The mrna expression downward (
P ﹤ 0.05), the losartan group has down regulation trend, but the obvious statistical significance of nothing (
P〉0.05).
Table 32 is respectively organized rat renal tubular epithelial cells TGF-β
1The expression of mRNA (n=6,
± s).
Compare with Normal group,
* P ﹤ 0.05, compare with the AA-Na group,
△ P＜0.05.
AA-Na(10 μ gml
-1) and after Contained Serum intervened, each organized the expression of rat renal tubular epithelial cells VEGF m RNA: be shown in Table 33.
After Contained Serum is intervened 24h, with Normal group relatively, AA-Na group rat renal tubular epithelial cells VEGF m rna expression significantly lower (
P ﹤ 0.01), compare with the AA-Na group, warming YANG blood circulation promoting recipe group (
P ﹤ 0.05) and the losartan group (
P ﹤ 0.01) VEGF m rna expression level significantly improves, and statistical significance is arranged.
Table 33 respectively organize rat renal tubular epithelial cells VEGF m RNA expression (n=6,
± s).
Compare with Normal group,
* P＜0.01, compare with the AA-Na group,
△ △ P＜0.01
AA-Na(10 μ gml
-1) and after Contained Serum intervened, each organized the expression of rat renal tubular epithelial cells ET-1 mRNA: be shown in Table 34.
After Contained Serum is intervened 24h, with normal group relatively, the expression of AA-Na group ET-1 mRNA significantly raise (
P ﹤ 0.01), compare with the AA-Na group, the expression downward of losartan group ET-1 mRNA (
P＜0.05), warming YANG blood circulation promoting recipe group has down regulation trend, but without obvious statistical significance.
Table 34 respectively organize rat renal tubular epithelial cells ET-1 mRNA expression (n=6,
± s).
Compare with Normal group,
* P＜0.01, compare with the AA-Na group,
△ △ P＜0.01
AA-Na(10 μ gml-1) and after Contained Serum intervenes, each organizes the expression of rat renal tubular epithelial cells BMP-7 mRNA: be shown in Table 35.
After Contained Serum is intervened 24h, with normal group relatively, AA-Na group BMP-7 mrna expression level significantly reduce (
p﹤ 0.01), compare with the AA-Na group, all significantly rises of warming YANG blood circulation promoting recipe group and losartan group expression (
p﹤ 0.01), statistical significance is arranged.
Table 35 respectively organize rat renal tubular epithelial cells BMP-7 mRNA expression (n=6,
± s).
Compare with Normal group,
* P＜0.01, compare with the AA-Na group,
△ △ P＜0.01
AA-Na(10 μ gml
-1) and after Contained Serum intervened, each organized rat renal tubular epithelial cells Caspases-3 mrna expression situation: be shown in Table 36.
After Contained Serum is intervened 24h, with Normal group relatively, AA-Na group Caspases-3 mrna expression level significantly raise (
P ﹤ 0.01), with AA-Na group relatively, warming YANG blood circulation promoting recipe group Caspases-3 mrna expression level significantly reduce (
P ﹤ 0.01), the downward of losartan group Caspases-3 mrna expression (
P ﹤ 0.05).
Table 36 respectively organize rat renal tubular epithelial cells Caspases-3 mrna expression situation (n=6,
± s).
Compare with Normal group,
* P＜0.01, compare with the AA-Na group,
△ P＜0.05,
△ △ P＜0.01.
ELISA AA-Na(10 μ gml
-1) and after Contained Serum intervened, each organized rat renal tubular epithelial cells COL-I expression: be shown in Table 37.
After Contained Serum is intervened 48h, with Normal group relatively, AA-Na group COL-I expression be significantly higher than Normal group (
P ﹤ 0.01), with AA-Na group relatively, warming YANG blood circulation promoting recipe group COL-I expression obviously lower (
P ﹤ 0.01).Losartan group COL-I expression obviously lower (
P ﹤ 0.01).
Table 37 respectively organize rat renal tubular epithelial cells COL-I expression (n=6,
± s).
Compare with Normal group,
* P＜0.01, compare with the AA-Na group,
△ △ P＜0.01
Three. conclusion.
Experiment in 1 body.
(1) Chinese herbal medicine that contains Aristolochic Acid such as Caulis Aristolochiae Manshuriensis can cause renal damage, causes the excretion level of renal hypofunction, anemia and urine protein, NAG and β 2-MG to raise.
(2) there is the downright bad apoptosis of the microvascular damage loss of kidney and renal tubules in Chronic Aristolochic Acid Nephropathy, this may be relevant with Ang-2 expression rising with nephridial tissue Ang-1, BMP-7, Tie-2, vegf expression minimizing and Caspase-3, and the above-mentioned factor finally starts and promoted progression of fibrosis jointly.
(3) the warming YANG blood circulation promoting recipe has the kidney protective effect to Chronic Aristolochic Acid Nephropathy; can improve renal function; alleviate anemia; reduce the excretion of urine protein, urine NAG and β 2-MG; can alleviate the Renal Morphology that Aristolochic Acid causes and change, its part protection mechanism may be relevant with the expression of the expression of its minimizing nephridial tissue Caspase-3, Ang-2mRNA and rising BMP-7, Ang-1mRNA, Tie-2mRNA, VEGFmRNA.
2 experiment in vitro.
(1) AA can suppress the NRK-52E cell proliferation, promotes its apoptosis, and this effect is dose dependent, and higher concentration (＞20 μ gml-1) has remarkable inhibition propagation and apoptosis-promoting effect.
(2) along with the rising of Aristolochic Acid concentration, the expression of α-SMA strengthens gradually, 10 μ gml
-1AA-Na can significantly raise the expression of COL-I, prolongation is in time expressed gradually and is strengthened, and illustrates that AA-Na has promoted Renal Tubular Epithelial Cells Transdifferentiationin, has accelerated the interstitial fibrosis process.
(3) AA-Na stimulates the expression of correlation factor after NRK-52E cell different time (12,24,48h) along with the prolongation of AA-Na stimulation time, and the COL-I expression rises gradually, is time dependence, TGF-β
1MRNA and ET-1mRNA expression when 24h is the highest, and downward trend is arranged during 48h, may be relevant with the toxicity of high concentration AA-Na.
(4) 10% Chinese medicines (warming YANG blood circulation promoting recipe) but the toxic action of Contained Serum group antagonism AA-Na; promote cell proliferation; having brought into play it plays a protective role to renal cells; 5% Contained Serum group has no significant effect cell, and 10% normal rat Contained Serum has no significant effect the effect that AA-Na suppresses cell proliferation.
(5) the warming YANG blood circulation promoting recipe can be lowered rat renal tubular epithelial cells TGF-β
1The expression of mRNA, ET-1 mRNA, Caspases-3 mRNA, COL-I; raise VEGF m RNA and BMP-7 mrna expression level; the warming YANG blood circulation promoting recipe may be by the regulation and control to these correlation factors; bring into play its antagonism Aristolochic Acid to the detrimental effect of renal cells; reduce extracellular matrix build-up; the protection blood vessel endothelium promotes epithelium regeneration, the effect of antagonism kidney region fibrosis.
(6) losartan group and alleviating the Aristolochic Acid nephrotoxicity, anti-interstitial fibrosis aspect also have curative effect in various degree.
Embodiment four: the intervention of warming YANG blood circulation promoting recipe to the IgAN glomerular sclerosis rat model.
IgAN glomerular sclerosis rat model for present Staphylococcal Enterotoxin B (SEB) MOI associating 5/6 nephrectomy making, the present invention delays by the warming YANG blood circulation promoting recipe Therapeutic Method that IgAN glomerular sclerosis model can provide a kind of novel therapeutic IgAN nephropathy, the method can better delay the progress of IgAN glomerular sclerosis model, improves renal function.
Choose 40 of healthy male SD rats, adaptability is divided into 5 groups according to body weight by table of random number after raising a week.Be Sham-operated control group (N); Model group (M); Warming YANG blood circulation promoting recipe group (W); Losartan group (L); Warming YANG blood circulation promoting recipe+losartan group (W+L).
Sham-operation is identical with embodiment two methods.
Model group: SEB MOI associating 5/6 nephrectomy.At first carry out MOI, namely after SD male rat adaptability raised for 1 week, give 40mg/ml BSA acidified aqueous solution gavage next day of beginning, 2-4 week is weekly from rat penile vein injection SEB 0.08mg/ml.qw with from 0.2ml/ .qw of lumbar injection complete Freund's adjuvant.Then carry out 5/6 nephrectomy, identical in method and embodiment two.
Sham-operated control group (N): with normal saline gavage (10ml.kg
-1.d
-1); Model group (M): method is the same; Warming YANG blood circulation promoting recipe group (W): on the model group basis, then give warming YANG blood circulation promoting recipe 30g.kg
-1.d
-1Gavage; Losartan group (L): on the model group basis, then give losartan 33.3mg.kg
-1.d
-1Gavage; Warming YANG blood circulation promoting recipe+losartan group (W+L): on the model group basis, then give (warming YANG blood circulation promoting recipe 30g.kg
-1.d
-1+ losartan 33.3mg.kg
-1.d
-1) gavage.
12 weekends, collect experimental rat blood, urine and renal tissue, inquire into the intervention effect of warming YANG blood circulation promoting recipe.Experiment flow is shown in Figure 20.
Experimental data and result are as follows.
1 blood/urine biochemistry detection result.
(1) respectively organize the variation of rat urine sediment RBC counting, 24h urine protein quantitation, urine CysC
Compare with sham operated rats: model group Urine sediments analyzer RBC measures showed increased, has significant statistical significance (P＜0.01); Compare with model group: warming YANG blood circulation promoting recipe group Urine sediments analyzer RBC measures minimizing, has significant statistical significance (P＜0.01), losartan group Urine sediments analyzer RBC measures also and reduces, and has statistical significance (P＜0.05), and combination of Chinese and Western medicine group not statistically significant (P〉0.05).See Table 38.
Table 38: rat urine sediment RBC counting (n=8, ± s).
| Grouping | (* 10 for arena RBC counting 4/ L) |
| The N group | 5.04 ± 0.62 |
| The M group | 15.50 ± 1.47 ★ ★ |
| The W group | 11.58 ± 1.00 △ △ |
| The L group | 14.15 ± 1.30 △ |
| The W+L group | 14.76 ± 1.57 |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
Compare with sham operated rats: model group 24h urine protein quantitation obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: losartan group and warming YANG blood circulation promoting recipe group 24h urine protein quantitation all reduce, and have statistical significance (P＜0.05), and combination of Chinese and Western medicine group 24h urine protein quantitation also reduces, and has significant statistical significance (P＜0.01).See Table 39.
Table 39: rat 24h urine protein (n=8, ± s).
| |
24h urinates albumen (mg/24h) |
| The N group | 5.46 ± 0.76 |
| The M group | 35.00 ± 4.25 ★ ★ |
| The W group | 30.34 ± 4.27 △ |
| The L group | 26.25 ± 4.34 △ △ |
| The W+L group | 27.25 ± 2.14 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
Compare with sham operated rats: model group urine CysC obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: warming YANG blood circulation promoting recipe group urine CysC reduces, has significant statistical significance (P＜0.01), losartan group urine CysC also reduces, and has significant statistical significance (P＜0.01), and combination of Chinese and Western medicine group not statistically significant (P〉0.05).See Table 40.
Table 40: rat urine CysC (n=8, ± s).
| Grouping | Urine Cys C (μ g/L) |
| The N group | 548.44 ± 22.05 |
| The M group | 798.11 ± 31.78 ★ ★ |
| The W group | 713.64 ± 26.00 △ △ |
| The L group | 718.33 ± 53.96 △ △ |
| The W+L group | 744.61 ± 38.44 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(2) respectively organize the variation of rat blood serum BUN, serum Scr, change of serum C ysC.
Compare with sham operated rats: model group serum BUN level obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: warming YANG blood circulation promoting recipe group serum Scr level reduces, has significant statistical significance (P＜0.01), combination of Chinese and Western medicine group serum Scr level also reduces, and has significant statistical significance (P＜0.01), and losartan group not statistically significant (P〉0.05).See Table 41.
Table 41: rat blood serum BUN (n=8, ± s).
| Grouping | Serum BUN (mmmol/L) |
| The N group | 5.40 ± 0.52 |
| The M group | 19.15 ± 1.42 ★ ★ |
| The W group | 15.25 ± 1.41 △ △ |
| The L group | 18.88 ± 0.76 |
| The W+L group | 14.15 ± 0.81 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
Compare with sham operated rats: model group serum Scr level obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group serum BUN level reduces, and has significant statistical significance (P＜0.01).See Table 42.
Table 42: rat blood serum Scr (n=8, ± s).
| Grouping | Serum Scr (μ mol/L) |
| The N group | 25.75 ± 1.83 |
| The M group | 67.13 ± 3.83 ★ ★ |
| The W group | 53.75 ± 5.87 △ △ |
| The L group | 49.50 ± 3.07 △ △ |
| The W+L group | 49.75 ± 4.43 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
Compare with sham operated rats: model group change of serum C ysC level obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group change of serum C ysC level all reduces, and has significant statistical significance (P＜0.01).See Table 43.
Table 43: rat blood serum CysC (n=8, ± s).
| Grouping | Urine Cys C (μ g/L) |
| The N group | 596.13 ± 34.28 |
| The M group | 1107.54 ± 98.01 ★ ★ |
| The W group | 891.95 ± 91.20 △ △ |
| The L group | 950.22 ± 97.35 △ △ |
| The W+L group | 956.49 ± 89.87 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
2 renal tissue morphological observation results.
(1) expression of immunofluorescence IgA, IgG, IgM, C3.
The sham operated rats immunofluorescence is observed IgA, IgG, IgM, the C3 expression in glomerule and renal tubules is all not obvious; Model group is observed and is shown with IgA in obviously enhancing of glomerular mesangium district's expression, the visible IgA in subregion deposits along Capillary loops, and can accompany and see that a small amount of IgM, IgG and C3 deposit expression in glomerule and renal tubules, the subregion can be accompanied and be seen that C3 is deposited on medium and small blood vessel wall; Each treatment group is observed and is shown that IgA, IgG, IgM, C3 deposit expression and all weaken to some extent than model group in glomerule, renal tubules and blood vessel.See Figure 21.
(2) om observation.
Sham operated rats glomerule, renal tubulointerstitial, kidney blood vessel structure normal; The visible focal or diffusivity mesangial region proliferation of mesangial cells of model group and extracellular matrix increase, mesangial region broadening, sacculus is glutinous to be connected, and the interior Capillary loops of glomerule subsides very downright bad, FSGS, the degeneration of renal cells cavity sample, visible focal renal tubules atrophy in matter between tubule, massive inflammatory cells infiltrated, renal cells atrophy, Tubulointerstitial fibrosis changes, and in the subregion, visible vessels intimal fibrosis and hyaloid thereof change.Each treatment group rats with focal or diffusivity mesangial region proliferation of mesangial cells and extracellular matrix increase, mesangial region broadening, sacculus is glutinous to be connected, in glomerule, Capillary loops subsides very downright bad, FSGS, the renal cells degeneration, necrosis comes off, and the visible inflammatory cell infiltration bead of renal tubular interstitium and Tubulointerstitial fibrosis degree alleviate than MXZ.See Figure 22.
The variation of PAS dyeing blood capillary openness and extracellular matrix relative area: compare with sham operated rats, model group blood capillary openness obviously descends, and the extracellular matrix relative area obviously increases, and has significant statistical significance (P＜0.01); With model group relatively, each treatment group blood capillary openness reduces, but each treatment group extracellular matrix relative area obviously reduces, and has significant statistical significance (P＜0.01).See Table 44.
Table 44: each organize blood capillary openness and extracellular matrix relative area variation (n=6, ± s).
| Grouping | The capillary openness | The extracellular matrix relative area |
| The N group | 9.52 ± 1.26 | 7.57 ± 0.95 |
| The M group | 3.76 ± 0.55 ★ ★ | 44.60 ± 5.42 ★ ★ |
| The W group | 7.24 ± 0.54 △ △ | 32.35 ± 3.40 △ △ |
| The L group | 6.14 ± 0.48 △ △ | 32.56 ± 3.90 △ △ |
| The W+L group | 6.25 ± 0.70 △ △ | 35.96 ± 5.52 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(2) transmission electron microscope observing.
sham operated rats glomerule clear in structure, filter membrane is complete, and the epithelial cell podocytic process is arranged in order, have no fusion, the subpodocytic space is obvious, and capillary lumen is full, basement membrane has no and thickens, and BC structure normal, epithelial cell podocytic process are arranged in order, amixis, extracellular matrix have no and increase, the renal tubules structural integrity, nucleus is justified greatly, microvillus clear in structure, more complete, and structure of mitochondria is complete, mitochondrial crista is abundant, and a small amount of endoplasmic reticulum is arranged in kytoplasm, the bending of model group glomerule local loop counterdie is homogenizing and thickens, fracture and flaggy sample change, the lumen of vessels obvious stenosis, podocyte swelling, most of podocytic process merges, flatten, and misaligned, the part podocytic process comes off, mesangial cell thickens, the extracellular matrix expansion, mesangial region broadening, mesangial region has the graininess electron-dense thing deposition of bulk, even the electron-dense thing of subcutaneous bulk deposition in visible, Capillary loops subsides, the subregion glomerular sclerosis, the swelling of part renal tubules, the nuclei dyeing chromaticness increases, microvillus comes off, the subregion mitochondrial crista destroys, visible a large amount of cavitys in the epithelial cell endochylema, as seen renal tubular epithelial cell exfoliation, the renal tubules atrophy, kidney region fibrosis, each treatment group ultrastructure in kidney changes lighter.See Figure 23.
3 SABC and Real-time PCR testing result.
(1) the 1 protein expression index of the TGF-β in nephridial tissue.
Compare with sham operated rats: model group TGF β 1 SABC index obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group TGF β 1 SABC index is lowered, and has statistical significance (P＜0.05 or P＜0.01).See Table 45 and Figure 24.
The variation of table 45:TGF β 1 IHC index (n=6,
± s).
| | TGF β | 1 IHC index |
| The N group | 2.68 ± 0.50 | |
| The M group | 4.88 ± 0.55 ★ ★ | |
| The W group | 2.93 ± 0.37 △ △ | |
| The L group | 4.45 ± 0.60 △ | |
| The W+L group | 2.94 ± 0.34 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(2) FN protein expression index in nephridial tissue.
Compare with sham operated rats: model group FN SABC index obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group FN SABC index is lowered, and has significant statistical significance (P＜0.01).See Table 46 and Figure 25.
The variation of table 46:FN IHC index (n=6,
± s).
| Grouping | FN IHC index |
| The N group | 1.86 ± 0.35 |
| The M group | 4.93 ± 0.79 ★ ★ |
| The W group | 3.23 ± 0.30 △ △ |
| The L group | 3.68 ± 0.39 △ △ |
| The W+L group | 2.89 ± 0.44 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(3) α in nephridial tissue-SMA protein expression index.
Compare with sham operated rats: model group α-SMA SABC index obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: losartan group and combination of Chinese and Western medicine group α-SMA SABC index is lowered, and has significant statistical significance (P＜0.01), and warming YANG blood circulation promoting recipe group α-SMA SABC index variation not statistically significant (P〉0.05).See Table 47 and Figure 26.
The variation of table 47: α-SMA IHC index (n=6, ± s).
| Grouping | α-SMA IHC index |
| The N group | 1.29 ± 0.19 |
| The M group | 4.23 ± 0.79 ★ ★ |
| The W group | 3.87 ± 0.65 |
| The L group | 2.41 ± 0.49 △ △ |
| The W+L group | 3.18 ± 0.44 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(4) PDGF protein expression index in nephridial tissue.
Compare with sham operated rats: model group PDGF SABC index obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: being combined and organizing α-SMA SABC index downward in warming YANG blood circulation promoting recipe group and Chinese and Western, has significant statistical significance (P＜0.01), and losartan group α-SMA SABC index variation not statistically significant (P〉0.05).See Table 48 and Figure 27.
The variation of table 48:PDGF IHC index (n=6, ± s).
| Grouping | PDGF IHC index |
| The N group | 2.09 ± 0.27 |
| The M group | 3.43 ± 0.55 ★ ★ |
| The W group | 3.00 ± 0.34 △ △ |
| The L group | 3.29 ± 0.50 |
| The W+L group | 2.83 ± 0.27 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(5) FGF-1 protein expression index in nephridial tissue.
Compare with sham operated rats: model group FGF-1 SABC index obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group FGF-1 SABC index is lowered, and has significant statistical significance (P＜0.01).See Table 49 and Figure 28.
The variation of table 49:FGF-1 IHC index (n=6, ± s).
| Grouping | FGF-1 IHC index |
| The N group | 1.67 ± 0.26 |
| The M group | 5.26 ± 0.79 ★ ★ |
| The W group | 2.71 ± 0.37 △ △ |
| The L group | 4.27 ± 0.56 △ △ |
| The W+L group | 2.67 ± 0.36 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(6) expression of TGF-β 1 mRNA in nephridial tissue.
Compare with sham operated rats: model group TGF β 1 mrna expression level obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group TGF β 1 mrna expression level is down regulation trend, has statistical significance (P＜0.05 or P＜0.01).See Table 50.
The variation of table 50:TGF β 1 mrna expression level (n=6, ± s).
| | TGF β | 1 mRNA |
| The N group | 0.0580 ± 0.0148 | |
| The M group | 0.5545 ± 0.0373 ★ ★ | |
| The W group | 0.1883 ± 0.0286 △ △ | |
| The L group | 0.3202 ± 0.0429 △ △ | |
| The W+L group | 0.1497 ± 0.0210 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(7) expression of FN mRNA in nephridial tissue.
Compare with sham operated rats: model group FN mrna expression level obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group FN mrna expression level is down regulation trend, has significant statistical significance (P＜0.01).See Table 51.
The variation of table 51:FN mrna expression level (n=6, ± s).
| Grouping | FN IHC mRNA |
| The N group | 0.0209 ± 0.0067 |
| The M group | 0.2410 ± 0.0354 ★ ★ |
| The W group | 0.1526 ± 0.0295 △ △ |
| The L group | 0.1722 ± 0.0328 △ △ |
| The W+L group | 0.1522 ± 0.0413 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(8) expression of α in nephridial tissue-SMA mRNA.
Compare with sham operated rats: model group α-SMA mrna expression level obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: losartan group α-SMA mrna expression level is down regulation trend, has statistical significance (P＜0.05), and warming YANG blood circulation promoting recipe group and combination of Chinese and Western medicine group α-horizontal not statistically significant of SMA mrna expression (P〉0.05).See Table 52.
The variation of table 52: α-SMA mrna expression level (n=6, ± s).
| Grouping | α-SMA mRNA |
| The N group | 0.0069 ± 0.0014 |
| The M group | 0.0612 ± 0.0088 ★ ★ |
| The W group | 0.0531 ± 0.0125 |
| The L group | 0.0491 ± 0.0091 △ |
| The W+L group | 0.0558 ± 0.0117 |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(9) expression of PDGF mRNA in nephridial tissue.
Compare with sham operated rats: model group PDGF mrna expression level obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: warming YANG blood circulation promoting recipe group and combination of Chinese and Western medicine group PDGF mrna expression level are down regulation trend, have significant statistical significance (P＜0.01), and the horizontal not statistically significant of losartan group PDGF mrna expression (P〉0.05).See Table 53.
The variation of table 53:PDGF mrna expression level (n=6, ± s).
| Grouping | PDGF mRNA |
| The N group | 0.0447 ± 0.0110 |
| The M group | 0.3230 ± 0.0441 ★ ★ |
| The W group | 0.2018 ± 0.0388 △ △ |
| The L group | 0.3146 ± 0.0518 |
| The W+L group | 0.2177 ± 0.0536 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
(10) expression of FGF-1 mRNA in nephridial tissue.
Compare with sham operated rats: model group FGF-1 expression obviously raises, and has significant statistical significance (P＜0.01); Compare with model group: each treatment group FGF-1 mrna expression level is down regulation trend, has significant statistical significance (P＜0.01).See Table 54.
The variation of table 54:FGF-1 mrna expression level (n=6, ± s).
| Grouping | FGF-1mRNA |
| The N group | 0.0280 ± 0.0065 |
| The M group | 0.3485 ± 0.0576 ★ ★ |
| The W group | 0.1077 ± 0.0205 △ △ |
| The L group | 0.1373 ± 0.0376 △ △ |
| The W+L group | 0.1867 ± 0.0462 △ △ |
Annotate: compare with the N group:
★ P＜0.05,
★ ★ P＜0.01; Compare with the M group:
△ P＜0.05,
△ △ P＜0.01.
Conclusion: the warming YANG blood circulation promoting recipe can delay the progress of IgAN glomerular sclerosis model, has certain kidney protective effect, can improve renal function, reduces Urine sediments analyzer RBC counting, blood/urine CysC level; Its part protection mechanism may be relevant with the expression of its minimizing nephridial tissue downward TGF-β 1, FN, PDGF, FGF-1.
The invention provides a kind of warming YANG Huoxue Herbs compound preparation be used to preventing and treating chronic kidney disease, and studied its clinical efficacy and safety, to seek the optimizing therapeutic regimen of chronic kidney disease.Also by this compound Chinese medicinal preparation on the impact of chronic renal failure rats renal function, inquired into it and delayed possible the mechanism of action of disease progression and the mechanism of preventing and treating Chronic Aristolochic Acid Nephropathy, with and to the retarding action of IgAN glomerular sclerosis model.
Although content of the present invention has been done detailed introduction by above preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.After those skilled in the art have read foregoing, for multiple modification of the present invention with to substitute will be all apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (5)
1. one kind is used for preventing and treating the warming YANG Huoxue Herbs compound preparation of chronic kidney disease, it is characterized in that, the raw material of this compound Chinese medicinal preparation comprises following composition:
Herba Cistanches 15-30g, Herba Epimedii 15-30g, Semen Persicae 10-15g, Flos Carthami 10-15g and Cortex Moutan 10-15g.
2. the warming YANG Huoxue Herbs compound preparation be used to preventing and treating chronic kidney disease as claimed in claim 1, is characterized in that, the raw material of described compound Chinese medicinal preparation consists of the following composition:
Herba Cistanches 30g, Herba Epimedii 15g, Semen Persicae 15g, Flos Carthami 15g and Cortex Moutan 15g.
3. the warming YANG Huoxue Herbs compound preparation be used to preventing and treating chronic kidney disease as claimed in claim 1, is characterized in that, the usage of described compound Chinese medicinal preparation is with being decocted in water for oral dose.
4. the purposes of the warming YANG Huoxue Herbs compound preparation be used to preventing and treating chronic kidney disease as described in any one in claim 1 ~ 3, is characterized in that, described purposes comprises for the pathogenetic research of Aristolochic acid nephropathy.
5. the purposes of the warming YANG Huoxue Herbs compound preparation be used to preventing and treating chronic kidney disease as claimed in claim 4, is characterized in that, described purposes also comprises for the IgAN glomerular sclerosis rat model is intervened.
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