CN100493539C - The application of the Tibetan picrorhiza rhizome in preparation the medicine for treating kidney disease - Google Patents
The application of the Tibetan picrorhiza rhizome in preparation the medicine for treating kidney disease Download PDFInfo
- Publication number
- CN100493539C CN100493539C CNB2006100371844A CN200610037184A CN100493539C CN 100493539 C CN100493539 C CN 100493539C CN B2006100371844 A CNB2006100371844 A CN B2006100371844A CN 200610037184 A CN200610037184 A CN 200610037184A CN 100493539 C CN100493539 C CN 100493539C
- Authority
- CN
- China
- Prior art keywords
- group
- treatment
- prevention
- rhizoma picrorhizae
- kidney
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
This invention relates to new use of the Tibetan picrorhiza rhizome in preparation the medicine for treating kidney disease. Say it in detail is the application of the Tibetan picrorhiza rhizome in preparation of the medicine for treating kidney disease. The said kidney disease in this invention includes of chronic kidney disease, diabetic nephropathy, kidney damage coursed by acute ischemia reperfusion. It is prepared by using abstracts of the distilled from Tibetan picrorhiza rhizome roots. The dosage form is oral dosage permitted in the pharmacy. It has been proved through various experiments on the animals that the Tibetan picrorhiza rhizome has nice curative effects to man various kidney disease. As the Tibetan picrorhiza rhizome has a broad resource, easy collect, preparing method of their abstracts is easy without costly and valuable instruments and reagents, so the invention has provided a kind of medicine for treating kidney disease.
Description
Technical field
The present invention relates to the new purposes of Rhizoma Picrorhizae, the particularly application of Rhizoma Picrorhizae in preparation treatment kidney disease medicine.
Background technology
It is very common kidney disease that chronic renal disease, diabetic nephropathy, acute ischemia pour into kidney injury of causing etc. again.In China, the crowd of diabetes, kidney disease is increasing, has become the most common disease.Therefore, the control of strengthening above-mentioned disease is China and even global problem demanding prompt solution.But, up to now, still do not have effective means of prevention and medicine at above-mentioned disease.Domestic and international research is found, the common Pathophysiology characteristics that above-mentioned disease all has promptly all exist oxidation reaction to strengthen, just oxidative stress status.Studies confirm that oxidative stress is not the satellite phenomenon of disease, but the infringement of mediation histoorgan participates in the key factor that development takes place pathological changes.
Rhizoma Picrorhizae is a kind of goatweed that originates in India, and its rhizome can be used as medicine.See Tang Materia Medica as Chinese crude drug beginning, have the infantile malnutrition effect that disappears of clearing away heat and cooling blood, dampness, be used for diseases such as pediatric epilepsy scared, infantile malnutrition, dysentery, hectic fever due to YIN-deficiency consumptive disease heat, spontaneous perspiration, night sweat, haematemesis.Before the seventies in 20th century, the Rhizoma Picrorhizae of China always the dependence on import main product in the Rhizoma Picrorhizae (Picrorhizakurrooa Royle ex Benth) of India.Nineteen sixty-five in the southeast, Tibet and northwestern Yunnan Province found Rhizoma Picrorhizae congener with India, aspects such as its crude drug form, tissue and nature and flavor and the former are closely similar, can replace India's Rhizoma Picrorhizae medication, called after Rhizoma Picrorhizae (Picrorhiza scrophulariifora Pennell)." Rhizoma Picrorhizae that records of Chinese pharmacopoeia is a Rhizoma Picrorhizae at present.Domestic research report and patent documentation about Rhizoma Picrorhizae all concentrates on its hepatoprotective effect, anti-diabetic activity etc., do not have this Chinese medicine is studied in the purposes aspect the kidney disease.
Summary of the invention
The object of the present invention is to provide the application of Rhizoma Picrorhizae extract in preparation treatment kidney disease medicine, opened up a kind of new purposes of Rhizoma Picrorhizae as Chinese medicine, also the treatment for kidney disease provides a kind of new approach.
The new application of Rhizoma Picrorhizae of the present invention is that Rhizoma Picrorhizae is used to prepare the medicine for the treatment of kidney disease as crude drug.Described kidney disease comprises that chronic renal disease, diabetic nephropathy, acute ischemia pour into the kidney injury that causes again.The main Rhizoma Picrorhizae rhizome that adopts also can adopt other effective sites of Rhizoma Picrorhizae plant.
Preferably adopt the alcohol extract of Rhizoma Picrorhizae rhizome.Perhaps adopt other organic solvent extraction, have similar drug effect.
Preferred production methods is: the Rhizoma Picrorhizae rhizome is ground into particulate powder after air-dry, is soaked in 50-95% ethanol or the methanol, ambient temperature overnight is purified, is filtered, and distillation is made the Rhizoma Picrorhizae extract through lyophilization.
The described Tibet density range of yellow extract recklessly is 1.0005～1.0269g/ml.
When the present invention was used for preparation treatment kidney disease medicine with Rhizoma Picrorhizae, the dosage form of medicine was the peroral dosage form that allows on the pharmaceutics.Specifically be with the Rhizoma Picrorhizae extract through conventional production process, add conventional peroral dosage form adjuvant, make various dosage forms such as tablet, capsule, granule, powder, oral liquid.Using method: adult 0.5～2g (in raw material), 2～3 times/day, oral.The child reduces by half.
Rhizoma Picrorhizae of the present invention can also with the medicine of present known other treatment kidney disease, make compound preparation as Radix Astragali etc., be used for the treatment of kidney disease.
The present invention is by various animal models and experiment showed, that Rhizoma Picrorhizae all has definite curative effect to various kidney diseases.Because Rhizoma Picrorhizae convenient sources, extensively, preparation method of extract is simple, does not need valuable instrument and equipment and reagent, therefore the invention provides a kind of easy, inexpensive, treat the medicine of kidney disease efficiently.
Description of drawings
Fig. 1 is the correlation analysis figure of 5/6 nephrectomy rat creatinine clearance rate and serum AOPP content.
Fig. 2 is the correlation analysis figure of 5/6 nephrectomy rat creatinine clearance rate and serum AGEs content.
Fig. 3 represents real-time quantitative fluorescence PCR analysis nephridial tissue ICAM-1mRNA expression.Fluorescent quantitation operating system gained data result Ct value (period that fluorescence signal is experienced when arriving the thresholding of setting) is according to 2
-Δ Δ CtMethod is analyzed comparison after handling.
Fig. 4 represents real-time quantitative fluorescence PCR analysis nephridial tissue MCP-1mRNA expression.Fluorescent quantitation operating system gained data result Ct value (period that fluorescence signal is experienced when arriving the thresholding of setting) is according to 2
-Δ Δ CtMethod is analyzed comparison after handling.
The specific embodiment
Embodiment one: the preparation of Rhizoma Picrorhizae extract
Rhizoma Picrorhizae extract of the present invention prepares by the following method: get Rhizoma Picrorhizae (crude drug) rhizome, air-dry back mechanical activation comminution becomes particulate powder, and (granular size is moderate, about 20mm
3About).The 350g raw material soaking places (150 rev/mins) on the shaking table in 50-95% ethanol 2000ml, and ambient temperature overnight is purified, negative pressure filtration, and distillation at least twice, after the Rhizoma Picrorhizae extract alcohol extract is made in lyophilization, close drying is preserved.The 1g alcohol extract is equivalent to the 3.5g crude drug.The density range of extract is 1.0005～1.0269g/ml.Soak the organic solvent that is adopted in the leaching process and also can adopt other alcohols, as methanol etc.
The Rhizoma Picrorhizae extract for preparing among the embodiment one is used for the experiment of following examples.
Embodiment two: the Rhizoma Picrorhizae extract delays the experiment of the progress of 5/6 nephrectomy rat chronic nephropathy
(1) materials and methods:
One, laboratory animal grouping
30 of male SD rats (Nanfang Medical Univ's Experimental Animal Center provides), body weight 231.56 ± 23.75g is divided into three groups at random.Rhizoma Picrorhizae treatment group (n=10) and operative control group (n=10), row 5/6 kidney surgery, 0.3% pentobarbital sodium 35mg/kg intraperitoneal injection of anesthesia excises the right kidney of left 2/3, one week of kidney back excision through the back; Sham operated rats (n=10) exposes kidney through back of the body otch, after the tractive kidney is torn kidney peplos, puts back to original position, sews up the incision.Postoperative one week beginning for the second time, Rhizoma Picrorhizae treatment group gives Rhizoma Picrorhizae extract and irritates stomach (400mg/kg/d), and operative control group and sham operated rats are given with the equal-volume normal saline and are irritated stomach.To the 9th week putting to death all rats.Put to death fasting the previous day, freely drink water, place metabolic cage and collect twenty-four-hour urine liquid.0.3% pentobarbital sodium intraperitoneal injection of anesthesia, abdominal aortic blood pours into kidney with the 50ml normal saline from ventral aorta, to remove erythrocyte in the circulation; Win left kidney, part is made 10% renal cortex homogenate, partial fixing with normal saline.
Two, urine, serological index detect
The ventral aorta blood sampling, 3000r/min is centrifugal.The blue bright method of coomassie
[1]Survey the twenty-four-hour urine protein content.Measure serum albumin (ALB), blood urea nitrogen (Bun), serum creatinine (Scr), urine creatine with automatic biochemistry analyzer, and calculate creatinine clearance rate (Ccr); The DTNB method
[2]Measure serum selenium glutathion peroxidase (SeGSHPx) activity; The thiobarbituric acid reaction method
[3]Measure urine malonaldehyde (MDA); The competitive ELISA method is surveyed serum advanced glycosylation end products (AGEs)
[4]Advanced oxidation protein products (AOPP) is measured the method for list of references
[5], be standard curve with the toluene-sodium-sulfonchloramide, measure 340nm place absorbance under the acid condition.
List of references:
[1]Lott?JA，Stephan?VA，Pritchard?KJ：Evaluation?of?the?Coomassie?Brilliant?Blue?G-250method?for?urinary?protein[J].Clin?Chem.1983，29：1946-1950
[2]Flohe?L，Gunzler?WA：Assays?of?glutathione?peroxidase[J].Methods?Enzymol.1984，105：114-120
[3]Obertop?H，Malt?RA：Lost?mass?and?excretion?as?stimuli?toparabiotic?compensatory?renalhypertrophy[J].Am?J?Physiol.1977.232：405-408
[4]Hou?FF，Boyce?J，Chertow?GM、Kay?J，et?al，Aminoguanidine?inhibits?advanced?glycation?endproducts?formationon?beta2-microglobulin[J].JAm?Soc?Nephrol.1998；9：277-283
[5]Yagi?K：A?simle?fluorometric?assay?for?lipoperoxide?in?blood?plasma.Biochem?Med15：212-216.1973
Three, PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM
The part nephridial tissue after 4% neutral formalin buffer is fixing, specimens paraffin embedding slices, PAS, Masson dyeing row light microscopy checking, double-blind method is observed histopathology and is changed.
The glomerular sclerosis degree adopts semiquantitative method (the Raij L of Reijis, Azar S, Keaue W:Mesangial immuneinjury, hypertention and progressive glomerular damage in Dahl rats[J] .Kidney Int.198426:137), be integrated into 0～4 grade by the hardened face of glomerule.0 grade: glomerule does not have sclerosis; 1 grade: glomerular sclerosis area≤25%; 2 grades: 26～50%; 3 grade 51%～75%; 4 grades: 76%～all glomerular sclerosis.Glomerular sclerosis index GI=((1n1+2n2+3n3+4n4)/observation glomerule sum) * 100%, n1, n2, n3 and n4 represent respectively and are classified as 1,2,3 and 4 glomerule number.
The evaluation of the ID reason extent of damage: divided 0～3 fen according to matter inflammatory cell infiltration degree between tubular ectasia, renal tubules atrophy, renal cells vacuolar degeneration, renal cells necrosis, interstitial fibrosis machine kidney: 0 minute: normal or pathological changes area＜25%; 1 minute: pathological changes area 25%～50%; 2 minutes: pathological changes area 50%～75%; 3 minutes: the pathological changes area〉75%.
Four, statistical method
Adopt 10.0 editions statistical packages of SPSS, the normal distribution measurement data is represented with X ± S.Three groups of above The data one factor analysis of variance relatively.Urine malonaldehyde (MDA) excretion rate, the relation of serum advanced oxidation protein products (AOPP), serum advanced glycosylation end products (AGEs) and serum selenium glutathion peroxidase (SeGSHPx) content and creatinine clearance rate adopts Bivariate analysis.
(2) interpretation of result:
One, ordinary circumstance
There was no significant difference between postoperative the 9th all Rhizoma Picrorhizae treatment group rat body weights and residual kidney/body weight and sham operated rats and the operative control group the results are shown in Table 1.
Rat body weight respectively organized by table 1 and residual kidney is heavy
Annotate: there was no significant difference between each group
Two, renal function and urine protein change
The Rhizoma Picrorhizae extract can obviously reduce by 5/6 nephrectomy rat twenty-four-hour urine protein content (P＜0.01), reduces serum blood urea nitrogen (Bun), serum creatinine (Scr) content, and the rising creatinine clearance rate, but difference does not have significance.Compare with sham operated rats, operative control group rat serum blood urea nitrogen (Bun) and serum creatinine (Scr) level obviously raise, and creatinine clearance rate reduces (P＜0.05), and the twenty-four-hour urine protein quantification significantly raises (P＜0.01), the results are shown in Table 2.
Table 2 is respectively organized the variation of kidney of rats function
Annotate: compare with sham operated rats,
*P＜0.05,
*P＜0.01; Compare with the treatment group,
▲P＜0.05,
▲ ▲P＜0.01
Three, kidney pathological change
The Rhizoma Picrorhizae extract can obviously alleviate 5/6 nephrectomy rat glomerular sclerosis and renal tubulointerstitial lesion degree, and its sxemiquantitative integration is starkly lower than operative control group rat (P＜0.05).Compare with sham operated rats, operative control group rat glomerular sclerosis and renal tubulointerstitial lesion degree significantly increase the weight of (P＜0.01).See Table 3.
Table 3 is respectively organized the matter extent of damage between kidney of rats bead hardenability value and kidney
| Grouping | Number of animals | The glomerular sclerosis index | The matter extent of damage between kidney |
| The |
10 | 3.95±0.23 ▲ | 0.16±0.03 ▲ |
| |
10 | 9.76±0.43 * | 0.48±0.07 * |
| Sham operated |
10 | 0.28±0.06 | 0.02±0.01 |
Annotate:
*Compare P＜0.01 with sham operated rats;
▲Compare P＜0.05 with the treatment group
Four, the change of oxidative stress level
Compare with operative control group, Rhizoma Picrorhizae extract 5/6 nephrectomy rat blood serum selenium glutathion peroxidase (SeGSHPx) active (P＜0.05) that can obviously raise, reduce urine malonaldehyde (MDA) excretion rate (P＜0.05), obviously reduce serum advanced oxidation protein products (AOPP), serum advanced glycosylation end products (AGEs) level (P＜0.05).Compare with sham operated rats, active significantly reduce (P＜0.05) of operative control group rat blood serum selenium glutathion peroxidase (SeGSHPx), reduce urine malonaldehyde (MDA) excretion rate and serum advanced oxidation protein products (AOPP), serum advanced glycosylation end products (AGEs) content and significantly raise (P＜0.01), the results are shown in Table 3.And creatinine clearance rate and serum advanced oxidation protein products (AOPP) (r=-0.483, P＜0.05) and serum advanced glycosylation end products (AGEs) (r=-0.686, P＜0.01) content are negative correlation.See Fig. 1 and Fig. 2.
Table 4 is respectively organized the variation of rat urine MDA excretion rate and blood plasma oxidative stress index
Annotate: compare with sham operated rats,
*P＜0.05,
*P＜0.01; Compare with the treatment group,
▲P＜0.05 conclusion:
The present invention shows that the Rhizoma Picrorhizae extract can obviously reduce by 5/6 nephrectomy rat twenty-four-hour urine protein content; alleviate 5/6 nephrectomy rat glomerular sclerosis and renal tubulointerstitial lesion degree; illustrate that Rhizoma Picrorhizae has the protection kidney, the effect of delaying chronic kidney progression of disease.Its mechanism may be when suppressing chronic kidney disease oxidative stress relevant.
Experimental example three: the Rhizoma Picrorhizae extract is to the kidney protective effect research of diabetes rat
(1) material and method
1, animal grouping and specimen are left and taken: 30 of cleaning level male SD rats, and body weight 120-140g (available from Nanfang Medical Univ's Experimental Animal Center), adaptability is fed after 3 days and is anaesthetized descending single kidney enucleation at 3% pentobarbital, and the back otch is extractd left kidney.Get 20 after 7 days at random, fasting after 16 hours the single intraperitoneal injection streptozotocin (use 0.1mol/L, the fresh preparation of the citrate buffer solution of pH4.5) 55mg/Kg is surplused 8 lumbar injection equivalent citrate buffer solutions as matched group, surveys blood glucose after 7 days〉16.7mmol/L is diabetes.To become the mould rat to be divided into 8 of diabetic groups at random by blood glucose and body weight, 8 of Rhizoma Picrorhizae extract-treated groups (300mg/Kg/d), treatment group every morning gives the Rhizoma Picrorhizae extract and irritates stomach, and diabetic groups is given the equivalent distilled water and is irritated stomach.Rat ad lib and drinking-water, leave and take twenty-four-hour urine and blood plasma after treating for 8 weeks, pour into kidney from ventral aorta with the ice normal saline, excise right kidney, part is made renal cortex homogenate, dehydration after the part neutral formalin is fixed 24 hours, waxdip, embedding, partial fixing is in 2.5% glutaraldehyde, and resin embedding is used for electron microscopic examination.
2, the detection of renal function and biochemical indicator: glucose oxidase method is surveyed blood glucose, automatic clinical chemistry analyzer is measured serum creatinine (SCr), blood albumin (ALB), cholesterol (CH), triglyceride (TG), urine creatine (UCr), and calculates creatinine clearance rate (CCr) urine creatine * urine amount/serum creatinine/rat body weight.Plasma A OPP measures: with the toluene-sodium-sulfonchloramide is standard curve, measures the absorbance of 340nm under the acid condition.Dtnb assay serum and kidney homogenate selenium glutathion peroxidase (SeGSHPx) activity; The thiobarbituric acid reaction method is measured serum, urine and kidney homogenate malonaldehyde (MDA); The Coomassie brilliant blue method detects the twenty-four-hour urine protein quantification; Colorimetry is surveyed urine N-acet-beta-amino glucosidase (NAG enzyme) (Shanghai Sun Bio-Tech Co., Ltd.'s test kit).
3, nephridial tissue pathological analysis: the capable PAS dyeing of nephridial tissue paraffin section 2 μ m, through image analysis system the PAS stained is analyzed, 30 complete glomerule are got in every section in the direction of the clock at random, the ratio of each glomerule PAS positive staining area of system-computed and whole glomerule area by analysis, ask the mean of 30 these ratios of glomerule then, be the relative amount of every section messangial cell epimatrix.Choose 3 rat specimen and amplify 300,000 times of photograph, applies image analysis system measurement base film thickness for every group under the Electronic Speculum.
4, statistical analysis: all data are represented with mean ± standard deviation, organize the relatively employing one factor analysis of variance of number, statistics application SPSS11.0 software analysis more.
(2) interpretation of result:
1, the Rhizoma Picrorhizae extract is to the influence of rat body weight and active state: compare with matched group, tangible polydipsia, polyphagia, polyuria symptom appear in diabetes rat, the mental status is poor, body weight all obviously alleviates (p all＜0.01), the Rhizoma Picrorhizae extract for treating can obviously improve the spiritual and movable of rat, and treatment group rat body weight alleviates more not treatment group few (p all＜0.05).See Table 5.
Table 5: the Rhizoma Picrorhizae extract is organized the influence of rat body weight, kidney performance figure, blood glucose, creatinine, albumin, T-CHOL, triglyceride, urine protein, urine NAG enzyme, creatinine clearance rate to each
d：P<0.05，c：P<0.01，vs?NS?group；e：P<0.05，f：P<0.01vs?DN?group
2, the Rhizoma Picrorhizae extract is to the influence of rat kidney pathological change: the kidney of diabetes rat all obviously increases, diabetic groups and Rhizoma Picrorhizae extract for treating group kidney performance figure are all apparently higher than matched group (p all＜0.01), but the Rhizoma Picrorhizae extract for treating can obviously reduce the kidney performance figure (p all＜0.01) of diabetes rat.The kidney pathologic finding then glomerular volume of visible diabetes rat obviously increases than matched group, and mesentery substrate obviously increases.The Rhizoma Picrorhizae extract for treating can alleviate the increase of glomerular volume, reduces the mesentery apposition.The visible diabetic glomeruli basement membrane of electron microscopic examination obviously thickens, and the Rhizoma Picrorhizae extract for treating can obviously alleviate thickening of basement membrane.
3, the Rhizoma Picrorhizae extract is to the influence of rat kidney function: the twenty-four-hour urine protein quantification of diabetes rat obviously raises than matched group during 8 weeks, urine N-acet-beta-amino glucosidase (NAG enzyme) also obviously raises (p all＜0.01), and the Rhizoma Picrorhizae extract for treating can obviously reduce the urine protein and the urine N-acet-beta-amino glucosidase (NAG enzyme) (p＜0.05) of diabetes rat.The creatinine clearance rate of diabetes rat obviously raises (p all＜0.05) than matched group, 8 weeks of Rhizoma Picrorhizae extract for treating the creatinine clearance rate to diabetes rat do not make significant difference.There is not significant difference between each group of serum creatinine.See Table 4.
4, the Rhizoma Picrorhizae extract is to the influence of rat blood sugar and blood fat: the Rhizoma Picrorhizae extract for treating can obviously reduce the blood glucose of diabetes rat, 150mg/Kg dosage group descends 18.88%, 300mg/Kg dosage group descends 26.67%, but does not make it to recover normal.Blood T-CHOL, the triglyceride of diabetes rat all obviously raise than matched group during 8 weeks, plasma albumin obviously descend (p all＜0.05), the T-CHOL of Rhizoma Picrorhizae extract 300mg/Kg treatment can obviously reduction diabetes rat, rising plasma albumin (p is all＜0.05), but blood triglyceride is not seen appreciable impact.See Table 4.
5, the Rhizoma Picrorhizae extract is to the influence of diabetes rat vivo oxidation product and antioxidase content: the blood antioxidase index GPx of diabetes rat is than matched group obviously descend (p all＜0.01) during 8 weeks, blood MDA, AOPP are then than matched group obviously raise (p all＜0.01), urine MDA excretion rate all obviously raises (p all＜0.01), and kidney homogenate MDA, GPx are all apparently higher than matched group.The Rhizoma Picrorhizae extract for treating can obviously reduce serum AOPP, the MDA level of diabetes rat, and rising GPx level reduces urine MDA excretion rate, reduces the MDA level that kidney raises, and improves the unusual rising (p all＜0.05) of GPx at kidney.See Table 6.
Table 6: the Rhizoma Picrorhizae extract is to plasma A OPP, MDA, GPx activity level, kidney homogenate MDA, GPx activity level, the influence of urine MDA level.
D:P＜0.05, c:P＜0.01, vs NS group; E:P＜0.05, f:P＜0.01 vs DN group conclusion:
The present invention has confirmed that first the Rhizoma Picrorhizae extract can significantly reduce diabetes rat urine protein and urine N-acet-beta-amino glucosidase (NAG enzyme) content, the rising plasma albumin, alleviate diabetic glomeruli basement membrane thickened and mesentery substrate hypertrophy, and blood glucose, the T-CHOL of reduction diabetes rat, show that Rhizoma Picrorhizae has preventive and therapeutic effect to the diabetes renal damage.
Experimental example four: the Rhizoma Picrorhizae extract pours into the protective effect of injury of kidney again to acute ischemia
(1) material and method
One, animal grouping and medication
Choose healthy male SD rat, (attached Nanfang Hospital of Nanfang Medical Univ Experimental Animal Center provides) is divided into following each group (n=8) at random about body weight 200～250g: 1. sham operated rats; 2. operative control group (simple ischemia 60min pour into 72h again): first three day of performing the operation given rat oral gavage 2rml normal saline, and for three days on end, 2h irritates stomach once before the art; 3. Rhizoma Picrorhizae extract various dose prevention+treatment group (40mg/kg, 80mg/kg, 160mg/kg): perform the operation and gave the rat oral gavage Rhizoma Picrorhizae extract in preceding 3 days, for three days on end, 2h irritates stomach once before the art, and postoperative is irritated stomach once every day; 4. Rhizoma Picrorhizae extract prevention group: perform the operation and gave rat oral gavage Rhizoma Picrorhizae extract (160mg/kg) in preceding 3 days, for three days on end, 2h irritates stomach once before the art, not administration of postoperative; 5. Rhizoma Picrorhizae extract is treated 1 group (early stage administration group): not administration before the operation, and postoperative 6h, 30h respectively irritate a Rhizoma Picrorhizae extract of stomach (160mg/kg); 6. Rhizoma Picrorhizae extract treatment II organizes (administration in late period group): not administration before the operation, postoperative is respectively irritated a Rhizoma Picrorhizae extract of stomach (160mg/kg) in 30h, 54h.
Two, the preparation of ischemia-reperfusion injury of kidney rat animal model
Acute ischemia pours into the preparation of injury of kidney animal model again: operation is preceding with rat fasting 12 hours, grasp rat, 3% pentobarbital sodium (30mg/kg) lumbar injection by the action main points, fully be fixed on the Mus plate after the anesthesia, under aseptic condition, get the abdominal part median incision and cut skin, passivity is separated abdominal muscle, the about 4-5cm of otch.After entering the abdominal cavity, expose right kidney earlier, passivity is separated perirenal fat and fascia, and the right kidney arteriovenous of dissociating with the double-deck ligation arteriovenous of stitching thread, is excised right kidney.Expose left kidney then, passivity is separated perirenal fat and fascia, and careful separation left side kidney arteriovenous and branch thereof open bulldog clamp after closing left renal artery 60min with noinvasive bulldog clamp folder, and the observation kidney becomes scarletly by pale, shows and pours into successfully.Feed 72h behind the sew up wound under normal operation, ad lib water, specimen taken at the appointed time.Sham operated rats exposes two kidneys and separates perirenal tissue, does not excise right kidney and folder and closes left renal artery, and all the other are handled with the operation group.
Three, the collection of specimen and processing
24h puts into metabolic cage with rat before the specimen taken at the appointed time, and the 24h urine is left and taken in 12h fasting before the specimen taken earlier, calculates the urine amount, after the packing-70 ℃ frozen.Be fixed on the Mus plate after at the appointed time rat fully being anaesthetized, abdominal aortic blood is got left kidney behind the perfused tissue, collects 4 ℃ of centrifugal 10min of 3000rpm in the blood specimen 30min, leaves and takes serum, after the packing-70 ℃ frozen.Get half nephridial tissue of the left nephridial tissue outside of belly, the about 0.3cm3 of following utmost point wedge type excision is cut into the 0.5-1mm3 size, and it is fixing to put into 2.5% glutaraldehyde phosphate buffer immediately, and remainder places 10% neutral formalin fixative fixing.Take by weighing nephridial tissue 50mg, add the 0.1M neutral phosphor phthalate buffer 3ml of pre-cooling, electric homogenizer grinds the preparation tissue homogenate, and microscopy is not seen intact cell centrifugal (2000rpm * 10min, 4 ℃) tissue homogenate, and it is standby to get supernatant-80 ℃ preservation.Other half nephridial tissue will be put into liquid nitrogen cryopreservation immediately.
Four, the nephridial tissue morphological change is observed in nephridial tissue PAS dyeing
After nephridial tissue is fixed more than 18 hours with 10% neutral formalin fixative, through the gradient ethanol dehydration: 75% ethanol 5min * 3 time → 85% ethanol 10min → 85% ethanol+Yihong 10min → 95% ethanol 10min * 2 time → dehydrated alcohol 10min * 2 time, the transparent 3min of dimethylbenzene * 2 times, soak cured (60 ℃ of soft waxs, 60 ℃ of 30min → hard waxes, 60min), embedding wax block, cut 2 υ m section, 75 ℃ of roasting sheet 45min.Tissue slice dewaxes to entry through dimethylbenzene, gradient ethanol.2% periodic acid 15min → washing → Schiff liquid 50min → washing → haematoxylin is redyed the transparent back of the anti-indigo plant of 15min → 0.5% hydrochloric acid-ethanol → washing differentiation → flowing water → gradient ethanol dehydration → dimethylbenzene mounting.Optical microscope is observed down and is taken a picture.And at microscopically according to PallerShi method evaluation injury of renal tubular mark: 10 * 20 times of light microscopic undertissue section picked at random are upper left, lower-left, upper right, bottom right and hit exactly 10 visuals field, 10 renal tubules are observed in each visual field, score according to following standard: renal cells endochylema cavity sample degeneration meter 1 minute, the flat meter of tubule epithelial cell 1 minute, brush border comes off and counted 1 fen, the downright bad meter of epithelial cell 2 minutes, various cast meters 2 minutes, interstitial edema meter 1 minute
Five, observe the nephridial tissue morphological change under the nephridial tissue Electronic Speculum
Nephridial tissue (0.5-1mm3 size) is fixed more than 12 hours before in 2.5% glutaraldehyde, and 0.1MPB phosphate buffer rinsing (PH7.4) 15min * 5 times fixes 1.5 hours behind 1% osmic acid (0.24M PH7.4), and 15min * 3 time are washed with buffer in fixing back; 30%, 50%, 70%, 90% acetone each 15min that dewaters step by step, 100% acetone dehydration 3 times, each 15 minutes, embedding medium: acetone=1:1 soaked into 1 hour, embedding medium (spur): acetone=1:2 soaked into 2 hours, and virgin resin soaks into embedding after 3 hours, and 60 ℃ of polymerizations are after 24 hours, repair piece, AO type microtome is cut into the 60mm section, chooses to be silver grey or xanchromatic ultrathin section uranium acetate dyeing 30 minutes, lead citrate dyeing 10-15 minute, the H-600 electron microscope observation.
Six, biochemical process detects serum creatinine, urine creatine
Leave and take blood, the urine specimen is sent clinical laboratory of Nanfang Hospital, directly detect with Toshiba's automatic clinical chemistry analyzer, and in conjunction with uri-meter calculation endogenous creatinine clearance rate (Ccr).Ccr=urine creatine/serum creatinine * 24 urine amount/1440 (ml/min)
Seven, kidney homogenate and blood plasma TBARS detect: adopting the thiobarbituric acid reaction method, is standard with the tetraethoxypropane, and the concentration of standard application liquid is 10nmol/ml.
The result calculates: MDA concentration=f/F * 10 (nmol/ml)
F: the optical density that records by sample liquid
F: the optical density that records by titer
Eight, kidney homogenate and blood plasma GSHPx detect: improvement DNTB colorimetry.
Spectrophotometer is measured in 423nm wavelength place and respectively managed absorbance A, unit definition: making GSH lowering of concentration 1 μ mol with every milliliter of serum in 37 ℃ of per minutes is an enzyme activity unit.The result calculates:
Serum GSHPx vigor (U/ml)=(the non-enzyme pipe of A-A measures pipe) the non-enzyme pipe of A * 100/3
Nine, immunohistochemistry detects nephridial tissue ICAM-1, MCP-1 and mononuclear phagocyte counting
Get paraffin organization and cut 5 μ m section, dewax to aquation through dimethylbenzene, gradient ethanol.Hatch 10min with protoenzyme in the deactivation with 3% hydrogen peroxide under the room temperature, wash 2min * 3 time with distilled water.To cut into slices and immerse in the citrate buffer, be heated to 96～100 ℃ of lasting 20min, cool off under the room temperature.Wash 5min * 3 time with PBS.Drip 1:50 normal goats serum confining liquid, hatch 20min under the room temperature.Drip the anti-Mus ICAM-1 of rabbit, the MCP-1 antibody of 1:150 dilution, drip the anti-Mus ED-1 of the rabbit antibody of 1:50 dilution, do negative control with non-immune rabbit igg, section places in the wet box, puts into 4 ℃ of refrigerator overnight.Wash 5min * 3 time with PBS, it is anti-to drip goat-anti rabbit-Evision two, hatches 30min for 37 ℃.Wash 5min * 3 time with distilled water, the DAB 5～10min that develops the color, microscopically is observed, and stops color reaction with the distilled water flushing.Haematoxylin is redyed 1min, 0.5% hydrochloric acid-ethanol differentiation, and warm water returns indigo plant, gradient ethanol dehydration, the transparent back of dimethylbenzene mounting.Microscopically is observed and is taken a picture.Nephridial tissue ICAM-1, MCP-1 SABC sxemiquantitative integral estimation are as a result carried out analyzing evaluation according to the dyeing scope: color range with reference to the Hugo standard, feminine gender is 0 minute, and color range≤25% is 1 minute,〉25%≤50% be 2 minutes, 50%≤75% be 3 minutes, 75% be 4 minutes.The mononuclear phagocyte counting adopts eyepiece grid location chi, and promptly (grid location chi area 25x25um2/ grid) 10 visuals field of picked at random under 400 times of light microscopics are counted all positive cell numbers and got its meansigma methods as analysis indexes.
Ten, real-time quantitative fluorescence PCR is measured nephridial tissue ICAM-1, MCP-1mRNA expression
11, statistical procedures
Each is organized data and represents with mean ± standard deviation, a plurality of sample averages relatively use One-Way ANOVA, multiple comparisons adopts the LSD check, P＜0.05 is for having statistical significance.All statistics are finished by SPSS statistical software 11.5.
(2) interpretation of result
One, acute ischemia pours into the foundation of injury of kidney rat animal model again
After the SD rat was excised right kidney noinvasive bulldog clamp folder and closes left renal artery 60min and pour into 72h again, serum creatinine (Scr), blood urea nitrogen (BUN) significantly raise, and urine creatine (Ccr) reduces, and creatinine clearance rate descends, and urine osmole reduces, and urine NAG enzyme raises.Perusal left side renomegaly is obvious, and weight increases, the cortex ischemia; It is obvious that mirror is observed kidney skin medullary substance intersection renal tubule degeneration and necrosis down, proves the modeling success.
Two, the Rhizoma Picrorhizae extract is to the influence of ischemia-reperfusion metanephros tissue morphology variation
1. under the light microscopic: behind the ischemia-reperfusion 72h, the all visible skin marrow intersection renal cells brush border of each administration group of Rhizoma Picrorhizae extract and operative control group comes off, epithelial cell degeneration, necrosis, come off, tubule intracavity cast forms, pathological changes such as interstitial edema, but each administration group nephridial tissue pathological change is lighter than operative control group.The matter scoring all is lower than operative control group (P is 0.000) (table 7) between each prevention+treatment group tubule, increase with dosage, the nephridial tissue pathological change alleviates gradually, matter scoring descends between tubule, between each dosage prevention+treatment group between tubule matter mark significant difference (P is 0.000 between each dosage group) arranged.Prevention group, treatment I group, treatment II group injury of renal tubular divide number average to be lower than operative control group (P=0.000 P=0.000 P=0.019), prevention group, treatment I group, treatment II group injury of renal tubular mark are higher than prevention+treatment 160mg/kg and organize (P=0.02 P=0.003 P=0.000), matter scoring number is lower than treatment II group (P=0.000 P=0.000) between prevention group, treatment I group tubule, there was no significant difference (P=0.479) between prevention group, the treatment I group.
2. under the Electronic Speculum: all visible renal cells microvillus swelling of each administration group of Rhizoma Picrorhizae extract and operative control group, come off, mitochondrial swelling, ridge vacuolar degeneration even disappear, the pathological changes of Rhizoma Picrorhizae extract administration group alleviates than operative control group.
Table 7 is respectively organized matter scoring between the kidney of rats tubule
| Group | Example number n | Matter scoring between tubule |
| Sham operated |
5 | 18.00±4.12 |
| Operative control group | 8 | 85.25±4.92 △ |
| Prevention+treatment 40mg/kg group | 8 | 76.75±4.26 △▲ |
| Prevention+treatment 80mg/kg group | 8 | 67.28±5.67 △▲ |
| Prevention+treatment 160mg/kg group | 8 | 63.12±3.22 △▲ |
| The prevention group | 8 | 67.75±1.67 △▲ |
| Treatment I group (early stage administration group) | 8 | 69.12±2.58 △▲ |
| Treatment II group (administration in late period group) | 8 | 80.43±3.15 △· |
△Compare P＜0.01 with sham operated rats;
▲Compare P＜0.01 with ischemia-reperfusion group;
Compare P＜0.05 with ischemia-reperfusion group; One-WayANOVA F value=161.391, P=0.000
Three, the Rhizoma Picrorhizae extract is to the influence of ischemia-reperfusion metanephros changes of function
Compare with sham operated rats, ischemia-reperfusion causes that Scr raises, and Ccr descends.Each prevention+treatment group Ccr is higher than operative control group (P=0.000 P=0.000 P=0.013) (table 8), and increase with dosage, Ccr raises gradually, between prevention+treatment 160mg/kg group and the prevention+treatment 40mg/kg group significant difference (P=0.013) is arranged, there was no significant difference (P=0.054) between prevention+treatment 160mg/kg group and the prevention+treatment 80mg/kg group; Prevention group, treatment I group, treatment II group Ccr are higher than operative control group (P=0.000 P=0.000 P=0.003); Ccr does not have remarkable significant difference (P=0.948 P=0.732 P=0.094) between prevention group, treatment I group, treatment II group and the prevention+treatment 160mg/kg group.
Table 8 is respectively organized rat Ccr and is changed
| Group | Example number n | Ccr(ml/mim) |
| Sham operated |
5 | 0.794±0.082 |
| Operative control group | 8 | 0.163±0.091 △ |
| Prevention+treatment 40mg/kg group | 8 | 0.315±0.102 △· |
| Prevention+treatment 80mg/kg group | 8 | 0.424±0.126 △▲ |
| Prevention+treatment 160mg/kg group | 8 | 0.462±0.171 △▲ |
| The prevention group | 8 | 0.458±0.120 △▲ |
| Treatment I group (early stage administration group) | 8 | 0.442±0.101 △▲ |
| Treatment II group (administration in late period group) | 8 | 0.357±0.114 △· |
△Compare P＜0.01 with sham operated rats;
▲Compare P＜0.01 with ischemia-reperfusion group;
Compare P＜0.05 with ischemia-reperfusion group; One-Way ANOVA F value=13.952, P=0.000
Four, the Rhizoma Picrorhizae extract to the ischemia-reperfusion rear oxidation stress index influence
1, the Rhizoma Picrorhizae extract is to the influence of nephridial tissue and blood plasma TBARS
Compare with sham operated rats, ischemia-reperfusion causes that nephridial tissue and blood plasma TBARS raise, after giving Rhizoma Picrorhizae extract, each prevention+treatment group kidney homogenate TBARS content is lower than operative control group (P is 0.000) (table 9), increase along with Rhizoma Picrorhizae extract dosage, kidney homogenate TBARS content reduces gradually, and kidney homogenate TBARS content has significant difference (P is 0.000 between each dosage group) between each dosage prevention+treatment group; Prevention group, treatment I group kidney homogenate TBARS are lower than operative control group (P=0.008 P=0.033), treatment II group kidney homogenate TBARS and operative control group there was no significant difference (P=0.190), prevention group, treatment I group, treatment II group kidney homogenate TBARS are higher than prevention+treatment 160mg/kg and organize (P is 0.000), and prevention group, treatment I group, treatment II organize there was no significant difference between three groups (P=0.563 P=0.184 P=0.433).After giving Rhizoma Picrorhizae extract, each prevention+treatment group blood plasma TBARS content is lower than operative control group (P is 0.000), increase along with Rhizoma Picrorhizae extract dosage, blood plasma TBARS content reduces gradually, between prevention+treatment 160mg/kg group and the prevention+treatment 40mg/kg group significant difference (P=0.000) is arranged, there was no significant difference (P=0.404) between prevention+treatment 160mg/kg group and the prevention+treatment 80mg/kg group; Prevention group, treatment I group, treatment II group blood plasma TBARS are lower than operative control group (P is 0.000), prevention group, treatment I group, treatment II group blood plasma TBARS are higher than prevention+treatment 160mg/kg and organize (P is 0.000), prevention group, treatment I group blood plasma TBARS are lower than treatment II group (P=0.000 P=0.000), there was no significant difference (P=0.855) between prevention group, the treatment I group.
Table 9 is respectively organized rat plasma and nephridial tissue TBARS level
| Group | ? n | Blood plasma TBARS (nmol/ml) | Nephridial tissue TBARS (nmol/mg albumen) |
| Sham operated |
5 | 6.26±0.19 | 2.24±0.19 |
| Operative control group | 8 | 13.29±0.48 △ | 6.13±0.38 △ |
| Prevention+treatment 40mg/kg group | 8 | 11.67±0.61 △▲ | 5.54±0.54 △· |
| Prevention+treatment 80mg/kg group | 8 | 9.74±054 △▲ | 4.97±0.41 △▲ |
| Prevention+treatment 160mg/kg group | 8 | 9.18±0.53 △▲ | 4.10±0.50 △▲ |
| The prevention group | 8 | 10.61±0.67 △▲ | 5.37±0.62 △▲ |
| Treatment I group (early stage administration group) | 8 | 10.67±0.69 △▲ | 5.53±0.58 △· |
| Treatment II group (administration in late period group) | 8 | 11.93±0.96 △▲ | 5.76±0.84 △ |
△Compare P＜0.01 with sham operated rats;
▲Compare P＜0.01 with ischemia-reperfusion group;
Compare P＜0.05 with ischemia-reperfusion group.One-Way?ANOVA，P＝0.000
2, the Rhizoma Picrorhizae extract is to the influence of nephridial tissue and blood plasma GSHPx
Compare with sham operated rats, ischemia-reperfusion causes that nephridial tissue and blood plasma GSHPx reduce, after giving Rhizoma Picrorhizae extract, each prevention+treatment group kidney homogenate GSHPx content is higher than operative control group (P is 0.000) (table 10), increase along with Rhizoma Picrorhizae extract dosage, kidney homogenate GSHPx content raises gradually, prevention+treatment 160mg/kg group kidney homogenate GSHPx content is higher than prevention+treatment 80mg/kg group and prevention+treatment 40mg/kg group (P=0.049P=0.000), does not have significant difference (P=0.083) between prevention+treatment 80mg/kg group and the prevention+treatment 40mg/kg group; Prevention group, treatment I group kidney homogenate GSHPx are higher than operative control group (P is 0.000), treatment II group kidney homogenate GSHPx and operative control group there was no significant difference (P=0.284), prevention group, treatment I group, treatment II group kidney homogenate GSHPx are lower than prevention+treatment 160mg/kg and organize (P=0.005 P=0.000 P=0.000), prevention group, treatment I group kidney homogenate GSHPx are higher than treatment II group (P=0.01 P=0.008), there was no significant difference (P=0.929) between prevention group, the treatment I group.After giving Rhizoma Picrorhizae extract, each prevention+treatment group blood plasma GSHPx content is higher than operative control group (P is 0.000) (table 10), increase along with Rhizoma Picrorhizae extract dosage, blood plasma GSHPx content reduces rising gradually, blood plasma GSHPx content there was no significant difference (P=0.424 P=0.206 P=0.667) between each dosage prevention+treatment group; Prevention group, treatment I group, treatment II group blood plasma GSHPx are lower than operative control group (P is 0.000), prevention group, treatment I group, treatment II group blood plasma GSHPx and prevention+treatment 160mg/kg organize there was no significant difference (P=0.403 P=0.114 P=0.082), prevention group, treatment I group blood plasma GSHPx are higher than treatment II group (P=0.013 P=0.002), there was no significant difference (P=0.448) between prevention group, the treatment I group.
Table 10 is respectively organized blood plasma and kidney homogenate GSHPx activity
| Group | n | GSHPx(U/ml) | GSHPx (U/mg albumen) |
| Sham operated |
5 | 21.66±0.91 | 5.36±0.50 |
| Operative control group | 8 | 6.24±1.42 △ | 323±0.43 △ |
| Prevention+treatment 40mg/kg group | 8 | 12.81±2.g6 △▲ | 3.82±0.39 △▲ |
| Prevention+treatment 80mg/kg group | 8 | 13.43±3.35 △▲ | 4.1g±0.42 △▲ |
| Prevention+treatment 160mg/kg group | 8 | 14.57±2.30 △▲ | 4.62±0.42 △▲ |
| The prevention group | 8 | 15.73±3.12 △▲ | 4.01±0.23 △▲ |
| Treatment I group (early stage administration group) | 8 | 16.78±2.25 △▲ | 4.03±0.31 △▲ |
| Treatment II group (administration in late period group) | 8 | 12.05±4.01 △▲ | 3.45±0.52 △ |
△Compare P＜0.01 with sham operated rats;
▲Compare P＜0.01 with ischemia-reperfusion group;
Compare P＜0.05 with ischemia-reperfusion group.One-Way?ANOVA，P＝0.000
Five, the Rhizoma Picrorhizae extract to ischemia-reperfusion after the inflammatory reaction parameter
1. the Rhizoma Picrorhizae extract is to the influence of nephridial tissue ICAM-1 protein expression
Sham operated rats renal tissue ICAM-1 does not see obvious expression, and operative control group kidney ICAM-1 expresses obviously, mainly be positioned at skin marrow intersection renal tubules and between matter, nearly song and Distal convoluted tubule are all seen positive staining, glomerule is expressed not obvious; Rhizoma Picrorhizae extract administration group kidney ICAM-1 positive staining obviously is weaker than operative control group.Semi-quantitative results shows, Rhizoma Picrorhizae extract 160mg/kg, the scoring of 80mg/kg prevention+treatment group ICAM-1 groupization are lower than operative control group (P=0.000P=0.000), ICAM-1 groupization scoring of 40mg/kg prevention+treatment group and operative control group there was no significant difference (P=0.188), increase along with Rhizoma Picrorhizae extract dosage, the scoring of ICAM-1 groupization reduces gradually, and significant difference (P=0.000 between each dosage group) (table 11) is arranged between each dosage group.Prevention group, the scoring of treatment I group ICAM-1 groupization are lower than operative control group (P=0.000P=0.000), treatment II group ICAM-1 groupization scoring and operative control group there was no significant difference (P=0.147), prevention group, treatment I group, the scoring of treatment II group ICAM-1 groupization are higher than 160mg/kg prevention+treatment group (P is 0.000), prevention group, the scoring of treatment I group ICAM-1 groupization are lower than treatment II group I (P=0.000 P=0.000), there was no significant difference (P=0.249) between prevention group, the treatment I group.
The scoring of table 11 nephridial tissue ICAM-1 groupization
| Group | Example number n | The scoring of ICAM-1 groupization |
| Sham operated |
5 | 0.34±0.09 |
| Operative control group | 8 | 3.60±0.09 △ |
| Prevention+treatment 40mg/kg group | 8 | 3.50±0.13 △ |
| Prevention+treatment 80mg/kg group | 8 | 3.21±0.09 △▲ |
| Prevention+treatment 160mg/kg group | 8 | 2.48±0.16 △▲ |
| The prevention group | 8 | 2.91±0.21 △▲ |
| Treatment I group (early stage administration group) | 8 | 3，00±0.17 △▲ |
| Treatment II group (administration in late period group) | 8 | 348±0.19 △ |
△Compare P＜0.01 with sham operated rats;
▲Compare P＜0.01 with ischemia-reperfusion group;
Compare P＜0,05 with ischemia-reperfusion group.One-Way ANOVA, F value=279.988, P=0.000
2 Rhizoma Picrorhizae extracts are to the influence of nephridial tissue MCP-1 protein expression
Sham operated rats renal tissue MCP-1 does not see obvious expression, and operative control group kidney MCP-1 expresses obviously, mainly be positioned at skin marrow intersection renal tubules and between matter, nearly song and Distal convoluted tubule are all seen positive staining, glomerule is expressed not obvious; Rhizoma Picrorhizae extract administration group kidney MCP-1 positive staining obviously is weaker than operative control group.Semi-quantitative results shows, each prevention+treatment group MCP-1 groupization scoring of Rhizoma Picrorhizae extract is lower than operative control group, and (P is 0.000, see Table 11), increase along with Rhizoma Picrorhizae extract dosage, the scoring of MCP-1 groupization reduces gradually, and significant difference (P=0.000 between each dosage group) is arranged between each dosage group.Prevention group, the scoring of treatment I group MCP-1 groupization are lower than operative control group (P=0.000), treatment II group MCP-1 groupization scoring and operative control group there was no significant difference (P=0.298), prevention group, treatment I group, the scoring of treatment II group MCP-1 groupization are higher than 160mg/kg prevention+treatment group (P is 0.000), prevention group, the scoring of treatment I group MCP-1 groupization are lower than treatment II group I (P=0.000 P=0.000), and the scoring of prevention group MCP-1 groupization is lower than treatment I group (P=0.000).
Table 11 is respectively organized kidney of rats and is organized the scoring of MCP-1 SABC
| Group | The example number | The scoring of MCP-1 groupization |
| Sham operated |
5 | 0.30±0.09 |
| Operative control group | 8 | 3.57±0.14 △ |
| Prevention+treatment 40mg/kg group | 8 | 3.22±0.22 △▲ |
| Prevention+treatment 80mg/kg group | 8 | 2.98±0.18 △▲ |
| Prevention+treatment 160mg/kg group | 8 | 2.23±0.14 △▲ |
| The prevention group | 8 | 2.58±0.21 △▲ |
| Treatment I group (early stage administration group) | 8 | 2.96±0.17 △▲ |
| Treatment II group (administration in late period group) | 8 | 348±0.69 △ |
△Compare P＜0.01 with sham operated rats;
▲Compare P＜0.01 with ischemia-reperfusion group;
Compare P＜0.05 with ischemia-reperfusion group.One-Way ANOVAF value=233.881, P=0.000
The influence that 3 Rhizoma Picrorhizae extracts are expressed nephridial tissue ICAM-1mRNA
FQ-RT-PCR result shows, the sham operated rats kidney of rats organizes the ICAM-1mRNA expression very low, operative control group nephridial tissue ICAM-1mRNA expresses obviously and raises, each Rhizoma Picrorhizae extract prevention+treatment group ICAM-1mRNA is lower than operative control group, and (P is 0.000, see Fig. 3, Fig. 4), along with the increase of Rhizoma Picrorhizae extract dosage, nephridial tissue ICAM-1mRNA expression reduces gradually, and significant difference (P=0.000 between each dosage group) is arranged between each dosage group.Prevention group, treatment I group, treatment II group ICAM-1mRNA expression are lower than operative control group (P is 0.000), prevention group, treatment I group, treatment II group ICAM-1mRNA expression are higher than 160mg/kg prevention+treatment group (P is 0.000), prevention group ICAM-1mRNA expression is lower than treatment I group, treatment II group (P=0.000 P=0.000), there was no significant difference (P=0.273 sees Table 12) between treatment I group, the treatment II group.
Table 12 is respectively organized the variation that kidney of rats is organized ICAM-1mRNA
| Group | The example number | △ CT ratio |
| Sham operated |
5 | 43.1±13.0 |
| Operative control group | 8 | 2806.5±183.4 * |
| Prevention+treatment 40mg/kg group | 8 | 321.2±30.7# |
| Prevention+treatment 80mg/kg group | 8 | 305.6±24.8# |
| Prevention+treatment 160mg/kg group | 8 | 138.0±11.5# |
| The prevention group | 8 | 2984±18.4# |
| Treatment I group (early stage administration group) | 8 | 1186.9±35.2# |
| Treatment II group (administration in late period group) | 8 | 1274.4±39.3# |
*Compare P＜0.01 with sham operated rats; # and operative control group be P＜0.01 relatively.
4, the Rhizoma Picrorhizae extract is to the influence of nephridial tissue MCP-1mRNA expression
FQ-RT-PCR result shows, the sham operated rats kidney of rats organizes the MCP-1mRNA expression very low, operative control group nephridial tissue MCP-1mRNA expresses obviously and raises, each Rhizoma Picrorhizae extract prevention+treatment group MCP-1mRNA is lower than operative control group, and (P is 0.000, see Fig. 3, Fig. 4), along with the increase of Rhizoma Picrorhizae extract dosage, nephridial tissue MCP-1mRNA expression reduces gradually, and significant difference (P=0.000 between each dosage group) is arranged between each dosage group.Prevention group, treatment I group, treatment II group MCP-1mRNA expression reduce (P is 0.000) than operative control group, prevention group, treatment I group, treatment II group MCP-1mRNA expression are higher than 160mg/kg prevention+treatment group (P=0.035 P=0.000 P=0.000), prevention group, treatment I group MCP-1mRNA expression are lower than treatment II group (P=0.000), prevention group MCP-1mRNA expression is lower than treatment I group (P=0.000 sees Table 13).
Table 13 is respectively organized the variation of MCP-1mRNA
| Group | The example number | △ CT ratio |
| Sham operated |
5 | 4.9±1.2 |
| Operative control group | 8 | 146.5±123 * |
| Prevention+treatment 40mg/kg group | 8 | 99.2±10.8# |
| Prevention+treatment 80mg/kg group | 8 | 36.4±4.8# |
| Prevention+treatment 160mg/kg group | 8 | 22.1±4.7# |
| The prevention group | 8 | 29.2±3.8# |
| Treatment I group (early stage administration group) | 8 | 40.6±5.2# |
| Treatment II group (administration in late period group) | 8 | 57.9±9.1# |
*Compare P＜0.01 with sham operated rats; # and operative control group be P＜0.01 relatively
5, the Rhizoma Picrorhizae extract is to the influence of nephridial tissue macrophages infiltration degree
Showed by immune group result, sham operated rats is not almost seen macrophages infiltration, respectively organizes all visible obviously macrophages infiltration of nephridial tissue behind the ischemia-reperfusion, give Rhizoma Picrorhizae extract after, each prevention+treatment group macrophage number is less than operative control group (P is 0.000, sees Table 14).Along with the increase of Rhizoma Picrorhizae extract dosage, the macrophage number reduces gradually in the nephridial tissue, and significant difference (P=0.000 between each dosage group) is arranged between each dosage group.Prevention group, treatment I group macrophage number are less than operative control group (P=0.000 P=0.000), treatment II group macrophage number and operative control group there was no significant difference (P=0.180), prevention group, treatment I group, treatment II group macrophage number are more than prevention+treatment 160mg/kg group (P is 0.000), treatment II group macrophage number is organized (P=0.000 P=0.000) more than prevention group, treatment I, there was no significant difference (P=0.086) between prevention group, the treatment I group.
Table 14 nephridial tissue macrophage counting
| Group | Example number n | Macrophage (individual/0.0625mm2) |
| Sham operated |
5 | 0.40±0.07 |
| Operative control group | 8 | 5.83±0.49 △ |
| Prevention+treatment 40mg/kg group | 8 | 4.03±0.37 △▲ |
| Prevention+treatment 80mg/kg group | 8 | 3.22±0.34 △▲ |
| Prevention+treatment 160mg/kg group | 8 | 2.55±0.43 △▲ |
| The prevention group | 8 | 3.73±0.19 △▲ |
| Treatment I group (early stage administration group) | 8 | 4.05±0.37 △▲ |
| Treatment II group (administration in late period group) | 8 | 5.58±0.33 △ |
△Compare P＜0.01 with sham operated rats;
▲Compare P＜0.01 with ischemia-reperfusion group;
Compare P＜0.05 with ischemia-reperfusion group.One-Way ANOVA, F value=144.255.P=0.000
Conclusion:
Rhizoma Picrorhizae extract is dose-dependence protection ischemia-reperfusion injury of kidney, and preventative or to give the protective effect of Rhizoma Picrorhizae extract in early days obvious than the later stage administration, and its mechanism of action is relevant with the inflammation-inhibiting reagentia with antioxidation.
Embodiment three: with the peroral dosage form of Rhizoma Picrorhizae preparation
Rhizoma Picrorhizae of the present invention can be mixed with peroral dosage forms such as tablet, capsule, granule, powder, oral liquid.
(1) make tablet:
With the extract of Rhizoma Picrorhizae after alcohol extraction, add the moderate lubrication agent and make soft material, granulate with 10 mesh sieves, drying, dry granular adds magnesium stearate, mixing, the tablet machine tabletting becomes tablet, film coating, packing promptly gets coated tablet.
(2) make capsule:
With the extract of Rhizoma Picrorhizae after alcohol extraction, spray drying is made powder, crosses about the 100-120 order and sieves, and encapsulated with the adjuvant mix homogeneously, packing promptly gets capsule.
(3) make granule:
With the extract of Rhizoma Picrorhizae after alcohol extraction, add excipient, to granulate, drying is crossed the 8-10 mesh sieve, and granule is made in packing.
(4) make powder:
Rhizoma Picrorhizae is pulverized, added mixing behind an amount of excipient, powder is made in packing.
(5) make oral liquid:
With the extract of Rhizoma Picrorhizae after alcohol extraction, be dissolved in water, add correctives, add the water pondage, make oral liquid.
(6) using method: adult 0.5～2g (in raw material), 2～3 times/day, oral.The child reduces by half.
Claims (6)
1, the application of Rhizoma Picrorhizae in preparation treatment chronic renal disease medicine.
2, application according to claim 1 is characterized in that: the alcohol extract that adopts the Rhizoma Picrorhizae rhizome.
3, application according to claim 1 and 2, it is characterized in that: adopt the Rhizoma Picrorhizae rhizome to be ground into particulate powder after air-dry, be soaked in 50-95% ethanol or the methanol, ambient temperature overnight is purified, filter, distillation, the Rhizoma Picrorhizae extract of making through lyophilization.
4, application according to claim 3 is characterized in that: the density range of described Rhizoma Picrorhizae extract is 1.0005～1.0269g/ml.
5, application according to claim 1 is characterized in that: the dosage form of medicine is the peroral dosage form that allows on the pharmaceutics.
6, application according to claim 5 is characterized in that: described peroral dosage form is selected from tablet, capsule, granule, oral liquid or powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100371844A CN100493539C (en) | 2006-08-24 | 2006-08-24 | The application of the Tibetan picrorhiza rhizome in preparation the medicine for treating kidney disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100371844A CN100493539C (en) | 2006-08-24 | 2006-08-24 | The application of the Tibetan picrorhiza rhizome in preparation the medicine for treating kidney disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1943671A CN1943671A (en) | 2007-04-11 |
| CN100493539C true CN100493539C (en) | 2009-06-03 |
Family
ID=38043465
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100371844A Expired - Fee Related CN100493539C (en) | 2006-08-24 | 2006-08-24 | The application of the Tibetan picrorhiza rhizome in preparation the medicine for treating kidney disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100493539C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX344305B (en) * | 2009-08-12 | 2016-12-13 | Munisekhar Medasani | Picrorhiza kurroa extract for prevention, elimination and treatment of infection diseases. |
| US9125921B2 (en) * | 2009-09-04 | 2015-09-08 | Munisekhar Medasani | Picrorhiza extract for prevention, elimination and treatment of infection diseases |
-
2006
- 2006-08-24 CN CNB2006100371844A patent/CN100493539C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| "高血糖-糖化氧化-系膜细胞肥大和基质积聚与糖尿病肾病及其高血糖记忆的细胞病理学关系的研究". 孙敏.. 2004 |
| "高血糖-糖化氧化-系膜细胞肥大和基质积聚与糖尿病肾病及其高血糖记忆的细胞病理学关系的研究". 孙敏.. 2004 * |
| 中药胡黄连的化学成分和药理作用的研究进展. 何薇，林江涛.中日友好医院学报，第19卷第6期. 2005 |
| 中药胡黄连的化学成分和药理作用的研究进展. 何薇，林江涛.中日友好医院学报，第19卷第6期. 2005 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1943671A (en) | 2007-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Loha et al. | Acute and subacute toxicity of methanol extract of Syzygium guineense leaves on the histology of the liver and kidney and biochemical compositions of blood in rats | |
| Lin et al. | Protective effect of Flos carthami extract against ethylene glycol-induced urolithiasis in rats | |
| Alshehri | Kaempferol attenuates diabetic nephropathy in streptozotocin-induced diabetic rats by a hypoglycaemic effect and concomitant activation of the Nrf-2/Ho-1/antioxidants axis | |
| Liu et al. | The amelioration of streptozotocin diabetes-induced renal damage by Wu-Ling-San (Hoelen Five Herb Formula), a traditional Chinese prescription | |
| Rashid et al. | Antioxidant, anti-inflammatory and hypoglycemic effects of Fagonia olivieri DC on STZ-nicotinamide induced diabetic rats-in vivo and in vitro study | |
| Hutadilok-Towatana et al. | Acute and subchronic toxicity evaluation of the hydroethanolic extract of mangosteen pericarp | |
| Orhan et al. | Preventive treatment of calcium oxalate crystal deposition with immortal flowers | |
| CN100493539C (en) | The application of the Tibetan picrorhiza rhizome in preparation the medicine for treating kidney disease | |
| Oyetunji et al. | Attenuating effect of polyalthia longifolia on cadmium sulfate-induced testicular toxicity | |
| CN106344832A (en) | Assistant blood lipid reducing tablets and preparation method thereof | |
| CN106074661A (en) | A kind of wheel blade Radix Codonopsis extract new application in preparation prevention injury of kidney medicine and health product | |
| Okafor et al. | Antidiabetic and hypolipidaemic effects of garden egg (Solanum aethiopicum) leaf extract in beta-cells of streptozotocin induced diabetic male wistar rats | |
| CN101897786B (en) | Application of total flavonoids of murraya paniculata leaves in preparing drugs for preventing and treating diabetic nephropathy | |
| CN103860706A (en) | Health-care pure traditional Chinese medicinal preparation with alcoholic liver injury protecting function | |
| Legbosi et al. | Sub-chronic toxicity of hydromethanolic stem bark extract of Musanga cecropioides (Urticaceae) in rat | |
| Najini et al. | The gastroprotective effects of canna edulis ker. tuber starch on peptic ulcer and the histopathological profile of rat stomach | |
| Kim et al. | Protective effects of the Chinese prescription Hachimi-jio-gan against diabetic oxidative stress | |
| Esquivel-Gutiérrez et al. | Effects of damiana (Turnera diffusa; var. diffusa and var. aphrodisiaca) on diabetic rats | |
| CN106188173A (en) | A kind of preparation method and application of high-purity compound syringoside | |
| Ukpanukpong et al. | Antidiabetic and antilipidemic effect of khaya senegalensis ethanolic bark extract in alloxan induced diabetic wistar rats | |
| Dhiaa et al. | VITAMIN E VERSUS PROPOLIS AS AN ADD-ON THERAPY TO SITAGLIPTIN/METFORMIN ON OXIDANT/ANTIOXIDANT STATUS AND LIPID PROFILE IN TYPE 2 DIABETIC PATIENTS. | |
| Jogdand et al. | Acute and sub-acute toxicity studies of hydro-alcoholic extract of dried fruits of Piper longum Linn in Wistar rats | |
| Obasi et al. | Phenolic leaf extract of Vitex doniana ameliorated hepatorenal tissues dysfunctions in diabetic Wistar rats | |
| CHRISTOPHE et al. | Anti-ulcers properties and safety profile assessment of aqueous stem bark extract of Alstonia boonei de wild (Apocynaceae) in rodents | |
| TWI653046B (en) | Herbal compound extract for improving diabetes with liver damage and liver fibrosis and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090603 Termination date: 20190824 |