CN1313096C - Asiaticoside use of medicinal preparation for treating and preventing chronic renal failure - Google Patents
Asiaticoside use of medicinal preparation for treating and preventing chronic renal failure Download PDFInfo
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- CN1313096C CN1313096C CNB2004100123582A CN200410012358A CN1313096C CN 1313096 C CN1313096 C CN 1313096C CN B2004100123582 A CNB2004100123582 A CN B2004100123582A CN 200410012358 A CN200410012358 A CN 200410012358A CN 1313096 C CN1313096 C CN 1313096C
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Abstract
The present invention discloses the application of asiaticoside in the preparation of medicines for preventing and treating chronic renal failure. The present invention verifies that asiaticoside can obviously relieve the urine protein within 24 hours, reduce serum creatinine and urea nitrogen, enhance plasma protein and glomerular filtration rate, reduce the deposition of extracellular matrixes in hardened areas, relieve the thickening of glomerular basement membranes, relieve the pathological damage of glomerulus and preventing and treating glomerular sclerosis and fibrosis. Thus, the asiaticoside can be completely applied to the preparation of medicines for preventing and treating chronic renal failure, and especially, the asiaticoside has more obvious significance to the preparation of medicines for preventing and treating membranous renal disease.
Description
Affiliated field
The present invention relates to the medical usage of active ingredients of plants, the application of Herba Centellae total glycosides in preparation control chronic renal failure medicine specifically.
Technical background
Herba Centellae is a Umbelliferae Centella plant, and herb is gone into grass, sweet, hot, cool, the heat reliving and toxin-eliminating of its property.Among the peoplely be usually used in treating flu, tonsillitis, infectious hepatitis, dysentery, traumatic injury, treatment pyogenic infections from tumour or sore, wound water outlet etc.Existing a lot of researchs of active princlple Herba Centellae total glycosides pharmacology of Herba Centellae and pharmaceutical use and report.For example this total glycosides has the clear infections effect of anti-stomach (" modern Application pharmacy " 1989 such as Pu training inflammation, b (1): 8).Hypermnesis effect Nallni k, etat Fitoterapia.1992,63 (3): 232); To the inhibitory action of nervus centralis (Piwan pv, et at.Fitoterapia 1991,62 (3): 253); Stimulating organism anabolic effect (Tenai R et at, ItalJ Biochem, 1988,37 (2): 69) or the like, be used for the treatment of the vein function clinically and do not conform to (MonleverdtA et al Aeta Ther, 1987.13 (6): 629) treatment microangiopathy (Pelearo G, et al Carr Ther Res ClinExp, 1989,46 (6)), treatment dermatosis and wound (CTiardinaA, etal, Cbrcn.bermalol, 1987,18 (Suppl, 1): 109) or the like.Present people are continuing research always and are inquiring after the how better new medical use of Herba Centellae total glycosides.
Summary of the invention
Purpose of the present invention just provides a kind of new medical use of Herba Centellae total glycosides.
The object of the present invention is achieved like this:
The inventor finds that through studying for a long period of time Herba Centellae total glycosides tool in the control chronic renal failure has certain effect.Invented its application in preparation control chronic renal failure medicine in view of this.
Chronic renal failure is the co-channel that various kidney diseases develop into whole latter stage, is the final outcome of the disease of matter between various glomerule, blood vessel and tubule own.Be under the effect of various virulence factors such as inflammation, various damage, medicine, diabetes and inherited genetic factors etc., Interstitial cell and cellular matrix increase, but substrate degradation is caused the fibrosis of matter between glomerular sclerosis that the bulk deposition of extracellular matrix causes and tubule, and companion's renal function is faded and lost.Western medicine does not still have good plan to the treatment of chronic renal failure at present.
The present invention studies have shown that Herba Centellae total glycosides has the effect of good anti-chronic renal failure.
Beneficial effect of the present invention has obtained proof by following examples.
Embodiment 1
Herba Centellae total glycosides is to the research of adenine renal failure
The experiment material male SD rat is about body weight 120g (Nanjing University of Traditional Chinese Medicine's Experimental Animal Center).Adenine (C5H5N5), molecular weight 135.13, Shanghai uncle bio tech ltd production difficult to understand.Immunohistochemistry main agents: an anti-goat-anti rat TGF-β 1, TIMP-1, PAI-1 polyclonal antibody; Two anti-rabbit igg immune reagent kits; Three anti-SABC test kits, TGF-β 1, TIMP-1 in situ hybridization test kit: doctor's moral biotech company.
Medication rat adaptability was fed 3 days, experimentized behind the weight increase.Experimental group content is that (dosage is 100mg/kg.d, 150mg/kg.d, 200mg/kg.d to 98.0% adenine suspension oral gavage, each successively) in a week, modeling gives Herba Centellae total glycosides 9mg/kg after 3 weeks, the blood sampling of 2 week of treatment back execution animal hearts detects hepatic and renal function, and get the nephridial tissue separated into two parts, a part is carried out pathologic finding, and another part carries out in situ hybridization and detects after paraformaldehyde is fixing, and matched group is irritated the distilled water of stomach equal volume.
The SABC method:
Section dewaxes to water 0.3%H
2O
2Deactivating endogenous peroxydase, 92 ℃ of antigen hot repairs are multiple, add TGF-β 1, TIMP-1, PAI-1 polyclonal antibody, and 4 ℃ are spent the night.Add with goat anti-rabbit igg-anti-, add the SABC complex, DAB-H
2O
2Colour developing.Haematoxylin is slightly redyed, conventional dehydration, transparent, mounting.Mirror is observed down and is taken pictures, and positive material is pale brown color.Blank and blood serum substituting contrast are established in this experiment, and each results of comparison is all negative.
In-situ hybridization method
Recover room temperature, 0.6%H
2O
2Deactivating endogenous peroxydase exposes the mRNA nucleic acid fragment, adds 20 μ l prehybridization solutions, 37 ℃ of-40 ℃ of constant temperature 3 hours add 20 μ l hybridization solutions, and 40 ℃ of hybridization are spent the night, washing, drip bovine serum albumin confining liquid and biotinylation mouse-anti digoxin, add SABC and biotinylation peroxidase, the DAB colour developing, haematoxylin is redyed, dehydration of alcohol, dimethylbenzene is transparent, mounting.Mirror film making is down observed, and nuclear indigo plant is dyed, and positive thing is pale brown color in the endochylema.Blank does not add hybridization probe, and the result is negative.
BJ University of Aeronautics ﹠ Astronautics's pathological image analytical system is adopted in result and analysis image analysis, and 6 specimen of every group analysis, each specimen are got 6 glomerule or 6 subregions; The content of 6 sectional positive reaction things of the every specimen of SABC and in situ hybridization picture analyzing.
The results are shown in Table 1, table 2:
Table 1: Herba Centellae total glycosides is to the influence of adenine chronic renal failure rat 24h protein quantification
| Group | n | 3 weekends | 4 weekends | 5 weekends |
| Normal group model group Herba Centellae group | 10 7 8 | 21.64±4.68 128.94±38.43 # 68.13±15.72 & | 24.32±2.62 187.75±30.42 # 79.82±16.43 & | 29.927.25 246±47.46 # 89.37±27.58 & |
Annotate ﹠amp; Compare p＜0.01 with model group, # compares p＜0.01 with normal group
Table 2: Herba Centellae total glycosides is to the influence of adenine chronic renal failure rats'liver renal function
| Group | n | Blood urea nitrogen (ρ/mmol.l -1) | Creatinine (ρ/μ mol.l -1) | Total protein (ρ/gl -1) | Albumin (ρ/gl -1) |
| Normal group model group Herba Centellae group | 10 7 8 | 4.85±1.48 36.10±7.32 # 16.52±1.60 & | 70.67±5.39 212.17±58.55 # 156.50±42.76 & | 72.04±1.82 53.79±2.02 # 65.04±2.08 & | 32.83±0.75 26±2.37 # 30.42±1.76 & |
Annotate ﹠amp; Compare p＜0.01 with model group, # compares p＜0.01 with normal group
Herba Centellae total glycosides is to the influence of adenine renal failure rat pathomorphology
Herba Centellae total glycosides group tubule epithelial cell vacuolar degeneration, accidental protein cast and local monocyte infiltration, the model group rat then sees the atrophy of many kitchen ranges of renal tubules property, hypertrophy, obvious protein cast, matter obvious fibrosis and edema, diffusivity monocyte infiltration.Model group shows the renal tubules arrangement disorder, the distortion of tube chamber severe, and the tubule basement membrane obviously thickens, the tubule cell disruption, form is unclear.Between the matter local edema, part fibroplasia is obvious, lymphocytic infiltration.Part glomerule fibrosis, form disappears, and the part glomerular volume is dwindled, and bag Man blister cavities is narrow, and basement membrane partly ruptures, adhesion.Mesentery broadening, disappears at the distortion of most of capillary lumen, and pathological changes gets involved＞and 80%.Part bead bag Man hypothallus thickens, and blister cavities broadens.
Herba Centellae total glycosides is to the influence of adenine renal failure rat TGF-β 1, TIMP-1, PAI-1
Experimental result trend shows: SABC and in situ hybridization result show that the albumen of PAI-1, TGF-β 1, TIMP-1 and mRNA positive reaction owner will be distributed in matter between kidney, and the response strength model group obviously is better than the Herba Centellae total glycosides group.
Transforming growth factor
1(TGF-β
1), be the mediation ECM sedimentary main factor, in the nephridial tissue damage, its concentration is expressed and is continued to increase, and parallels with the increase of ECM composition.Matrix metalloproteinase (MMP
s) be one of three main systems of ECM degraded (serine protease, cysteine proteinase, MMP
sOne of), the concentration of MMP mortifier (TIMP) and fibrinolysin system and MMP
sAct on closely related.Activator of plasminogen (PA) is the main member of serine stretch protein enzyme system, in the banking process of EMC, it is t-PA and the equilibrated change of Plasminogen activator-1 (PAI-1) that the adjusting of protease changes, in the substrate amplification procedure, may tend to PAI-1, and the activity of PA is relevant with PAI activity and concentration.In view of important function and the influence each other relation of three in nephridial tissue EMC banking process, be MAIN OUTCOME MEASURES so this research turns into these three kinds of molecular changes.
The relation of TGF-β 1, PAI-1, TIMP-1 and CRF.The existing lot of documents report of the effect of TGF-β in kidney inflammation, sclerosis and repair process, its hypertrophy to kidney cell, hypertrophy, apoptosis and part and systemic immune response all play an important role, but the most outstanding be the metabolism regulating action of ECM.TGF-β 1 is the important medium of glomerular sclerosis.Have among the people, mice, Rat Mesangial, epithelial cell of experiment confirm In vitro culture, TGF-β 1 can induce the expression of ECM heterogeneity.Simultaneously, TGF-β 1 still comprise PAIS, TIMPS multiple molecule upstream element and play multiple regulating action.PAI and PA regulate the active important substance of fibrinolytic system.Itself being had the effect of degraded ECM some composition by the activated fibrinolysin of PA, as fibronectin, laminin and fibrin etc., and is the activator of matrix metalloproteinase.In vitro study proves, the intrinsic cell of kidney (mesangial cell, glomerular epithelium cell, endotheliocyte, renal cells) can synthesize PA, PAI-1, reach the U-PA receptor, PAI-1 plays an important role in glomerular sclerosis and kidney region fibrosis process as the PAI main component, and the rising of its concentration is to cause one of kidney ECM composition secretion increase, sedimentary key factor.TIMP-1 is one of four kinds of TIMP, is distributed widely in tissue and the body fluid, can participate in the generation evolution of multiple disease, and Nakamura finds in experiment, and rat model nephridial tissue TIMP-1mRNA level increases, then collagen protein, laminin showed increased.Experiment confirm is also arranged, add in the hypercholesterolemia rat model at unilateral nephrectomy, it is relevant that kidney region fibrosis and TGF-β 1, TIMP-1mRNA level increase jointly, can obviously suppress interstitial fibrosis and give antioxidant.
Herba Centellae total glycosides can stop the CRF PD, improves the laboratory animal life quality, reduces laboratory animal blood BUN and Scr level.
Target molecule mRNA expression all is lower than model group in varying degrees behind the Herba Centellae total glycosides therapeutic intervention, can reduce glomerule, renal tubules TGF-β 1, PAI-1, TIMP-1mRNA expression, suppresses the ECM deposition, delays the renal fibrosis process.
Embodiment 2
Herba Centellae total glycosides is to the experimentation of 5/6 nephrectomy rat chronic renal failure
Experiment material: male SD rat, about body weight 120g (Nanjing University of Traditional Chinese Medicine's Experimental Animal Center).The anti-Mus ColIV of rabbit, FN polyclonal antibody.
Animal Model Making is according to 5/6 nephrectomy method.
After adapting to 1 week of nursing, the Monday in the 2nd week is extractd rats with left 2/3 kidney, and the Monday in the 3rd week is extractd rat right side kidney.The Monday in operation the 4th week of back is divided at random by the serum creatinine level of animal: (1) model group; (2) Herba Centellae group.After the grouping, gastric infusion every day begins to divide into groups: (1) normal group, model group are given distilled water 3ml/d; (2) the Herba Centellae total glycosides group is pressed 9mg/kg and is irritated stomach.Treat when examining extremely after 12 weeks, arteria caudalis is got blood, measures serum creatinine and urea nitrogen levels, and gets nephridial tissue row pathological examination.And in 4,8,12,16 week of operation back mensuration 24h urine protein quantitation.
The nephridial tissue morphologic detection:
Get fresh nephridial tissue, 10% neutral formalin is fixed, and paraffin embedding is done HE, PAS dyeing.Glomerular sclerosis is carried out the sxemiquantitative classification.Get 6 animals for every group.20 glomerule of every sections observation.Calculate the percentage ratio that the glomerular matrix area accounts for the glomerule area, the PAS stained is analyzed with HPIAS-1000 pathology image analysis system.Simultaneously each group rat interstitial disease of renal tubule is carried out semi-quantitative analysis.Promptly normal, tubule dilatation, little shrink tube, cast, cell infiltration, interstitial fibers according to matter between tubule be divided into normally (-), light (+), in 4 ranks of (++), weight (+++).
SABC detects:
Section dewaxes to water 0.3%H
2O
2Deactivating endogenous peroxydase, 92 ℃ of antigen hot repairs are multiple, add ColIV, FN polyclonal antibody, and 4 ℃ are spent the night.Add and use goat anti-rabbit igg two anti-, add the SABC complex, DAB-H
2O
2Colour developing.Haematoxylin is slightly redyed, conventional dehydration, transparent, mounting.Mirror is observed down and is taken pictures, and positive material is pale brown color.Blank and blood serum substituting contrast are established in this experiment, and each results of comparison is all negative.
The result:
Ordinary circumstance: the obvious malnutrition of model group animal, lethargy, movable slow, inappetence, fluffy, the withered tarnish of fur, mortality rate 20%.The Herba Centellae group is better, and mortality rate is 8%.
Herba Centellae total glycosides sees Table 3 to the influence of 5/6 nephrectomy rat 24h protein quantification
Table 3 Herba Centellae total glycosides is to the influence of twenty-four-hour urine protein quantification
| Group | n | The 4th week | The 8th week | The 12nd week | The 16th week |
| Normal group model group Herba Centellae group | 9 6 8 | 6.74±2.36 # 19.32±4.38 17.38±3.85 | 6.98±1.64 # 51.86±5.12 26.23±4.49 # | 7.58±2.41 # 94.43±8.13 35.51±4.77 # | 7.84±2.79 # 263.29±26.34 74.68±19.97 # |
Annotate: # compares P＜0.01 with model group
Since the 8th week, the Herba Centellae group is compared with model group, and significant difference p＜0.01 is arranged, and the prompting Herba Centellae total glycosides can obviously reduce the urine protein of model group rat.
Herba Centellae total glycosides is to the influence (seeing Table 4) of 5/6 nephrectomy kidney of rats function and plasma protein
Table 4 Herba Centellae total glycosides is to the influence of 5/6 nephrectomy kidney of rats function and plasma protein
| Group | n | Blood urea nitrogen (ρ/mmol.l -1) | Creatinine (ρ/μ mol.l -1) | Total protein (ρ/gl -1) |
| Normal group model group Herba Centellae group | 9 6 8 | 7.2±1.26 # 43.28±14.57 28.86±9.32 # | 95.2±5.8 # 281.8±113.45 142.4±48.57 # | 71.26±4.86 # 62.9±2.98 66.87±2.45 # |
Annotate # and compare with model group, p＜0.05 or 0.01 illustrates that Herba Centellae total glycosides can improve the renal function of model group rat, improves plasma protein.
The kidney pathological examination sees Table 5, table 6
Model group: the glomerular mesangium district be focal or diffusivity in severe broadening, increase based on mesentery substrate, proliferation of mesangial cells is not obvious, capillary lumen is narrowed down by the mesentery compressing of broadening and is or inaccessible.Part glomerular capillary button loop and renal capsule adhesion, the glomerular sclerosis that has, atrophy, renal capsule thickens, even combines together with fibrosis around the bead.Proximal tubular epithelial cells degeneration swelling around the pathological changes glomerule diminishes tube chamber, the visible protein cast of part tubule intracavity.Between matter the proliferation of fibrous tissue that is dispersed in is arranged, especially with around the hardened glomerule obviously, there is a large amount of proliferations of fibrous tissue the subregion, and see have be dispersed in or the focal inflammation cellular infiltration, based on lymphocyte.
The Herba Centellae group: the glomerular capillary chamber is open, and most glomerular mesangiums district is slight broadening, also has minority glomerular mesangium district to become slight or severe broadening, also increases accidental sacculus adhesion based on mesentery substrate.The degeneration of minority proximal tubular epithelial cells is arranged, and a matter has a spot of proliferation of fibrous tissue and cell infiltration.
Table 5 kidney pathological examination results
| Normal group | Model group | The Herba Centellae group | |
| Substrate percent | 0.26±0.059 # | 1.65±0.249 | 1.28±0.186 # |
Annotate # and compare p＜0.05 or 0.01 with model group
Qualitative change situation between table 6 renal tubules
| Group | n | - | + | ++ | +++ |
| Normal group model group Herba Centellae group | 9 6 8 | 9 0 4 | 1 3 5 | 0 5 1 | 0 2 ## 1 #& |
Notes are compared #p＜0.05, ##p＜0.01 with normal group
﹠amp; Compare p＜0.05 with model group
SABC result
The expression of ColIV mainly is distributed in positions such as GCBM, mesangial region and renal capsule basement membrane; The expression of FN mainly is distributed in the glomerular mesangium district, and expression is also arranged on the basement membrane, but painted light, compares with normal group, and the expression scope of model group and intensity obviously enlarge and strengthens, and the Herba Centellae total glycosides group is compared with model group, significantly alleviates.See Table 7
Table 7 is respectively organized the comparison of FN in the mesentery substrate, ColIV relative amount
| n | Normal group | Model group | The Herba Centellae group | |
| FN content ColIV content | 6 6 | 0.023±0.017 0.031±0.019 | 0.271±0.032 * 0.325±0.112 * | 0.102±0.025 # 0.11±0.014 # |
Annotate * and compare p＜0.01 with normal group
# and model group p＜0.05 or p＜0.01
This experiment is made the chronic renal failure model by utilizing 5/6 nephrectomy, gives the Herba Centellae total glycosides treatment, found that Herba Centellae total glycosides can significantly alleviate the twenty-four-hour urine albumen of model group rat, reduces serum creatinine, blood urea nitrogen, improves plasma protein.Reduce the deposition of extracellular matrix, control glomerular sclerosis, Fibrotic formation at hardening region.Herba Centellae total glycosides is to preventing the great important that has of end-stage renal failure thus.
Embodiment 3
Herba Centellae total glycosides is to the influence of chronic serum sickness mesangial proliferative nephritis
Laboratory animal and grouping
50 of the healthy male SD rats of outbreeding system, body weight 150 ± 21 grams in 10 ages in week, are provided by Nanjing University of Traditional Chinese Medicine's Experimental Animal Center., after animal via normal diet adaptability fed for 1 week, urine protein qualitative results feminine gender was divided into normal group at random, model contrast, Herba Centellae total glycosides group.Every group 10.
Main agents
Complete Freund's adjuvant and incomplete Freund's adjuvant (GIBCO company), bovine serum albumin (BSA) and escherichia coli endotoxin (SIGMA company).The one anti-anti-Mus TGF of the rabbit β that comprises
1Polyclonal antibody, mouse anti P of Rats DGF monoclonal antibody (Beijing Zhong Shan biotech company), Tris alkali, 30%H
2O
2(auspicious biotech company is created in Nanjing).SABC immunohistochemical staining test kit (Wuhan doctor's moral biotech company).
Rat experiment mesangial proliferative nephritis replication of Model
Adopt the chronic serum sickness MsPGN model of improvement
[1], 40 male SD rats, ketamine intravenous anesthesia is after excision left kidney in back is carried out pre-immunity after 1 week of recuperating.Add BSA3mg at every the subcutaneous branch injection of rat back complete Freund's adjuvant 0.1ml, in 1 weekend, 2 weekend back subcutaneous injection incomplete Freund's adjuvant 0.1ml add BSA3mg, 3 weekends, continuous 4 times of lumbar injection BSA, each 1h at interval, every of injected dose is respectively 0.5,1,1.5,3.0mg; Every of next day is at lumbar injection BSA2mg.Formal immunity: after pre-immunity is finished first day, hocket the next day of tail vein and lumbar injection, every caudal vein is injected BSA dosage from 0.5mg, each 0.5mg that increases, till 2.5mg, continue dosage 0.5mg weekly, till every day consumption 5mg.The lumbar injection amount is 1 times of tail vein injection amount, till every day consumption 10mg.2 week of immunity back tail vein injection escherichia coli endotoxin 100 μ g/ only.All rat is put to death with formal immunity 8 all backs and gets nephridial tissue.
Biochemistry and pathological examination
Biochemical analysis: (1) twenty-four-hour urine protein quantification; (2) urine C3, NAG; (3) blood urea nitrogen, creatinine; (4) total serum protein, albumin.Semi-automatic biochemical analyzer detects
The morphologic inspection of renal tissue
SABC detects
Section dewaxes to water 0.3%H
2O
2Deactivating endogenous peroxydase, 92 ℃ of antigen hot repairs are multiple, add TGF-β 1, PDGF polyclonal antibody, and 4 ℃ are spent the night.Add and use goat anti-rabbit igg two anti-, add the SABC complex, DAB-H
2O
2Colour developing.Haematoxylin is slightly redyed, conventional dehydration, transparent, mounting.Mirror is observed down and is taken pictures, and positive material is pale brown color.Blank and blood serum substituting contrast are established in this experiment, and each results of comparison is all negative.
The result
Damp and hot rat model urine protein and the pharmaceutical intervention situation of being impairment of the kidney
From the 6th week of modeling, model group and Herba Centellae group all have albuminuria in various degree, and pass in time and increase the weight of gradually, and during the 6th, eight, ten weekends, each Herba Centellae group of twenty-four-hour urine protein quantification significantly is lower than model group.Herba Centellae total glycosides group prompting Herba Centellae total glycosides can obviously alleviate the albuminuria of MsPGN rat.See Table 8
Table 8 24h urine protein quantitation situation of change at the 6th, eight, ten weekends (mg/24h)
| Group | n | Six weekends | Eight weekends | Ten weekends |
| Normal group model group Herba Centellae group | 9 6 7 | 19.64±4.48 156.94±42.84 # 64.02±13.31 △ | 25.32±2.76 195.75±31.63 # 80.95±15.56 △ | 33.92±9.25 260±57.37 # 89.41±29.69 △ |
Annotate # and compare p＜0.01 with normal group, △ compares p＜0.01 with model group
The damp and hot rat model serum creatinine that is impairment of the kidney, blood urea nitrogen and plasma protein situation of change
During the tenth weekend, blood CRE, the BUN of model group is all apparently higher than normal group (p＜0.01, Herba Centellae group blood CRE, BUN have compared utmost point significant difference (p＜0.01 with model group, illustrate that Herba Centellae total glycosides can improve the renal function of MsPGN rat, during the tenth weekend, Herba Centellae group plasma protein has been compared utmost point significant difference, and (p＜0.01 illustrates that Herba Centellae total glycosides can obviously improve total protein, the albumin of MsPGN rat with model group.
See Table 9
Table 9: the tenth weekend serum creatinine, blood urea nitrogen and plasma protein situation of change
| Group | n | Blood urea nitrogen (ρ/mmol.l -1) | Creatinine (ρ/mol.l -1) | Total protein (ρ/gl -1) | Albumin (ρ/gl -1) |
| Normal group model group Herba Centellae group | 9 6 7 | 4.85±1.48 16.10±1.32 # 12.22±1.60 & | 70.67±5.39 122.17±11.55 # 96.50±4.76 & | 72.04±1.82 53.79±2.02 # 64.04±2.08 & | 32.83±0.75 26±2.37 # 30.42±1.76 & |
Annotate # and compare p＜0.01 ， ﹠amp with normal group; Compare p＜0.01 with model group
The interstitial disease of renal tubule situation of the damp and hot rat model that is impairment of the kidney
The tenth weekend, urine NAG, the UUC3 of model group obviously raise, and compared with normal group when examining extremely, and utmost point significant difference (p＜0.01 is arranged.The Herba Centellae group can reduce urine NAG, UUC3.See Table 10
Table 10 is urinated the situation of change of NAG, UUC3 the tenth weekend
| Group | N | NAG | UUC3 |
| Normal group model group Herba Centellae group | 9 6 7 | 13.77±4.64 # 147.89±52.70 58.25±20.59 # | 1.78±1.38 # 35.17±4.62 14.85±4.09 # |
Annotate: # compares P＜0.05 or P＜0.01 with model group
Nephridial tissue is checked
Light microscopy checking model group 6 weeks of immunity: the part glomerule increases, and glomerular capillary is obviously expanded, endotheliocytic swelling, and cell infiltration, renal cells is granule and vacuolar degeneration, has red cell cast and hyaline cast to form.8 weeks of immunity: the focal segmental hypertrophy of mesangial cell, the adhesion of part renal capsule, cell infiltration.Immunity mainly shows as indivedual glomerule 10 weeks and atrophy occurs and the fibrosis that arrives, majority presents the mesenteric tissue hypertrophy that the weight that fills the air does not wait, most of zone is the moderate hypertrophy, show as mesangial region broadening, mesangial cell and mesentery substrate all have hypertrophy in the mesangial region of broadening, and the severe hypertrophy appears in individual areas, and capillary lumen is narrow and small, discrete structure destroys, and blood vessel disappears.Inflammatory cell infiltration is obvious, the slight hypertrophy of Herba Centellae total glycosides group individual areas mesangial cell, and the slight broadening of mesentery substrate, tube chamber does not have the extruding phenomenon.See Table 11.
The pathology detection result of table 11 rat
| Group | N | Residual kidney mesangial region/glomerule area | Residual kidney mesangial cell number |
| Normal group model group Herba Centellae group | 6 6 6 | 7.06±2.12 △ 29.29±9.94 14.04±3.81 △ | 11.69±3.53 △ 35.17±8.70 20.53±5.05 △ |
Annotate: △ compares P＜0.01 with model group
The visible glomerular mesangium fauna of Electronic Speculum model group theca cell hypertrophy, mesentery substrate increases, the visible segmental podocytic process of epithelial cell merges or fine hair becomes, the vaporific electron-dense thing deposition of mesangial region visible cloud is compared the Herba Centellae total glycosides group and is not seen that obvious proliferation of mesangial cells and mesentery substrate increase phenomenon with model group.
Immunofluorescence normal group glomerular mesangium district does not have IgG, C
3Deposition, the model group immunity showed as in 6 weeks fine particulate IgG in the glomerule (+~ ++) and C3 (+~ ++) deposit at mesangial region, immunity has in 8 all glomerule the IgG (++ ~ +++) of coarse granule shape and C3 (++ ~ +++) to deposit at mesangial region, and IgG when examine immune 10 weekends extremely (+++~ ++ ++) and C3 (+++~ ++ ++) are the big particle shape and deposit at mesangial region.Herba Centellae total glycosides group IgG (+~ ++) and C3 (+~ ++) deposition obviously reduces, and is to be dispersed in graininess.
SABC result
The result shows normal group TGF β
1Glomerule has extremely faint expression, and is many in glomerule in the distribution of matter between kidney.Model group is TGF β when the 6th, 8 weeks
1Mainly be distributed in the basement membrane of epithelium of bag Man capsule, glomerule, the distribution of while matter between kidney is showed increased also.Model group can be seen TGF β when examining extremely in the 10th week
1Be distributed in bag Man capsule, the basement membrane of epithelium of glomerule, blood vessel endothelium in a large number, the distribution of while matter between kidney is showed increased also, and part is lumps.Herba Centellae total glycosides TGF β
1Compare obvious minimizing p＜0.01=explanation Herba Centellae total glycosides than model group in the distribution of glomerule and can obviously reduce TGF β in the MsPGN kidney of rats tissue
1Distribution.
The result shows that normal group has the basal expression of PDGF, and visible PDGF mainly is distributed in matter position between the epithelial cell of mesentery substrate and some renal capsule and tubule in the 6th, 8 whens week.The PDGF of model group dyeing obviously strengthened when the 10th week was examined extremely, mainly be distributed in matter position between the epithelial cell of mesentery substrate and renal capsule and tubule, compare with normal group, the PDGF dyeing scope of model group significantly strengthens (p＜0.01=, the dyeing scope of the PDGF of Herba Centellae total glycosides is compared than model group, significantly reduce that (p＜0.01=, prompting Herba Centellae total glycosides can obviously reduce in the MsPGN rat mesentery substrate PDGF in the distribution of glomerule.See Table 12
Table 12PDGF and TGF-β
1Immunohistochemical analysis
| Group | N | The content of PDGF | TGF-β 1Content |
| Normal group model group Herba Centellae group | 6 6 6 | 0.326±0.145 0.089±0.021 # 0.089±0.021 # | 0.027±0.018 # 0.245±0.091 0.095±0.021 # |
Annotate: # compares P＜0.01 with model group
In kidney disease, cytokine participates in pathological process by two kinds of approach at least: the one, as the aggressivity inflammatory mediator, mainly contain IL-2, TNF-α, IL-1 β, IFN-γ, PDGF, TGF β
1Deng; The 2nd, as anti-inflammatory cytokines, participate in the egodefense of kidney disease, promote the dissipation and the reparation of inflammation, mainly contain IL-4, IL-10, IL-13, IL-1ra etc.Cause the balance of inflammatory and anti-inflammatory cytokines, determine lapsing to of nephropathy.
PDGF is the strongest a kind of mitogenic factor of mesangial cell, at animal and human's class MsPGN all to find having PDGF-A or PDGF-BmRNA and PDGF-B receptor mrna and proteic high expressed in the nephridial tissue.PDGF is when influencing the inflammation breeding, and the expression that may also influence cytoskeletal protein changes and reorganization.Have and discover that the antagonist and the pdgf receptor tyrosine kinase inhibitor STI571 that use PDGF-B can alleviate matter destruction between glomerular sclerosis, tubule, glomerule and a matter position III, the gathering of IV collagen and the overexpression of TGF, prompting can the delay of progression nephropathy especially make the progress of mesangial proliferative nephritis by antagonism PDGF-B.
Transforming growth factor-beta
1(TGF β
1) be a kind of multi-functional cytokine, its overexpression can cause the fibrosis of histoorgan.A large amount of clinical and experiment showed, TGF β
1With the glomerular sclerosis substantial connection, TGF β
1By impelling mesangial cell secretion generation IV Collagen Type VI and fibronectin to cause extracellular matrix to be assembled.TGF β
1Can stimulate synthetic the increasing of ECM of mesangial cell, epithelial cell and the proximal tubular epithelial cells of cultivation.Use TGF β
1Specific inhibitor Decorin transfection people mesangial cell comes the antagonism renal fibrosis.TGF β thus
1Proceeding in the hardened process at renal glomerular disease is the mediation factor of a key.
Experimental result shows, at normal glomerule TGF β
1Be distributed between kidney matter than being many in the glomerule, consistent with bibliographical information.And at the 6th, 8 all TGF β of model group
1Distribution at glomerule increases gradually, TGF β when examine for 10 weekends extremely
1The main matter position between glomerular basement membrane, mesangial region, renal capsule parietal layer epithelium and tubule of distribution, Herba Centellae total glycosides can obviously reduce PDGF and TGF β
1In the especially distribution at matter position between renal tubules of glomerule, reduce its mRNA level, illustrate that Herba Centellae total glycosides can be by suppressing PDGF and TGF β
1Albumen and thereby mRNA express to suppress proliferation of mesangial cells and substrate increases.
Embodiment 4
Herba Centellae total glycosides is to the influence of membranous nephropathy rat
Laboratory animal: select 40 of healthy SD rats, age in days 46 days, body weight 200 ± 20 grams are purchased the Experimental Animal Center in Nanjing University of Traditional Chinese Medicine.Experimental session gives the rat full-valence pellet feed and freely drinks water.Keep 18～24 ℃ of temperature, relative air humidity 40～70%.
Main agents and instrument: cationization bovine serum albumin (C-BSA) is provided for oneself, incomplete Freund (U.S. Gibco company), and goat-anti rat I, III Collagen Type VI (Foochow steps Newbiotics Inc), Herba Centellae total glycosides is pressed 9mg/kg dosage and is irritated stomach.ACETM automatic clinical chemistry analyzer (U.S. international nuclear power group product), BHS-313 fluorescence microscope (Japanese OLYMPUS product).
Experimental technique
Antigen preparation: according to the isoelectric point, IP of literature method change N-BSA,, record C-BSA PI 〉=10 after the lyophilization, be stored in-80 ℃ of ultra cold storage freezers standby with distilled water (4 ℃) dialysis 48h.
Modelling: the reference literature method is improved the back a little and is made rat membranous nephropathy (MN) animal model.1.1mg-BSA is dissolved in the 0.55ml normal saline, with equivalent IFA fully emulsified after, it be pre-immune giving rat multiple spot subcutaneous injection; After 1 week, begin the immunity of causing a disease, be i.e. once inject (iv) C-BSA4mg the next day, continuous 4 weeks to rat tail vein.
Grouping and administration
The SD rat is bought the back back and adapts to 1 week of raising, urine examination albumen total negative earlier.After the pre-immunity, be divided into 4 groups at random according to rat body weight, every group each 10, promptly normal group, model group, Herba Centellae are organized.
Normal group: give distilled water 3ml ig once a day, afterbody iv 1ml next day of behind the 1h.
Model group: give distilled water 3ml ig once a day, an afterbody iv C-BSA 4mg (C-BSA being used the solution that the is mixed with 5mg/ml) next day of behind the 1h.
The same model group of Herba Centellae group modeling, drug treatment simultaneously.
Observation index
Ordinary circumstance is observed: the mental status, active situation, body weight, chaeta, diet and the defecation etc. of observing rat.
Lab testing: 24h urine protein quantitation (24hUTP), serum albumin (ALB), blood fat (TC, TG) and renal function (Scr, BUN, Ccr).The ACE automatic clinical chemistry analyzer detects These parameters.
The renal tissue pathological examination: light microscopic is done two kinds of dyeing of HE, PAS; Immunofluorescence.
The SABC method
Section dewaxes to water 0.3%H
2O
2Deactivating endogenous peroxydase, 92 ℃ of antigen hot repairs are multiple, add sheep anti mouse Col III and Col I polyclonal antibody, and 4 ℃ are spent the night.Add and use goat anti-rabbit igg two anti-, add the SABC complex, DAB-H
2O
2Colour developing.Haematoxylin is slightly redyed, conventional dehydration, transparent, mounting.Mirror is observed down and is taken pictures, and positive material is pale brown color.Blank and blood serum substituting contrast are established in this experiment, and each results of comparison is all negative.
Computer image analysis
Adopt HPIAS-1000 pathological image analytical system, measure the positive material area painted in each glomerule and the area of whole glomerule, with the relative amount of every kind of composition of ratio value representation in glomerule of the two, each specimen is got 3 glomerule, 6 specimen of every group analysis.
General changed condition
The normal group reaction is quick, and is movable normal, hair color gloss, and diet is normal with drinking-water, and stool is graininess, and about 24h urine amount 10ml, the urine color is yellowish.All the other 3 groups at experimental session, lassitude occurs, bradykinesia; Hair is owed gloss, and Juan Dai Curled crouches, and the happiness hogback flocks together; Diet reduces with drinking-water, and activity is few, and body weight gain is slow; Hypourocrinia, loose stool etc.Above-mentioned performance is the most obvious with model group, and the appearance abdominal part that has swells, the perineal position edema, during the execution that has, as seen cut open the belly has a large amount of ascites.
The variation of urine amount
The variation of 24h urine amount, except that normal group, all the other respectively organize rat urine amount all minimizing, and reduces the most obvious with model group; Credit is analysed rat urine quantitative change situation there were significant differences (P＜0.05) by statistics, sees Table 13.
Table 13 Herba Centellae total glycosides to the influence of membranous nephropathy rat urine amount (X ± SD, ml/24h)
| Before the treatment | After the treatment | |
| Normal group (n=10) model group (n=7) Herba Centellae group (n=8) | 12.51±3.43 13.29±5.02 13.01±4.96 | 12.43±2.75 7.30±3.90 12.95±2.97 △ |
△ and model group are than P＜0.05;
Lab testing
The variation of 24h urine protein
Pathogenic immune 2 weekends, except that normal group, all the other 2 rat 24h urine protein all obviously increase, and obvious and the highest with the model group increase especially, pathogenic immune 4 weekends, except that normal group, model group rat 24h urine protein increases obviously.Through variance analysis, 2 weekends and 4 weekends are respectively organized rat urine protein situation of change, and significant differences (P＜0.01) is all arranged, and see Table 14.
Table 14 Herba Centellae total glycosides to the influence of membranous nephropathy rat 24h urine protein (X ± SD, mg/24h)
| Group | The 24h urine protein quantitation | |
| 2 weekends | 4 weekends | |
| Normal group (n=10) model group (n=7) Herba Centellae group (n=8) | 6.44±1.39 * 178.76±26.18 48.97±9.07 * | 4.18±3.4 * 240.93±31.38 32.75±5.92 * |
*With model control group than P＜0.01;
Serum albumin changes
Pathogenic immune 2 weekends, except that normal group, all the other 2 rat blood serum albumin all have reduction, reduce obvious and minimum, pathogenic immune 4 weekends, still minimum with model group especially with model group.Through variance analysis, 2 weekends and the albuminous situation of change of 4 Herba Centellae group rat blood serums at weekend have significant differences (P＜0.01), see Table 15
Table 15: Herba Centellae total glycosides to the albuminous influence of membranous nephropathy rat blood serum (X ± SD, g/L)
| Group | ALB | |
| 2 weekends | 4 weekends | |
| Normal group (n=10) model group (n=7) Herba Centellae group (n=8) | 36.50±0.43 * 21.25±2.82 30.00±2.57 * | 38.13±0.73 * 25.13±2.32 34.13±2.37 * |
*With model group than P＜0.01;
The plasma cholesterol situation
Pathogenic immune 2 weekends, except that normal group, all the other 2 rat plasma cholesterol all obviously raise, especially it is obvious and the highest to raise with model group, pathogenic immune 4 weekends, still the highest with model group, through variance analysis, 2 weekends and 4 weekend Herba Centellae group rat plasma cholesterol situation of change, significant differences (P＜0.01) is all arranged, see Table 16.
Table 16 Herba Centellae total glycosides to the influence of membranous nephropathy rat plasma cholesterol (X ± SD, mmol/L)
| Group | TC | |
| 2 weekends | 4 weekends | |
| Normal group (n=10) model group (n=7) Herba Centellae group (n=8) | 1.77±0.35 * 5.09±1.03 2.12±0.35 * | 1.48±0.09 * 5.06±0.45 1.99±0.46 * |
*With model group than P＜0.01;
The situation of change of plasma triglyceride level
Pathogenic immune 2 weekends, except that normal group, all the other 2 groups of rat plasma triglycerides all obviously raise, especially raise obvious and the highest with model group, pathogenic immune 4 weekends, still the highest with model group, 2 weekends and 4 weekend Herba Centellae group rat plasma triglyceride situation of change, significant differences (P＜0.01) is all arranged, see Table 17.
Table 17 Herba Centellae total glycosides to the influence of membranous nephropathy rat plasma triglyceride (X ± SD, mmol/L)
| Group | TG | |
| 2 weekends | 4 weekends | |
| Normal group (n=10) model group (n=7) Herba Centellae group (n=8) | 1.19±0.16 * 6.96±4.32 1.53±0.57 * | 1.31±0.28 * 7.47±3.53 1.52±0.38 * |
*With model control group than P＜0.01
The situation of change of serum creatinine
Pathogenic immune 2 weekends, except that normal group, all the other 2 groups of rat serum creatinines all have obvious rising, especially raise obviously with model group, cause a disease immune 4 weekends, still the highest with model group, through variance analysis, 2 weekends and 4 weekend Herba Centellae group rat serum creatinine situation of change, significant differences (P＜0.01) is all arranged.See Table 18
Table 18 Herba Centellae total glycosides is to the influence (X ± SD, μ mol/L) of membranous nephropathy rat serum creatinine
| Group | Scr | |
| 2 weekends | 4 weekends | |
| Normal group (n=10) model group (n=7) Herba Centellae group (n=8) | 93.63±6.72 *134.50±24.71 108.63±5.58 * | 95.50±4.00 * 162.25±14.38 114.88±10.89 *□ |
*With model control group than P＜0.01.
The blood urea nitrogen situation of change
Immune 2 weekends, except that normal group, all the other 2 groups of rat serum blood urea nitrogens all have obvious rising, especially it is obvious and the highest to raise with model group, causes a disease immune 4 weekends, still the highest with model group, through variance analysis, 2 weeks not with 4 weekend Herba Centellae group rat serum blood urea nitrogen situation of change, significant differences (P＜0.01) is all arranged, see Table 19.
Table 19 Herba Centellae total glycosides to the influence of membranous nephropathy rat serum blood urea nitrogen (X ± SD, mmol/L)
| Group | BUN | |
| 2 weekends | 4 weekends | |
| Normal group (n=10) model group (n=7) Herba Centellae group (n=8) | 4.08±0.63 * 25.32±3.18 10.54±4.61 *△ | 4.14±1.42 * 37.72±9.57 13.29±5.59 *△ |
*With model group than P＜0.01.
Renal tissue pathological change situation
Light microscopy checking
Normal group: the HE visible glomerule morphosis that dyes is normal, and the glomerular capillary loop is open good, GBM do not have thicken, epithelial cell podocytic process form is normal, mesangial cell and mesentery substrate do not have and increase.
Model group: carry out HE, PAS, dyeing is observed under mirror, visible glomerular volume increases slightly, and GBM obviously thickens, and cell number slightly increases, and substrate increases slightly;
Herba Centellae group: glomerular volume increases a little, and GBM does not have obviously and thickens, and cell number slightly increases, with normal control group no significant difference.
The SABC testing result
Normal group Col-III mainly be expressed in the renal cells basement membrane and between the matter place, and dyeing more shallow.Model group Col-III expresses significantly and strengthens,
Membranous nephropathy is one of adult's nephrotic syndrome common pathological type, mainly is a kind of typical autoimmune disease that forms owing to original position immune complex deposit on the glomerular basement membrane.Its pathological characteristic is to have majority to be positioned under the glomerular epithelium cell or the immune complex deposit in the basement membrane outside, thereby shows as the GCBM diffuse thickening.Then show as a large amount of albuminuria or nephrotic syndrome clinically.
Urine protein of this experiment Herba Centellae group obviously reduces than model group, infers that Herba Centellae total glycosides may be by alleviating excretion quantity of urinary protein, improving the purpose that hemodynamics and hypermetabolism state in the little blood vessel of kidney reach the protection renal tubules.The improvement of blood urea nitrogen and serum creatinine also points out the Herba Centellae group can improve glomerular filtration rate, promotes the discharge of blood urea nitrogen and creatinine, improves renal function.Find that simultaneously Herba Centellae total glycosides has the minimizing immune complex deposit, alleviates glomerular basement membrane and thickens, alleviate the effect of glomerule pathology infringement.
This experiment immunization group result shows, normal group Col-III mainly be expressed in the renal cells basement membrane and between the matter place, and dyeing is more shallow.Model group Col-III expresses significantly and strengthens, and the Herba Centellae group relatively has significant difference with model group.The prompting Herba Centellae total glycosides can significantly reduce the expression of Col-III matter between renal tubules, delays the generation of MN rat model kidney region fibrosis.
In sum, Herba Centellae total glycosides can significantly alleviate the twenty-four-hour urine albumen of model group rat, reduces serum creatinine, blood urea nitrogen, improves plasma protein, improves glomerular filtration rate; Reduce the deposition of extracellular matrix, alleviate glomerular basement membrane and thicken, alleviate the infringement of glomerule pathology, control glomerular sclerosis, Fibrotic formation at hardening region; So Herba Centellae total glycosides can be applied in preparation control chronic renal failure medicine fully, particularly the application that prevents and treats in the membranous nephropathy medicine in preparation has more significance.
The Herba Centellae total glycosides crude drug can be buied on medical market.With it is active component, can be prepared into the various pharmaceutical preparatioies that are used to prevent and treat chronic renal failure or prevent and treat membranous nephropathy.
Following examples have exemplified several dosage forms of Herba Centellae total glycosides:
Embodiment 5
The asiatic centella total glucoside oral liquid:
With 250mg Herba Centellae total glycosides (), be dissolved in the 1000ml water, make the aqueous solution of 0.25% concentration, heating for dissolving, mix homogeneously in the 20ml medicine bottle of packing into, seals, sterilizes.
Instructions of taking: oral, take Herba Centellae total glycosides 50-100mg every day.
Embodiment 6
The asiatic centella total glycoside injection liquid:
Get Herba Centellae total glycosides 2000mg, press the conventional preparation method of injection, make the injection that 4mg/2ml/ props up concentration, steam circulation sterilization 30 minutes.
Usage and dosage: intramuscular injection or intravenous injection, every day 20-100mg.
Embodiment 7
The Herba Centellae total glycosides tablet:
Get Herba Centellae total glycosides 2000mg, method for preparing tablet thereof adds starch routinely, and dextrin, magnesium stearate etc. are mixed and made into wet grain, and the machine punching press is in blocks, and every contains Herba Centellae total glycosides 25mg.
Usage and dosage: oral, every day 2-3 time, each 25mg-100mg.
Pharmaceutical dosage form of the present invention is not limited solely to this, and it can be prepared into more dosage form, as drop pill, capsule, soft capsule, sustained-release preparation or the like.
Drug usage consumption of the present invention also is not limited solely to this.The clinicist also can determine the usage and dosage of medicine according to patient's concrete condition.
Claims (1)
1, the application of Herba Centellae total glycosides in preparation control chronic renal failure medicine.
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| CN1377649A (en) * | 2002-04-16 | 2002-11-06 | 中国人民解放军第二军医大学 | Use of hydroxy asiaticoside for preparingm edicine for curing depression |
| CN1387868A (en) * | 2001-05-29 | 2003-01-01 | 中国人民解放军第二军医大学 | Prepn process and new use of general asiaticoside |
| CN1491656A (en) * | 2002-10-22 | 2004-04-28 | 上海海天医药科技开发有限公司 | Medicinal use of asiatic centella extract containing asiaticoside and hydroxy asiaticoside |
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| CN1387868A (en) * | 2001-05-29 | 2003-01-01 | 中国人民解放军第二军医大学 | Prepn process and new use of general asiaticoside |
| CN1377649A (en) * | 2002-04-16 | 2002-11-06 | 中国人民解放军第二军医大学 | Use of hydroxy asiaticoside for preparingm edicine for curing depression |
| CN1491656A (en) * | 2002-10-22 | 2004-04-28 | 上海海天医药科技开发有限公司 | Medicinal use of asiatic centella extract containing asiaticoside and hydroxy asiaticoside |
Non-Patent Citations (1)
| Title |
|---|
| 积雪草的研究进展 赵宇新等，中国中医药信息杂志，第9卷第8期 2002 * |
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