| Propose: Diabetic nephropathy(DN)is a clinical common and severe chronic microvascular complications of,the main clinical features for protein leakage of urine,renal function deterioration of and pathological changes mainly changes of glomerular filtration rate,basement membrane thickening,widened mesangial cells,until the glomerular sclerosis,develop to renal reactive power failure.With the increase of the number of patients with diabetes,the incidence rate is increasing,which is a serious threat to human health.Diabetic nephropathy pathogenesis is complex and,in recent years,many studies have shown that,for various reasons resulting in glomerular filtration membrane and podocyte injury in diabetic nephropathy plays a great role in the development of generation,so the research on podocyte improve podocyte function of drug treatment has become the focus of diabetic nephropathy.The professor mentors Nan Zheng have thought,“toxin impairing renal collaterals,hidden in pleurodiaphragmatic spaces” theory is The main pathogenesis of diabetic nephropathy,proposed “Detoxification and dredging collaterals,nourishing kidney spaces-tonifying,sparse benefit pleurodiaphragmatic in terspace” Treatment ideas and method.ZhenHuaXiaoShenAn capsule is professor mentors Nan Zheng frequently used experience in the treatment of diabetic nephropathy,In the clinical curative effect is remarkable.Through the experimental observation experimental diabetic rats kidney and Podocyte Associated Protein CD2 AP 、 podocin 、 α-atinin-4 、 podocalyxin 、 α3β1-integrin’s Expression effect,indicate” ZhenHuaXiaoShenAn capsule”treatment of diabetic nephropathy effectiveness.Method:1 Adaptability of 70 male wistar rats fed a week,were randomly divided into normal control group(10)and module(60),made by intraperitoneal injection of module 1% STZ 50 mg/kg in the preparation of experimental diabetic rats models,72 h after STZ injection,tail vein blood sampling,measuring ≥16.7mmol/L as building success.Building successful 51 rats were randomly divided into 11 DM model group,ZhenHuaXiaoShenAn capsule high,medium and low dose group,Irbesartan tablet group 10 each.High,medium and low dose group with 1 g/kg/d respectively,0.5 g/kg/d,0.25 g/kg/d,to the ZhenHuaXiaoShenAn capsule suspension gavage;Positive control group to the Irbesartan suspension 20 mg/kg/d gavage,continuous gavage observation 12 weeks.To observe the general condition of rats,record 24 hours urine volume,weight;The end of the experiment records kidney weight/body weight,detection of fasting plasma glucose,24 h urine trace albumin;Detection of cholesterol(TC),triglyceride(TG),serum creatinine(Scr),urea nitrogen(BUN).2 Observed renal ultrastructure by optical microscope and transmission electron microscopy,and observed the renal organism pathological structure changes and kidney podocytes changed for ZhenHuaXiaoShen capsule on diabetic rats kidney pathological morphological structure.3 Use immunohistochemical method observation podocyte slit diaphragm CD2AP、podocin protein expression;use fluorescence quantitative PCR technology detection CD2AP、podocin m RNA expression.4 Use immunohistochemical method observation podocyte podocytic process and adhesion molecule α-atinin-4、podocalyxin protein expression;use real-time quantitative PCR technology detection α-atinin-4、podocalyxin、α3β1-integrin m RNA expression.Result:1 Mental state of rats in normal control group good,hair luster,responsive,weight continued to increase.mental state of rats in DM model group is not good,dull hair、 erected,slowness of movements,polydipsia、 polyphagia、 polyuria、 emaciation obvious symptoms,few rats some parts of the body appear abscess、fester,experimental period death 4 rats.Rat in traditional chinese medicine high,medium and low dose group and western medicine control group mental state、activity reaction cannot do better than rats in normal control group,weight increases in speed slower,urinary production of later stage thanprophase reduce,experimental period traditional chinese medicine high,medium and low dose group every death 2 rats,western medicine control group death 2 rats.2 Renal tissue observed by light microscopy : DM model group: rat glomerular mesangial area diffuse broadening,MC mild hyperplasia,Partial capillary Compression and cave,GBM thickening,glomeruli of kidney ECM increase,renal interstitial Inflammatory cell infiltration,interstitial fibrous tissue hyperplasia glomerular capsule adhesion.Traditional chinese medicine high,medium and low dose group and western medicine control group: glomeruli of kidney structure also has similar Pathological changes,but than DM model group significantly alleviate,rat glomeruli of kidney ECM increase and MC hyperplasia than DM model group significantly alleviate,most Capillary lumen are open state,Interstitial fibrous tissue hyperplasia than DM model group reduce.Traditional chinese medicine high,medium and low dose group and western medicine control group compared with DM model group,renal tissue injury all significantly alleviate(P<0.05)。 Electron microscope observation DM model group: Rat rlomerular filtration membrane obvious change,GBM diffuse thickening,endothelial cells hyperplasia,endothelial cells extensive conciliatory podocyte Decrease in number,podocyte podocytic process extensive conciliatory,Mesangial region enlarge,mesangial matrix increased efficiently.Traditional chinese medicine high,medium and low dose group and western medicine control group: The above pathological changes all different degrees alleviate,Rat glomerular Capillary endothelial cells normal,podocytic process Partial fusion,GBM no thickening,mesangial region no obvious expansion.3 The body weight and urine volume of chinese medicine in high,medium and low dose group were significantly different than those in DM model group(P<0.01);And there were significant differences among high,medium and low dose groups of chinese medicine(P<0.01);The urine volume of high dose group of traditional Chinese medicine is better than western medicine control group(P<0.01).Detection in 4,8,12 weeks rats in each group 24 h urine micro albumin content(ALB).DM model group,each dose group of chinese medicine,western medicine control group rat ALB content increased gradually,and each period was significantly higher than the normal control group(P<0.01);Chinese medicine high,medium and low dose group and western medicine control group,at 4 weeks the content of ALB was significantly lower than that of DM model group(P<0.01);Comparison of traditional chinese medicine high dose group and western medicine control group,there was significant difference(P<0.05).DM model group,urea nitrogen in chinese medicine high,medium and low dose group and western medicine control group(BUN)、Serum creatinine(Scr)level are significantly higher than the normal control group(P<0.01);Chinese medicine high,middle and low dose group and western medicine control group the level of BUN and Scr in rats were significantly lower than that of DM model group(P<0.01);Chinese medicine high,medium and low dose group between was a significant difference(P<0.01);Comparison of traditional chinese medicine high dose group and western medicine control group no obvious difference(P>0.05)。4 DM model group,chinese medicine high,medium and low dose group,western medicine control group rats blood glucose all significantly higher than the normal control group(P<0.01);Traditional chinese medicine high,medium and low dose group,western medicine control group,blood glucose were significantly lower than the DM model group(P<0.01);Chinese medicine high,middle dose group of fasting blood glucose significantly lower than western medicine control group(P<0.01);There was no significant difference between the western medicine control group and the low dose group of chinese medicine(P>0.05);Traditional chinese medicine high dose group and low dose group between was a significant difference(P<0.01).5 Determination of cholesterol(TC),triglyceride(TG)content in rats of each group.DM model group,chinese medicine high,medium and low dose group,western medicine control group rats TC content was significantly higher than that in normal control group(P<0.01);Chinese medicine high,medium and low dose group TC content compared with the DM model group,the group was significantly reduced(P<0.01);Between Western medicine control group and DM model group no obvious difference(P>0.05);Between traditional chinese medicine high,middle dose group and low dose group There were significant differences(P<0.01);Between traditional chinese medicine high,middle dose group and Western medicine control group have obvious difference(P<0.01).DM model group,Chinese medicine high,medium and low dose group,western medicine control group rats the content of TG was higher than that of the normal control group(P<0.01);Chinese medicine high,medium and low dose group,western medicine control group rats TG content was significantly lower than that of DM model group(P<0.01);Between traditional chinese medicine high,middle dose group and low dose group There were significant differences(P<0.01);Between chinese medicine High,medium and low dose group and Western medicine control group There were significant differences(P<0.01).6 Apply IHC(immunohistochemistry)and actual time ration test group express in rat nephridial tissue.Seen under the light microscopy: CD2 AP in the normal control group were distributed along the capillary in the glomerular.In the cytoplasmic side of the foot cell express,Expressed obviously;CD2AP expression in DM model group was significantly less,Partial regional segmental missing;Chinese medicine high,medium and low dose group and Western medicine control group compared with CD2 AP,the expression of DM model group was significantly improved.Gray value detection results:DM model group and High,medium,low dose group of Chinese Medicine,The gray value of CD2 AP in western medicine control group was lower than that in normal control group(P<0.01).Traditional Chinese medicine high,medium and low dose group and Western medicine control group rats were higher than the DM model group(P<0.01).Chinese medicine high does group and Chinese medicine low does group between significant difference(P<0.01);Chinese medicine high does group and Chinese medicine low does group between No obvious difference(P>0.05).DM model group,Chinese medicine high,medium and low dose group,western medicine control group M RNA CD2 AP content was rat significantly lower than the normal control group(P<0.01);Chinese medicine high,medium and low dose group,western medicine control group M RNA CD2 AP content was rat significantly higher than the DM control group(P<0.01);There were significant differences among high,medium and low dose groups of Chinese Medicine(P<0.01);There was no significant difference between the traditional Chinese medicine high dose group and Western medicine control group(P>0.05).7 Podocin expression in renal tissues of rats in each group were detected by immunohistochemistry and real-time PCR.Seen under the light microscopy: Normal control group of podocin,In the glomerular along the GBM is linear distribution of uniform;The expression of podocin in DM model group was significantly decreased;Chinese medicine high,medium and low dose group,western medicine control group podocin expression than the DM model group were significantly improved.Gray value detection results: DM model group,Chinese medicine high,medium and low dose group,western medicine control group podocin gray values were lower than the normal control group(P<0.01).There was a significant difference between the traditional Chinese medicine high dose group and the low dose group(P<0.01);There was no significant difference between the traditional Chinese medicine high dose group and Western medicine control group(P>0.05).Real time quantitative PCR results: DM model group,Chinese medicine high,medium and low dose group,western medicine control group M RNA podocin group(P>0.01);Chinese medicine high,medium and low dose group,western medicine control group rats M RNA podocin content was significantly higher than the DM model group(P>0.01);There were significant differences among high,medium and low dose groups of Chinese Medicine(P>0.01);Hazel takes renal capsule high dose group and the control group had no significant difference(P>0.05).8 The expression of α-actinin-4 in renal tissues of rats was detected by immunohistochemistry and real-time quantitative PCR.Seen under light microscope: Normal control group of the alpha-actinin-4 protein along the glomerular basement membrane capillary like point linear distribution of uniform;The expression of DM protein in the α-actinin-4 model group was significantly higher than that in the control group;The expression of α-actinin-4 protein in high,medium and low dose group and Western medicine control group was lower than that of DM model group.Gray value detection results: DM model group,Chinese medicine high,medium and low dose group,western medicine control group,the gray value of alpha α-actinin-4 were higher than the normal control group(P>0.01).Traditional Chinese medicine high,medium and low dose group and Western medicine control group,the gray values were lower than the DM model group(P<0.01).There were significant differences between the Chinese medicine high,medium and low dose groups(P<0.01);There was a significant difference between high dose group and Western medicine control group(P<0.05).DM model group,Chinese medicine high,medium and low dose group,western medicine control group,the m RNA α-actinin-4 content was significantly higher than the normal control group(P<0.01);Chinese medicine high,middle and low dose group and Western medicine control group alpha m RNA α-actinin-4 4 content is lower than the DM model group(P<0.01);There was no significant difference between the traditional Chinese medicine high dose group and Western medicine control group(P>0.05);There were significant differences between the Chinese medicine high,medium and low dose groups(P<0.01).9 Apply immunohistochemistry and actual time ration PCR test group express in rat podocalyxin nephridial tissue.Seen under the light microscopy: In the normal control group,the podocalyxin was distributed evenly along the GBM in the glomerular.In DM model group,the expression of podocalyxin protein in the kidney was missing.Traditional Chinese medicine high,medium and low dose group,western medicine control group,slight loss,But compared with the DM model group.Gray value detection results: DM model group,Chinese medicine high,medium and low dose group,western medicine control group podocalyxin gray values were lower than the normal control group(P<0.01).Traditional Chinese medicine high,medium and low dose group,western medicine control group of gray values were higher than the DM model group(P<0.01).There was a significant difference between the Chinese medicine high dose group and low dose group(P<0.01);There was a significant difference between the Chinese medicine high dose group and Western medicine control group(P<0.05).10 Apply immunohistochemistry and actual time ration PCR test group express in rat α3β1 integrin nephridial tissue.DM model group,Chinese medicine high,medium and low dose group,western medicine control group,the α3β1 integrin RNA m content was significantly lower than the normal control group(P<0.01);Chinese medicine high,medium and low dose group,western medicine control group α3β1 integrin RNA m content is higher than the DM model group(P<0.01);There were significant differences between the Chinese medicine high,medium and low dose groups(P<0.01);There was no significant difference between the Chinese medicine high dose group and Western medicine control group(P>0.05)Conclusion:This experimental study result explain,ZhenHuaXiaoShenAn capsule can improve the experimental DM rats the general state and clinical symptoms;alleviate renal tissue pathological structure,reduce urine micro albumin excrete;Improving glucose and lipid metabolism.Meanwhile,improve podocyte associated protein CD2 AP 、 podocin 、podocalyxin expression,inhibition α-actinin-4 protein expression,thus alleviate damage of renal tissue,improve renal function,delay DM development.The professor mentors Nan Zheng have thought,the key pathogenesis of diabetes nephropathy is “toxin impairing renal collaterals,hidden in pleurodiaphragmatic spaces”.This experiment according to the key pathogenesis use “Detoxification and dredging collaterals,nourishing kidney spaces-tonifying,sparse benefit pleurodiaphragmatic in terspace” theory and Western Medicine mechanism of diabetic nephropathy,glomerular filtration membrane and Podocyte structure and its function together.ZhenHuaXiaoShenAn capsule in treatment method Under the guidance of detoxification and dredging collaterals,nourishing kidney spaces-tonifying,sparse benefit pleurodiaphragmatic in terspace,through the intervention of podocyte associated protein CD2AP、podocin、α-actinin-4、podocalyxin、α3β1-integrin expression and bring into play treatment of diabetic nephropathy effect. |
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