| The first partObservation of therapeutic effect of jianpi xiaozhong granule on PHN rats and its effect on podocyteObjective(1)To observe the effect of jianpi xiaozhong granule on PHN rats;(2)To observe the effect of jianpi xiaozhong granule on podocyte and functional proteins.Methods: Healthy male SPF Wistar rats were selected and randomly selected from the normal group for modeling.The normal group received a one-time intraperitoneal injection of distilled water(1ml/100 gand the model group received a one-time intraperitoneal injection of the same amount of sheep anti-fx1 a anti-serum.After successful modeling,the regional group was randomly divided into model group and jianpi detumescence granule group.Blood creatinine,plasma albumin,cholesterol and triglyceride were detected with a special detection kit for rats.Pathological staining methods(such as HE staining,PASM staining,et immunofluorescence staining and immunohistochemistry were used to observe the pathological changes and podocyte injury of rats.The m RNA expressions of functional proteins nephrin,podocin and podoplanin expressed in podocytes were determined by real-time fluorescence quantitative PCR method to compare the functional changes of podocytes after treatment with jianpi xiaozhong granules.Results:(1)After 2 weeks of administration,the urine protein level in the spleen-strengthening and detumescence treatment group was significantly lower than that in the model group at 24 h,and the difference was statistically significant(P<0.05).After 4 weeks of administration,the urine protein content in the model group was significantly decreased at24 h compared with that in the model group at 0 weeks.However,after 4 weeks and 6 weeks of administration,the urine protein content in the treatment group was still significantly lower than that in the model group(P<0.05).After 8 weeks of administration,the urine protein content in the spleen-strengthening and detumescence group was significantly lower than that in the model group;(2)Biochemical indexes: the results showed that there was no statistically significant difference in serum creatinine between the normrol group,model group and spleen strengthening and detumescence group(P>0.05).The injection of anti-Fx1 A serum did not affect renal function in rats.At the albumin level,after the injection of anti-fx1 a serum,the model group was significantly lower than the normrol group,and the difference was statistically significant(P <0.05).However,there was no significant difference in albuminlevelbetween the treatment group and the normal group(P > 0.05);CHOL,LDL levels in the model group and spleen strengthening and detumescence group were significantly higher than those in the normal control group,P < 0.05,and the difference was statistically significant.TG level in the model group was significantly higher than that in the normal control group,and the difference was statistically significant(P< 0.05).There was no significant difference between the spleen strengthening and detumescence group and the normal control group(P>0.05).(3)Pathology: there was no significant thickening of basement membrane with PASM staining in the normal group,but there was a small amount of nailing process formation in the basement membrane thickening in the model group,and there was a certain improvement in the spleen strengthening and detumescence group compared with the model group.In the model group,some mesangial stroma had granular resinophilic deposits and some interstitial fibrous tissue had mild hyperplasia.No obvious fibrous tissue hyperplasia was found in the spleen-strengthening and detumescence oup.(4)Immunohistochemistry: WT1 expression in the model group and spleen detumescence treatment group decreased compared with that in the normal group,and the difference was statistically significant(P < 0.05).Compared with the model group,WT1 expression in the spleen detumescence treatment group increased significantly,and the difference was statistically significant(P < 0.05).The expression of podocin in the functional protein model group of podocytes was significantly higher than that in the normal group,and the difference was statistically significant(P < 0.05),while there was no significant difference between the spleen strengthening and detumescence group and the normal group(P>0.05).There was no significant difference between the expression of nephrin in the model group,the splenic detumescence group and the normal group(P> 0.05),and no significant difference between the splenic detumescence treatment group and the normal group(P> 0.05).(5)PCR: compared with normal control group,spleen detumescence group of nephrin and podocin,the index of podoplanin expression and no difference between the normal group(P>0.05),the index expression abnormal model group,model group podocin m RNA expression significantly increased compared with normal group,the difference was statistically significant(P < 0.05),and the rest two indicators higher than normal group have no obvious difference,but there was a significant increase trend..(5)PCR: compared with nontrol group,spleen detumescence nephrin and podocin,the index expression of podoplanin no obvious difference with the normal group,(P > 0.05),the index expression abnormal model group,model group podocin m RNA expression significantly increased compared with normal group,the difference was statistically significant,P < 0.05,and the rest two indicators higher than normal group have no obvious difference,but there was a significant increase trend.Conclusion :(1)Jianpi xiaozhong granule reduced the urine protein level of PHN model rats and increased the albumin level of PHN moderats.(2)Jianpi xiaozhong granule can alleviate the pathological injury of PHN model rats,inhibit the thickening of basement membrane and delay the development of sease.(3)Spleen-strengthening and detumescent granules inhibit the development of the disease by protecting the foot cells.The second partQuantitative analysis of protein group in PHN rats treated with jianpi xiaozhong granuleObjective To explore the drug target of jianpi xiaozhong granule in the treatment of membranous nephropathy and the biomarker to evaluate the therapeutic effect by extracting the key nodes and determining the key proteins through protein spectrum analysis,and to clarify the mechanism of the effective intervention of jianpi xiaozhong granule.Methods In vitro labeling(TMT)was used to analyze the changes of protein in rats treated with jianpi xiaozhong granule from the perspective of proteomics.The whole process strictly controlled the false positive rate of identified proteins.Results(1)In all rat samples,there were 25077 peptides,3107 proteins,23948 peptides and3064 proteins with quantitative information,and 1602 proteins with quantitative information in all channels.(2)Clustering analysis method is applied to the main component analysis,PCA,by using analysis method of HCA and Fuzzy c-means to analyze protein differences between groups,the results showed that between two groups of comparison,there are differences in at least a group of a total of 628 proteins,differences in protein 374 normal control group and model group,normal control group and spleen detumescence particle group differences in protein 260,model group and spleen detumescence particle group differences in protein 298;(3)David was used to annotate and analyze the biological functions of the differential proteins in GO(gene ontology).A total of 132 biological pathways with significant enrichment of differentially expressed proteins in the model group and the normal group were selected for pathways with P<0.05,and the pathways with high correlation between significant enrichment of differentially expressed proteins in the model group and the normal group were selected as follows:Complement activation,classical pathway,complement activation,The alternative pathway),oxidative stress(response to oxidative stress),glutathione metabolism(glutathione metabolic process),epithelial cell transdifferentiation(epithelial cell differentiation),REDOX process(oxidation-reduction process),the peptide enzyme activity of the negative control(negative Regulation of endopeptidase activity,apoptotic mitochondrial changes,negative regulation of blood coagulation,negative regulation of extrinsic apoptotic signaling by the receptors of the death domain Pathway via death domain receptors),etc.There were 80 significantly enriched biological pathways in the normal group and the spleen invigorating and detumescence group with P<0.05,and the pathways with high correlation were:REDOX pathways(oxidation-reduction process),aging pathway(aging),cell adhesion pathways(cell-cell adhesion),oxidative stress(response to oxidative stress),epithelial cell transdifferentiation(pithelial cell differentiation),osteoblast differentiation(osteoblast Differentiation),retinoic acid metabolic process,cellular oxidant detoxification,kidney development pathway,glutathione metabolic process,etc.There were 140 biological pathways significantly enriched in the model group and the spleen strengthening and detumescence group with P<0.05,and the pathways significantly enriched and correlated were:Negative regulation of endopeptidase activity,oxidation-reduction process,negative regulation of apoptotic process,response to oxidative stress,and acute phase reaction Response),inflammation(inflammatory response),complement activation classical pathway(complement activation,classical pathway),complement activation bypass channels(complement activation,the alternative pathway),complement activation(complement activation),etc.(4)The intrinsic components of complement C3,C4,REDOX enzyme SOD2,gsh-px,cytoskeleton protein transgelin,plectin,etc.were significantly regulated.Conclusion(1)The PHN model activates the classical complement pathway and the bypass pathway and is under oxidative stress.(2)The traditional Chinese medicine jianpi xiaozhong granule plays a role in inhibiting complement activation and improving oxidative stress. |
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