Data Mining And Mechanism Study Of Tan Jinchuan’s Experience In Treating Membranous Nephropathy

Part one The efficacy and safety of Chinese herbal compound in the treatment of membranous nephropathy:a systematic reviewObjective:To systematically evaluate the clinical efficacy and safety of Chinese herbal compound in the treatment of membranous nephropathy.Methods:CNKI,Wanfang database,Weipu database,China Biomedical Database,Pub Med,Embase,Cochrane database were searched to collect clinical studies on the adjuvant treatment of MN with traditional Chinese medicine compound.Duplicate documents were deleted,the documents that did not meet the inclusion and exclusion criteria were eliminated by reading the title,abstract and full text,data extraction and quality evaluation of the included documents were carried out,and the Rev Man5.3 software was used for Meta analysis.Stata 14.0 software was used to draw a funnel chart to judge publication bias.The prescriptions of traditional Chinese medicines included in the study were entered into an Excel table,the frequency and efficacy classification information of the medicines were counted,and the association rule analysis of the prescriptions of traditional Chinese medicines was performed using R3.5 software.Results:1.Finally,19 RCT studies of Chinese medicine compound+basic treatment vs basic treatment were included,a total of 1022 MN patients.31RCT studies of traditional Chinese medicine compound+basic treatment+hormones+immunosuppressive agents vs basic treatment+hormones+immunosuppressive agents,a total of 2017 MN patients.2.On the basis of conventional treatment,the use of traditional Chinese medicine compound to treat patients with MN can improve the clinical efficacy[OR=3.47,95%CI（2.44,4.93）,P<0.00001]and the efficacy of TCM symptoms[OR=8.21,95%CI（2.66,25.32）,P=0.0002],improve TCM syndrome score[SMD=1.11,95%CI（0.68,1.54）,P<0.00001],reduce 24-hour urine protein level[SMD=-1.15,95%CI（-1.54,-0.76）,P<0.00001],increase plasma albumin level[SMD=1.02,95%CI（0.56,1.48）,P<0.00001],reduce TC level[MD=-0.78,95%CI（-1.35,-0.20）,P=0.008]and TG level[MD=-0.73,95%CI（-0.88,-0.58）,P<0.00001],but it is effective in improving renal function（BUN,Scr）and reducing adverse reactions The effect is not obvious.3.On the basis of hormone and immunosuppressive therapy,the use of Chinese medicine compound to treat patients with MN can improve the clinical efficacy[OR=2.90,95%CI（2.27,3.70）,P<0.00001]and the efficacy of TCM symptoms[OR=3.10,95%CI（1.91,5.05）,P<0.00001],improve TCM syndrome score[SMD=-0.93,95%CI（-1.34,-0.52）,P<0.00001],reduce24-hour urine protein level[SMD=-0.90,95%CI（-1.23,-0.57）,P<0.00001],increase plasma albumin level[SMD=1.22,95%CI（0.86,1.58）,P=0.000],reduce blood lipid TC level[SMD=-0.69,95%CI（-0.98,-0.39）,P<0.00001],reduce blood lipid TG level[SMD=-0.75,95%CI（-1.09,-0.41）,P<0.0001],improve renal function BUN level[SMD=-0.56,95%CI（-0.97,0.14）,P=0.008]and Scr level[SMD=-0.58,95%CI（-0.89,-0.27）,P=0.0002]to reduce the occurrence of adverse reactions Rate[OR=0.32,95%CI（0.22,0.44）,P<0.00001].4.On the basis of conventional treatment,the traditional Chinese medicine compound was used in the treatment of MN patients.The top 6Chinese medicines used in the traditional Chinese medicine prescription are Astragalus,Poria,Chinese Yam,Angelica,Codonopsis and Atractylodes macrocephala.The treatment should bupiyishen.On the basis of hormone and immunosuppressive therapy,the Chinese medicine compound is used to treat MN patients.The top 6 Chinese medicines used in the Chinese medicine prescription are Astragalus,Poria,Atractylodes,Danshen,Angelica and Chinese yam.The treatment should yiqihuoxue.Summary:1.Chinese herbal compound can improve the therapeutic effect of membranous nephropathy and reduce the incidence of adverse reactions.2.Astragalus is the core of the traditional Chinese medicine used in the treatment of MN.It is recommended to bupiyishen in the early stage,and to yiqihuoxue in the middle and late stages.Part two Professor Tan Jinchuan’s experience in the treatment of membranous nephropathyObjective:To excavate and analyze Professor Tan Jinchuan’s medication experience in the treatment of membranous nephropathy,and to summarize the characteristics and rules of his medication.Methods:Collect the outpatient medical records of Professor Tan Jinchuan’s treatment of membranous nephropathy.Epi Data 3.1 software package was used for data entry,and Excel 2013,SPSS19.0 and R3.5 software packages were used to perform statistical analysis and data mining on the Chinese medicine prescriptions included in the medical records.Results:1.353 Chinese medicine prescriptions were included,including 163Chinese medicines,with a total frequency of 5,273.Traditional Chinese medicines with warm nature,sweet taste,and liver meridian are used most frequently,while tonic medicines,blood activating and blood stasis medicines,rheumatism-relieving medicines,and diuretic and dampening medicines was used frequently.2.According to cluster analysis,Chinese medicines are divided into 5categories:Class I is spleen-tonifying drugs,Class II is kidney-tonifying drugs,Class III is collateral-relief drugs,Class IV is dampness drugs,and Class V is blood-activating drugs.3.The results of association rules show that Astragalus,Angelica,Chuanxiong,Dilong,Qingfengteng,Poria and Licorice are closely related.Summary:1.Astragalus is the core traditional Chinese medicine for the treatment of membranous nephropathy by Professor Tan Jinchuan and often appears with Chuanxiong,Angelica,Poria and Qingfengteng.2."Jianpiyishen,huayuqushi,gongbujianshi"is Professor Tan Jinchuan’s method of treating membranous nephropathy.Part three Analysis of the Mechanism of Yishentongluo Recipe in Treatment of Membranous NephropathyObjective:Based on network pharmacology,the mechanism of yishentongluo recipe in the treatment of membranous nephropathy is explored,which opens up new ideas for follow-up experimental research and provides scientific basis for clinical application.Method:By searching the TCMSP and TCMID databases,the active ingredients of yishentongluo recipe and their targets were screened,and then MN-related targets were collected,and the target points of the Chinese medicine compound and the MN targets were intersected,and the intersection genes were subjected to PPI network Topological analysis and screening of core targets.Use R software to perform GO and KEGG enrichment analysis on the core targets,and analyze the potential signal pathways of yishentongluo recipe in the treatment of MN.Use Cytoscape 3.7.2 software to construct a network diagram of drugs,targets,diseases,and signaling pathways.Result:1.There are 70 active ingredients and 237 targets in yishentongluo recipe.2.There are 2022 targets related to membranous nephropathy.3.There are 130 potential targets of yishentongluo recipe in the treatment of MN.4.21 core targets and 5 gene clusters were screened out by PPI network topology analysis.5.The target of yishentongluo recipe for treating MN is significantly enriched in biological processes such as oxidative stress,metal ions,lipopolysaccharide,and tissue oxygen levels,in molecular functions such as phosphatase binding,heme binding site,protein phosphorylation binding,nuclear receptor,steroid hormone receptor activity,cytokine receptor in cellular components such as Cell membrane,membrane microdomain,cyst cavity,serine/threonine protein kinase complex,mitochondrial outer membrane,cytoplasmic cyst cavity.6.The target of yishentongluo recipe for treating MN is significantly enriched in signal pathways such as diabetic complications,endocrine resistance,IL-17,TNF,Th17 cell differentiation,apoptosis,measles,PI3K/Akt,MAPK,VEGF,etc.Summary:1.Yishentongluo recipe can mediate cellular immune inflammation and apoptosis by regulating a variety of biological processes and multiple signal pathways,and improve MN kidney injury.2.Yishentongluo recipe has the characteristics of"multi-component,multi-target,and multi-pathway"in the treatment of MN.Part four Construction of mi RNA-m RNA regulatory network in the treatment of MN with yishentongluo recipeObjective:Construct the mi RNA-m RNA regulatory network of yishentongluo recipe in the treatment of MN,explore the regulatory effect of traditional Chinese medicine on mi RNA in patients with MN,and explain the mechanism of the treatment of MN by traditional Chinese medicine compound prescriptions,and provide new ways and new ideas for the study of Chinese medicine in the prevention and treatment of membranous nephropathy.Method:Kidney tissue from patients with membranous nephropathy and the adjacent normal tissues of patients undergoing nephrectomy were collected.The expression of mi RNA and m RNA in the kidney tissues of the two groups of patients was detected through high-throughput sequencing.The active ingredients and targets of yishentongluo recipe was collected.The Venny 2.1software performs Venn analysis on the target of the active ingredients in the yishentongluo recipe and the differential m RNA of MN patients obtained by sequencing,and predicts the downstream target genes of the differential mi RNA through mi RDB,mi RTar Base and Target Scan databases,and uses Venny 2.1 to obtain the intersection m RNA For overlapping genes with differentially expressed mi RNA targets,a PPI network is established through the String database,and a PPI network diagram is established using Cytoscape.The target is analyzed by GO and KEGG enrichment analysis by using R software.The mi RNA-m RNA regulatory network of yishentongluo recipe in the treatment of MN was constructed using Cytoscape 3.7.2 software.Result:1.Compared with the control group,there are 240 differentially expressed m RNAs（DEG）in the kidney tissue of MN patients,including 102up-regulated DEG and 138 down-regulated DEG.2.12 potential targets（overlapping genes）of yishentongluo recipe for treating MN were obtained.3.Compared with the control group,there are 90 differential mi RNAs in the kidney tissue of MN patients,of which 54 mi RNAs are up-regulated and36 mi RNAs are down-regulated.4.54 up-regulated mi RNAs predicted 10,215 target genes,and 36down-regulated mi RNAs predicted 10,659 target genes.5.Finally,8 overlapping genes between the potential targets of yishentongluo recipe and the differential mi RNA target genes were screened out.6.There are 15 mi RNAs potentially regulated by yishentongluo recipe.7.Target genes are significantly enriched in biological processes such as mi RNA transcription,adrenaline,steroid hormones,in cellular components such as membrane microdomains,nuclear transcription factor complexes,nuclear euchromatin and in molecular functions such as SMAD binding,RNA polymerase II activation transcription factor binding,G Protein-coupled receptor binding.8.Target genes are significantly enriched in signal pathways such as TNF,IL-17,B cell receptor,and Erb B.Summary:1.The mi RNA-m RNA network is regulated by yishentongluo recipe for the treatment of membranous nephropathy.2.The mi RNA-m RNA regulatory network can provide ideas and research foundation for follow-up research.Part five Effect of yishentongluo recipe on mi R-514a-5p/TNFSF15pathway in MN rats and its mechanismObjective:Based on the mi R-514a-5p/TNFSF15 signaling pathway to explore the mechanism of yishentongluo recipe in inhibiting glomerular podocyte apoptosis in rats with membranous nephropathy.On the one hand,the possible mechanism of yishentongluo recipe in the treatment of MN is explained.It also further verifies the accuracy of the results of the fourth part.Method:A rat model of membranous nephropathy was established through tail vein injection of cationized bovine serum albumin（C-BSA）.The successfully modeled MN rats were randomly divided into model groups（physiological saline,equal volume）and the high,medium and low dose groups of yishentongluo recipe(yishentongluo recipe,26.44,13.22,6.61 g·kg^-1)and the Benazepril group(Benazepril hydrochloride,10 mg·kg^-1),Healthy rats were taken as the normal group（normal saline,equal volume）,10 rats in each group.Corresponding dose of medicine was given to the stomach,once a day,for 4 weeks of continuous intervention.After the administration,the 24-hour urine protein（UTP）level of the rat was detected,and the total protein（TP）,albumin（ALB）,creatinine（Scr）and urea nitrogen（BUN）levels in the serum were measured.The pathological changes of rat kidney were observed by PASM,transmission electron microscope,Ig G,C3 immunofluorescence.The expression levels of mi R-514a-5p and TNFSF15 m RNA in rat kidney tissue were detected by RT-q PCR.The protein expression levels of TNFSF15,Podocin,Nephrin,Podocalyxin,Synaptopodin,BAD,Bax,Bcl-2 and Bcl-XL in rat kidney tissue were detected by Western blot and immunohistochemical.The apoptosis rate of rat kidney tissue was detected by Tunnel.The binding ability of mi R-514a-5p and TNFSF15 was detected by the dual luciferase reporter experiment.Result:1.Compared with the normal group,the UTP level of rats in the model group decreased（P<0.05）.Compared with the model group,the UTP level of rats in the Benazepril and yishentongluo recipe groups decreased to varying degrees（P<0.05）.2.The glomerular basement membrane of rats in the model group showed varying degrees of thickening and vacuolar degeneration,Ig G and C3 granular deposition along the glomerular capillary wall,electron dense deposits of varying sizes were seen,podocyte foot processes Broad integration.After the intervention of yishentongluo recipe and Benazepril Hydrochloride,the pathological changes of rat kidney tissue were alleviated.3.Compared with the normal group,the serum levels of TC and TG in the model group increased,while the levels of TP and ALB decreased（P<0.05）,and the levels of BUN and Scr had no significant changes.Compared with the model group,the levels of TC and TG in the serum of rats in the Benazepril group and the yishentongluo recipe group decreased,while the levels of TP and ALB increased（P<0.05）,and the levels of BUN and Scr had no significant changes.4.Compared with the normal group,the level of mi R-514a-5p in the kidney tissue of the model group was significantly reduced,and the level of TNFSF15 m RNA was significantly increased（P<0.05）.Compared with the model group,the benazepril group and the yishentongluo recipe group was significantly increased,and the level of TNFSF15 m RNA was significantly decreased（P<0.05）.5.The results of immunohistochemistry showed that the positive signals of Podocin,Nephrin,Podocalyxin and Synaptopodin were brown-yellow.Compared with the normal group,the expression of the podocyte marker proteins Nephrin,Podocin,podocalyxin,and Synaptopodin in the kidney tissue of the model group was weakened;compared with the model group,the yishentongluo recipe and the benazepril group rat podocyte marker Nephrin,Podocin,podocalyxin,and Synaptopodin protein expression were all enhanced to varying degrees.6.Compared with the normal group,the apoptosis in the kidney tissue of the model group was significantly increased.Compared with the model group,the apoptosis in the kidney tissue of the rats in the yishentongluo recipe and Benazepril group was significantly reduced（P<0.05）.7.The results of immunohistochemistry showed that the positive signals of TNFSF15,Bax,BAD,Bcl-2,and BCL-XL were brown-yellow.Compared with the normal group,the expression of TNFSF15,Bax and BAD protein in the kidney of the model group increased,and the expression of Bcl-2 and BCL-XL protein decreased（P<0.05）.Compared with the model group,the expression of TNFSF15,Bax,and BAD protein in the kidney of rats in the yishentongluo recipe and Benazepril group was weakened,and the protein expression of Bcl-2 and BCL-XL was increased（P<0.05）.8.Western blot results showed that compared with the normal group,the expression levels of TNFSF15,Bax and BAD proteins in the kidneys of the model group increased,while the expression levels of Bcl-2 and BCL-XL proteins decreased（P<0.05）,and compared with the model group,the expression levels of TNFSF15,Bax and BAD proteins in the kidneys of the yishentongluo recipe and Benazepril groups decreased,while the expression levels of Bcl-2 and BCL-XL proteins increased（P<0.05）.9.Compared with the mi R-NC group,mi R-514a can significantly reduce the relative fluorescence activity of podocytes transfected with TNFSF-3’-UTR wt（P<0.05）,while the relative fluorescence of TNFSF-3’-UTR mut The activity has no obvious effect（P>0.05）.Summary:1.Yishentongluo recipe reduce the kidney injury of MN rats by inhibiting the apoptosis of podocytes.2.Yishentongluo recipe reduces the renal injury of MN rats by inhibiting the mi R-514a-5p/TNFSF15 pathway.Conclusions:1.The traditional Chinese medicine compound with astragalus as the main medicine has a definite effect in the treatment of MN,which can significantly improve the effect of basic western medicine or hormone immunosuppressive treatment and reduce adverse reactions.2."Jianpiyishen,huayuqushi,gongbujianshi"is Professor Tan Jinchuan’s experience in the treatment of MN.3."multi-component,multi-target,multi-pathway"is the characteristics of Yishentongluo recipe in the treatment of MN.4.The mi RNA-m RNA network is regulated by yishentongluo recipe for the treatment of membranous nephropathy.5.Yishentongluo recipe reduce the kidney injury of MN rats by inhibiting the apoptosis of podocytes.6.Yishentongluo recipe reduces the renal injury of MN rats by inhibiting the mi R-514a-5p/TNFSF15 pathway.