Research On Mechanism Of Longqi Fang On Diabetic Kidney Disease Based On GEO Chip And Network Pharmacology

Background:DKD is one of the most important complications of DM,and it is also the main cause of ESRD and final death.At present,the pathogenesis of DKD is not completely clear,mainly related to genetic and environmental factors,but also related to a variety of mechanisms such as persistent hyperglycemia,inflammatory response,oxidative stress,renal fibrosis and so on.At present,the treatment of DKD is mainly to control blood glucose and RAAS inhibitors,but it can not effectively curb the ESRD stage of the course of disease.Therefore,how to effectively prevent/delay the development of DKD is the most urgent problem to be solved at present.Modern traditional Chinese medicine experts believe that DKD belongs to the category of"Xiaoke nephropathy".They believe that the main pathogenic factors of DKD are congenital deficiency,overeating fat and sweet,viscera deficiency and kidney injury due to fatigue.The pathogenesis is the combination of phlegm and blood stasis,yin deficiency and blood stasis.According to the characteristics of the pathogenesis,Professor Guo Hongmin self-made "Longqi Fang" has significant clinical efficacy in symptomatic treatment,which can effectively control the further deterioration of blood glucose and renal function in patients with DKD,but there is a lack of systematic research on the mechanism of Longqi Fang on DKD.However,because Longqi Fang is composed of many traditional Chinese medicines,the effective components and pharmacodynamic substances are complex.Therefore,how to simplify the effective components of Longqi Fang and understand the role of Longqi Fang and the core target of DKD is the key of this study.At the same time,it also provides new research ideas for the research and development of new traditional Chinese medicine and anti DKD treatment.Purpose:1.Based on GEO chip,network pharmacology method and molecular docking technology,the molecular biological mechanism of Longqi Fang in the treatment of DKD was discussed,and the signal pathway,core target and effective components of Longqi Fang in the treatment of DKD were screened.2.Verify the selected core targets from the perspective of animal experiment,and further confirm the systematic study of the action mechanism of Longqi Fang on DKD.Research methods:1.Search the GEO chip database for the disease "diabetic kidney disease of DNA",obtain the relevant data of GSE30528 and GSE30529,and use R language and Perl software to obtain the differential genes;At the same time,the core targets of DKD are obtained through Genecards database,and the core targets obtained by the two are summarized to obtain the final DKD targets.The effective components and action targets of 9 traditional Chinese medicines in"Longqi Fang" were searched in TCMSP database and literature.The intersection target of Longqi Fang and DKD is obtained through Venny software,Cytoscape 3.7.1 construct the network visualization of "DKD intersection target active component Longqi Fang",and obtain the main components of Longqi Fang and DKD;Upload the obtained intersection targets to the STRING platform,obtain the PPI protein interaction network diagram,and screen the main core targets through R software;Go and KEGG enrichment analysis of intersection targets was carried out by R software;Finally,the main components and core targets were verified by molecular docking analysis.2.The obtained core targets were verified by animal experiments.40 rats were randomly divided into 5 groups according to the random number table method,including 8 in the normal group and 32 in the model group.DKD rat model was established in 32 rats;After successful modeling,they were randomly divided into model group,Longqi Fang high dose group,medium dose group and low dose group,with 8 rats in each group.The model group did not do any intervention since the modeling.The other three groups were given Longqi Fang by gavage according to high dose,medium dose and low dose for 8 weeks.The general state of rats after 8 weeks was observed,and the blood glucose,insulin,urea nitrogen,creatinine,urinary microalbumin and urinary protein/creatinine ratio were measured;The levels of inflammatory factors IL-6,TNF-α and MCP-1 by ELISA;After the rats were killed,the right kidney was taken out and sectioned for HE staining to observe the changes of pathological structure;The expression of protein and mRNA of IL-6,TNF-α,MCP-1 and RAGE in kidney tissues were detected by Western blot and qRT-PCR,and it was found that the administration of Chinese medicine "Longqi Fang" could effectively reduce the level of inflammation,with the most significant in the high-dose group.Result:1.Network pharmacologyThe chips GSE30528 and GSE30529 of DKD renal tubules and glomeruli were obtained through GEO chip database.542 differential genes of GSE30528 and 484 differential genes of GSE30529 were obtained through R language screening;DKD targets were obtained through Genecards database,and 165 DKD targets were obtained by setting the screening condition correlation score≥50.By searching TCMSP platform,only all components of Panax notoginseng,lotus leaf,Pueraria lobata,Ligusticum chuanxiong,wolfberry and Polygonatum were obtained.According to the standard of OB≥30%and DL≥0.18,the corresponding effective components were screened,including 7 effective components of Panax notoginseng,14 effective components of lotus leaf,6 effective components of chuanxiong,35 effective components of wolfberry,3 effective components of Pueraria lobata and 9 effective components of Polygonatum.Its corresponding Chinese medicine targets were 223 for Panax ginseng,39 for Chuanxiong,321 for Lycium barbarum,358 for lotus leaf,76 for Pueraria lobata,and 133 for Huangjing.Rhodiola rosea,Rhodiola rosea,Dilong,and Shengdihuang were searched through the literature and obtained 47 active ingredients of Rhodiola rosea,36 active ingredients of Dilong,and 52 active ingredients of Shengdihuang through the Swiss ADME platform,with 1008 core targets of Shengdihuang,1092 of Rhodiola rosea,and 931 of Dilong.The targets obtained from Longqi Fang and DKD were uploaded to VENNY software,and 104 intersection targets were obtained,with 124 major active ingredients mapped.Pass the intersection target obtained Cytoscape 3.7.1 software constructs the network diagram of "DKD intersection target active component Longqi Fang",and the main components are quercetin,epigallocatechin gallate,kaempferol,jinshengcao,wheat flavone,dihomo gamma linolenic acid,docosapentaenoic acid,Dihydrocapsaicin,baicalein and wild zizanic acid;Upload the intersection targets to the STRING platform to obtain the protein interaction network diagram,and draw the core targets TNF,IL-6,AKT1,IL1β,TP53 and VEGFA by R language;Go and KEGG were analyzed by R language,and 2069 biological processes,42 cell components and 113 molecular functions closely related to DKD were obtained;We obtained 151 signaling pathways for DKD,including AGEs-RAGE signaling pathway,hypoxia inducible factor 1 signaling pathway and PI3K-Akt signaling pathway.The optimal conformation of binding energy was obtained by molecular docking with autodock Vina software,mainly TNF protein and baicalein,IL-6 and quercetin,AKT1 protein and epigalocatechin gallate,IL1β Protein and quercetin,TP53 and baicalein,VEGFA and epigalocatechin gallate.2.Animal experimentThe established DKD rat model was intervened by different concentrations of traditional Chinese medicine "Longqi Fang".The general state of rats in the high-dose Longqi Fang group was better than that in the medium dose,low-dose and model groups;The blood glucose and insulin levels of "Longqi Fang" high-dose group were better than those of medium dose,low-dose and model group,which was statistically significant;In terms of renal function,UAlb and UACR in urine in "Longqi Fang" high-dose group were better than those in medium dose,low-dose and model group;It was found that the intervention of high-dose "Longqi Fang" could effectively improve the state,blood glucose and renal function of DKD rats.The histopathological observation of kidney in this experiment found that there were significant changes in the structure of renal tubules and glomeruli in DKD rats compared with the normal group,and there were a large number of inflammatory cells.After the intervention of traditional Chinese medicine "Longqi Fang",it was found that the high-dose group could significantly improve the glomerulus and renal tubules and effectively inhibit inflammatory factors.The core inflammatory target of Longqi Fang intervention in DKD obtained through network pharmacology was analyzed for experimental inflammation,and IL-6 and TNF in serum were detected by ELISA-α、The levels of MCP-1,IL-6 and TNF in renal tissue were detected by Western blot and qRT-PCR-α、It was found that "Longqi Fang" could effectively reduce the level of inflammation,especially in the high-dose group.Conclusion:1.Through the research of GEO chip and network pharmacology,it is found that the main core targets of Longqi Fang and DKD are TNF,IL-6,AKT1,IL1β,TP53,VEGFA;The main effective ingredients are quercetin,epigallocatechin gallate,kaempferol,chrysoeriol,trici,Dihomo-gamma-linolenic-acid,Docosapentaenoicacid,Dihydrocapsaicin,Baicale in andaeginetic acid docosahexenoic acid,two hydrogen capsaicin,baicalein,wild acid;The main signaling pathways are AGEs-RAGE,HIF-1 and PI3K-Akt signaling pathway.It shows that Longqi Fang has renal protective effect on DKD mainly through multiple targets,multiple components and multiple signal pathways.2.Longqi Fang can effectively reduce the indexes of renal function injury(UALB,UACR)and blood glucose metabolism(TBG,insulin)in DKD rats;At the same time,renal histopathological examination found that after Longqi Fang intervention,the renal structure of DKD rats was arranged completely,and the structural damage of renal tubules and glomeruli was less,indicating that Longqi Fang can effectively improve blood glucose metabolism and renal function of DKD rats,effectively improve the renal structure of DKD rats and play a renal protective role.3.IL-6,TNF-α and MCP-1 were detected by serum ELISA,Western blot protein expression and qRT-PCR mRNA gene.Longqi Fang may protect the kidney of DKD rats through anti-inflammatory effect.