| Background:Diabetic nephropathy(DN)is a common microvascular complication of diabetes mellitus,which has high morbidity and mortality worldwide.The quality of life of DN patients will be severely reduced.The pathogenesis of DN is very complicated,such as genetics,obesity,abnormal blood lipid metabolism,and gut microbiota disorder will cause the occurrence and development of DN.There have been more in-depth studies on the pathogenesis of DN.However,the specific mechanism has not yet been fully elucidated.Therefore,there is no clinically approved "effective drug" directly used in the treatment of DN.Rhizoma Coptidis(RC)has a long history of treating diabetes and its complications,but current research mainly focuses on berberine,and there is little research on whether other alkaloids have the effect of treating DN.Epiberberine(EPI)is an isoquinoline alkaloid derived from RC.It is an isomer with berberine,which has a variety of pharmacological effects,such as hypoglycemic,hypolipemic,anti-inflammatory,and regulating gut microbiota.However,there is no report about the improvement effect and mechanism of EPI on DN and the influence of gut microbiota.Objective:1.To screen out the alkaloids of RC that have therapeutic effects on DN and explore the improvement effects of this alkaloid on DN in vivo and in vitro.2.Based on transcriptomics and bioinformatics analysis,investigate the mechanism of EPI in improving DN and find its potential targets.3.Exploring the effect of EPI on the gut microbiota of DN mice,which is expected to provide new ideas for the research and development of DN drugs.Methods and results:1.Preliminary screening of alkaloids in RC for treatment of DN(1)Six active protoberberine ingredients of RC were screened by TCMSP and other databases,including berberine,berberrubine,EPI,palmatine,coptisine,and worenine.A total of 418 related targets of these six active components were obtained.(2)A total of 892 targets related to DN were collected from Gene Cards and other databases,among which 79 targets were common targets related to active ingredients of RC,mainly including interleukin-6(IL-6),fibronectin 1(FN1),and other targets.(3)The GO and KEGG enrichment analysis of common targets was carried out by the Metascape database.The GO analysis results showed that the improvement of DN was mainly related to the regulation of oxidative stress and the endoplasmic reticulum cavity.The results of KEGG analysis showed that the improvement of DN by RC alkaloids was mainly related to AGE-RAGE,PI3K-Akt,and other signaling pathways.(4)"Ingredient-target-pathway" showed that berberine,EPI,palmatine,and coptidine were the four main active ingredients in the alkaloids of RC in the treatment of DN,and MAPK1,Akt1,and IL-6 were the main targets of RC alkaloids in the treatment of DN.It was suggested that RC alkaloids might play a role in renal protection through anti-inflammatory,anti-oxidative stress,and anti-fibrosis.(5)EPI had a stronger inhibitory effect than berberine on the proliferation of glomerular mesangial cells(GMCs)induced by high-glucose(HG).(6)Male db/db mice were used as DN models and db/m mice as the normal control group(NC).Model mice were randomly divided into the model group,berberine 100 mg/kg group,palmatine 100 mg/kg group,coptidine 100 mg/kg group,and EPI 100 mg/kg group.Mice in the drug group were given a corresponding dose of alkaloid intragastric administration,while the NC group and DN group were given an equal volume of normal saline intragastric administration once a day for 8 weeks.(7)After 8 weeks of administration,berberine,coptidine,and EPI could significantly reduce the bodyweight of db/db mice.(8)Both berberine and EPI could significantly reduce FBG in db/db mice.(9)RC alkaloids have the effect of improving lipid metabolism,among which berberine and EPI have a better effect on lowering blood lipid.(10)Both berberine and EPI could improve renal function injury in DN mice,and EPI is significantly better than the other three alkaloids in reducing the key index UACR of DN.2.Effects of EPI on improving the damage of HG-induced GMCs(1)The results of MTT showed that EPI could significantly inhibit the proliferation of GMCs induced by HG.(2)The immunofluorescence results of Ki67 and PCNA showed that EPI could reduce the expression of Ki67 and PCNA in GMCs induced by HG.(3)Flow cytometry showed that EPI arrested the HG-induced GMCs in the G2/M phase of cells,and down-regulated the expression of Cyclin E and CDK2.(4)EPI could significantly down-regulate the expression of ROS in GMCs induced by HG.(5)It was found that EPI significantly reduced the expression of AGEs and RAGE in GMCs induced by HG.3.The improvement effect of EPI on db/db mice(1)Male db/db mice were used as the DN model,and db/m mice were regarded as the normal control group(NC group).Model mice were randomly divided into the model group(DN group),EPI 50 mg/kg,100 mg/kg,and 200 mg/kg groups,and γsecretase inhibitor(DAPT)5 mg/kg group(DAPT group).Mice in the EPI group were given a corresponding dose of EPI intragastrically,while mice in the NC group and DN group were given an equal volume of normal saline intragastrically,once a day for 8weeks.DAPT group was given 5 mg/kg subcutaneously for 5 consecutive days per week,without treatment for 2 days for 8 weeks.(2)During administration,the db/m mice in the NC group were in good mental state,lively,and their hair was shiny.The db/db mice in the DN group were lethargic,had loose and dull hair.After 8 weeks of administration,the state of mice in each EPI group was improved.(3)EPI significantly decreased the 24 h food intake and water intake of db/db mice,and significantly reduced the body weight and FBG of db/db mice.(4)EPI significantly improved lipid metabolism and renal function in db/db mice.(5)EPI significantly alleviated renal histopathological injury in db/db mice.It also reversed the renal ultrastructural changes in db/db mice induced by long-term hyperglycemia.(6)EPI reduced AGEs content in serum and kidney,IL-6,and TNF-α content in the serum of mice.(7)Compared with mice in the DN group,there was no statistical difference in the organ coefficients of mice in each administration group4.Exploring the mechanism of EPI in improving DN based on transcriptomics(1)RNA-seq method was used to analyze the renal transcriptome of the NC group,DN group,and EPI 200 mg/kg group(EPI group)to find out the Differentially expressed genes(DEGs).Compared with the NC group,2084 DEGs were up-regulated and 2006 DEGs were down-regulated in the DN group.Compared with the DN group,there were only 662 DEGs in the EPI group,among which 315 DEGs were up-regulated and 347 DEGs were down-regulated.(2)A total of 267 DEGs with opposite trends between DN/NC and EPI/DN were screened by Venn diagram,and these 267 DEGs were considered as potential targets of EPI in the treatment of DN.(3)Four important modules were obtained through the Cytoscape,and 10 Hub genes were obtained through the MCC calculation method,which are Agt,Apoa2,C3,Fgg,Fbxo44,Cckar,Gna1,Prok1,Spsb1,and Asb11.(4)The results of qRT-PCR showed that the changes of Agt,Fgg,C3,and Jchain in kidney tissue were consistent with the trend of kidney transcriptome.(5)Molecular docking results showed that EPI can only form hydrogen bonds with LEU141 and SER116 in Agt,indicating that Agt may be the most critical target gene for EPI to improve DN.(6)EPI down-regulated the expression of Agt,TGFβ,and Smad2 proteins in the kidney and HG-induced GMCs.(7)EPI decreased the expression of Collagen IV in mouse kidneys in a dose-dependent manner.(8)After the expression of Agt was down-regulated,EPI failed to activate the Agt-TGFβ/Smad2 signaling pathway.5.Effects of EPI on the gut microbiota of db/db mice(1)After administration,the feces of the NC group,DN group,and EPI 200mg/kg group(EPI group)were analyzed by 16 S r DNA high-throughput sequencing technology,and the effects of EPI on the gut microbiota of DN mice were investigated.The Venn diagram showed that there are 342 common OTUs in the NC group,DN group,and EPI group.Among them,the NC group has 34 unique OTUs,the DN group has 107 unique OTUs,and the EPI group has 25 unique OTUs.(2)Compared with the NC group,the proportion of Firmicutes in the DN group increased,and the proportion of Bacteroides decreased;EPI could reverse this change.(3)The results of the UPGMA clustering tree showed that the composition of the gut microbiota of the EPI group and the NC group were more similar.(4)The LEf Se analysis showed that the structure of the gut microbiota of the NC and DN groups was significantly different,while the structure of the gut microbiota of the EPI group and the NC group was slightly different.Conclusion:1.It was screened out that EPI is the alkaloid with the best effect in treating DN among the alkaloids of RC.2.EPI has a protective effect on the damage of HG-induced GMCs and reduces kidney damage in db/db mice.3.The mechanism of EPI in improving in db/db mice is closely related to Agt and its downstream TGFβ/Smads signaling pathway.4.EPI has a certain regulatory effect on the gut microbiota of DN mice... |
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