Effect Of Dioscin On Gd-IgA1 Secretion By Human B Cells And Its Mechanism

Background:Immunoglobulin A nephropathy(IgAN)is the most common primary glomerulonephritis in the world.At present,there is no specific treatment,and it ranks first in the end stage renal disease caused by primary glomerulonephritis.As an immune complex-mediated glomerulonephritis,IgAN has complex pathological changes and different clinical manifestations.However,the current treatment of IgAN focuses on the regulation of downstream immune and inflammatory events,rather than the specific treatment of IgAN.Therefore,it is urgent to explore specific treatment methods from the pathogenesis of IgAN.Many studies have confirmed that the increase of circulating galactose deficient IgAl(Gd-IgA1)is a key link in the pathogenesis of IgAN,and the elevated level can predict the progression of the disease.The IgA1 molecules deposited in the Mesangial area of IgAN patients also have the characteristics of galactose deletion.The O-glycosylation modification of IgAl molecule is closely related to the pathogenesis of IgAN.In patients with IgAN,the deletion of Gal of IgA1 molecule leads to the exposure of GalNAc,which binds to IgG antibodies that recognize glycosyl groups to form pathogenic complexes.The starting glycosyl group of the O-sugar chain is GalNAc.With the help of its molecular chaperone Cosmc,C1GALT1 connects the Gal group to the core GalNAc in the way of β1,3 bond to complete the glycosylation modification of IgA1.When the expression and activity of C1GALT1 and Cosmc are decreased,Gd-IgA1 increases.After years of clinical observation,practice and experience summary,the TCM circle has gradually formed a more unified understanding of the disease name,core pathogenesis and syndrome differentiation of IgAN.IgAN has no specific TCM disease name,and mostly belongs to the categories of "kidney wind","hematuria","edema","low back pain","fatigue" and so on.Outside of evil reality,wind-heat and damp-heat of middle/lower jiao are the most common.Traditional Chinese medicine is a national treasure of our country,which has been more and more recognized at home and abroad in thousands of years of practice and innovation,and has been widely used in clinic,especially in the treatment of complex diseases,which has the characteristics of remarkable curative effect and few side effects.Dioscorea nipponica Makion has the functions of dispelling wind and relieving pain,relaxing muscles and dredging collaterals,relieving cough and asthma,etc.Modern pharmacological studies have shown that Dioscorea nipponica Makion has many effects,such as immune regulation,reducing blood sugar,relieving cough,relieving asthma and expectorant,improving cardiovascular function,anti-tumor and so on.In recent years,there are many reports about Dioscorea nipponica Makion or its extract in the treatment of IgAN.Yiqi Qingre ointment,a preparation containing traditional Chinese medicine,is safe and effective in the treatment of IgAN,but its material basis and mechanism of action are not clear.Dioscin is the main active component and active ingredient of Dioscorea nipponica Makion,and its therapeutic effect in kidney disease has been paid more and more attention.Dioscin can protect cells by inhibiting wnt pathway and reducing the expression of TGF-β1.In the model of renal ischemia/reperfusion injury,dioscin can obviously down-regulate the levels of BUN and Cr in serum of rats,and can alleviate the renal injury.Therefore,Dioscin may have a novel mechanism,inhibit the excessive secretion of Gd-IgA1 by B cells and play a certain role in renal protection,and is expected to become a potential drug for the prevention and treatment of IgAN.Objective:1.Through network pharmacology,the active components,potential targets and signal pathways in Dioscorea nipponica Makion were predicted and screened,and the key active components,key targets and signal pathways in the treatment of IgAN were analyzed,so as to screen intervention drugs for subsequent cell experiments in vitro.2.Through in vitro cell experiments,in-depth study of the effect of dioscin on the secretion of Gd-IgA1 by human B cells and its mechanism,in order to further confirm the therapeutic effect of dioscin on IgAN and provide new therapeutic ideas for the prevention and treatment of IgAN with traditional Chinese medicine.Methods:1.Network pharmacology:The main active components of Dioscorea nipponica Makion were obtained by network pharmacology,the action targets of these active components were predicted,and the disease targets of IgAN were obtained,and the disease and drug prediction targets were intersected to screen out common potential therapeutic targets.The interaction network diagram of "Dioscorea nipponica Makion-component-IgAN-target" was constructed,and the active components with the highest dregee value were selected as the key components of Dioscorea nipponica Makion in the treatment of IgAN.Then the PPI network of protein-protein interaction was constructed by STRING database,and the drug-disease common target was analyzed by R software for functional enrichment of key target genes GO and KEGG.2.1n vitro cellular test:To observe the intervention effect of dioscin on human B cells and the detection of related indexes.Human B cell stably secreting IgA and Gd-IgA1 were used as carriers,stimulated by LPS and intervened by different concentrations of dioscin.The proliferation of DAKIKI cells in each group was detected by CCK8 method,the effect of dioscin on IgA and Gd-IgA1 secretion by DAKIKI cells was detected by ELISA method,and the mRNA and protein levels of glycosyltransferase C1GALT1 and its molecular chaperone Cosmc in human B cell DAKIKI were detected by qRT-PCR and Western blot respectively.Results:1.Network pharmacology:(1)12 potentially active components(11 of which are related to IgAN related targets and signal pathways)were screened from TCMSP and BATMAN-TCM databases.(2)121 action targets of these active components were predicted by PharmMapper database,and 1346 disease action targets of IgAN were retrieved through Genecards and OMIM database.45 potential targets of Dioscorea nipponica Makion were screened out by intersecting drug prediction targets and disease targets.(3)The action targets related to IgAN of the potential active components of Dioscorea nipponica Makion were introduced into Cytoscape software for visualization.The results showed that the potential active molecules of the top 5 of dregee were Diosgenin palmitate,Delta3,5-Deoxytigogenin,Dioscin,7-Epitaxol,DIOSGENIN,DEHYDRO.Compared with Delta3,5-Deoxytigogenin,dioscin and diosgenin play a more important role in the traditional Chinese medicine Dioscorea nipponica Makion.(4)The PPI network of protein interaction in Dioscorea nipponica Makion was constructed,and it was concluded that Dioscorea nipponica Makion may play a role in the treatment of IgAN by acting on the key targets ALB,CASP3,EGFR,ESR1,HSP90AA1 and so on.(5)The functional enrichment analysis of key target genes GO and KEGG was carried out on the common target of Dioscorea nipponica Makion and IgAN.GO enrichment analysis selected three parts:biological process,cell composition and molecular function.A total of 408 biological processes,13 cell composition and 39 molecular functions were obtained.65 signal pathways were obtained by KEGG pathway analysis.2.In vitro cellular test:(1)The results of CCK8 test showed that compared with the blank control group,LPS40 and 80μg/mL could stimulate the proliferation of DAKIKI cells(P<0.05),and the effect of 40μg/mL was the most obvious.Compared with LPS 40μg/mL stimulation group,dioscin 0.25,0.5,1.0μg/mL could inhibit the proliferation of DAKIKI cells(P<0.05),and it was positively correlated with the concentration of dioscin.(2)The results of ELISA experiment showed that compared with the blank control group,the secretion of IgA and Gd-IgA1 increased in LPS 40μg/mL stimulation group(P<0.01,P<0.1),while dioscin 0.25,0.5,1.0μg/mL could inhibit the secretion of IgA and Gd-IgA1 in different degrees(P<0.05).(3)Compared with the blank control group,the relative expression of mRNA of C1GALT1 and Cosmc in LPS 40g/mL stimulation group was down-regulated(P<0.01).Compared with the group stimulated by LPS,different concentrations of dioscin could up-regulate the relative expression of mRNA of C1GALT1 and Cosmc,and 1.0μg/mL of dioscin could significantly up-regulate the relative expression of mRNA of C1GALT1 and Cosmc(P<0.05).(4)The results of Western Blot experiment showed that compared with the blank control group,the protein expression of C1GALT1 and Cosmc in LPS 40μg/mL stimulation group decreased(P<0.05).Compared with the LPS stimulation group,the protein expression of C1GALT1 and Cosmc was up-regulated after the intervention of different concentrations of dioscin,and dioscin 1.0μg/mL could significantly up-regulate the protein expression of C1GALT1 and Cosmc(P<0.05).Conclusion:1.The network pharmacological research suggests that dioscin is probably the key active ingredient in the treatment of IgAN.2.Dioscin can inhibit the proliferation of DAKIKI cells and the increase of IgA and Gd-IgAl secretion induced by LPS stimulation,and its mechanism may be related to the up-regulation of the mRNA and protein expression of C1GALT1 and Cosmc,suggesting that dioscin may intervene in the initial stage of IgAN by reducing the production of Gd-IgA1.