| Background:Ig A nephropathy(Ig AN)is the most common primary glomerular disease over the world.Currently,there is no specific therapy for Ig AN.The proliferation of glomerular mesangial cells is the most basic pathological feature and plays a key role in early pathological changes of Ig AN,but the regulation mechanism is still unclear.Our previous studies found that autophagy was involved in the pathogenesis of Ig AN.Autophagy activation could be a potential therapy for kidney disease,but there are few studies in Ig AN.Tripterygium wilfordii Hook.F.(Tw HF)is a traditional Chinese herbal medicine with immunosuppressive and anti-inflammatory effects.Tw HF preparations are used to reduce proteinuria and hematuria,inhibit inflammation,and protect renal function in glomerulonephritis including Ig AN.However,the composition of Tw HF is complicated,and the toxicity and side effects limit applications.Our previous study found that triptolide(TP)was the main active ingredient of Tw HF.by the“compound-targets-disease” analysis based on network pharmacology.TP has a potency 100-200 times higher than tripterygium glycosides,improving bioavailability and reducing side effects.In various diseases,TP regulated cell proliferation,apoptosis,differentiation,invasion,and immune response by inducing autophagy.However,the specific mechanism of TP in Ig AN is not yet clear.Caspase recruitment domain-containing protein 9(CARD9)was a risk gene for Ig AN found in the Genome-Wide Association Study(GWAS).However,there was no research about the specific role of CARD9 in the pathogenesis of Ig AN.CARD9 is a downstream effector molecule of pattern recognition receptors(PRRs),which is involved in the regulation of immune homeostasis,inflammatory response,autophagy,and proliferation in infection,inflammation,tumor,and autoimmune diseases.CARD9 was a therapeutic target for cardiovascular disease by regulating autophagy.p38 MAPK is a transcription factor regulated by CARD9 and has been suggested to negatively regulate autophagy in various diseases.Notably,we predicted that CARD9 was the target of TP by Molecular Docking software.In summary,we speculated that TP promotes autophagy via the CARD9/p38 MAPK pathway to inhibit mesangial cell proliferation,thereby improving Ig AN.Objective:In this study,we established a TP-treated Ig AN mouse model and produced Ig A1-induced human mesangial cells(HMCs)to investigate the therapeutic effects of TP and the role of CARD9 in Ig AN,and the mechanism by which TP regulates autophagy and proliferation of mesangial cells through the CARD9/p38 MAPK pathway.The findings provide new theoretical bases for mesangial cell proliferation and suggests new ideas for future targeted therapy of Ig AN.Methods:1.Ig AN mouse model was induced by oral mucosal immunization and received intragastric TP treatment.To investigate the efficacy and safety of TP and its role in autophagy and proliferation of mesangial cells.The mice were divided into Control,TP,Ig AN,and Ig AN+TP groups for the following experiments:(1)Renal Ig A immunofluorescence staining;(2)HE and PAS staining;(3)Detection of liver and kidney function and urine protein/creatinine ratio levels;(4)The expressions of PCNA,Cyclin D1,P62,and LC3 II were detected by immunohistochemistry and Western Blot.2.The HMCs were induced by poly-Ig A1 to investigate whether TP inhibited HMCs proliferation by promoting autophagy.The HMCs were divided into Control,TP,Ig A1,and Ig A1+TP groups for the following experiments:(1)CCK8 assay and Western Blot were used to screen the optimal concentration of TP to inhibit HMCs proliferation;(2)Cell immunofluorescence and Western Blot were used to detect the expressions of PCNA,Cyclin D1,P62,and LC3 II;(3)Cell viability was assessed by CCK8 assay;(4)Flow cytometry analysis of cell cycle in HMCs;(5)m RFP-GFP-LC3 dual-fluorescence system was used to observe the autophagy flow in HMCs;(6)The autophagy inhibitor 3-MA was applied,and HMCs were divided into Ig A1,Ig A1+3-MA,Ig A1+TP,Ig A1+TP+3-MA groups.Western Blot was used to detect the expression of PCNA,Cyclin D1,P62,and LC3 II.3.To investigate the expression and clinical relevance of CARD9 in Ig AN patients,and its role in HMCs autophagy and proliferation.(1)The GSE116626 dataset of renal biopsies from Ig AN patients was analyzed;(2)Double immunofluorescence staining was used to detect the expression of Gd-Ig A1(KM55)and CARD9 in renal biopsies;(3)The correlation between the degree of mesangial cells proliferation and the expression of CARD9 in Ig AN patients was analyzed;(4)CARD9 si RNA(si CARD9)was constructed,and the HMCs were divided into Control,Ig A1,Ig A1+si Ctrl,and Ig A1+si CARD9 groups.The levels of autophagy and proliferation indexes of HMCs were detected by Western Blot,CCK8 assay,and flow cytometry;(5)CARD9overexpression plasmid(Exp-CARD9)was constructed,and HMCs were divided into Control,Ig A1,Ig A1+ NC,Ig A1+Exp-CARD9 groups.The levels of autophagy and proliferation indexes of HMCs were detected by Western Blot and CCK8 assay.4.Ig AN mice and poly-Ig A1 induced HMCs were used to investigate whether TP promoted autophagy to inhibit mesangial cell proliferation by CARD9/p38 MAPK.(1)Molecular docking software to analyze the binding of TP to CARD9;(2)The mice were divided into Control,TP,Ig AN and Ig AN+TP groups,the expression of renal CARD9 was detected by immunofluorescence,Western Blot,and q RT-PCR,and the level of p-p38MAPK/p38 MAPK was detected by Western Blot;(3)The HMCs were divided into Control,TP,Ig A1,and Ig A1+TP groups.The expression of CARD9 was detected by Western Blot and q RT-PCR,and the level of p-p38 MAPK/p38 MAPK was detected by Western Blot;(4)The HMCs were divided into Control,Ig A1,Ig A1+si Ctrl,and Ig A1+si CARD9 groups,and the levels of CARD9 and p-p38 MAPK/p38 MAPK were detected by Western Blot;(5)The HMCs were divided into Ig A1,Ig A1+TP,Ig A1+TP+NC,Ig A1+TP+Exp-CARD9 groups.Western Blot,CCK8 assay,and flow cytometry were used to detect the levels of autophagy and proliferation indexes;and Western Blot was used to detect the levels of CARD9 and p-p38 MAPK/p38 MAPK.Results:1.In Ig AN mice,Ig A was significantly deposited in the mesangium;mesangial cell proliferation was increased;PCNA,Cyclin D1,and P62 increased while LC3 II decreased.In Ig AN+TP mice,mesangial Ig A deposition and cell proliferation were significantly reduced;PCNA,Cyclin D1,and P62 decreased while LC3 II increased.The hepatorenal functions of the mice after TP treatment remained within the normal range.2.The optimal concentration of TP to inhibit the HMCs proliferation was 20ng/ml;in Ig A1-induced HMCs,PCNA,Cyclin D1,and P62 increased while LC3 II decreased;the autophagic flow was blocked,HMCs proliferation was increased,and the cell cycle was accelerated.In Ig A1+TP group,PCNA,Cyclin D1,and P62 decreased while LC3 II increased;the autophagic flow was promoted,HMCs proliferation was decreased,and the cell cycle was blocked;the anti-proliferative effect of TP was inhibited by the autophagy inhibitor 3-MA.3.The level of CARD9 m RNA in patients with Ig AN was significantly increased;CARD9 was mainly expressed in the mesangium and overlapped with Gd-Ig A1;a significant positive correlation was observed between CARD9 intensity and mesangial proliferation;CARD9staining in renal biopsies with MCD,FSGS,DN,and MPGN was negative while that of HSPN and MN was positive.CARD9 knockdown promoted autophagy and inhibited the proliferation of Ig A1-induced HMCs,while CARD9 overexpression inhibited autophagy and promoted cell proliferation.4.CARD9 was a target of TP;TP inhibited CARD9 expression in Ig AN mice and Ig A1-induced HMCs;CARD9 upregulation inhibited autophagy and promoted the proliferation of HMCs,reducing the anti-proliferative effect of TP;p38 MAPK pathway was activated in Ig AN mice and Ig A1-induced HMCs,TP inhibited p38 MAPK phosphorylation;CARD9 knockdown downregulated p-p38 MAPK/p38 MAPK in Ig A1-induced HMCs;CARD9 overexpression reduced the inhibitory effect of TP on the p38 MAPK pathway.Conclusion:1.TP treatment applied to Ig AN mice inhibited proliferation and promoted autophagy in mesangial cells was safe and effective.2.Mesangial cell proliferation was increased and autophagy was suppressed in Ig A1-induced HMCs,TP inhibited Ig A1-induced HMCs proliferation by promoting autophagy.3.High expression of CARD9 in Ig AN was positively correlated with the severity of HMCs proliferation,with CARD9 knockdown promoting autophagy and inhibiting HMCs proliferation.4.CARD9/p38 MAPK pathway was activated in Ig AN;TP promoted autophagy to inhibit HMCs proliferation by downregulating the CARD9/p38 MAPK pathway in Ig AN.25 figures,20 tables,178 references... |
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