TLR Mediates HMGB1-induced Lupus Nephritis Glomerular Extracellular Matrix Deposition Via Activation Of The Myd88/NF-?B Signaling Pathway

Objective:Lupus nephritis?LN?is one of the importantcomplications of systemic lupus erythematosus?SLE?.The incidence rate increases every year,however the cure rate is very low.Therefore,it is particularly important to explore its pathogenesis and then find theeffective therapeutic targets.Renal failure induced by glomerulosclerosis is the final stage of most LN kidney injury.Inhibiting or slowing the deposition of glomerular extracellular matrix and thus improving glomerulosclerosis may be an effective way to delay the progression of renal injury.Exploring the reason of the deposition of extracellular matrix in globules might be an important entry point for relieving the development of LN.In this study,renal specimens from LN patients and human mesangial cells?HMC?were studied.HMC stimulated with replacement plasma,obtaited from LN patients,or rHMGB1 were used to investigate whether TLR2 participates in the extracellular matrix deposition by binding to HMGB1 and activating Myd88/NF-?B signal.The role of TLR2 in the extracellular matrix deposition of LN glomeruli was verified in MRL/lprtlr2^-/-mice.Methods:1.Renal specimens from LN patients and normal renal tissues remoted to the para-carcinoma were collected.The expression of TLR2 and Collagen IV were evaluated by immunohistochemistry.2.Replacement plasma from LN patients and TLR2 antibody,were used to stimulate HMC.ELISA was used to detect the secretion of FN protein in culture supernatant of HMC.3.Glycyrrhizic acid?Gly,HMGB1 specific activity inhibitor?,were used to pretreat HMC before plasma or rHMGB1 stimulation.ELISA was used to detect the secretion of FN protein in culture supernatant of HMC.4.Western blot technique was used to detect the Myd88 expression level and P65 expression was detected by immunofluorescence.5.TLR2 antibody,ST2825?Myd88 specific inhibitor?,PDTC?NF-?B specific inhibitor?were used to pretreat HMC before rHMGB1 stimulation.ELISA was used to detect the secretion of FN protein in culture supernatant of HMC.6.The expression of TLR2 and FN in renal tissue of MRL/lprtlr2^-/-,MRL/lpr and MRL/MPJ mice was detected by immunofluorescence.7.The expression of Myd88 and NF-?B in renal tissue of MRL/lprtlr2^-/-,MRL/lpr and MRL/MPJ mice was detected by immunohistochemistry.Results:1.Up-regulation of TLR2 and Collagen IV protein expression in glomerular cells in patients with LN.The results of immunohistochemistry showed that the expression of TLR2 and Collagen IV proteins in the glomeruli of renal tissue from LN patients was significantly higher than that in normal kidney tissues remoted to para-cancer,and there was a positive correlation between TLR2 and Collagen IV protein in glomeruli of LN patients.2.TLR2 mediates upregulation of FN secretion in HMC treated with replacement plasma from LN patients.Western blot results showed that compared with the control group,the expression of TLR2 protein in plasma-stimulated group gradually increased and peaked at 45 min.The results of ELISA showed that compared with the control group,the FN protein level in the culture supernatant of plasma-stimulated group increased;while FN protein level in plasma+TLR2 antibody group was lower than that in plasma-stimulated group.3.HMGB1 mediates upregulation of FN secretion in HMC stimulated with replacement plasma from LN patients.ELISA results showed that compared with the control group,the levels of FN protein in the culture supernatants of plasma-stimulated group and rHMGB1-stimulated group were significantly increased.Compared with plasma-stimulated group,the FN protein level was down-regulated in plasma+Gly group.4.rHMGB1 activates the TLR2/Myd88/NF-?B signaling pathway in HMC exposed to LN plasma.Western blot results showed that compared with the control HMCs,the expression of TLR2 protein in rHMGB1-stimulated HMCs was significantly increased at 45 min and the level of Myd88 protein was significantly increased at 75 min.The results of immunofluorescence showed that the positive signal of P65 protein was maily located in cytoplasm in control group,while the positive signal of P65 was located in nuclei,and the nuclear expression of P65protein in HMC cells increased significantly after treatment with replacment plasma for 90 min.However,the nuclear expression of P65 protein in plasma+TLR2 antibody group was lower than that in plasma-stimulated group.5.FN secretion induced by rHMGB1 is mediated by the TLR2/Myd88/NF-?B signaling pathway.The ELISA results showed that compared with the rHMGB1 stimulation group,FNproteinlevelintheculturesupernatantofTLR2antibody+rHMGB1 stimulation group,ST2825+rHMGB1 stimulation group,and PDTC+rHMGB1 stimulation group were significantly decreased.6.The FN protein expression in glomerular of MRL/lprtlr2^-/-mice decreased.The PCR results showed that the Fas wild DNA and TLR2 wild DNA were negative and the Fas mutant DNA and TLR2 mutant DNA were positive in MRL/lprtlr2^-/-mouse.Immunofluorescence results showed that compared with the control group,the expression of TLR2 and FN protein in glomerular cells of MRL/lpr mice was significantly increased;however,TLR2 and FN protein expression levels were downregulated in MRL/lprtlr2^-/-micecompared with MRL/lpr mice.7.The expression of Myd88 and NF-?B protein in MRL/lprtlr2^-/-mice glomerular decreased.Immunohistochemistry results showed that compared with the control group,the expression levels of Myd88 and P65 proteins in glomerular cells of MRL/lpr mice were significantly increased;however,the expression of Myd88and P65 protein were downregulated in MRL/lprtlr2^-/-mice compared with MRL/lpr mice.Conclution:TLR2 might participate in LN by modulating the extracellular matrix deposition of mesangial cells induced by HMGB1partly through activation of Myd88/NF-?B signaling pathway.