The Effect Of HMGB1 And TLR/STAT Pathway On Lupus Nephritis

Objective: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease affecting multiple organs including the kidneys, and lupus nephritis (LN) is a capital cause of death and disability. Therefore, it is very important for us to study pathogenesis and search for new therapy methods of LN to improve life quality of the patients.The main pathologic changes of LN are proliferation of mesangial and stroma cells, infiltration of lymphocytes and mononuclear cells. Recent evidence has revealed that many cytokines may be involved in lupus nephritis, and the balance between proinflammatory cytokines and anti-inflammatory cytokines plays an important role in the immune response. High mobility group protein box 1 (HMGBl), an intranuclear architerctural protein is well known for its nuclear funetions. Recently, it has been identified as a potent proinflammatory cytokine. HMGB1 could control the activation and chemotaxis of inflarnmatory cells, and stimulates proinflammatory cytokine synthesis in inflammatory cells. Also, it is an endogenous immune adjuvant and endogenous signal for dendritic cell maturation and Thl polarization, which can promote an effective inflammatory immune response. Many studies have showed that HMGB1 was involved in autoimmune diseases, and it is a new therapy target for arthritis. However, the effect of HMGB1 in LN pathogenesis have not been clarified. Therefore the objective of this study was to determine the role of the novel proinflammatory cytokine HMGB1 in lupus nephritis.Methods:1 The measurement of HMGB1 in the peripheral blood of the patients with LNSamples of peripheral blood were obtained from 34 patients with SLE including 16 patients without LN, 18 patients with LN, the samples were also obtained from 12 healthy volunteers as controls. All SLE patients met the 1987 revised diagnostic criteria of the American College of Rheumatology without smoking habits, malignant tumors and infection diseases. Enzyme linked immunosorbent assay (ELISA) was used to detect serum concentration of HMGB1. Reverse transcriptase-polymerse chain reaction (RT-PCR) was used to detect the expression of HMGB1 mRNA and flow cytometry (FCM) was used to measure the expression of Toll-like receptor 4 (TLR4) on peripheral blood mononuclear cell (PBMC). Meanwhile, the related clinical and lab indexes were recorded.2 Culture of human mesangial cells and detection of proliferating statusHuman mesangial cells were inoculated in the dose of 1×104mL-1. After 24h, cells were cultured with standard medium as control group or with medium supplemented with 10μg.L-1 human recombinant protein HMGB1 as trial group in vitro. Then cells were collected in 6h, 12h and 24h respectively as well as normal control group cells. Immunocytochemical staining was adopted to detect PCNA proteins expressions in human mesangial cells. Immunocytochemical staining and FCM were performed to detect the expression of TLR2 protein. RT-PCR was used to dectect STAT1/3 mRNA expressions.Results:1 Expression of HMGB1 in the peripheral blood of patients with LN and its clinical significance(1) The changes of HMGB1 mRNA detected by RT-PCR in PBMCRT-PCR showed that expression of HMGB1 mRNA on PBMC were higher in patients with LN than these in SLE and healthy people. There was no statistically significant difference between SLE group and healthy controls.(2) Expression of HMGB1 proteins in the peripheral blood The level of HMGB1 proteins in serum was higher in patients with LN than those without LN and healthy people[(9.79±1.76 vs 7.55±1.67, 7.79±0.75)ng.mL-1](P<0.01). There was no statistically significant difference between SLE group and healthy controls.(3) Changes of TLR4 expression on PBMCFCM showed that the expression of TLR4 in CD14+ monocytes of patients with LN was higher than that with SLE and healthy people, while there were no significance in CD3+ T cells among LN, SLE and healthy people.(4) Level of HMGB1 protein in serum of LN was positively correlated with the proteinuria. The expression of TLR4 in CD14+ monocytes of patients with LN was positively correlated with the proteinuria.2 Proliferating status and changes of human mesangial cells that were stimulated by HMGB1(1) The changes of PCNA protein expression on human mesangial cellsImmunocytochemical staining showed that positive expression of PCNA were observed in nuclei of human mesangial cells. The positive expression cell of PCNA was observed in control group, the rate of positive expression was ( 48.61±1.23 )%. HMGB1 increased the positive expression of PCNA in time-dependent manner. After 24h, the rate of positive expression was (98.28±1.86)%.(2) The changes of TLR2 protein expression on human mesangial cellsImmunocytochemical staining indicated that positive stainings for TLR2 were observed in both cytolemma and cytoplasm of human mesangial cells. There were negative staining for TLR2 in control group, while enhanced stainings were observed in HMGB1 groups.FCM showed that HMGB1 could significantly up-regulate the expression of TLR2 protein in time-dependent manner .(3) The changes of signal transducer and activator of transcription (STAT) signal pathwayRT-PCR indicated that HMGB1 could significantly up-regulate the expressions of STAT1 and STAT3 mRNA in time-dependent manner.(4) There were markedly positive correlation between the expression of PCNA , TLR2 and STAT1 mRNA(r=0.817, P=0.000; r=0.830, P=0.000), as well as PCNA, TLR2 proteins and STAT3 mRNA (r=0.863, P=0.000; r=0.926, P=0.000).Conclusions:1 Level of HMGB1 protein in serum of LN patients was higher in patients with LN than those without LN and healthy people. That was also positively correlated with the level of proteinuria. These revealed that HMGB1 was one of the important cytokines in LN.2 HMGB1 could bind to cell-surface receptors-TLR2 of mesangial cell and effectively activate STAT, further promote cell proliferation, which may be one of the mechanisms in renal injure in LN.In conclusion, the present experiment showed that HMGB1 may play an important role in the pathogenesis of LN, which may be a considerable new therapeutic target molecule in LN. But our research was confined to the effect of HMGB1 on mesangial cell proliferation, whose exact mechanism in LN pathogenesis needs further investigation.