The Effect Of High Mobility Group Box 1 On Cell Cycle Distribution And Expression Of Cell Cycle Protein In Mouse Mesangial Cell

Objective: At this study we detected the distribution of cell cycle and the expression of PCNA, CyclinD1, CDK4 and p16 in MMC changes and analyze the correlation among them in order to explore the possible effect of HMGB1 on the generation of MMC.Systemic lupus erythematosus (SLE) is a systemic autoimmune disease affecting multiple organs including the kidney, and lupus nephritis (LN) is a capital cause of death and disability. Although the pathogenesis of SLE has not yet entirely clear, immune dysfunction induced by lymphocyte dysfunction and unbalanced cytokine secretion is the core of the pathogenesis. As an important late inflammatory mediator, HMGB1 involved in the occurrence and development of rheumatoid arthritis, SLE, inflammatory diseases and other autoimmune diseases [2-3]. Our early experiments revealed that the level of HMGB1 mRNA and protein in the peripheral blood of patients with lupus nephritis increased, which meaned that HMGB1 is one of the important inflammatory cytokines in the pathogenesis of lupus nephritis, and related to the mesangial cell proliferation [8].However, the possible mechanism how HMGB1 effct on cell generation is still uncertain.Methods: MMC were obtained from the Department of Medical pathology of the Hebei University. MMC selected for the study were randomly divided into control group and HMGB1 stimulation group(50μg/L). The cells were collected after 4, 8, 12h . Cells' cycle distribution were detected by Flow Cytomtery; Immunocytochemiacal stain were used to dectct the level of PCNA protein in MMC; the expression of CyclinD1 mRNA were detected by PT-PCR; Western Blot were used for the detection of leves of CyclinD1,CDK4 and p16 protein.Results : 1HMGB1 induced MMC proliferation and promted the cell cycle transduction from the G1 Stage to S Stage of MMCCompared with normal control group, the ratio of cells in G0/G1 phase decreased and ratio of S phase increased at the 4h, 8h simulated by HMGB1, at the same time , the proliferation index were significantly higher than that in control group(64.6±0.23; 69.3±0.15 vs 57.8±0.32 ) .(P<0.05)2 HMGB1 up-regulated the levels of PCNA protein in MMCPositive expression of PCNA protein was expressed in nucleus of the normal MMC by immunocytochemistry. The expression of PCNA was increased significantly in MMC stimulated by HMGB1, peaked at 4h, gradually decreased to baseline at 12 h .3The Cyclin D1mRNA and protein increased in HMGB1-stimulated groupThe results showed that HMGB1-induced time-dependant mRNA levels of cyclinD1 were evaluated by RT-PCR analysis. As shown, the increased expression of cyclin D1 was detected at 0 h and peaked at 4 h, return to baseline at 12h.The synthesis of cyclin D1 protein in HMGB1-stimulated cells was analyzed by Western blot. The expression level of cyclinD1 protein increased, peaked at 4 h, and gradually decreased to baseline at 12 h4 HMGB1 up-regulated the expression of CDK4 protein in MMCCompared with normal control group(0.67±0.02), the expression of CDK4 protein in MMC was significantly increased after 4h' stimulation(0.88±0.03). But the level of CDK4 protein decreased when cells were stimulated for 8~12 hours(0.45±0.02 , 0.42±0.01), there was no significant difference compared with control group(P>0.05).5 HMGB1 decreased the expression of p16 protein in MMCCompared with normal control group(1.11±0.02), the expression of p16 protein in MMC was significantly decreased at 4,8 and 12 h stimulated by HMGB1 ( 0.47±0.02, 0.46±0.06, 0.14±0.02 respectively ) in time-dependent fashion (P<0.05).Conclusions:HMGB1 could induce mouse mesangial cells proliferation and promote the transition of cell cycle from G1 stage to S stage by up-regulating Cyclin D1/CDK4/p16 pathway, which might be a effect mechanism of HMGB1 in lupus nephritise pathogenesis.