| BackgroundSystemic lupus erythematosus(SLE) is one kind of complex autoimmune disease, characterized by high level of autoantibody, B cell hyperresponsiveness and multiple organs damage. Currently, clinicians often use corticosteroids, non-steroidal anti-inflammatory drugs, immunosuppressants and other non-specific immunosuppressive therapies to treat SLE, which suppress autoimmune response inevitably bring about liver toxicity, gastroenteritis and other side effects at the same time. Autoantibody is an important risk factor of SLE, plasma cell is the direct source of antibody, indicating that treatment of SLE by targeting plasma cell may directly inhibit or reduce the production of autoantibody. B lymphocyte induced maturation protein 1(Blimp1), a key transcription factor in inducing mature and development of plasma cell, also plays an important role in maintaining survival and antibody-secreting ability of plasma cell, our previous experiments confirmed that the high expression of Blimp1 in SLE was positively correlated with the SLE disease activity, the titer of autoantibody and the number of plasma cell, which suggested Blimp1 may become a new index for diagnosing SLE, inhibition of Blimp1 may become a new direction for the treatment of SLE by targeting plasma cell.ObjectiveIn this study, RNA interference and small molecule chemical drug 2-methoxyestradiol were used to inhibit the high expression of Blimp1 in MRL-lpr lupus mice, after Blimp1 expression was suppressed, the changes of characteristics of SLE, such as 24-hour urine protein, peripheral blood anti-double stranded DNA antibody, pathological changes of kidney, pathological enlargement of lymphoid organs, numbers of plasma cell and double-negative T cell were observed, the expression changes of antibody production associated transcription factor BCMA, XBP-1 and J-chain were also analysed, so as to explore the effect and possible molecular mechanism of inhibition Blimp1 in SLE.Methods(1) Lentiviral-mediated Blimp1 si RNA(experimental group) and empty vector(control group) were constructed, then packaged and titration lentivirus, intravenous injection of MRL-lpr lupus mice with Blimp1 si RNA and empty vector for three weeks. Blimp1, BCMA, XBP-1, J-chain and C-myc m RNA expression levels in the PBMC were analyzed by RT-PCR, the protein expression and tissue distribution of Blimp1 in liver, kidney, spleen and lymph node were analysed by western blot and IHC. Meanwhile, coomassie blue staining was used to analyse the 24-hour urine protein levels of mice in experimental and control groups, peripheral blood anti-double stranded DNA antibody level was analysed by ELISA, renal pathological changes were evaluated by HE staining.(2) MRL-lpr lupus mice and normal B6 mice were randomly divided into experimental group(2-ME2 treatment) and control group(DMSO treatment), mice of each group were administered orally for four weeks. Body weight, the area of skin ulcers, random urine protein and urine creatinine levels of mice were analysed per week, then the mice were sacrificed, the weights of mouse lymphoid organs, the scores of back lesion area and lymph node swelling were recorded, Blimp1 m RNA and protein expression were analysed by q RT-PCR and western blot, flow cytometry was used to analyse the changes of mouse spleen lymphocyte subsets.Results(1) Blimp1 m RNA and protein expression in peripheral blood, spleen, kidney and lymph node of MRL-lpr lupus mouse were up-regulation, after Blimp1 si RNA injection three weeks, Blimp1 m RNA expression level and anti-double stranded DNA antibody level in peripheral blood of lupus mice were decreased 78% and 28%(p<0.05), respectively, accompanied by antibody secretion related transcription factor XBP-1, J-chain and BCMA m RNA levels reduced. Compared to the control group, Blimp1 protein levels in the liver of experimental group lupus mice did not show statistical difference, but decreased 95%, 72% and 47% in the kidney, spleen and lymph node(p<0.05), respectively. In addition, due to the effect of Blimp1 si RNA, kidney disease was mitigated and 24-hour urinary protein level was also significantly decreased(p<0.05) in lupus mice.(2) MRL-lpr lupus mouse peripheral blood, kidney and spleen had a high level of Blimp1, 2-ME2 could significantly inhibit Blimp1 m RNA expression in peripheral blood(ΔCt value 6.99±0.69 vs 7.36±0.50, p<0.05), Blimp1 protein expression in kidney(OD value 0.46±0.07 vs 0.15±0.16, p<0.05) and spleen(OD value 0.88±0.11 vs 0.26±0.18, p<0.05) of lupus mice, and reduce urine protein/creatinine ratio(0.77±0.29 vs 0.27±0.17, p<0.05), inhibit the excessive proliferation of spleen(spleen/body weig ht ratio 11.91±2.59 vs 7.04±3.30, p<0.05), reduce the number of pathogenic T cell(CD3+CD4-CD8- cell 11.93±2.70 x106 vs 2.13±1.74 x106, p<0.05), meanwhile, 2-ME2 could reduce the size of skin lesions on mice back and the score of lymphadenopathy, alleviate kidney disease of lupus mice.ConclusionsOur previous experiments found the high expression of Blimp1 in SLE was positively correlated with the SLE disease activity,the titer of autoantibody and the number of plasma cell.This study confirmed Blimp1 expression is up-regulation in MRL-lpr lupus mice,Blimp1 si RNA can significantly reduce Blimp1 m RNA and protein level,24-hour urine protein and peripheral anti-double stranded DNA level,alleviate kidney disease in lupus mice.2-ME2 can inhibit the high expression of Blimp1 in kidney,spleen and peripheral blood,reduce urine protein/creatinine ratio,suppress excessive proliferation of spleen and lymph node,reduce the number of pathogenic T cell in spleen and mitigate kidney pathological changes of MRL-lpr lupus mice,suggesting that inhibition of Blimp1 can relieve the symptoms of SLE lupus mice,the mechanism of its action may through inhibiting Blimp1 thereby inhibiting plasma cell differentiation and maturation related transcription factor XBP-1,BCMA,J-chain expression,resulting in disorder of plasma cell maturation,reducing the number of plasma cell,declining level of autoantibody secretion.Based on the above evidence,Blimp1 may become a new laboratory index to diagnose SLE,inhibition Blimp1 may provide a new strategy for the treatment of SLE.Tris美国BBI公司Tween-20美国Sigma公司Blimp1一抗(大鼠抗小鼠)美国Santa Cruz公司XBP-1一抗(兔抗鼠)美国Santa Cruz公司C-myc一抗(兔抗鼠)美国Santa Cruz公司GAPDH单抗(兔抗鼠)美国Sigma公司小鼠抗双链DNA抗体ELISA试剂'美国Alpha Diagnostic公司HF212UV CO2孵箱香港Heal Force公司MDF-382E超低温冰箱日本Panasonic公司而Blimp1 si RNA处理3周后的实验组MRL-lpr狼疮小鼠PBMC中Blimp1 m RNA水... |
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