| BackgroundsSystemic lupus erythematosus(SLE) is a kind of severity autoimmune disease.The prevalence of SLE is about 1/1000 in Chinese.To now,there are not effect therapeutic tool.In 20 century,5 years' survival rate of SLE is 20%.For the past few years,survival rate of SLE has improved,for immune examination and to correctly use corticosteroids and immunodepressants.And now,5 years' survival rate of SLE is 90-98%,10 years' survival rate of SLE is 80-84%.It has been confirmed that cyclophosphamide(CTX) is effected to lupus nephritis(LN) by many clinical researches.But the more the total dosage of CTX,the more effect to LN,and the more side effect.CTX has many side effect about infections,sex gland inhibition,and tumor.Recently many new methods were discovered,such as new immunodepressants,biological agents,plasmapheresis,and stem-cellular transplant.But the effect of those methods had been not confirmed,and the cost of those methods was very expensive.So,for reference successfully combination therapy in leucemia,we first based on theory cell proliferation,sequential combined vincristine(VCR) with low dosage CTX intermittently to treat severe SLE.CTX is a cell cycle nonspecial stage agent,while VCR as an cell cycle special stage agent.And VCR was used to SLE thrombocytopenia.During the last decade,the new combination therapy has effective and safetive through we carried on clinicl practice.We chose murine chronic graft-versus-host disease(cGVHD) as a model for lupus nephritis.And we selected two biomarkers for lupus nephritis,MCP-1 and TGFβ1,and two immune markers for SLE,BAFF and BAFFR.ObjectivesFrom clinically to theoretically,to explore the effects and possible mechanisms of sequential combined VCR combined with low dosage CTX intermittently to treat severe SLE, by observing the changes of Urine protein,serum creatinine,HE staining of kidney, proinflammaton cytokines—MCP-1 and TGFβ1 expression in kidney,and BAFF and BAFFR expression in spleen.It is hoped that new promising combination therapy on SLE,the overall prognosis and outcome of SLE would be favorably improved accordingly. MethodsThe study was divided to two parts.First part:to set up murine chronic graft-versus-host disease model for study.experimental animal:6-8 week-age female(C57BL/6J×DBA/2) F1 murine and 6-8 week-age female DBA/2 routine.Weight(18-20)g.to create murine chronic graft-versus-host disease model was 36.Second part:the effect and safety of VCR combined with low dosage CTX intermittently to treat murine cGVHD model.After the murine cGVHD model was set up successfully,they were randomly divided into blank group,VCR pause group,CTX pause group,CTX every other day(EOD) group, VCR+CTX combination group.There are 24 weeks from set up model to sacrifice model.Detection item1 General state of health:weight,mental state,survival2 Blood detection:peripheral blood leucocyte,ALT,Cr,anti-dsDNA,ELISA for MCP-1, TGFβ1,BAFF3 Urine detection:24-hour urine protein quantitation,ELISA for MCP-1,TGFβ14 Liver:HE staining of liver5 Kidney:HE,Masson,PSAM,IF staining of kidney,immunohistochemical method to MCP-1,TGFβ1 in kidney6 RT-PCR to MCP-1,TGFβ1 mRNA7 Spleen:immunohistochemical method to BAFF,BAFFR in spleen8 RT-PCR to BAFF,BAFFR mRNAResults1 General state of health:①After cGVHD models were set up 6 week,the models had 24-hour urine protein quantitation 5.02±0.88mg,anti-dsDNA positive,andⅣLN pathology.So cGVHD models were successful.②It exhibited a increase in weight,ALT,and liver pathology,statistical significance were found in CTX EOD group and other groups.(P＜0.05)2 Kidney index①It exhibited a decrease in 24-hour urine protein quantitation,statistical significance were found in CTX EOD group,VCR+CTX combination group and other groups.(P＜0.01) blank group＜VCR pause group＜CTX pause group＜CTX EOD group≈VCR+CTX combination group.It exhibited a decrease in Cr,AI,anti-dsDNA,statistical significance were found in CTX EOD group,VCR+CTX combination group,CTX pause group and other groups.(P＜0.05)②It exhibited a decrease in ELISA for urine MCP-1 and TGFβ1,statistical significance were found in CTX EOD group,CTX pause group,VCR+CTX combination group and other groups.(P＜0.01)③MCP-1 and TGFβ1'expression in model kidney were reduced in CTX EOD group, VCR+CTX combination group and had statistical significance in CTX EOD group, VCR+CTX combination group with blank group,VCR pause group,CTX pause group.④24-hour urine protein quantitation was closely correlated with blood Cr,urine MCP-1,and MCP-1 expression in tubular cells and glomcrulus cells.⑤24-hour urine protein quantitation was closely correlated with blood Cr,urine TGFβ1,and TGFβ1 expression in tubular cells and glomcrulus cells.3 Immune system index①Blood BAFF were reduced in CTX EOD group,VCR+CTX combination group and had statistical significance in CTX EOD group,VCR+CTX combination group with blank group,VCR pause group,CTX pause group.②BAFF expression in model spleen were reduced in CTX EOD group,VCR+CTX combination group and had statistical significance in CTX EOD group,VCR+CTX combination group with blank group,VCR pause group,CTX pause group.③Blood BAFF was closely correlated with blood anti-dsDNA,BAFF expression in spleen.And BAFF expression in spleen was not correlated with BAFFR expression in spleen.4 Interaction of VCR and CTX①The result of ANOVA of factorial design show therapeutic alliance of VCR and CTX had interaction in 24-hour urine protein quantitation,blood Cr,and urine MCP-1(P＜0.05), and did not have interaction in urine TGFβ1(P=0.122).②The result of ANOVA of factorial design show therapeutic alliance of VCR and CTX had interaction in blood BAFF(P＜0.05),and did not have interaction in blood WBC (P=0.085).Conclusions1 The murine cGVHD model hadⅣLN pathology and the increase in expression of MCP-1,TGFβ1,BAFF.2 The combination of VCR and CTX had synergistic effect in decrease in 24-hour urine protein quantitation,Cr,and the expression of MCP-1,BAFF.3 The adverse effect of VCR+CTX combination group was similar to VCR pause group and CTX pause group.4 BAFF/BAFFR system may be a pathway that VCR+CTX combination group treat the murine cGVHD model.
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