目的探讨补体C5a/C5aR通路在结肠癌发病中对纤维介素蛋白-2凝血酶原酶(fibrinogen-like protein 2,FGL2)的调节机制,明确补体系统在结肠癌发病中的功能和作用。方法收集2013年12月至2015年7月第三军医大学西南医院普外科、新桥医院消化...目的探讨补体C5a/C5aR通路在结肠癌发病中对纤维介素蛋白-2凝血酶原酶(fibrinogen-like protein 2,FGL2)的调节机制,明确补体系统在结肠癌发病中的功能和作用。方法收集2013年12月至2015年7月第三军医大学西南医院普外科、新桥医院消化科15例经病理活检确诊的住院临床结肠癌患者癌及癌旁组织。采用免疫组织化学方法检测结肠癌患者癌及癌旁组织中补体活化片段C5b-9、活化C3及C5aR的表达,佐证补体系统在结肠癌中的活化状态;进一步通过体内外实验(Western blot)验证结肠癌患者癌及癌旁组织MAPK信号通路中P38的磷酸化水平及FGL2的沉积,明确结肠癌发病中补体C5a/C5aR通路与二者的调节关系。结果免疫组织化学及Western blot实验证实结肠癌患者癌组织C5b-9、活化C3及C5aR的表达明显高于癌旁组织,FGL2的沉积亦显著高于癌旁组织;体外结果提示C5a能促进巨噬细胞系Raw264.7MAPK通路中P38的磷酸化水平(P=0.0013)及FGL2的产生能力(P=0.0071),加入C5aR阻断剂之后,二者表达随之减弱。结论 C5a/C5aR通路能通过MAPK信号途径中的P38信号完成对FGL2的调节作用进而参与结肠癌的发病进程。展开更多
AIM To investigate the role of the complement 5a(C5a)/C5 a receptor(C5a R) pathway in the pathogenesis of acute liver failure(ALF) in a mouse model.METHODS BALB/c mice were randomly assigned to different groups, and i...AIM To investigate the role of the complement 5a(C5a)/C5 a receptor(C5a R) pathway in the pathogenesis of acute liver failure(ALF) in a mouse model.METHODS BALB/c mice were randomly assigned to different groups, and intraperitoneal injections of lipopolysaccharide(LPS)/D-galactosamine(D-Gal N)(600 mg/kg and 10 μg/kg) were used to induce ALF. The KaplanMeier method was used for survival analysis. Serum alanine aminotransferase(ALT) levels, at different time points within a 1-wk period, were detected with a biochemistry analyzer. Pathological examination of liver tissue was performed 36 h after ALF induction. Serum complement 5(C5), C5 a, tumor necrosis factor-α(TNF-α), interleukin(IL)-1β, IL-6, high-mobility group protein B1(HMGB1) and sphingosine-1-phosphatelevels were detected by enzyme-linked immunosorbant assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of C5 a R, sphingosine kinase 1(Sph K1), p38-MAPK and p-p38-MAPK in liver tissue, peripheral blood mononuclear cells(PBMCs) and peritoneal exudative macrophages(PEMs) of mice or RAW 264.7 cells was analyzed by western blotting. C5 a R m RNA levels were detected by quantitative real-time PCR.RESULTS Activation of C5 and up-regulation of C5 a R were observed in liver tissue and PBMCs of mice with ALF. Blockade of C5 a R with a C5 a R antagonist(C5a Ra C5 a Ra) significantly reduced the levels of serum ALT, inflammatory cytokines(TNF-α, IL-1β and IL-6) and HMGB1, as well as the liver tissue damage, but increased the survival rates(P < 0.01 for all). Blockade of C5 a R decreased Sph K1 expression in both liver tissue and PBMCs significantly at 0.5 h after ALF induction. C5 a Ra pretreatment significantly downregulated the phosphorylation of p38-MAPK in liver tissues of ALF mice and C5 a stimulated PEMs or RAW 264.7 cells. Moreover, inhibition of p38-MAPK activity with SB203580 reduced Sph K1 protein production significantly in PEMs after C5 a stimulation.CONCLUSION The C5a/C5 a R pathway is essential for up-regulating Sph K1 expression through p38 MAPK activation in ALF in mice, which provides a potential immunotherapeutic strategy for ALF in patients.展开更多
基金Supported by the National Natural Science Foundation of China,No.81260455 and No.81160065
文摘AIM To investigate the role of the complement 5a(C5a)/C5 a receptor(C5a R) pathway in the pathogenesis of acute liver failure(ALF) in a mouse model.METHODS BALB/c mice were randomly assigned to different groups, and intraperitoneal injections of lipopolysaccharide(LPS)/D-galactosamine(D-Gal N)(600 mg/kg and 10 μg/kg) were used to induce ALF. The KaplanMeier method was used for survival analysis. Serum alanine aminotransferase(ALT) levels, at different time points within a 1-wk period, were detected with a biochemistry analyzer. Pathological examination of liver tissue was performed 36 h after ALF induction. Serum complement 5(C5), C5 a, tumor necrosis factor-α(TNF-α), interleukin(IL)-1β, IL-6, high-mobility group protein B1(HMGB1) and sphingosine-1-phosphatelevels were detected by enzyme-linked immunosorbant assay. Hepatic morphological changes at 36 h after ALF induction were assessed by hematoxylin and eosin staining. Expression of C5 a R, sphingosine kinase 1(Sph K1), p38-MAPK and p-p38-MAPK in liver tissue, peripheral blood mononuclear cells(PBMCs) and peritoneal exudative macrophages(PEMs) of mice or RAW 264.7 cells was analyzed by western blotting. C5 a R m RNA levels were detected by quantitative real-time PCR.RESULTS Activation of C5 and up-regulation of C5 a R were observed in liver tissue and PBMCs of mice with ALF. Blockade of C5 a R with a C5 a R antagonist(C5a Ra C5 a Ra) significantly reduced the levels of serum ALT, inflammatory cytokines(TNF-α, IL-1β and IL-6) and HMGB1, as well as the liver tissue damage, but increased the survival rates(P < 0.01 for all). Blockade of C5 a R decreased Sph K1 expression in both liver tissue and PBMCs significantly at 0.5 h after ALF induction. C5 a Ra pretreatment significantly downregulated the phosphorylation of p38-MAPK in liver tissues of ALF mice and C5 a stimulated PEMs or RAW 264.7 cells. Moreover, inhibition of p38-MAPK activity with SB203580 reduced Sph K1 protein production significantly in PEMs after C5 a stimulation.CONCLUSION The C5a/C5 a R pathway is essential for up-regulating Sph K1 expression through p38 MAPK activation in ALF in mice, which provides a potential immunotherapeutic strategy for ALF in patients.
