G蛋白不同亚型在严重烫伤小鼠补体活化巨噬细胞分泌功能中的作用

The role of different subtypes of G protein in the secretory function of macrophages stimulated by activated complement following severe burn injury

目的观察严重烫伤后活化的补体对小鼠腹腔巨噬细胞(PM)分泌一氧化氮(NO)和肿瘤坏死因子(TNF)α的影响,探讨信号传递途径中不同G蛋白亚型的作用。方法血浆采集分组:补体血浆组,采用小鼠18%TBSAⅢ度烫伤模型;去补体血浆组,先在小鼠腹腔注射眼镜蛇毒素因子(CVF)去补体后再按上述标准烫伤。伤后6h分别收集两组小鼠全血制备血浆,用于培养正常小鼠的PM及经抑制型G蛋白(Gi)阻断剂百日咳毒素(PT)预处理的PM、经刺激型G蛋白(Gs)激活剂霍乱毒素(CT)预处理的PM,观察各组细胞培养上清液中NO及TNF-α含量的变化。结果补体血浆组培养上清液中的NO和TNF-α含量分别为(80±12)μmol/L和(46±6)%,明显高于去补体血浆组的(34±5)μmol/L和(26±5)%(P<0.01).PT预处理后补体血浆组PM产生的NO明显下降[(45±10)μmol/L,P<0.01],而TNF-α活性[(58±10)%]增加(P<0.05),CT预处理后补体血浆组PM产生的NO增加[(105±18)μmol/L,P<0.05],TNF-α的活性[(27±6)%]降低(P<0.01).结论严重烫伤后活化补体引起PM分泌NO和TNF-α增多这一现象,至少部分是通过G蛋白途径实现的。其中对PM生成NO的调控主要是通过Gi蛋白途径发挥作用,对PM分泌TNF-α的调控则以Gs蛋白信号通路为主。

Objective To investigate the influence of activated complement on the secretory function of peritoneal macrophage （PMφ） in the production of nitric oxide （NO） and tumor necrosis factor-or （TNF- α） , especially in the role of different G-protein subtypes in this process after burns. Methods The mice inflicted by 18% TBSA full-thickness scald was established and employed as the model. And the mice were divided into A （the complements were preserved and activated） and B （with intraperitoneal injection of CVF to deplete complement before scald） groups. The plasma of the mice in the two groups was collected at 6 postburn hour（ PBH ） and cultured with PMφ from normal mice. The PMφ were pretreated with pertussis toxin （PT） and with cholera toxin （ CT）. The NO and TNF-α levels in the supernatant of normal PMφ culture with different pretreatment were measured by Greiss assay. Results The NO and TNF-α contents in group A [ （80±12）μmol/L,（46±6）% ]were obviously higher than those in group B[ （34±5）μmol/L, （26±5）%, P 〈 0.01]. The NO content produced by PMφ（45±10μmol/L） in A group decreased （ P〈0.01 ） , while the TNF-α activity（58 ±10）% increased by PT pretreatment（ P〈0.05 ）. On the contrary, the NO content produced by PMφ（ 105±18umol/L） in group A increased （ P 〈 0.01 ）, while the TNF-α activity（ 27±6 ） % decreased by CT pretreatment （ P〈 0.01 ）. Conclusion These results indicates that the secretory function of normal PMφ can be enhanced by complement activation after thermal injury, which might partly be due to the effect of activated complement conponents through complement receptor coupled Gprotein. In the secretory function of complement stimulated Mφ, Gi protein has a major role in the production of NO, Gs protein is mainly involved in the secretion of TNF-α.