Exendin-4对烧伤小鼠脾脏单核细胞免疫反应的影响及其调控机制

Effects of exendin-4 on immune function of splenic monocytes and its regulatory mechanism： experiment with mice after scalding

目的阐明肠促胰岛素长效类似物Exendin-4（Ex-4）对烧伤小鼠脾脏单核细胞免疫调节功能的影响，并探讨其可能信号转导机制。方法BALB／C／Ix鼠随机（随机数字法）分为烫伤组（n=30）和假伤组（n=10），其中烫伤组动物背部按照15％总体表面积（TBSA）浸于94℃热水中8s造成烫伤，而假伤组动物背部皮肤则浸于37℃水中8s。伤后24h取假伤组和烫伤组脾脏分离单核细胞进行培养，分别分为空白组（LPS-0）、脂多糖（LPS）组、LPS加低Ex-4组、LPS加中Ex-4组、LPs加高Ex-4组，均培养24h。用酶标仪检测吸光度值测定细胞活性。用ELISA法检测培养上清肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、IL-10、转化生长因子-β（TGF-β）水平。采用Western印迹法检测单核细胞胰高血糖素样肽1受体（GLP-1R）、GPCR调节激酶2（GRK-2）、刺激性G蛋白亚基Ⅸ（Gcts）和抑制性G蛋白亚基α（Gαi）的表达。使用cAMP和PKA活性试剂盒检测细胞内cAMP和cAMP依赖蛋白激酶（PKA）活性。结果LPS加中、高剂量Ex-4假伤组单核细胞活性显著低于假伤LPS组（均P〈O．05），而烫伤组细胞增殖活性变化不明显。Ex-4作用后，烫伤组单核细胞分泌TNF-α增加（P〈0．05），而假伤组没有显著改变。两组内单核细胞IL-6的分泌均呈现剂量依赖性增加，但两组之间差异无统计学意义。两组IL-10的分泌均呈剂量依赖性增加，假伤组中浓度Ex-4促进效应已很明显，但烫伤组仅高浓度Ex-4具有促进作用，Ex-4对两组TGF—β分泌均起显著抑制作用，在假伤组呈显著的剂量依赖性（P〈0．05），但是在烫伤组不同浓度Ex-4的抑制效应差异无统计学意义。烫伤后脾脏单核细胞Ex-4信号转导通路分子包括GLP-1R、GRK-2和G仅s含量均下降（均P〈0．05），而Gcd含量升高（P〈0．05）。高剂量Ex-4对烫伤组单核细胞cAMP的促进作用显著低于假伤组（P〈0．05），但是烫伤组PKA水平高于假伤组（P〈0．05），结论严重烫伤后Ex-4信号转导通路发牛显著改变，导致Ex-4对单核细胞功能的调控发生部分抵抗甚至异常，Ex-4对烧伤小鼠单核细胞抗炎与抑炎反应均具有明显调节作用。

Objective To investigate the effects of the incretin mimetic exendin-4 （Ex-4） on the immune functions of splenic monocytes, and explore its potential regulatory mechanism in signal transduction. Methods Thirty BALB/C mice underwent scalding with their back skin immersed in 94℃ hot water for 8s so as to establish the scalding model. Other 10 mice had their back skin immersed in 37℃ water for 8s （pseudo-scalding model）. LPS group Twenty-four hours later the mice were killed with their spleens taken out. Monocytes were separated by adhesion method to be cultured in the presence of pure lipopolysaccharide （LPS） and LPS plus Ex-4 at low, moderate, and high concentrations respectively for 24 h. Enzyme linked immunosorbent assay （ELISA） was used to determine the absorbance so as to assess the cell viability. And the supernatant was collected to measure by ELISA the levels of cytokines： tumor necrosis factor-α （TNF-α）, monocyte chemotaetic protein 1 （MCP-1）, interleukin-6（IL-6）, IL-10, and transforming growth factor-β （TGF-β）. The expression levels of glucagon-like peptide 1 receptor （GLP-1R）, G-protein coupled receptor kinase 2 （GRK-2）, stimulating G protein ot-subunit （Got s）, and inhibitory G protein ot-subunit （Got i） were detected by Western blotting, cAMP activity assay kit and PKA activity assay kit were used to detect the activity of the cyclic adenosine monophosphate （cAMP） and cyclic-AMP dependent protein kinase A （PKA）. Results The absorbanee levels of different subgroups in the psedo-scalding group were all lower than those in the scalding groupo, especially those of the LPS＋moderate or LPS＋high Ex-4 subgroups （both P 〈0.05）. The TGF-β level of the LPS subgroup was significantly higher in the scalding group than in the the pseudoscalding group （P 〈 0.05）. The TGF-β levels of different LPS ＋ Ex4 subgroups in both group were all significantly lower in the scalding group than in the the pseudoscalding group （all P 〈0.05）. The cAMP levels of the different Ex-4 subgroups in the scalding group did not change significantly. However, the cAMP level of the high Ex-4 subgroup in the scalding group was significantly higher than that of the control group （P 〈0.05）. The PKA levels of the high Ex-4 subgroups in both group were both significantly higher than those of the control group （both P〈 0.05）, and the PKA level of the high Ex-4 subgroup in the scalding groupo was significantly higher than that of the psudoscalding group （P 〈0.05）. Conclusion Scalding elicits significant changes in Ex-4 signaling pathway in monoeytes, thus leading to partial resistance and even abnormality in response to Ex-4. Thus, Ex-4 exhibits both anti- and pro-inflammatory effects in monocytes secondary to acute insults.