游离脂肪酸受体2对小鼠肠缺血再灌注损伤的作用及机制

Effect and mechanism of free fatty acid receptor 2 on intestinal ischemia reperfusion injury in mice

目的:研究过表达游离脂肪酸受体2(FFAR2)能否减轻小鼠肠缺血再灌注损伤。方法:将24只C57BL/6小鼠随机分为假手术组(Sham组)、肠缺血再灌注组(IIR组)、FFAR2部分激动剂4-CMTB组(IIR+4-CMTB组)、FFAR2完全激动剂醋酸钠组(IIR+SA组),每组6只小鼠。IIR+4-CMTB组尾静脉注射4-CMTB(10 mg/kg),注射24 h后,建立小鼠IIR损伤模型;IIR+SA组饮水中添加5%醋酸钠溶液,喂养1周后,建立小鼠IIR损伤模型。造模成功后,HE染色观察肠组织病理损伤;检测肠组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、髓过氧化物酶(MPO)的含量;GC-MS/MS法分析小鼠肠道内短链脂肪酸(SCFA)含量;RT-PCR检测肠组织中TNF-α、IL-1β、IL-6、FFAR2、Reg3γ和β防御素1、3和4的含量;Western Blot检测肠组织中紧密连接蛋白ZO-1、Occludin、Claudin-1及p-MAPK和p-ERK1/2蛋白表达水平。结果:与Sham组相比,IIR小鼠肠组织病理学评分、MDA、MPO和炎性细胞因子水平显著升高,SOD活性和FFAR2水平显著降低,小鼠肠道屏障功能损伤显著加重,肠道内SCFA发生变化,MAPK和ERK蛋白的磷酸化水平显著降低(P<0.05);与IIR组比较,FFAR2激动剂组的病理学评分、炎性细胞因子水平、MDA、MPO水平显著降低,SOD活性和FFAR2水平显著升高(P<0.05),小鼠肠道屏障功能损伤显著降低,MAPK和ERK蛋白的磷酸化水平显著升高;两组激动剂组间上述指标无统计学意义(P>0.05)。结论:过表达FFAR2通过MAPK/ERK信号通路抑制炎症反应,保护肠道屏障的完整性,进而减轻小鼠肠缺血再灌注损伤。

Objective:To investigate whether overexpression of free fatty acid receptor 2(FFAR2)can reduce intestinal ischemia-reperfusion(IIR)injury in mice.Methods:Twenty-four C57BL/6 mice were randomly divided into sham operation group(Sham group),intestinal ischemia-reperfusion group(IIR group),FFAR2 partial agonist 4-CMTB group(IIR+4-CMTB group),and FFAR2 full agonist sodium acetate group(IIR+SA group),with 6 mice in each group.In the IIR+4-CMTB group,4-CMTB(10 mg/kg)was injected into the tail vein,and 24 h after injection,mice IIR injury models were established;In the IIR+SA group,5%sodium acetate solution was added to the drinking water,and after one week of feeding,mice IIR injury models were established.After the model was successfully established,HE staining was used to observe the pathological damage of intestinal tissues;the contents of MDA,SOD and MPO in intestinal tissues were detected;the contents of short-chain fatty acid(SCFA)in the intestinal tract of mice were analyzed by GC-MS/MS;the contents of TNF-α,IL-1β,IL-6,FFAR2,Reg3γ,andβ-defensins 1,3 and 4 in intestinal tissues were detected by RT-PCR;and the expression levels of tight junction proteins ZO-1,Occludin,and Claudin-1,and proteins p-MAPK and p-ERK1/2 in intestinal tissues were detected by Western Blot.Results:Compared with sham group,IIR mice had significantly increased intestinal histopathological score,MDA,MPO and inflammatory cytokine levels,significantly decreased SOD activity and FFAR2 levels,significantly aggravated intestinal barrier function injury,changed SCFA in the intestine,and significantly decreased phosphorylation levels of MAPK and ERK proteins(P<0.05);compared with IIR group,FFAR2 agonist group had significantly decreased pathological scores,inflammatory cytokine levels,MDA and MPO levels,significantly increased SOD activity and FFAR2 levels(P<0.05),significantly decreased intestinal barrier function injury,and significantly increased phosphorylation levels of MAPK and ERK proteins;while the above parameters showed no statistically significant difference between the two agonist groups(P>0.05).Conclusion:Overexpression of FFAR2 inhibits the inflammatory response through MAPK/ERK signaling pathway and protects the integrity of the intestinal barrier,which in turn attenuates intestinal ischemia-reperfusion injury in mice.