p38促分裂原活化的蛋白激酶信号调控铁死亡参与运动改善2型糖尿病小鼠非酒精性脂肪性肝病

Exercise Improves Nonalcoholic Fatty Liver Disease in T2DM Mice by Inhibiting Ferroptosis Through p38 MAPK Signaling Pathway

目的从运动调控细胞铁死亡的角度,探究8周跑台运动改善2型糖尿病(diabetes mellitus type 2,T2DM)合并非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)小鼠肝细胞脂肪变性的分子机制。方法选取8只8周龄健康m/m小鼠作为对照组(Con),32只同周龄db/db小鼠被随机分为db/db组、db+Exe组、db+SB203580组及db+Exe+SB203580组,每组8只。db+Exe组、db+Exe+SB203580组进行持续8周的中等强度跑台运动干预(40 min/d,5 d/周),db+SB203580组及db+Exe+SB203580组进行8周的p38促分裂原活化的蛋白激酶(mitogen-activated protein kinase,MAPK)抑制剂SB203580腹腔注射处理(5 mg/kg,5 d/周),实验期间每周定时检测小鼠体重及空腹血糖。8周干预结束后,油红O及苏木精-伊红(hematoxylin-eosin,HE)染色法观察小鼠肝脏病理性改变状况,相应的酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)试剂盒测定血脂、肝功能、肝脏铁离子及氧化应激水平,定量反转录PCR(quantitative reverse transcriptase-mediated PCR,qRT-PCR)测定脂肪合成及氧化应激相关mRNA转录水平,免疫组织化学染色观察铁死亡相关蛋白质的表达水平,蛋白质印迹(Western blot,WB)法检测肝组织中脂质合成及铁死亡相关蛋白质表达水平。结果8周跑台运动降低了db/db小鼠体重、血糖、肝指数及血脂水平,改善其肝脏脂肪变性,下调肝组织Fe^(2+)、MDA、MCP-1、IL-6、SREBF1、ACC1表达水平,并上调了db/db小鼠肝组织中GSH含量及铁死亡相关蛋白质NRF2、HO-1、SLC7A11、GPX4蛋白表达水平。运动联合p38 MAPK抑制剂干预显著下调db/db小鼠肝组织ACC1、MCP-1、IL-6的mRNA及Fe^(2+)、MDA水平,并上调GSH水平。然而,与跑台运动组相比,运动联合p38 MAPK抑制剂组小鼠肝脏内Fe^(2+)、MDA、MCP-1、SREBF1表达显著上升,GSH及铁死亡相关蛋白质表达水平显著下降。此外,与单纯抑制剂组相比,运动联合抑制剂干预组小鼠肝组织铁蓄积及脂质过氧化现象明显改善。结论8周跑台运动可能是通过p38 MAPK依赖途径抑制肝细胞铁死亡,缓解肝细胞脂质过氧化及肝脏脂肪变性,从而改善T2DM小鼠的NAFLD。

Objective To explore the mechanism of treadmill exercise against type 2 diabetes mellitus(T2DM)with non-alcoholic fatty liver disease(NAFLD)based on the regulator effects of exercise on ferroptosis.Methods Eight 8-week-old male m/m mice were used as control group(Con,n=8),and db/db mice of the matched age were randomly divided into T2DM model group(db/db,n=8),exercise group(db+Exe,n=8),p38 mitogen-activated protein kinase(MAPK)inhibitor group(db+SB203580,n=8)and exercise combined with p38 MAPK inhibitor group(db+Exe+SB203580,n=8).After one-week adaptive feeding,the mice in the db+Exe group and db+Exe+SB203580 group underwent moderate intensity treadmill exercise for 40 min/d,5 d/week lasting 8 weeks.The db+SB203580 group and db+Exe+SB203580 group were treated with SB203580(a specific inhibitor of p38 MAPK)with a dose of 5 mg/kg,5 d/week for 8 weeks.And the exercise intervention was performed 2 h later after the intraperitoneal injection of SB203580.The body weight and fasting blood glucose of mice were measured regularly every week during the experiment.After 24 h of the last intervention,the mice were weighted,the liver tissues were taken,weighted and the liver index was calculated.The pathological changes of liver were determined by Oil Red O and hematoxylin-eosin(HE)staining.The levels of blood lipids,liver function,Fe^(2+)and oxidative stress markers of liver were measured by enzyme linked immunosorbent assay(ELISA).The related mRNA expression levels of lipogenesis and inflammation were evaluated by quantitative reverse transcriptase-mediated PCR(qRT-PCR).The related protein expression levels of lipogenesis and ferroptosis in liver were determined by immunohistochemical(IHC)staining and Western blot.Results The body weight,fasting blood glucose,liver index,blood lipid and transaminase levels in the db/db group were significantly increased compared with the Con group.HE and Oil Red O staining showed severe lipid accumulation and ballooning change in the liver of db/db mice.Biochemical tests showed that Fe^(2+)and MDA level of liver constitution homogenate increased,while GSH level decreased significantly.The results of qRTPCR showed that the mRNA levels of MCP-1,IL-6,SREBF1 and ACC1 in liver tissue of db/db mice were all significantly increased.Western blot results showed that the expression levels of SREBF1,ACC1 increased,ferroptosis relative proteins were significantly decreased.The 8 weeks of exercise significantly reduced the rise in body weight,blood glucose,liver index and blood lipid levels in db/db mice.Exercise intervention also alleviated hepatic steatosis and reduced the expression levels of Fe^(2+),MDA,MCP-1,IL-6,ACC1 and SREBF1,upregulated the expression levels of GSH,NRF2,HO-1,SLC7A11 and GPX4 in liver tissue of db/db mice.The intervention of exercise combined with SB203580 significantly down-regulated the mRNA expression levels of ACC1,MCP-1,IL-6,reduced the levels of Fe^(2+)and MDA,and up-regulated the level of GSH in db/db mice.Compared with the db+Exe group,the expression of Fe^(2+),MDA,MCP-1,and SREBF1 in the liver of the db+Exe+SB203580 group mice significantly increased,while the expression level of GSH and expression levels of ferroptosis relative proteins also significantly decreased.In addition,compared with db+SB203580 group,the iron accumulation and lipid peroxidation in the liver of db+Exe+SB203580 group were significantly improved.Conclusion The 8-week treadmill exercise can effectively alleviate liver injury and steatosis,and its mechanism may be related to the inhibition of hepatocyte ferroptosis through p38 MAPK signal.