RYGB手术对2型糖尿病大鼠的肝脂质代谢途径及关键酶的影响

Effects of Roux-en-Y gastric bypass surgery on hepatic lipid metabolism pathway and key enzymes in type 2 diabetes rats

目的探讨Roux-en-Y胃旁路手术(RYGB)对肥胖型2型糖尿病大鼠肝脏的肝脂肪代谢途径及关键酶的影响。方法将40只SD大鼠随机分为正常对照组(control组)、糖尿病模型组(DM组)、糖尿病假手术组(DM+sham组)和RYGB手术组(RYGB组)。DM组、DM+sham组和RYGB组大鼠均接受高脂肪饮食饲养6周,并于6周末进行尾静脉注射链脲佐菌素(25 mg/kg)建立糖尿病大鼠模型,正常对照组大鼠接受标准饮食饲养。RYGB组大鼠建模成功后再接受Roux-en-Y胃旁路手术处理。术后0-8周每周定期采用双能X线骨密度仪检测大鼠体脂和去脂体质量,通过血糖仪分析血糖水平。术后2周、4周和8周断头法处死各组大鼠并分离和收集大鼠肝组织,油红O染色检测肝脏脂质含量,甘油三酯(TG)含量检测试剂盒检测大鼠肝脏甘油三酯水平,肝脏胰岛素敏感性指数(HISI)评估大鼠肝脏胰岛素敏感性,Western blot检测mTOR/P70S6K通路mTOR、p-mTOR、p70S6K和p-70S6K蛋白的表达水平。结果术后0-8周,与DM组和DM+sham组相比,RYGB组大鼠的血糖水平、体质量和体脂含量明显下降(P<0.05),而去脂体质量无明显变化(P>0.05)。油红O染色结果显示,与DM+sham相比,RYGB组大鼠肝脏脂肪中性脂质染色阳性百分率明显降低(P<0.05),脂滴大小和数量明显减少(P<0.05),肝脏甘油三酯含量显著降低(P<0.05),肝脏胰岛素敏感性指数显著升高(P<0.05)。Western blot检测结果显示,术后2周,与DM+sham组相比,RYGB组大鼠DGAT2、ATGL和P62的表达水平明显降低(P<0.05),而p-HSL和MTP的表达水平明显升高(P<0.05),自噬相关蛋白LC3Ⅱ/LC3Ⅰ比值显著降低(P<0.05),大鼠肝组织的p-mTOR和p-p70S6K表达水平显著降低(P<0.05)。在术后4周,与DM+sham组相比,RYGB组大鼠LC3Ⅱ/LC3Ⅰ比值显著升高,p62、p-mTOR和p-p70S6K表达水平显著降低(P<0.05),DGAT2、ATGL、p-HSL和MTP的表达水平无明显变化(P>0.05)。术后8周,各组间自噬相关蛋白表达水平和mTOR和p70S6K磷酸化水平差异均无统计学意义(P>0.05)。结论RYGB可能通过激活肝脂水解途径,抑制肝脂合成途径,从而降低肝脂毒性和改善胰岛素敏感性。

Objective To investigate the effects of Roux-en-Y gastric bypass surgery(RYGB)on the hepatic fat metabolism pathway and the key enzymes in obese type 2 diabetic(T2DM)rats.Methods Forty SD rats were randomly divided into control group,diabetes model group(DM group),diabetes+sham operation group(DM+sham group)and RYGB group.The rats in DM group,DM+sham group and RYGB group were fed with a high-fat diet for 6 weeks,and were injected with streptozotocin(25 mg/kg)via the tail vein at the end of the 6 th week to establish the diabetic rat model.The rats in control group received a standard diet.Rats in RYGB group received Roux-en-Y gastric bypass surgery after successful modeling.At week 0-8 after operation,the body fat and the fat-free body weight of rats were detected by dual energy X-ray absorptiometry,and the blood glucose level was analyzed by the blood glucose meter.At week 2,4,8 after operation,rats in each group were killed by decapitation,and liver tissues were separated and collected.Liver lipid content was detected by oil red O staining,and triglyceride(TG)content was detected by triglyceride content detection kit.Hepatic insulin sensitivity of rats was evaluated by insulin sensitivity index(HISI)method.The expression levels of mTOR,p-mTOR,p70S6K and p-70S6K proteins in the mTOR/P70S6K pathway were detected by Western blot.Results Within 0-8 weeks after operation,compared with DM group and DM+sham group,the blood glucose level,the body weight and the body fat of rats were decreased significantly in RYGB group(P<0.05),but the fat-free body weight showed no significant change(P>0.05).The results of oil red O staining showed that compared with DM+sham group,the positive percentage of neutral lipid staining,the size and the number of lipid droplets,the content of triglyceride were decreased significantly in RYGB group(P<0.05),and HISI was significantly increased in RYGB group(P<0.05).Western blot results showed that compared with DM+sham group,the expression levels of DGAT2,ATGL and p62 were significantly decreased in RYGB group(P<0.05),the expression levels of p-HSL and MTP were significantly increased(P<0.05),the ratio of LC3Ⅱ/LC3Ⅰwas significantly decreased(P<0.05),and the expression levels of p-mTOR and p-p70S6K in rat liver tissue were significantly decreased(P<0.05).At week 4 after the operation,compared with DM+sham group,the ratio of LC3Ⅱ/LC3Ⅰwas significantly increased in RYGB group(P<0.05),the expression levels of P62,p-mTOR and p-p70S6K were significantly decreased(P<0.05),and the expression levels of DGAT2,ATGL,p-HSL and MTP showed no significant change(P>0.05).At week 8 after the operation,there was no significant difference in the expression levels of autophagy-related proteins and the phosphorylation levels of mTOR and p70S6K between the groups(P>0.05).Conclusion RYGB may reduce the hepatic lipid toxicity and improve the insulin sensitivity by activating hepatic lipid hydrolysis pathway and inhibiting hepatic lipid synthesis pathway.