短链脂肪酸对他克莫司相关高血糖的影响及相关机制研究

The effect and mechanism of short-chain fatty acid regulate tacrolimus-related hyperglycemia in mice

目的探讨口服补充短链脂肪酸(SCFAs)对他克莫司相关高血糖的影响及其作用机制。方法2019年6月至2020年6月利用C57BL/6小鼠建立各组实验模型:对照组(口服无菌水)、他克莫司组(每日口服他克莫司10 mg/kg)、SCFAs组(每日口服他克莫司和150 mmol/L丁酸钠、异戊酸钠混合溶液0.5 ml)、抗生素组(每日口服广谱抗生素)、他克莫司抗生素组(每日口服他克莫司和广谱抗生素)。干预8周后,各组均检测空腹血糖、口服糖耐量试验(OGTT)、糖化血红蛋白(HbA1c),对照组、他克莫司组、SCFAs组检测回盲部SCFAs浓度、血清胰高血糖素样肽-1(GLP-1)、空腹血清胰岛素、肠黏膜GLP-1表达量。结果他克莫司组空腹血糖[(7.31±0.97)mmol/L]和HbA1c[(8.34±1.12)%]均显著高于对照组[(5.23±0.30)mmol/L、(4.32±0.80)%,P<0.05];抗生素组、他克莫司抗生素组、SCFAs组的空腹血糖[(4.92±0.31)、(5.95±0.37)、(5.87±0.68)mmol/L]和HbA1c[(5.61±0.98)%、(4.56±0.26)%、(5.07±1.79)%]与对照组的差异均无统计学意义(P>0.05)。他克莫司组OGTT提示口服糖耐量受损,其余各组未见明显异常。他克莫司组的小鼠回盲部丁酸、异戊酸浓度分别为(722.3±262.2)、(10.0±5.1)μg/g,显著低于对照组[(1321.3±165.5)、(19.7±3.6)μg/g,均P<0.05];SCFAs组的丁酸、异戊酸浓度[(1375.7±451.6)、(24.5±11.5)μg/g]与对照组差异无统计学意义(P>0.05)。他克莫司组空腹血清胰岛素显著低于对照组[(3.2±0.6)mIU/L与(4.4±0.9)mIU/L];SCFAs组空腹血清胰岛素[(7.0±1.1)mIU/L]与对照组的差异无统计学意义(P>0.05)。血清GLP-1检测结果显示他克莫司组显著低于对照组[(4.7±2.9)pg/ml与(33.1±9.1)pg/ml,P<0.05],而SCFAs组[(42.5±19.9)pg/ml]与对照组差异无统计学意义。免疫组化染色检查示他克莫司组回盲部肠黏膜GLP-1表达较对照组和SCFAs组均显著下降。结论口服他克莫司后小鼠血糖升高,体内部分SCFAs(丁酸、异戊酸)显著降低;补充外源性SCFAs可升高肠道中GLP-1并促进机体胰岛素的分泌,显著缓解他克莫司对血糖的影响。

Objective To investigate the effect and mechanism of short-chain fatty acids(SCFAs)on the side-effect of tacrolimus on blood glucose.Methods The C57BL/6 mice were treated with tacrolimus orally(10 mg/kg,tacrolimus group),tacrolimus plus 150 mmol/L sodium butyrate and isovalerate mixed solution(SCFAs group),broad-spectrum antibiotics(antibiotic group),and tacrolimus plus broad-spectrum antibiotics(tac&abx group).After 8 weeks intervention,the fasting blood glucose(FBG),oral glucose tolerance test(OGTT),hemoglobin A1C(HbA1c)were tested as indicators of glucose metabolism,and the gut microbiota,SCFAs concentration in the ileocecal,serum glucagon-like peptide-1(GLP-1),fasting serum insulin,and GLP-1 expression in intestinal mucosa were performed for intestinal-glucose metabolism mechanism.Results The FBG and HbA1c were significantly increased in tacrolimus group[(7.31±0.97)mmol/L,(8.34±1.12)%]than control group[(5.23±0.30)mmol/L,(4.32±0.80)%,all P<0.05],which remained normal in antibiotic group[(4.92±0.31)mmol/L,(5.61±0.98)%],tac&abx group[(5.95±0.37)mmol/L,(4.56±0.26)%]and SCFAs groups[(5.87±0.68)mmol/L,(5.07±1.79)%].The OGTT in the tacrolimus group showed glucose tolerance impairment,while other groups remained normal.The ileocecal butyric acid and isovaleric acid concentrations in the tacrolimus group were(722.3±262.2)μg/g and(10.0±5.1)μg/g,lower than the control group[(1321.3±165.5)μg/g,(19.7±3.6)μg/g,P<0.05].The above acids in the SCFAs group remained normal as in the control group[(1375.7±451.6)μg/g,(24.5±11.5)μg/g].The fasting serum insulin in the tacrolimus group decreased significantly to[(3.2±0.6)mIU/L,compared with control(4.4±0.9)mIU/L]and SCFAs groups[(7.0±1.1)mIU/L].The GLP-1 test indicated a significant decrease in the tacrolimus group[(4.7±2.9)pg/ml,P<0.05]compared with the SCFAs group and control group[(42.5±19.9)pg/ml,(33.1±9.1)pg/ml].Conclusions Tacrolimus affects glucose metabolism through the SCFAs-associated GLP-1 pathway in the intestine,and oral supplementation with mixed SCFAs provides a new insight for the prevention and treatment of tacrolimus-induced hyperglycemia in transplant recipients.