紫檀芪对糖尿病性白内障大鼠氧化应激和炎症反应的影响及机制

Influence of pterostilbene on oxidative stress and inflammatory reaction in diabetic cataract rats and its mechanism

目的 探讨紫檀芪经腺苷酸活化蛋白激酶(AMPK)/沉默信息调节因子1(SIRT1)/过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)信号通路对糖尿病性白内障(DC)大鼠氧化应激和炎症反应的影响。方法 取64只SD大鼠经腹腔注射链脲佐菌素诱导构建DC模型,造模成功60只,将其随机分为模型组及紫檀芪低剂量组、紫檀芪中剂量组、紫檀芪高剂量组、紫檀芪高剂量+AMPK抑制剂(Dorsomorphin)组各12只,另取12只正常大鼠作为对照组。紫檀芪低、中、高剂量组分别给予2、4、8 mg/mL紫檀芪溶液灌胃(10 mL/kg),同时给予0.9%氯化钠溶液尾静脉注射(10 mL/kg);紫檀芪高剂量+Dorsomorphin组给予8 mg/mL紫檀芪溶液灌胃(10 mL/kg),同时给予0.02 mg/mL Dorsomorphin溶液尾静脉注射(10 mL/kg);模型组、对照组均给予0.9%氯化钠溶液灌胃(10 mL/kg),同时给予0.9%氯化钠溶液尾静脉注射(10 mL/kg)。各组大鼠均每天按上述方式给予药物干预1次,共干预14 d。干预后,测量大鼠空腹血糖;采用裂隙灯观察其晶状体混浊情况并评分;苏木精-伊红染色观察大鼠晶状体组织结构变化;比色法检测大鼠晶状体组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px);酶联免疫吸附法检测大鼠血清IL-6、IL-17、IL-10;免疫印迹法检测大鼠晶状体组织中上皮间质转化标志蛋白[波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)、E-钙黏蛋白(E-cadherin)]及AMPK/SIRT1/PGC-1α信号通路蛋白表达。结果 与对照组比较,模型组大鼠晶状体上皮细胞呈片状、条索状向晶状体成纤维细胞迁移聚集,空腹血糖、晶状体混浊情况评分、血清IL-6水平、血清IL-17水平、晶状体组织MDA与Vimentin、N-cadherin蛋白表达均高(P均<0.05),晶状体组织中SOD与GSH-Px水平、血清IL-10水平、p-AMPK/AMPK及SIRT1、PGC-1α、E-cadherin蛋白表达均低(P均<0.05)。与模型组比较,紫檀芪低、中、高剂量组大鼠晶状体上皮细胞上述形态改变及迁移聚集现象得到改善,空腹血糖、晶状体混浊情况评分、血清IL-6水平、血清IL-17水平、晶状体组织中MDA与Vimentin、N-cadherin蛋白表达均低(P均<0.05),晶状体组织中SOD与GSH-Px水平、血清IL-10水平、p-AMPK/AMPK及SIRT1、PGC-1α、E-cadherin蛋白表达均高(P均<0.05),且呈剂量依赖性(P均<0.05)。与紫檀芪高剂量组比较,紫檀芪高剂量+Dorsomorphin组大鼠晶状体上皮细胞上述形态改变及迁移聚集现象加重,空腹血糖、晶状体混浊情况评分、血清IL-6与IL-17水平、晶状体组织中MDA与Vimentin、N-cadherin蛋白表达高(P均<0.05),晶状体组织中SOD与GSH-Px水平、血清IL-10水平、晶状体组织中p-AMPK/AMPK及SIRT1、PGC-1α、E-cadherin蛋白表达均低(P均<0.05)。结论 紫檀芪可通过激活AMPK/SIRT1/PGC-1α信号通路减轻DC大鼠氧化应激与炎症反应。

Objective To investigate the influence of pterostilbene on oxidative stress and inflammatory reaction in diabetic cataract(DC) rats by regulating AMP-activated protein kinase(AMPK)/silent information regulator 1(SIRT1)/peroxisome proliferator activated receptor γ coactivator-1α(PGC-1α) signaling pathway.Methods Sixty-four SD rats were injected intraperitoneally with streptozotocin to establish the DC models,and they were randomly divided into the model group,low-dose pterostilbene group,medium-dose pterostilbene group,high-dose pterostilbene group,and highdose pterostilbene+AMPK inhibitor(Dorsomorphin) group,with 12 DC model rats in each group;another 12 SD normal rats were injected intraperitoneally with an equal volume of citrate buffer as the control group.Rats in the low-dose,medium-dose and high-dose perostilbene groups were given 2,4 and 8 mg/mL perostilbene solution by gavage(10 mL/kg),and 0.9% sodium chloride solution by tail vein injection(10 mL/kg).The rats in the high-dose perostilbene+Dorsomorphin group were given 8 mg/mL perostilbene solution by gavage(10 mL/kg),and 0.02 mg/mL Dorsomorphin solution by tail vein injection(10 mL/kg).Rats in both the model group and the control group were given 0.9% sodium chloride solution by gavage(10 mL/kg),and 0.9% sodium chloride solution by tail vein injection(10 mL/kg).The rats in each group were given drug intervention once a day in the above manner for 14 days.After corresponding intervention in each group,the fasting blood glucose of rats was measured;the lens opacity was observed and scored by slit lamp;hematoxylin-eosin staining was used to detect the changes of lens tissue structure of rats;the colorimetry was used to measure the malondialdehyde(MDA),superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) levels in the lens tissues of rats;the enzyme-linked immunosorbent assay was used to measure the serum interleukin(IL)-6,IL-17,and IL-10 levels;Western blotting was used to detect the expression levels of epithelial-mesenchymal transition marker proteins(Vimentin,N-cadherin,and E-cadherin) and AMPK/SIRT1/PGC-1α pathway proteins in lens tissues of rats.Results Compared with the control group,the lens epithelial cells in the model group migrated and aggregated to the lens fibroblasts in the form of sheets and cords;the fasting blood glucose,lens opacity score,serum levels of IL-6 and IL-17,and protein expression levels of MDA,Vimentin and N-cadherin in lens tissues increased significantly(all P<0.05),the levels of SOD and GSH-Px in lens tissues,serum level of IL-10,and the protein expression levels of p-AMPK/AMPK and SIRT1,PGC-1α and E-cadherin in the lens tissues decreased significantly(all P<0.05).Compared with the model group,the above-mentioned morphological changes,migration and aggregation of lens epithelial cells in the low-dose,medium-dose and high-dose pterostilbene groups were improved,the fasting blood glucose,lens opacity score,serum levels of IL-6 and IL-17,and protein expression levels of MDA,Vimentin and N-cadherin in lens tissues decreased(all P<0.05),the levels of SOD and GSHPx in lens tissue,serum level of IL-10,and the protein expression levels of p-AMPK/AMPK and SIRT1,PGC-1α and Ecadherin in lens tissues increased(all P<0.05),with a dose-dependent manner(P<0.05).Compared with the high-dose pterostilbene group,the above-mentioned morphological changes and migration and aggregation of the lens epithelial cells in the high-dose pterostilbene+Dorsomorphin group were aggravated,the fasting blood glucose,lens opacity score,serum levels of IL-6 and IL-17,and protein expression levels of MDA,Vimentin and N-cadherin in lens tissues increased(all P<0.05),the levels of SOD and GSH-Px in lens tissues,serum level of IL-10,and the protein expression levels of p-AMPK/AMPK and SIRT1,PGC-1α and E-cadherin in the lens tissues decreased significantly(all P<0.05).Conclusion Pterostilbene can reduce the oxidative stress and inflammatory response in DC rats by activating AMPK/SIRT1/PGC-1αsignaling pathway.