基于AMPK/mTOR/ULK1自噬相关通路探讨左归降糖通脉方对AGEs合并缺糖缺氧星形胶质细胞炎性损伤的影响  

Effect of Zuogui Jiangtang Tongmai Prescription on Astrocyte Injury by AGEs Combined with Oxygen-glucose Deprivation Based on AMPK/mTOR/ULK1 Pathway Related to Autophagy

作　　者：李钰佳 李定祥[1] 彭珣 邓奕辉[1] LI Yujia;LI Dingxiang;PENG Xun;DENG Yihui*(Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学,长沙410208

出　　处：《中国实验方剂学杂志》2022年第16期90-99,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81874416);湖南省科技创新团队项目(2020RC4050)。

摘　　要：目的:探讨左归降糖通脉方对糖基化终末产物(AGEs)合并缺糖缺氧(OGD)损伤后星形胶质细胞(AS)的影响及干预机制。方法:使用细胞增殖与活性检测试剂盒(CCK-8)确定AGEs作用浓度及OGD时间,选择左归降糖通脉方(ZGJTTMP)含药血浆的作用浓度;将星形胶质细胞随机分为空白组、模型(AGEs+OGD)组、ZGJTTMP组、腺苷酸活化蛋白激酶(AMPK)抑制剂(Compound C)组、AMPK激动剂(AICAR)组、ZGJTTMP+AICAR组,倒置显微镜下观察各组细胞形态结构,CCK-8法检测各组细胞存活率,酶联免疫吸附测定法(ELISA)检测各组细胞中白细胞介素^(-1)β(IL^(-1)β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的含量;电镜下观察各组细胞内自噬小体的数目,免疫荧光染色法观察各组细胞中微管相关蛋白1轻链3(LC3)的表达情况,蛋白免疫印迹法(Western blot)检测各组细胞LC3、p62、磷酸化(p)-AMPK、AMPK、磷酸化(p)-哺乳动物雷帕霉素靶蛋白(mTOR)、mTOR、磷酸化(p)-UNC-51样激酶1(ULK1)、ULK1蛋白的表达情况。结果:选择AGEs 200 mg·L^(-1)合并OGD 6 h作为最适造模条件,5%作为ZGJTTMP含药血浆的最佳作用体积分数。倒置显微镜下见造模后细胞受损严重,ZGJTTMP组与Compound C组细胞损伤得到明显改善;ELISA结果示模型组IL^(-1)β、IL-6、TNF-α的含量显著增加(P<0.01),ZGJTTMP组与Compound C组炎症因子含量显著下降(P<0.01);电镜下可见模型组细胞内自噬小体数目明显增多,免疫荧光结果示LC3荧光表达面积显著增加(P<0.01),ZGJTTMP组与Compound C组细胞内自噬小体数目明显减少,LC3表达面积显著减少(P<0.01);Western blot结果显示,与空白组比较,模型组细胞LC3Ⅱ/LC3Ⅰ、p-AMPK/AMPK蛋白表达显著升高(P<0.01),p62、p-mTOR/mTOR、p-ULK1/ULK1显著下降(P<0.01),与模型组比较,ZGJTTMP组与Compound C组细胞内LC3Ⅱ/LC3Ⅰ、p-AMPK/AMPK蛋白表达显著下降(P<0.01),p62、p-mTOR/mTOR、p-ULK1/ULK1显著升高(P<0.01)。结论:ZGJTTMP对AGObjective:To explore the effect and mechanism of Zuogui Jiangtang Tongmai prescription(ZGJTTMP)on astrocytes(ASs)injured by advanced glycation end products(AGEs)combined with oxygenglucose deprivation(OGD).Method:Cell counting kit-8(CCK-8)was used to determine the optimal concentration of AGEs and the action time of OGD,and the optimal blood concentration of ZGJTTMP was selected for follow-up experiments.ASs were divided into normal group,model group(AGEs+OGD),ZGJTTMP group,an adenosine 5'-monophosphate-activated protein kinase(AMPK)inhibitor(Compound C)group,AMPK activator(AICAR)group,and combination group(ZGJTTMP+AICAR).The morphological changes in ASs in each group were observed under an inverted microscope.The cell survival rate in each group was detected by CCK-8.The content of interleukin^(-1)β(IL^(-1)β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)was detected by enzyme-linked immunosorbent assay(ELISA).The number of autophagosomes in each group was counted under an electron microscope.The expression of microtubule-associated protein light chain 3(LC3)was observed by immunofluorescence.The protein expression of LC3,p62,p-AMPK,AMPK,p-mammalian target of rapamycin(mTOR),mTOR,p-UNC-51 like kinase 1(ULK1),and ULK1 was detected by Western blot.Result:According to the results of cell survival rate,200 mg·L^(-1)AGEs and OGD for 6 h were selected as the optimal modeling conditions for the model group,and 5%was selected as the optimal blood concentration of ZGJTTMP.Under the inverted microscope,the cells were severely damaged after modeling,but the cell injury in the ZGJTTMP group and the Compound C group was significantly improved.As revealed by ELISA results,the content of IL^(-1)β,IL-6,and TNF-αin the model group increased(P<0.01),and the content of inflammatory factors in the ZGJTTMP group and the Compound C group decreased(P<0.01).Under the electron microscope,the number of autophagosomes in the model group increased significantly.The immunofluorescence results showed that the expression area o

关 键 词：星形胶质细胞 自噬 左归降糖通脉方 腺苷酸活化蛋白激酶(AMPK) 哺乳动物雷帕霉素靶蛋白(mTOR) UNC-51样激酶1(ULK1) 

分 类 号：R2-0[医药卫生—中医学] R33R289R587.1