黄蜀葵花总黄酮通过激活AMPK/mTOR通路调控自噬改善克罗恩病肠道纤维化  

TFA Regulating Autophagy through Activation of AMPK/mTOR Pathway to Improve Intestinal Fibrosis of CD

作　　者：张慧翔 张丹[2] 钱海华[2] 王雅丽 曾莉[1] ZHANG Huixiang;ZHANG Dan;QIAN Haihua;WANG Yali;ZENG Li(First School of Clinical Medicine,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Province Hospital of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学第一临床医学院,江苏南京210023 [2]江苏省中医院,江苏南京210023

出　　处：《中医药信息》2022年第7期1-10,26,共11页Information on Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81904203)。

摘　　要：目的:观察黄蜀葵花总黄酮(total flavone of Abelmoschus manihot,TFA)对肠道成纤维细胞合成Ⅰ型胶原蛋白(Collagen Ⅰ)的影响,探讨TFA治疗克罗恩病肠道纤维化的机制。方法:从SD大鼠中分离原代肠道成纤维细胞(intestinal fibroblasts,IFs),使用免疫荧光法鉴定细胞特征。利用胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)构建IFs细胞模型,随机分为对照组、模型组和TFA低、中、高剂量(5、10、50μg/mL)组。CCK-8法检测TFA及雷帕霉素(rapamycin,RAPA)对IFs细胞最佳干预条件。通过免疫荧光法检测LC3Ⅱ、Beclin-1、ATG16L1及p62等自噬有关蛋白表达,使用mRFP-GFP-LC3实验检测自噬流水平。采用Western blot和qRT-PCR法测定Collagen Ⅰ水平。用AMPK抑制剂化合物C(Compound C,CC)、mTOR抑制剂RAPA联合TFA处理IGF-1刺激的IFs细胞,将细胞随机分为对照组、模型组、TFA(10μg/mL)组、TFA+CC组、TFA+RAPA组。采用免疫荧光及mRFP-GFP-LC3实验检测自噬水平。运用Western blot和qRT-PCR法评估Collagen Ⅰ水平。采用Western blot法检测p-AMPK、p-MTOR、p-p70S6K和p-4EBP1蛋白表达。结果:免疫荧光结果显示,原代细胞具有Vimentin(+),α-SMA(-)的特征,证明其为肠道成纤维细胞。CCK-8结果提示,TFA最佳干预浓度为5、10、50μg/mL,时间为48 h;RAPA最佳干预浓度为5 nmol/mL,时间为48 h。与对照组相比,模型组的Collagen Ⅰ蛋白及mRNA水平上升(P <0.01),自噬有关蛋白LC3Ⅱ、Beclin-1、ATG16L1等表达减弱,p62表达上升,自噬流减少,p-AMPK/AMPK比值降低,p-mTOR/mTOR、p-p70S6K/p70S6K和p-4EBP1/4EBP1比值升高(P <0.01)。与模型组相比,TFA各剂量组Collagen Ⅰ水平降低(P <0.01),自噬水平上升,其中高剂量组最明显;TFA(10μg/mL)组p-AMPK/AMPK比值上调,p-mTOR/mTOR、p-p70S6K/p70S6K和p-4EBP1/4EBP1比值下调(P <0.01)。与TFA组相比,TFA+CC组中CollagenⅠ水平升高(P <0.01),自噬水平下降,p-AMPK/AMPK比值下降,p-mTOR/mTOR、p-p70S6K/p70S6K和p-4EBP1/4EBP1比值Objective:To observe the effect of total flavone of Abelmoschus manihot(TFA)on the synthesis of collagen I in intestinal fibroblasts(IFs)and to investigate its mechanism in treating intestinal fibrosis of Crohn’s disease(CD). Methods:Primary IFs were isolated from SD rats and the feature of the primary cells were identified by immunofluorescence staining. The IGF-1 stimulated IFs cell model was established and randomly divided into the control group,the model group and the TFA groups of low-dose,medium-dose and high-dose(5 μg/mL,10 μg/mL,50 μg/mL). CCK-8 was used to detect the optimal intervention conditions for IFs by TFA and rapamycin(RAPA). The expressions of autophagy-related proteins,such as LC3,Beclin-1,ATG16L1 and p62 were measured by immunofluorescence. mRFP-GFP-LC3 was used to detect the level of autophagic flow. IGF-1 stimulated IFs were intervened by AMPK inhibitor Compound C(CC),mTOR inhibitor RAPA combined with TFA,and were randomly divided into the control group,the model group,the TFA group(10 μg/mL),the TFA plus CC group and the TFA plus RAPA group.Immunofluorescence and mRFP-GFP-LC3 were used to detect the level of autophagy. The protein and mRNA levels of Collagen Ⅰ were assessed by Western blot and qRT-PCR. The protein expressions of pAMPK,p-MTOR,p-p70S6K and p-4EBP1 were detected by Western blot. Results:Immunofluorescence results showed that the primary cells were characterized by Vimentin(+)and α-SMA(-),indicating that they were IFs. CCk-8 results showed that the optimal intervention concentrations for TFA were 5 μg/mL,10 μg/mL and 50 μg/mL for 48 h. The optimal intervention concentration for RAPA was 5 nmol/mL for 48 h.Compared with those in the control group,the protein and mRNA expressions of Collagen Ⅰ increased(P <0. 01),the expressions of autophagy-related proteins of LC3,Beclin-1 and ATG16L1 weakened,p62expression increased,autophagic flow decreased,p-AMPK/AMPK ratio decreased,and the ratios of pmTOR/mTOR,p-p70S6K/p70S6K and p-4EBP1/4EBP1 elevated in the model group(P <

关 键 词：肠道纤维化 克罗恩病 黄蜀葵花总黄酮 肠道成纤维细胞 自噬 

分 类 号：R285[医药卫生—中药学]