大黄素联合HIF-1α与KGF通过自噬途径对肠上皮细胞低氧应激损伤的保护作用  

Emodin combined with HIF-1αand KGF protects intestinal epithelial cells from hypoxia induced cell damage through autophagy

作　　者：王晓辉 哈小琴 郑英 杨志华 李兵 邢媛 张媛媛 贾庆华 WANG Xiaohui;HA Xiaoqin;ZHENG Ying;YANG Zhihua;LI Bing;XING Yuan;ZHANG Yuanyuan;JIA Qinghua(Department of Clinical Laboratory,The People's Liberation Army Joint Service Support Unit 940 Hospital,Lanzhou 730050,China;Key Laboratory of Stem Cells and Gene Drug of Gansu Province,Lanzhou 730050,China)

机构地区：[1]解放军联勤保障部队第九四○医院检验科,甘肃兰州730050 [2]甘肃省干细胞与基因药物重点实验室,甘肃兰州730050

出　　处：《西部医学》2025年第4期483-489,共7页Medical Journal of West China

基　　金：甘肃省自然科学基金项目(24JRRA1130);兰州市科技支撑计划项目(2024-3-72)。

摘　　要：目的探讨大黄素联合低氧诱导因子-1α(HIF-1α)、角质细胞生长因子(KGF)在低氧引起的肠应激损伤中保护肠上皮细胞的作用机制。方法将大鼠小肠上皮细胞(IEC-6)分为BC组(常氧条件下正常培养的IEC-6细胞)、NC组(低氧条件下培养的IEC-6细胞)、HK组(低氧条件下培养的HIF-1α及KGF双表达重组腺病毒感染的IEC-6细胞)、Emodin组(低氧条件下5μmol/L大黄素培养基培养的IEC-6细胞)以及HKE组(低氧条件下HIF-1α和KGF双表达重组腺病毒感染的IEC-6细胞,并加入大黄素同时培养)。各组细胞培养24 h后,分别通过倒置显微镜观察各组细胞形态;扫描电镜观察各组形成自噬的情况;MTT检测各组细胞的增殖;流式细胞仪检测各组细胞的DNA含量;免疫荧光染色法检测腺病毒转染后HIF-1α、KGF蛋白的表达;Real Time-PCR法检测Beclin-1、LC3基因mRNA表达;Western blot检测Beclin-1、LC3蛋白水平。结果培养24 h后,倒置显微镜下显示,BC细胞状态良好,呈短梭形紧密排列;NC组细胞状态差,异形明显、稀疏悬浮;HK组、Emodin组及HKE组细胞状态较好,多为长梭形有聚团现象。透射电镜下电镜显示,培养24 h后,BC组细胞形态结构完整、胞质均匀;NC组细胞溶解,胞核不完整、胞膜内陷、胞质分裂,含大量溶酶体等;HK组、Emodin组及HKE组细胞呈现早期凋亡特征,胞质中可见自噬小体。与HK组、Emodin组相比,HKE组能够明显的促进IEC-6细胞的增殖。与BC组相比较,NC组细胞G0/G1期百分比为显著增加,G2期、S期细胞百分比显著降低(P<0.05)。HKE组的细胞HIF-1α、KGF蛋白表达量最高,荧光强度最强。与BC组比较,HK组、Emodin组及HKE组细胞中Beclin-1、LC3蛋白表达均增加,且HKE组蛋白及mRNA表达水平明显高于HK组和Emodin组(P<0.05)。结论大黄素联合HIF-1α及KGF通过自噬机制对低氧应激引起的IEC-6细胞损伤有保护作用。Objective To investigate the protective effect of emodin combined with cytokines on intestinal stress injury induced by hypoxia through autophagy.Methods Rat intestinal epithelial cells(IEC-6)were divided into five groups:BC group(IEC-6 cells cultured under normoxic conditions),NC group(IEC-6 cells cultured under hypoxic conditions),HK group(IEC-6 cells transfected with recombinant adenovirus expressing both HIF-1αand KGF under hypoxia),Emodin group(IEC-6 cells were cultured with 5μmol/L emodin medium under hypoxia conditions)and HKE group(IEC-6 cells expressing both HIF-1αand KGF were cultured with 5μmol/L emodin medium under hypoxic conditions).After 24 hours of cell culture,the proliferation of IEC-6 cells in each group was detected by MTT method.The DNA content of cells in each group was detected by flow cytometry.The formation of autophagy was observed by scanning electron microscope.The expression of HIF-1αand KGF protein after adenovirus transfection was detected by immunofluorescence staining.The mRNA expressions of beclin-1 and LC3 genes were detected by real time PCR.The protein expressions of beclin-1 and LC3 were detected by Western blot.Results Compared with HK group,Emodin group and HKE group,HKE group could significantly promote the proliferation of IEC-6 cells.The expression of HIF-1αand KGF protein was the highest and the fluorescence intensity was the strongest in HKE group.Compared with BC group,the percentage of cells in G0/G1 phase increased significantly in NC group,and the percentage of cells in G2 and S phase decreased significantly(P<0.05).The protein expression of beclin-1 and LC3 increased in HK group,Emodin group and HKE group,and the protein and mRNA expression levels of HKE group were significantly higher than those of HK group and Emodin group(P<0.05).Conclusion Emodin combined with HIF-1αand KGF can protect IEC-6 cells from hypoxia stress through autophagy.

关 键 词：低氧诱导因子-1α 角质细胞生长因子 大黄素 自噬 低氧 IEC-6细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R965[医药卫生—基础医学]