世界中医药2025，Vol.20Issue(13)：2256-2265,10.DOI:10.3969/j.issn.1673-7202.2025.13.005
益气养阴通络方含药血清通过调控叉头盒蛋白K2和自噬抑制凋亡
Yiqi Yangyin Tongluo Formula-medicated Serum Inhibits Apoptosis by Regulating FOXK2 and Autophagy
摘要
Abstract
Objective:To investigate the inhibitory effect of the Yiqi Yangyin Tongluo Formula on apoptosis of diabetic renal tubu-lar epithelial cells by regulating forkhead box protein K2(FOXK2)and related autophagy pathways.Methods:Serum containing the Yiqi Yangyin Tongluo Formula was prepared from rats and used at a concentration of 20％for in vitro experiments.Human kidney epithelial cells(HK-2)in the logarithmic growth phase were divided into six groups:control group,model group,medicated serum group,FOXK2 knockdown control group(sh-NC),FOXK2 knockdown group(sh-FOXK2),and medicated serum+FOXK2 knock-down group(medicated serum+sh-FOXK2).A diabetic model was induced under high-glucose conditions.The medicated serum group and medicated serum+sh-FOXK2 group received 20％serum containing Yiqi Yangyin Tongluo Formula,while the sh-FOXK2 and medicated serum+sh-FOXK2 groups were transfected with FOXK2 knockdown plasmids.The positive rate of apoptotic cells was detected by TUNEL assay,and the apoptosis rate was detected by flow cytometry.Western blot(WB)was used to detect FOXK2,apoptosis-related proteins(Caspase-3,cleaved Caspase-3,Bcl-2,and Bax),and autophagy-related proteins(LC3,mTOR,p62).Immunofluorescence was used to detect LC3 and p62 expression,and qRT-PCR was employed to measure FOXK2 mRNA levels.Results:Compared to the control group,the model group exhibited significantly higher positive rate of apoptotic cells and ap-optosis rate(P＜0.05).Compared to the model group,the medicated serum group showed significantly lower positive rate of apop-totic cells and apoptosis rate(P＜0.05).Compared to the control group,the model group exhibited higher FOXK2,Caspase-3,cleaved Caspase-3,Bax,mTOR,and p62 protein expression,while Bcl-2 and LC3 protein expression was significantly reduced(P＜0.05).LC3 fluorescence intensity was weaker and p62 fluorescence stronger(P＜0.05).Compared with the model group,the med-icated serum group showed significantly lower expression of FOXK2,Caspase-3,cleaved Caspase-3,Bax,mTOR,and p62,and in-creased levels of Bcl-2 and LC3(P＜0.05).LC3 fluorescence was enhanced,and p62 fluorescence was reduced(P＜0.05).FOXK2 protein(in nucleus,cytoplasm,and total cell)and mRNA levels were significantly higher in the model and sh-NC groups compared to the control(P＜0.05),and significantly reduced in the sh-FOXK2 group compared to the model group(P＜0.05).Compared to the control group,the model group and the sh-NC group exhibited higher positive rates of apoptotic cells and apoptosis rates(P＜0.05).Compared to the model group,the sh-FOXK2 group showed lower positive rates of apoptotic cells and apoptosis rates(P＜0.05).Compared to the control group,the model group and sh-NC group exhibited higher relative expression levels of FOXK2,Caspase-3,cleaved Caspase-3,Bax,mTOR,and p62 proteins(P＜0.05),while the relative expression levels of Bcl-2 and LC3 proteins were lower(P＜0.05),LC3 fluorescence intensity was weaker(P＜0.05),and p62 fluorescence intensity was stron-ger(P＜0.05).Compared to the model group,the sh-FOXK2 group showed lower relative expression levels of Caspase-3,cleaved Caspase-3,Bax,mTOR,and p62 proteins(P＜0.05),higher relative expression levels of Bcl-2 and LC3 proteins(P＜0.05),stron-ger LC3 fluorescence intensity(P＜0.05),and weaker p62 fluorescence intensity(P＜0.05).The positive rate of apoptotic cells and the apoptosis rate were lower in the medicated serum group and the medicated serum+sh-FOXK2 group,with the lowest values observed in the medicated serum+sh-FOXK2 group(P＜0.05).In both the medicated serum group and the medicated serum+sh-FOXK2 group,the relative expression levels of FOXK2,Caspase-3,cleaved Caspase-3,Bax,mTOR,and p62 proteins were lower,while those of Bcl-2 and LC3 proteins were higher(P＜0.05).LC3 fluorescence intensity was stronger,and p62 fluorescence inten-sity was weaker(P＜0.05),with the most significant differences from the model group observed in the medicated serum+sh-FOXK2 group(P＜0.05).Conclusion:Yiqi Yangyin Tongluo Formula inhibits apoptosis in diabetic renal tubular epithelial cells by downregulating FOXK2 expression,promoting autophagy,and modulating apoptosis-and autophagy-related pathways.关键词
益气养阴通络方/糖尿病/肾小管上皮细胞/叉头盒蛋白K2/细胞凋亡/细胞自噬/自噬相关蛋白/凋亡相关蛋白Key words
Yiqi Yangyin Tongluo Formula/Diabetes/Renal tubular epithelial cell/FOXK2/Apoptosis/Autophagy/Autophagy-related proteins/Apoptosis-related proteins分类
医药卫生引用本文复制引用
宇汝翠,李金虎,吴瑞影,陈亭亭,汪星星..益气养阴通络方含药血清通过调控叉头盒蛋白K2和自噬抑制凋亡[J].世界中医药,2025,20(13):2256-2265,10.基金项目
国家自然科学基金面上项目(82274426) （82274426）
国家自然科学基金项目(81904155) （81904155）
2024年度安徽省中医药学会中医药科研项目(2024ZYYXH028) （2024ZYYXH028）
安徽省高等学校省级质量工程项目(2021JYXM1763) （2021JYXM1763）
