左归降糖舒心方含药血浆对ox-LDL诱导J774A.1巨噬细胞焦亡及凋亡的影响  

Effects of Zuogui Jiangtang Shuxin formula medicated plasma on pyroptosis and apoptosis induced by ox-LDL in J774A.1 cells

作　　者：杨金伟 喻嵘 吴勇军[4] 肖凡[5] 刘秀 邓奕辉[1] Yang Jinwei;Yu Rong;Wu Yongjun;Xiao Fan;Liu Xiu;Deng Yihui(College of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Hunan 410208,China;Graduate School,Hunan University of Chinese Medicine,Hunan 410208,China;Key Laboratory of Colleges and Universities,Traditional Chinese Medicine Prescription and Transformation of Medical Laboratory,Hunan 410208,China;School of Pharmacy,Hunan University of Chinese Medicine,Hunan 410208,China;School of International Education,Hunan 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院,湖南410208 [2]湖南中医药大学研究生院 [3]中医方证研究转化医学湖南省重点实验室 [4]湖南中医药大学药学院 [5]湖南中医药大学国际教育学院

出　　处：《北京中医药大学学报》2021年第4期332-339,共8页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(No.81573956,82074400);国家重点研发计划中医药现代化研究重点专项(No.2018YFC1704300);湖南省重点研发计划项目(No.2020SK2101);湖南省教育厅科研项目(No.18A220,20K094);湖南省“国内一流建设学科”中医学开放基金项目(No.2018ZYX44)。

摘　　要：目的研究左归降糖舒心方含药血浆对氧化低密度脂蛋白(ox-LDL)诱导巨噬细胞焦亡及凋亡状态的影响,探讨该方治疗糖尿病动脉粥样硬化斑块的新靶点。方法 SD大鼠随机分成3组,连续5 d灌胃相应的药物制备含药血浆:空白组(灭菌超纯水)、左归降糖舒心方组(25.92 g/kg)、西药联合用药组(二甲双胍0.83 g/kg+阿托伐他汀钙12.45 mg/kg)。应用ox-LDL诱导细胞焦亡及凋亡状态。通过CCK-8法比较各组J774A.1细胞增殖情况,计算细胞存活率,筛选含药血浆及ox-LDL干预浓度。将分化完全的J774A.1巨噬细胞随机分为空白血浆组、模型组、左归降糖舒心方含药血浆组、西药联合含药血浆组,每组6个复孔。诱导及给药24 h后,LDH释放实验检测细胞膜完整性及细胞焦亡水平;流式细胞仪检测细胞凋亡率;Western blot法检测焦亡相关蛋白NOD样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)、gasdermin D(GSDMD)蛋白、凋亡相关蛋白半胱氨酸蛋白酶-3(Caspase-3)及B细胞白血病淋巴瘤相关蛋白基因(Bax)蛋白的表达水平。结果含药血浆及空白血浆在浓度为10%水平对细胞增殖无明显抑制作用,作为含药血浆实验干预浓度。ox-LDL在100 mg/L浓度下,可明显降低细胞存活率,故考虑以100 mg/L ox-LDL为诱导细胞焦亡及凋亡的造模浓度。与空白组比较,模型组细胞存活率明显降低,LDH释放率增高(P<0.01);同时细胞凋亡明显增多(P<0.01);模型组在ox-LDL诱导下,细胞焦亡相关蛋白NLRP3、Caspase-1、GSDMD表达上调,促进凋亡相关因子Caspase-3及Bax表达增强(P<0.01)。与模型组比较,左归降糖舒心方含药血浆组细胞存活率增高,LDH释放率及细胞凋亡率明显降低(P<0.01);左归降糖舒心方含药血浆可显著抑制ox-LDL所致的NLRP3、Caspase-1、GSDMD、Caspase-3及Bax表达水平上调(P<0.01)。而且,左归降糖舒心方含药血浆组阻断ox-LDL引起的焦亡过程细胞膜破坏及凋亡Objective To investigate the effects of Zuogui Jiangtang Shuxin(kidney-supplementing glucose-lowering heart-soothing) formula medicated plasma on ox-LDL-induced pyroptosis and apoptosis and to explore new targets in the treatmert of diabetic atherosclerosis with this formula. Methods SD rats were administered intragastrically with 7.2 g/kg Zuogui Jiangtang Shuxin formula or 0.83 g/kg metformin combined with 12.45 mg/kg atorvastatin calcium to prepare drug-containing plasma. Zuogui Jiangtang Shuxin formula medicated plasma was then extracted for J774 A.1 cells treatment. J774 A.1 cells were pretreated with ox-LDL to establish the experimental model of pyroptosis and apoptosis. CCK-8 essay was used to meausre the effect of medicated plasma or ox-LDL on proliferation of J774 A.1 cells and to determine the optimal concentration. J774 A.1 cells were randomly divided into 4 groups: normal group, model group, Zuogui Jiangtang Shuxin medicated plasma group and Western medicine combination(metformin+atorvastatin calcium) medicated plasma group. After inducting and administration for 24 hours, LDH releasing test was used to evaluate the cell membrane integrity. Cell apoptosis were measured by using flow cytometry. The expression of NLRP3, Caspase-1,GSDMD, Caspase-3 and Bax were detected by using Western blot assay. Results 10% medicated plasma and blank plasma showed no inhibition of cells, the concetnration of which was chosen to be suitable for experiment. The cell survival rate decreased when J774 A.1 cells were treated with 100 mg/L ox-LDL for the induction concentration. Compared with the blank group, 100 mg/L ox-LDL suppressed the growth of J774 A.1 cells. It was also shown with LDH releasing test that LDH activity and apoptosis rate increased in the model group cells significantly(P<0.01). Moreover, ox-LDL could up-regulate the expression of NLRP3, Caspase-1,GSDMD, Caspase-3 and Bax in the model group(P<0.01). Compared with the model group, Zuogui Jiangtang Shuxin medicated plasma reduced inhibition rate, LDH releas

关 键 词：左归降糖舒心方 含药血浆 J774A.1细胞 细胞焦亡 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]