摘要
目的:研究补阳还五汤对脂多糖(LPS)诱导的巨噬细胞RAW264.7重度内质网应激(ERS)的抗凋亡作用及其机制。方法:Western bolt法检测不同浓度、不同时间LPS诱导下巨噬细胞RAW264.7 ERS的标志蛋白GRP78的表达,建立重度ERS模型。将细胞分别分为空白对照组(20%胎牛血清)、模型对照组(20%胎牛血清+LPS 40μg/ml)、空白血清组(20%空白血清+LPS 40μg/ml)、15 g/kg补阳还五汤5%、10%、20%含药血清组、2 mmol/L 4-苯基丁酸组、2 mmol/L 4-苯基丁酸联合15 g/kg补阳还五汤20%含药血清组。用流式细胞术、荧光素FITC标记的AnnexinV与碘化丙啶(PI)结合染色检测细胞凋亡情况。Western bolt检测补阳还五汤含药血清对LPS诱导的巨噬细胞RAW264.7重度ERS相关蛋白葡萄糖调节蛋白78(GRP78)、蛋白激酶样内质网激酶(PERK)、内切核糖核酸酶肌醇需要酶1(IRE1)表达的影响。用RT-PCR实验方法检测补阳还五汤含药血清对凋亡通路中含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase3)、含半胱氨酸的天冬氨酸蛋白水解酶12(Caspase12)、Fas、Fas配体(Fasl)、B-淋巴细胞瘤2(Bcl-2)表达的影响。结果:与空白对照组比较,模型对照组早凋率、晚凋率及总凋亡率均显著升高(P<0.01);与模型对照组比较,15 g/kg补阳还五汤10%、20%含药血清组与2 mmol/L 4-苯基丁酸组均能明显降低重度ERS时引起的细胞凋亡(P<0.01);与空白对照组比较,模型对照组GRP78、IRE1、PERK蛋白表达均有显著上调(P<0.01);与模型对照组比较,15 g/kg补阳还五汤各含药血清组可不同程度降低GRP78的蛋白表达(P<0.05或P<0.01),15 g/kg补阳还五汤10%、20%含药血清组可不同程度下调PERK、IRE1的蛋白表达(P<0.01);2 mmol/L 4-苯基丁酸组可有效下调GRP78、IRE1、PERK的蛋白表达(P<0.01);与空白对照组比较,模型对照组中Caspase12、Fas、Fasl的mRNA表达显著上调(P<0.01),Bcl-2的mRNA表达显著下调(P<0.01);与模型对照组比较,补阳还五汤各含药血清组及2 mmol/L 4-苯基丁酸组能显著下调Fas的mRNA表达,上调Bcl-2的mRNA表达,15 g/kg补阳还五汤10%、20%含药血清组能显著下调Caspase12、Fasl的mRNA表达(P<0.05或P<0.01)。结论:补阳还五汤改善巨噬细胞RAW264.7重度ERS的状态,通过Caspase12、Bcl-2、Fas-Fasl信号通路调控细胞在重度ERS状态下出现的细胞凋亡。
Objective: To study the anti apoptotic effect of Buyang Huanwu Decoction on LPS(Lipopolysaccharide) induced severe endoplasmic reticulum stress(ERS) in RAW264.7 macrophages and its mechanism. Methods: The severe ERS model was established in RAW264.7 induced by LPS at different concentrations and at different times. Western blot was used to detect the expression of GRP78, a marker protein of RAW264.7 ERS. The cells were divided into the control group(20% fetal bovine serum),the model group(20% fetal bovine serum + 40 μg/ml LPS), 15 g/kg distilled water + serum group(20% serum + 40 μg/ml LPS), 15 g/kg Buyang Huanwu Decoction groups(5%, 10% and 20% medicated serum), 2 mmol/L 4-phenylbutyric acid group, and 2 mmol/L 4-phenylbutyric acid combined with serum containing 15 g/kg Buyang Huanwu decoction group. The cell apoptosis was detected by flow cytometry, annexin V labeled with FITC and propidium iodide(PI) staining. Western bolt was used to detect the effect of serum containing Buyang Huanwu decoction on the expressions of GRP78, PERK and IRE1 in RAW264.7 macrophages induced by LPS. The effect of serum containing Buyang Huanwu decoction on the expressions of Caspase3, caspase12, Fas, FasL and Bcl-2 in apoptosis pathway were detected by RT-PCR. Results: Compared with the control group, the early apoptosis rate, late apoptosis rate and total apoptosis rate were significantly increased in the model group(P<0.01). Compared with the model group, the apoptosis rate induced by severe ERS was decreased in serum(10%、20%) containing 15 g/kg Buyang Huanwu Decoction groups and 2 mmol/L 4-phenylbutyric acid group(P<0.01). Compared with the control group, the expressions of GRP78, IRE1 and PERK in severe ERS model group were significantly up regulated in the model group(P<0.01). Compared with the model group, the protein expression of GRP78 was down regulated in all doses of Buyang Huanwu Decoction groups to different degrees(P<0.05 or P<0.01),while the protein expressions of PERK and IRE1 were down regulated in serum(10%、20%) containing drug groups to different degrees(P<0.01), the protein expressions of GRP78, IRE1 and PERK were significantly decreased in 2 mmol/L 4-phenylbutyric acid group. Compared with the control group, the mRNA expressions of caspase12, Fas and Fasl genes were significantly increased in the model group(P<0.01),while the mRNA expression of Bcl-2 gene was significantly decreased(P<0.01).Compared with the model group, all doses of serum containing Buyang Huanwu decoction groups and 2 mmol/L 4-phenylbutyric acid group reduced the mRNA expression of Fas gene and increased the mRNA expression of Bcl-2 gene in different degrees. The mRNA expression of caspase12 and Fasl gene were decreased in serum(10%、20%)containing Buyang Huanwu decoction groups(P<0.05 orP<0.01). Conclusion: Buyang Huanwu Decoction can improve the severe ERS status of macrophages RAW264.7, and regulate the cell apoptosis in severe ERS through caspase 12, Bcl-2 and Fas-Fasl signaling pathways.
作者
张路煜
游宇
李林
肖雄
侯贝贝
王海贝
刘玉晖
Zhang Luyu;You Yu;Li Lin;Xiao Xiong;Hou Beibei;Wang Haibei;Liu Yuhui(Jiangxi University of Traditional Chinese Medicine,Nanchang 330004;The First Affiliated Hospital of Nanchang University,Nanchang 330006)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2021年第1期22-28,共7页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然科学基金项目(编号:81860712、81460621)。
关键词
补阳还五汤
巨噬细胞
内质网应激
细胞凋亡
信号通路
Buyang Huanwu Decoction
macrophages
endoplasmic reticulum stress
apoptosis
signaling pathway
