机构地区:[1]江西中医药大学中药资源与民族药研究中心,江西南昌330004 [2]江西中医药大学科技学院,江西南昌330004 [3]江西中医药大学药学院,江西南昌330004
出 处:《中药新药与临床药理》2021年第4期467-472,共6页Traditional Chinese Drug Research and Clinical Pharmacology
基 金:国家自然科学基金项目(81860739);江西省卫生计生委中医药科研项目(2017A286);国家重点研发计划项目(2019YFC1712300);江西省双一流学科(中药学)建设项目(JXSYLXK-ZHYAO034)。
摘 要:目的观察无尾果不同提取部位对人风湿性关节炎成纤维滑膜细胞(MH7A)相关细胞因子分泌及凋亡的影响,初步筛选其抗类风湿关节炎(RA)的有效部位。方法采用肿瘤坏死因子α(TNF-α)诱导MH7A细胞建立类风湿关节炎滑膜细胞模型,并以无尾果总提物、水部位、正丁醇部位、乙酸乙酯部位和石油醚部位进行干预。采用CCK8法检测MH7A细胞的增殖活性,并计算半数抑制浓度(IC_(50));流式细胞术检测MH7A细胞凋亡;ELISA法检测TNF-α诱导MH7A细胞分泌白细胞介素(IL)-6、IL-4、IL^(-1)β、血管内皮生长因子(VEGF)、NF-κB受体活化因子配体(RANKL)水平;Western Blot法检测MH7A细胞的凋亡相关蛋白表达水平。结果无尾果总提物、水部位、正丁醇部位、乙酸乙酯部位和石油醚部位对MH7A细胞的IC_(50)值分别为223.9、99.4、123.7、497.9、>1000μg·m L^(-1)。与正常对照组比较,模型组细胞上清液中的IL-6、IL^(-1)β、IL-4、VEGF和RANKL含量显著升高(P<0.01),凋亡细胞显著增多(P<0.01),促调亡因子Bax、Caspase-9、Caspase-3和抗凋亡因子Bcl-2的蛋白表达水平均显著升高(P<0.01)。与模型组比较,无尾果总提物组、水部位组和正丁醇部位组均可显著诱导MH7A细胞凋亡(P<0.01);显著降低IL-6、IL^(-1)β、VEGF和RANKL水平(P<0.01),升高IL-4水平(P<0.01);水部位组和正丁醇部位组能明显上调促凋亡蛋白Bax、Caspase-9和Caspase-3的表达(P<0.01),抑制抗凋亡蛋白Bcl-2的表达(P<0.01)。结论无尾果提取物诱导MH7A细胞凋亡及抗炎作用可能是其抗RA的机制之一,水部位和正丁醇部位为其主要药效部位。Objective The effects of different extracts of coluria longifolia Maxim on cytokines secretion and apoptosis of human rheumatoid arthritis fibroblast synoviocyte MH7 A were investigated,to preliminarily screen the effective fraction against rheumatoid arthritis.Methods Total extract and the fractions of water,n-butanol,ethyl acetate and petroleum ether from the C.longifolia Maxim were prepared and applied to TNF-αinduced rheumatoid arthritis fibroblast synoviocyte MH7 A cells.The proliferation of MH7 A cells was detected by CCK8 method and IC_(50) was calculated;the apoptosis of MH7 A cells was detected by flow cytometry;the levels of IL-6,IL-4,IL^(-1)β,vascular endothelial growth factor(VEGF)and NF-κB receptor activating ligand(RANKL)induced by TNF-αwere detected by ELISA;the expression of apoptosis related proteins in MH7 A cells was detected by Western Blot.Results The IC_(50) values of the total extract and the fractions of water,n-butanol,ethyl acetate and petroleum ether from the C.longifolia Maxim on MH7 A cells were 223.9,99.4,123.7,497.9 and>1000μg·mL1,respectively.Compared with the normal control group,the levels of IL-6,IL^(-1)β,IL-4,VEGF and RANKL in the model group were significantly increased(P<0.01),ratio of apoptotic cells and the expression of apoptosis related proteins including Bax,Caspase-9,Caspase-3 and Bcl-2 were also significantly increased(P<0.01).Compared with the model group,the total extract group,water extract group and n-butanol fraction group of C.longifolia Maxim could significantly induce the apoptosis of MH7 A cells(P<0.01),significantly reduce the levels of IL-6,IL^(-1)β,VEGF and RANKL(P<0.01),and increase the level of IL-4(P<0.01).Furthermore,compared with the model group,the expression of pro-apoptotic factors Bax,Caspase-9 and Caspase-3 were significantly increased(P<0.01),and the expression of anti-apoptotic protein Bcl-2 was significantly decreased(P<0.01).Conclusion The inhibition of inflammation and induction of apoptosis in MH7 A cells induced by the extract of C.
关 键 词:无尾果 人风湿性关节炎成纤维滑膜细胞(MH7A) 细胞因子 细胞凋亡 类风湿关节炎 有效部位 水部位 正丁醇部位
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