龙鳖胶囊含药血清对YAP抑制剂诱导人软骨细胞凋亡的保护作用及机制研究
x

请在关注微信后,向客服人员索取文件

篇名: 龙鳖胶囊含药血清对YAP抑制剂诱导人软骨细胞凋亡的保护作用及机制研究
TITLE: Study on Protective Effects of Longbie Capsule Contained Serum on the Apoptosis of Chondrocytes Induced by YAP Inhibitor
摘要: 目的:探索龙鳖胶囊含药血清(后文简写为“LBJN”)对Yes结构结合蛋白(YAP)抑制剂维替泊芬(verteporfin)诱导软骨细胞凋亡的保护作用及机制。方法:通过两步酶消化法提取原代人膝骨性关节炎软骨细胞,利用甲苯胺蓝染色法和Ⅱ型胶原蛋白免疫荧光染色法对细胞进行鉴定。采用流式细胞术检测2、5μmol/Lverteporfin单用和分别与5%LBJN联用48h后对细胞凋亡的影响,并设置溶剂对照[0.1%二甲基亚砜(DMSO)]和5%LBJN对照。采用Westernblot法检测0.1%DMSO(溶剂对照)、2μmol/Lverteporfin、2μmol/Lverteporfin+5%LBJN和0(空白对照)、2.5%LBJN、5%LBJN处理48h后细胞中凋亡相关蛋白[YAP、B细胞淋巴瘤2(Bcl-2)、活化胱天蛋白酶3(cleaved-caspase-3)]的表达水平;采用Westernblot法检测0(空白对照)、2.5%、5%LBJN处理48h后细胞中自噬相关蛋白[哺乳动物雷帕霉素靶蛋白(mTOR)、Beclin-1、LC3A/B]的表达水平。结果:分离出的细胞符合软骨细胞的特征。与0.1%DMSO比较,2、5μmol/Lverteporfin作用后细胞的凋亡率均显著升高(P<0.05),且两个浓度的作用效果相当(P>0.05);与单用verteporfin比较,2、5μmol/Lverteporfin与5%LBJN联用后细胞的凋亡率均显著降低(P<0.05)。与0.1%DMSO比较,2μmol/Lverteporfin作用后细胞中YAP、Bcl-2蛋白表达水平均显著降低(P<0.05),而cleaved-caspase-3蛋白表达水平显著升高(P<0.05);与2μmol/Lverteporfin比较,2μmol/Lverteporfin+5%LBJN作用后细胞中YAP、Bcl-2蛋白的表达水平均显著升高(P<0.05),而cleaved-caspase-3蛋白的表达水平显著降低(P<0.05)。与空白对照比较,2.5%、5%LBJN作用后细胞中YAP、Bcl-2、Beclin-1蛋白的表达水平和LC3A/B-Ⅱ/LC3A/B-Ⅰ比值均显著升高(P<0.05),而cleaved-caspase-3、mTOR蛋白的表达水平均显著降低(P<0.05)。结论:LBJN具有阻断YAP抑制剂verteporfin诱导的软骨细胞凋亡的作用,其机制可能与调节软骨细胞中凋亡相关蛋白的表达和增强软骨细胞的自噬有关。
ABSTRACT: OBJECTIVE:To ex plore the protective effects of Longbie capsule contained serum (called“LBJN”for short )on the apoptosis of chondrocytes induced by YAP inhibitor verteporfin and its mechanism. METHODS :Primary human knee osteoarthritis(OA)chondrocytes were extracted by two-step enzymatic digestion ,and then identif ied by toluidine blue staining and type Ⅱ collagen immunofluorescence staining. The effects of 2,5 μmol/L verteporfin alone or combined with 5%LBJN on cell apoptosis were detected by flow cytometry. Solvent control (0.1% DMSO)and 5% LBJN were set. Western blot assay was adopted to detect the expression of apoptosis related proteins (YAP,Bcl-2,cleaved-caspase-3) after treated with 0.1%DMSO(solvent control ),2 μmol/L verteporfin,2 μmol/L verteporfin+5%LBJN 和 0(blank control ),2.5% LBJN and 5% LBJN for 48 h. The expression of autophagy related proteins (mTOR,Beclin-1,LC3A/B) after treated with 0 (blank control ),2.5%,5% LBJN for 48 h were det ected by Western blot assay. RESULTS :The isolated cells accorded with the characteristics of chondrocytes. Compared with 0.1%DMSO, the apoptosis rates of cells were increased significantly after treated with 2,5 μmol/L verteporfin(P<0.05),and the effects of the two concentrations were similar (P>0.05). Compared with verteporfin alone ,2,5 μmol/L verteporfin combined with 5%LBJN could significantly decrease the apoptotic rate of cells (P<0.05). Compared with 0.1%DMSO,the protein expression of YAP and Bcl-2 were decreased significantly after treated with 2 μ mol/L verteporfin (P<0.05), while the protein expression of cleaved-caspase-3 were increased significantly (P<0.05). Compared with 2 μmol/L verteporfin,protein expression of YAP and Bcl-2 were increased significantly after treated with 2 μmol/L verteporfin+5%LBJN(P<0.05),while the protein expression of cleaved-caspase-3 were decreased significantly (P<0.05). Compared with blank control ,the protein expression of YAP ,Bcl-2 and Beclin-1 were increased significantly after treated with 2.5%,5%LBJN(P<0.05),while protein expression of cleaved-caspase- 3 and mTOR were decreased significantly (P<0.05). CONCLUSIONS :LBJN can block the apoptosis of chondrocytes induced by YAP inhibitor verteporfin ,and its mechanism may be related to regulating the expression of apoptosis related proteins and enhancing autophagy of chondrocytes.
期刊: 2021年第32卷第12期
作者: 梁桂洪,黄和涛,潘建科,曾令烽,杨伟毅,罗明辉,杨园,陈红云,韩燕鸿,赵金龙,刘军
AUTHORS: LIANG Guihong,HUANG Hetao,PAN Jianke,ZENG Lingfeng, YANG Weiyi,LUO Minghui,YANG Yuan,CHEN Hongyun,HAN Yanhong,ZHAO Jinlong,LIU Jun
关键字: 龙鳖胶囊;含药血清;骨性关节炎;软骨细胞;Yes结构结合蛋白抑制剂;凋亡;自噬
KEYWORDS: Longbie capsule ;Contained serum ;Osteoarthritis;Chondrocytes;YAP inhibitor ;Apoptosis;Autophagy
阅读数: 268 次
本月下载数: 4 次

* 注:未经本站明确许可,任何网站不得非法盗链资源下载连接及抄袭本站原创内容资源!在此感谢您的支持与合作!