基于PI3K/AKT信号通路探究活血通络法对硝普钠诱导的关节软骨细胞凋亡的分子作用机制  

Discussion on the Molecular Mechanism of Blood Circulation and Blood Circulation Method for Chondrocyte Apoptosis Induced by Sodium Nitroprusside Based on PI3K/Akt Signaling Pathway

作　　者：廖建青 马富文 冯晋 谢邦 吕静 韦佳佳 王琦 LIAO Jianqing;MA Fuwen;FENG Jin(Yunnan University of Traditional Chinese Medicine,Kunming Yunruin 650500,China;Yunnan Hospital of Traditional Chinese Medicine,Kunming Yunnan 650021,China)

机构地区：[1]云南中医药大学,云南昆明650500 [2]云南省中医医院,云南昆明650021

出　　处：《四川中医》2021年第9期33-38,共6页Journal of Sichuan of Traditional Chinese Medicine

基　　金：国家自然科学基金项目:活血通络法介导PI3K/AKT信号通路调控膝骨关节炎软骨细胞凋亡的机制研究(编号:81560781);云南省内设研究研究基金:云南省中医医院院内制剂骨痹合剂的安全性评价研究(编号:2018NS0038);云南中医药大学2020年度研究生科学研究基金:三七总皂苷(PNS)治疗KOA模型小鼠的淋巴回流相关性研究(编号:2020Y09)。

摘　　要：目的:基于PI3K/AKT信号通路探究具有活血通络作用的骨痹合剂对硝普钠诱导的软骨细胞凋亡模型可能的分子作用机制。方法:体外培养小鼠软骨传代细胞,建立硝普钠诱导的细胞凋亡模型。细胞传代成功后,根据分组分别加入5%、10%、20%浓度的骨痹合剂、氨糖美辛、生理盐水含药血清。采用MTT检测细胞存活和生长情况,运用流式细胞仪(Flow Cytometry,FCM)、PCR仪、蛋白质免疫印迹法(Western blotting)检测在硝普钠诱导干预下3组含药血清的小鼠膝关节软骨细胞相关蛋白水平的表达。结果:①对照组软骨细胞形态饱满,整体呈现梭形;而硝普钠组细胞变细、变长,细胞状态变差,细胞凋亡比例增加;②相对于正常对照组的软骨细胞,硝普钠处理的软骨细胞Bad、Caspase-3、Caspase-9、NF-κB表达明显上调(P<0.01),而骨痹合剂组和氨糖美辛组细胞内的Bad、Caspase-3、Caspase-9、NF-κB蛋白的表达被抑制(P<0.01)。③与正常对照组比较,硝普钠组PI3K/AKT和mTOR蛋白表达量均上升,与硝普纳诱导的生理盐水组比较,硝普纳诱导的骨痹合剂组PI3K/AKT和mTOR蛋白的表达量均下降。结论:①硝普纳对细胞具有损伤作用,能促进和诱导软骨细胞凋亡;②具有活血通络作用的骨痹合剂能通过调控PI3K/AKT信号通路,抑制NF-κB、Bad、caspase3、caspase9蛋白的表达,从而减少硝普纳诱导的软骨细胞凋亡。Objective:Based on PI3 K/AKT signaling pathway,to explore the possible molecular mechanism of activating blood circulation on chondrocyte apoptosis model of mice knee joint induced by sodium nitroprusside.Methods:Mice cartilage passage cells were cultured in vitro,the apoptosis model induced by sodium nitroprusside is set up.After successful cell passage,the drug serum containing Gubi Mixture,Glucosamine Indomethacin Entric-coated Tablets and NS concentration of 5%,10%,20%were added according to their groups.Cell survival and growth were measured by MTT,by using PCR and Flow Cytometry,Western blotting detection in the induction of intervention of sodium nitroprusside three groups of medicine serum of mice with knee joint cartilage cells related to the expression of protein levels.Results:1.The chondrocytes in the controlled group were plump and fusiform,however,after treatment with sodium nitroprusside,the cells became thinner and longer,and the cell state became worse,the apoptosis rate of chondrocytes was increased.2.Compared with the chondrocytes in the normal controlled group,the chondrocytes treated with sodium nitroprusside were Bad,Caspase–3,Caspase–9,NF-KB significantly raised(P<0.01),while the intracellular Bad,Caspase 3,Caspase-9,NF-KB protein expression were inhibited(P<0.01),in the Gubi Mixture and the Glucosamine Indomethacin group.3.Compared with the normal controlled group,the sodium group of PI3 K/AKT and mTOR protein expression are rising,and compared with the sodium nitroprusside induced normal saline group,sodium nitroprusside induced the Gubi Mixture group P13 K/AKT and mTOR protein expression decreased.Conclusion:1.Sodium nitroprusside can damage cells and promote or induce apoptosis of chondrocytes.2.Gubi Mixture can inhibit the expression of NF-KB,Bad,Caspase-3 and Caspase-9 proteins by regulating the P13 K/AKT signaling pathway,so as to reduce the apoptosis of chondrocytes induced by sodium nitroprusside.

关 键 词：膝关节骨性关节炎 原代软骨细胞 活血通络法 骨痹合剂PI3K/AKT信号传导通路 

分 类 号：R285.5[医药卫生—中药学]