摘要
目的:探讨淫羊藿苷(ICA)对脂多糖(LPS)诱导成骨细胞凋亡及葡萄糖调节蛋白78/蛋白激酶R样内质网激酶/C/EBP同源蛋白(GRP78/PERK/CHOP)通路的影响。方法:采用大鼠骨髓基质干细胞定向诱导分化为成骨细胞的方法,将成骨细胞随机分成5组:对照组(只含成骨细胞)、LPS组(成骨细胞中加入终浓度为500 ng·ml^-1LPS)、ICA低、中、高剂量组(分别在LPS组基础上加入终浓度为1,10,100μmol·L^-1ICA)。采用CCK-8法检测细胞增殖情况,采用对硝基苯棕榈酸酯(PNPP)法检测成骨细胞碱性磷酸酶(AKP)活性的变化情况,采用ELISA法检测成骨细胞Ⅰ型胶原蛋白表达情况,采用Annexin V/PI双染法流式细胞术检测细胞凋亡情况,采用Western blot检测凋亡及GRP78/PERK/CHOP通路相关蛋白表达情况。结果:骨髓基质干细胞经定向诱导后,符合成骨细胞形态学表现,AKP染色呈阳性反应,Ⅰ型胶原蛋白免疫组织化学染色结果显示:细胞质染色呈棕黄色;与对照组相比,LPS组细胞48 h时的吸光度(A)值、AKP活性、Ⅰ型胶原蛋白和B淋巴细胞瘤-2基因(Bcl-2)蛋白表达水平显著降低(P<0.05),细胞凋亡率、活化的天冬氨酸特异性半胱氨酸蛋白酶-3(cleaved-caspase-3)、Bax、GRP78、磷酸化的PERK(p-PERK)、磷酸化α亚基的真核起始因子2(p-eIF2α)、CHOP蛋白表达水平显著升高(P<0.05);与LPS组相比,ICA各剂量组细胞48 h时的A值、AKP活性、Ⅰ型胶原蛋白和Bcl-2蛋白表达水平显著升高(P<0.05),细胞凋亡率、cleaved-caspase-3、Bax、GRP78、p-PERK、p-eIF2α、CHOP蛋白表达水平显著降低(P<0.05),且呈剂量依赖性(P<0.05)。结论:淫羊藿苷可能通过抑制GRP78/PERK/CHOP信号通路活化,抑制LPS诱导的成骨细胞凋亡。
Objective:To investigate the effects of icariin(ICA)on the lipopolysaccharide(LPS)-induced apoptosis of osteoblasts and GRP78/PERK/CHOP pathway.Methods:Rat bone marrow stromal stem cells were induced to differentiate into osteoblasts by directional induction,and the osteoblasts were randomly divided into five groups:the control group(only osteoblasts),LPS group(the above cells treated with LPS with the final concentration of 500 ng·ml^-1),ICA low,medium and high dose groups(ICA respectively at the final concentration of 1,10 and 100μmol·L^-1 was added to LPS group).The CCK-8 method was used to detect the cell proliferation,the AKP activity of osteoblasts was detected by PNPP method,the expression of typeⅠcollagen in osteoblasts was detected by ELISA,Annexin V/PI double staining was used to detect the apoptotic cells by flow cytometry,and Western blot was used to detect the apoptosis and expressions of GRP78/PERK/CHOP pathway-related proteins.Results:After the directional induction,BMSCs conformed to the morphological features of osteoblasts,the alkaline phosphatase staining was positive,and the immunohistochemical staining of collagen type I showed that the cytoplasm was brown.Compared with the control group,in LPS group,the A value at 48 h,AKP activity,expressions of collagen type I and Bcl-2 protein decreased significantly(P<0.05),and the apoptotic rate,expression levels of cleaved-caspase-3,Bax,GRP78,p-PERK,p-eIF2αand CHOP protein increased significantly(P<0.05).Compared with LPS group,in all ICA groups,the A value at 48 h,AKP activity,expressions of collagen type I and Bcl-2 protein increased significantly(P<0.05),and the apoptotic rate,expression levels of cleaved-caspase-3,Bax,GRP78,p-PERK,p-eIF2αand CHOP protein decreased significantly(P<0.05).The effects of ICA groups were dose-dependent(P<0.05).Conclusion:Icariin may inhibit the LPS-induced apoptosis of osteoblasts by inhibiting the activation of GRP78/PERK/CHOP signaling pathway.
作者
唐晓悦
朱浩
曹灿
Tang Xiaoyue;Zhu Hao;Cao Can(Department of Stomatology,Peace Branch,General Hospital of the Northern War Zone of the PLA,Shenyang 110000,China;Department of Oral and Maxillofacial Surgery;Department of Stomatology,General Hospital of the Northern War Zone of the PLA)
出处
《中国药师》
CAS
2019年第12期2177-2182,共6页
China Pharmacist
基金
辽宁省科学技术计划项目(编号:201601404)
