摘要
目的研究艾拉莫德通过靶向组蛋白去乙酰化酶1(HDAC1)调控类风湿关节炎滑膜成纤维细胞的生长的作用机制。方法对滑膜成纤维细胞(FLS)进行培养,用0,25,50,100μg·mL^(-1)艾拉莫德处理细胞,并将其分为对照组、艾拉莫德组(100μg·mL^(-1)艾拉莫德)、艾拉莫德+pcDNA组(100μg·mL^(-1)艾拉莫德+转染pcDNA)及艾拉莫德+HDAC1组(100μg·mL^(-1)艾拉莫德+转染HDAC1)。以细胞计数试剂盒-8(CCK-8)法检测各组滑膜成纤维细胞的增殖情况;以蛋白质印迹(Western blot)法及实时荧光定量逆转录聚合酶链反应(RT-qPCR)法检测滑膜成纤维细胞中HDAC1的表达情况;以流式细胞术检测滑膜成纤维细胞凋亡情况;以Transwell检测滑膜成纤维细胞侵袭情况。结果与对照组比较艾拉莫德组细胞在0 h时光密度(OD)值未见明显的变化,在24 h、48 h及72 h时OD值明显下降(均P<0.05);与艾拉莫德+pcDNA组比较,艾拉莫德+HDAC1组细胞在0 h时及24 h时OD值未见明显的变化,在48 h及72 h时OD值明显上升(均P<0.05)。对照组、艾拉莫德组、艾拉莫德+pcDNA组、艾拉莫德+HDAC1组的HDAC1蛋白表达水平分别为0.96±0.09,0.43±0.05,0.42±0.06,0.80±0.09;72 h OD值分别为1.07±0.09,0.55±0.09,0.52±0.07,0.86±0.09;细胞凋亡率分别为(4.46±0.44)%,(19.13±1.43)%,(20.29±1.35)%,(10.59±1.68)%;细胞侵袭数目分别为(114.00±12.98),(61.20±9.20),(56.00±5.00),(92.40±7.33)个。与对照组比较,艾拉莫德组细胞的HDAC1 mRNA和HDAC1蛋白表达水平明显下降(P<0.05),与艾拉莫德+pcDNA组比较,艾拉莫德+HDAC1组细胞的HDAC1 mRNA和HDAC1蛋白表达水平明显上升(P<0.05)。与对照组比较,艾拉莫德组细胞凋亡率、Cl-caspase-3及Bax蛋白表达水平均明显上升,细胞侵袭数目、MMP-2和MMP-9蛋白表达水平均明显下降(均P<0.05);与艾拉莫德+pcDNA组比较,艾拉莫德+HDAC1组细胞凋亡率、Cl-caspase-3及Bax蛋白水平均明显下降,侵袭数目、MMP-2和MMP-9蛋白表达水平均明显上升(均P<0.05)。结论艾拉莫德通过靶向组蛋白去乙酰化酶1,能够调控类风湿关节炎滑膜成纤维细胞的生长。
Objective To investigate the mechanism of Iguratimod regulating the growth of rheumatoid arthritis synovial fibroblasts cells by targeting histone deacetylase 1.Methods Synovial fibroblasts(FLS)were cultured,and were treated with 0,25,50,100μg·mL^(-1) iguratimod,and cells were divided into control group,Iguratimod group(100μg·mL^(-1) Iguratimod),Iguratimod+pcDNA group(100μg·mL^(-1) Iguratimod+transfer pcDNA)and Iguratimod+HDAC1 group(100μg·mL^(-1)Iguratimod+transfer HDAC1).Cell counting kit-8(CCK-8)was used to detect the proliferation of synovial fibroblasts in each group.The expression of HDAC1 in synovial fibroblasts was detected by Western blot and real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).The apoptosis of synovial fibroblasts was detected by flow cytometry;Transwell was used to detected the invasion of synovial fibroblasts.Results Compared with control group,the OD value of cells in Iguratimod group did not change significantly at 0 h,and decreased significantly at 24 h,48 h and 72 h(P<0.05).Compared with Iguratimod+pcDNA group,the OD value of cells in Iguratimod+HDAC1 group did not change significantly at 0 h and 24 h,the OD value increased significantly at 48 h and 72 h(P<0.05).The HDAC1 protein expression levels of control group,Iguratimod group,Iguratimod+pcDNA group and Iguratimod+HDAC1 group were 0.96±0.09,0.43±0.05,0.42±0.06 and0.80±0.09;72 h OD values were 1.07±0.09,0.55±0.09,0.52±0.07,0.86±0.09;the apoptosis rates were(4.46±0.44)%,(19.13±1.43)%,(20.29±1.35)%,(10.59±1.68)%;the number of invasive cells were114.00±12.98,61.20±9.20,56.00±5.00,92.40±7.33,respectively.Compared with control group,the expression levels of HDAC1 mRNA and HDAC1 protein in Iguratimod group were significantly decreased(P<0.05),while the expression levels of HDAC1 mRNA and HDAC1 protein in Iguratimod+pcDNA group were significantly increased compared with Iguratimod+pcDNA group(all P<0.05).Compared with control group,the apoptosis rate,the expression levels of Cl-caspase-3 and Bax protein in Iguratimod group were significantly increased,the number of cell invasion,the expression levels of MMP-2 and MMP-9 protein were significantly decreased(all P<0.05).Compared with Iguratimod+pcDNA group,the apoptosis rate,Cl-caspase-3 and Bax protein levels in Iguratimod+HDAC1 group were significantly decreased,and the number of invasion,MMP-2 and MMP-9 protein expression levels were significantly increased(all P<0.05).Conclusion Iguratimod can regulate the growth of rheumatoid arthritis synovial fibroblasts by targeting histone deacetylase 1.
作者
翁永平
朱峰
汪越
WENG Yong-ping;ZHU Feng;WANG Yue(Department of Medical,The First People’s Hospital of Jiande,Hangzhou,Hangzhou 311600,Zhejiang Province,China;Department of Rheumatology and Immunology,The First People’s Hospital of Jiande,Hangzhou,Hangzhou 311600,Zhejiang Province,China;Department of Pharmacy,The First People’s Hospital of Jiande,Hangzhou,Hangzhou 311600,Zhejiang Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2022年第16期1920-1924,共5页
The Chinese Journal of Clinical Pharmacology
