瑞香素通过含半胱氨酸的天冬氨酸蛋白水解酶通路对胶原诱导性关节炎大鼠成纤维样滑膜细胞增殖与凋亡的影响  

Effects of daphnetin on the proliferation and apoptosis of fibroblast-like synoviocytes in rats with collagen-induced arthritis through the cysteine-containing aspartate proteolytic enzyme pathway

作　　者：郑茂 邹玉[1] 王洁莹 况南珍 傅颖媛[2] ZHENG Mao;ZOU Yu;WANG Jieying;KUANG Nanzhen;FU Yingyuan(Department of Clinical Laboratory,People's Hospital of Deyang City,Deyang,Sichuan 618000,China;Basic Medical College,Nanchang University,Jiangxi,Nanchang 330006,China;Department of Clinical Laboratory,The Second Affiliated Hospital of Hainan Medical University,Haikou,Hainan 570100,China)

机构地区：[1]德阳市人民医院检验科,四川德阳618000 [2]南昌大学基础医学院,江西南昌330006 [3]海南医学院第二附属医院检验科,海南海口570100

出　　处：《安徽医药》2022年第11期2198-2202,I0001,共6页Anhui Medical and Pharmaceutical Journal

基　　金：国家自然科学基金(31460239);德阳市科技计划重点研发项目(2020SZZ087)。

摘　　要：目的探讨瑞香素(DAP)通过含半胱氨酸的天冬氨酸蛋白水解酶(caspase)通路对胶原诱导性关节炎(CIA)大鼠成纤维样滑膜细胞(FLS)增殖与凋亡的影响。方法体外培养CIA-FLS,选取对数生长期细胞以不同浓度DAP(0、5、10、20、40、80 mg/L)作用48 h后,倒置显微镜下观察细胞生长状态,并通过人胆囊收缩素/缩胆囊素八肽(CCK-8)检测CIA-FLS细胞活力;采用DAP(40 mg/L)作用48 h后,通过Real-time PCR检测CIA-FLS中caspase-3、caspase-8、caspase-9 mRNA相对表达量;采用特异性不可逆的caspase抑制剂(Casp3-In、Casp8-In、Casp9-In、Pancasp-In)以不同浓度、不同时间预处理CIA-FLS后,通过CCK-8检测DAP(40 mg/L)对CIA-FLS细胞活力的影响;采用不同caspase抑制剂预处理CIA-FLS 2 h后,再与DAP(40 mg/L)共处理48 h,通过Hoechst 33258荧光染色观察CIA-FLS凋亡形态以及Annexin V-FITC/PI双染流式细胞术检测CIA-FLS凋亡率。结果经DAP作用后CIA-FLS细胞活力下降,增殖明显受抑,并与DAP作用浓度呈剂量依赖性;经DAP作用后CIA-FLS中caspase-3、caspase-8、caspase-9 mRNA相对表达量分别为(3.07±0.43)、(1.67±0.09)、(2.35±0.20),与对照组相比明显增加(P<0.05);各caspase抑制剂预处理均在不同程度上减弱DAP抑制CIA-FLS增殖的效应,但Casp8-In不如Casp3-In、Casp9-In、Pancasp-In效果明显;荧光染色显示,经DAP作用后CIA-FLS细胞核形状大小不一,细胞核固缩、碎裂明显,出现较多浓染致密的颗粒状、块状荧光,Casp3-In、Casp9-In、Pancasp-In预处理组,呈均匀蓝色荧光的正常细胞核较DAP组明显增多,Casp8-In预处理组与DAP组差异不明显;流式结果显示,经DAP作用后CIA-FLS凋亡率为(34.80±3.90)%,与对照组相比明显升高(P<0.05),Casp3-In、Casp8-In、Casp9-In、Pancasp-In预处理组CIA-FLS凋亡率分别为(18.09±2.50)%、(29.46±2.16)%、(20.41±3.53)%、(10.17±1.91)%,均明显低于DAP组(P<0.05),但Casp8-In预处理组CIA-FLS凋亡率明显高于Casp3-In、Casp9-In、Pancasp-In�Objective To explore the effect of daphnetin(DAP)on the proliferation and apoptosis of fibroblast-like synoviocytes in collagen-induced arthritic rats(CIA-FLS)through the cysteinyl aspartate specific proteinase(caspase)pathway.Methods CIA-FLSs were cultured in vitro,and cells in logarithmic growth phase were selected and treated with different concentrations of DAP(0,5,10,20,40,80 mg/L)for 48 h.The cell growth state was observed under an inverted microscope,and the cell viability of CIA-FLSs was detected by the CCK-8 method.After treatment with DAP(40 mg/L),the relative expression levels of caspase-3,caspase-8 and caspase-9m RNA in CIA-FLSs were detected by real-time PCR.After CIA-FLS were pretreated with specific irreversible caspase inhibitors(Casp3-In,Casp8-In,Casp9-In,and Pancasp-In)at different concentrations and for different times,the effect of DAP(40 mg/L)on the viability of CIA-FLS cells was detected by the CCK-8 method.CIA-FLSs were pretreated with different caspase inhibitors for 2 h,and then cotreated with DAP(40 mg/L)for 48 h.The apoptosis morphology of CIA-FLSs was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining.Results The cell viability of CIA-FLSs decreased and the proliferation was significantly inhibited after DAP treatment,and the concentration was in a dose-dependent manner.The relative expression levels of caspase-3,caspase-8,and caspase-9 m RNA in CIA-FLSs after DAP treatment were 3.07±0.43,1.67±0.09,and 2.35±0.20,respectively,which were significantly higher than those in the control group(P<0.05).Pretreatment with each caspase inhibitor attenuated the effect of DAP on inhibiting the proliferation of CIA-FLSs to varying degrees,but Casp8-In was not as effective as Casp3-In,Casp9-In,or Pancasp-In.Fluorescence staining showed that after DAP treatment,the CIA-FLS nuclei had different shapes and sizes,with obvious pyknosis and fragmentation of the nuclei,and there was more dense granular and massive fluorescence,Casp3-In,Casp9-In and Pancasp-In.Compared

关 键 词：关节炎 类风湿 关节炎 实验性 瑞香素 成纤维样滑膜细胞 含半胱氨酸的天冬氨酸蛋白水解酶 增殖 凋亡 

分 类 号：R285.5[医药卫生—中药学]