富硒黄芪皂苷对大鼠退变软骨细胞增殖的影响及其机制研究  

Effect and mechanisms of seleno enriched astragalus saponin on the proliferation of degenerative chondrocytes in rats

作　　者：夏鹏飞 颜春鲁 安方玉 赵磊 金华 孙仕华[7] 柳鹏瑶 张永花 XIA Peng-fei;YAN Chun-lu;AN Fang-yu;ZHAO Lei;JING Hua;SUN Shi-hua;LIU Peng-yao;ZHANG Yong-hua(Scientific Research and Experiment Center,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China;Teaching Experiment Training Center,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China;College of Pharmacy,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China;School of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China;Clinical College of Traditional Chinese Medicine,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,Gansu Province,China;Gansu Provincial Key Laboratory of Chemical and Quality Control of Chinese and Tibetan Medicine,Lanzhou 730000,Gansu Province,China;Department of Orthopaedics,The Second People's Hospital of Lanzhou^Lanzhou 730000,Gansu Province,China)

机构地区：[1]甘肃中医药大学科研实验中心,甘肃兰州730000 [2]甘肃中医药大学教学实验实训中心,甘肃兰州730000 [3]甘肃中医药大学药学院,甘肃兰州730000 [4]甘肃中医药大学公共卫生学院,甘肃兰州730000 [5]甘肃中医药大学中医临床学院,甘肃兰州730000 [6]甘肃省高校中(藏)药化学与质量研究省级重点实验室,甘肃兰州730000 [7]兰州市第二人民医院骨科,甘肃兰州730000

出　　处：《中国临床药理学杂志》2020年第6期639-642,共4页The Chinese Journal of Clinical Pharmacology

基　　金：甘肃省中医药管理局科研基金资助项目(GZK-2017-2);甘肃省高校中(藏)药化学与质量研究省级重点实验室开放基金资助项目(zzy-2015-04);甘肃省高等学校科研基金资助项目(2018A-043);兰州市科技局基金资助项目(2017-4-58)。

摘　　要：目的建立白细胞介素-1β(IL-1β)诱导退变的软骨细胞模型,研究富硒黄芪皂苷调节软骨细胞增殖治疗膝骨关节炎(KOA)的作用及其机制。方法将培养的第3代大鼠软骨细胞分为7组:空白对照组、模型组和更低、低、中、高和更高5个剂量实验组。除空白对照组(加入软骨细胞)以外,其他各组分别用IL-1β10 ng·mL^-1诱导48 h,获得退变软骨细胞模型。空白对照组和模型组均加入含10%小牛血清的DEME培养基,5个剂量实验组分别加入富硒黄芪皂苷25,50,100,200,300 mmol·L^-1。经验证选择干预48 h的中剂量实验组进行后续实验。用噻唑蓝法检测细胞增殖(OD值)情况,用免疫组化法检测Ⅱ型胶原、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白的表达水平(灰度值),实时荧光定量-PCR法检测磷脂酰肌醇-3激酶(PI3K)和蛋白激酶B(Akt)基因表达水平(2-ΔΔCt)。结果空白对照组、模型组和中剂量实验组于48 h的细胞增殖水平分别为0.41±0.04,0.12±0.02和0.38±0.02;这3组的Ⅱ型胶原蛋白表达水平分别为153.21±48.91,82.15±34.22和213.74±52.37;这3组的Bcl-2蛋白表达水平分别为78.43±15.33,36.27±10.43和58.27±11.59;这3组的Bax蛋白分别为25.41±8.14,52.53±12.32和30.91±7.18;这3组的Caspase-3蛋白表达水平分别为31.25±8.25,80.72±12.46和52.71±10.53;这3组的PI3K基因表达水平分别为1.00±0.03,2.75±0.02和1.14±0.01;这3组的Akt基因表达水平分别为1.00±0.04,1.83±0.07和1.13±0.04。上述指标:模型组与空白对照组比较,差异均有统计学意义(均P<0.05);中剂量实验组与模型组比较,差异均有统计学意义(均P<0.05)。结论富硒黄芪可调节软骨细胞增殖,这可能通过PI3K/Akt途径发挥作用。Objective To study regulation effect of seleno enriched astragalus saponin on proliferation in degenerated chondrocytes induced by interleukin-1β(IL-1β).Methods The third generation of 48 h chondrocytes was divided into seven groups:Blank control group,model group,and very low dose experiment group(experimental-VL group),low dose experiment group(experimental-L group),middle dose experiment group(experimental-M group),high dose experiment group(experimental-H group),very high dose experiment group(experimental-VH group).Degenerative chondrocytes were established by IL-1β(10 ng·m L^-1)as model group.The blank control group and model group were given the deme medium containing 10%calf serum;five dose experiment groups were intervened 25,50,100,200,300 mmol·L^-1 astragalus saponin.After 48 h of intervention,the experiental-M group was selected for follow-up experiment.The cell proliferation(OD value)was analyzed by MTT.The proteins expression(gray level)of collagen-Ⅱ,B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2 associated X protein(Bax)and cysteine asparate protease-3(Caspase-3)were detected by immunohistochemistry.The genes expression level(2-ΔΔCt)of phosphatidylinositol 3-kinase(PI3 K)and protein kinase B(Akt)in cartilage cell was detected by fluorescence quantitative-PCR.Results The proliferation level in blank control group,model group,experimental-M group were 0.41±0.04,0.12±0.02,0.38±0.02;the collagen typeⅡprotein in the three groups were 153.21±48.91,82.15±34.22,213.74±52.37;the Bcl-2 protein in the three groups were 78.43±15.33,36.27±10.43,58.27±11.59;the Bax protein in the three groups were 25.41±8.14,52.53±12.32,30.91±7.18;the Caspase-3 protein in the three groups were31.25±8.25,80.72±12.46,52.71±10.53;the PI3 K gene expre-ssion in the three groups were 1.00±0.03,2.75±0.02,1.14±0.01;the Akt gene expression in the three groups were 1.00±0.04,1.83±0.07,1.13±0.04.Comparison between model group and blank control group,the difference of the factors were significant(all P<0.05);compari

关 键 词：富硒黄芪皂苷 白细胞介素-1Β 退变软骨细胞 磷脂酰肌醇-3激酶/蛋白激酶B-B细胞淋巴瘤信号通路 增殖 

分 类 号：R28[医药卫生—中药学]