痹痿同治法对脂多糖诱导大鼠退变软骨细胞炎症和凋亡的影响  

Effect of arthralgia-flaccidity simultaneous treatment on inflammation and apoptosis of lipopolysaccharide-induced rat degenerative chondrocytes

作　　者：耿秋东 牛素生 张燕 王和鸣[1,2] 李楠 GENG Qiudong;NIU Susheng;ZHANG Yan;WANG Heming;LI Nan(College of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou350122,China;Key Laboratory of Orthopedics&Traumatology of Traditional Chinese Medicine and Rehabilitation of Ministry of Education,Fuzhou 350122,China)

机构地区：[1]福建中医药大学中医学院,福州350122 [2]中医骨伤及运动康复教育部重点实验室

出　　处：《北京中医药大学学报》2022年第11期1142-1150,共9页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(No.81973880);福建省自然科学基金项目(No.2019J01349)。

摘　　要：目的 探讨痹痿同治法方药化湿定痛汤合龟鹿二仙胶对骨关节炎的可能作用机制。方法 制备化湿定痛汤合龟鹿二仙胶含药血清及空白血清。采用4周龄SD大鼠膝关节软骨建立稳定的软骨细胞体外培养体系,采用免疫组化染色法对F2代软骨细胞进行鉴定。鉴定成功后分别用4个体积分数的含药血清(5%、10%、15%、20%)和空白血清处理软骨细胞(5×103个/孔),培养24、48、72 h后,CCK-8法检测软骨细胞活性,选择提升软骨细胞活性的最佳含药血清体积分数进行后续实验。用10 mg/L脂多糖诱导F2代软骨细胞,复制退变软骨细胞模型,将细胞分为空白组、模型组、化湿定痛汤合龟鹿二仙胶含药血清组(简称“含药血清组”)。干预24 h后,采用CCK-8法检测各组软骨细胞活性,ELISA法检测各组细胞培养液上清中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的水平,qRT-PCR法检测各组软骨细胞B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)mRNA的表达,Western blotting检测各组软骨细胞Bcl-2、Bax、切割型Caspase-3蛋白的表达情况。结果 与空白组比较,模型组软骨细胞活性降低(P<0.01),细胞上清液中IL-1β、IL-6、TNF-α水平升高(P<0.01),Bax、Caspase-3 mRNA的表达及Bax、切割型Caspase-3蛋白的表达上调(P<0.01),Bcl-2 mRNA和蛋白的表达下调(P<0.01)。与模型组比较,含药血清组软骨细胞活性升高(P<0.05),细胞上清液中IL-1β、IL-6、TNF-α水平下降(P<0.05),Bax、Caspase-3 mRNA的表达及Bax、切割型Caspase-3蛋白的表达下调(P<0.05),Bcl-2 mRNA和蛋白的表达上调(P<0.05)。结论 痹痿同治法能改善软骨细胞生存的炎症微环境,提升软骨细胞活性,改善软骨细胞凋亡情况,进而发挥对大鼠退变软骨细胞的保护作用,延缓骨关节炎发病进程。Objective To explore the possible mechanism of arthralgia-flaccidity simultaneous treatment recipe Huashi Dingtong decoction and Guilu Erxian glue on osteoarthritis.Methods Huashi Dingtong decoction and Guilu Erxian glue medicated serum and blank serum were prepared. A stable in vitro culture system of chondrocytes was established using 4-week-old SD rat knee articular cartilage, and the F2 generation chondrocytes were identified by immunohistochemical staining. After successful identification, four volume fractions of medicated serum(5%, 10%, 15%, and 20%) and blank serum were used to treat chondrocytes(5×10~3 cells/well). After culturing for 24 h, 48 h, and 72 h, the CCK-8 method was used to detect chondrocyte activity, and the best medicated serum volume fraction was selected to enhance chondrocyte activity for follow-up experiments. The F2 generation chondrocytes were induced to replicate the degenerated chondrocyte model with 10 mg/L lipopolysaccharide, and the cells were divided into the following groups: the blank group, model group, Huashi Dingtong decoction and Guilu Erxian glue medicated serum group(medicated serum group). After 24 h of intervention, the chondrocyte activity of each group was detected using the CCK-8 method. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the supernatant of cell culture medium were detected by ELISA. qRT-PCR method was used to detect the mRNA expression of chondrocyte B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and cysteine protease-3(Caspase-3) in each group. Western blotting was used to detect the expression of Bcl-2, Bax and Cleaved Caspase-3 proteins in the chondrocytes of each group.Results Compared with the blank group, the activity of chondrocytes in the model group decreased(P<0.01), the levels of IL-1β, IL-6 and TNF-α in the cell supernatant increased(P<0.01), the mRNA expressions of Bax and Caspase-3 and the protein expressions of Bax and Cleaved Caspase-3 were up-regulated(P<0.01), and the

关 键 词：痹痿同治法 化湿定痛汤 龟鹿二仙胶 软骨细胞 骨关节炎 大鼠 

分 类 号：R285.5[医药卫生—中药学]