地黄梓醇调控ATDC5软骨细胞的衰老  

Catalpol from Rehmannia glutinosa regulates senescence in ATDC5 chondrocytes

作　　者：贾瑞英 梅杰 何强 李丹 孙欣[3] 钱卫庆[3] 刘振[1] Jia Ruiying;Mei Jie;He Qiang;Li Dan;Sun Xin;Qian Weiqing;Liu Zhen(Heze Municipal Hospital,Heze 274000,Shandong Province,China;Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250011,Shandong Province,China;Department of Orthopedics and Traumatology,Nanjing Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine,Nanjing 210022,Jiangsu Province,China)

机构地区：[1]菏泽市立医院,山东省菏泽市274000 [2]山东中医药大学附属医院,山东省济南市250011 [3]南京中医药大学附属南京中医院骨伤科,江苏省南京市210022

出　　处：《中国组织工程研究》2024年第34期5467-5472,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(3217100541),项目参与者:李丹;江苏省科技厅社会发展面上项目(BE20211612),项目负责人:孙欣;江苏省重点研发计划(社会发展)项目(BE2020625),项目负责人:钱卫庆。

摘　　要：背景:课题组前期体内、体外研究结果表明地黄梓醇能够显著降低膝骨关节炎大鼠滑膜组织中炎症指标水平,同时能够延缓膝骨关节炎进展,但是否通过影响软骨细胞衰老进而延缓膝骨关节炎的进展尚未明确。目的:探讨地黄梓醇能否调控ATDC5软骨细胞衰老及可能的机制。方法:将ATDC5软骨细胞分为空白组(0.1%牛血清白蛋白)、模型组(0.1%牛血清白蛋白+1μmol/L阿霉素)、地黄梓醇低剂量组(0.1%牛血清白蛋白+1μmol/L阿霉素+20μmol/L地黄梓醇)及地黄梓醇高剂量组(0.1%牛血清白蛋白+1μmol/L阿霉素+80μmol/L地黄梓醇)。应用阿霉素诱导构建ATDC5软骨细胞衰老模型,按上述分组予以对应的处理。CCK-8法检测地黄梓醇对ATDC5软骨细胞活力的影响,筛选地黄梓醇最佳给药浓度。相应处理后应用β-半乳糖苷酶染色法检测各组ATDC5软骨细胞衰老情况;实时荧光定量PCR法检测相关基因表达(P21、P53、Ⅱ型胶原、基质金属蛋白酶13、白细胞介素6);Western blot检测P21、P53、Ⅱ型胶原、基质金属蛋白酶13、白细胞介素6的表达水平;免疫荧光法检测P21、P53和Ⅱ型胶原表达情况;流式细胞仪检测各组细胞凋亡情况。结果与结论:(1)经鉴定成功诱导ATDC5软骨细胞并诱导衰老模型;(2)地黄梓醇浓度在0,20,40,80μmol/L时对细胞活力均无明显影响,提示地黄梓醇对细胞无毒性,可安全使用(P> 0.05);当浓度≥100μmol/L时,细胞活力降低,提示可能存在毒性,故选择80μmol/L作为高剂量进行后续实验;(3)与空白组β-半乳糖苷酶阳性细胞百分率(17.32±0.72)%比较,模型组(86.93±2.18)%显著升高(P <0.05);与模型组比较,地黄梓醇低、高剂量组(57.28±1.73)%、(27.18±0.97)%均显著降低(P <0.05);(4)与模型组比较,地黄梓醇低、高剂量组的P21、P53、基质金属蛋白酶13、白细胞介素6 mRNA和蛋白相对表达量均显著下调,而Ⅱ型胶原的mRNA和蛋白相对表达量显著上�BACKGROUND:The results of in vivo and in vitro studies showed that catalpol from Rehmannia glutinosa can significantly reduce the level of inflammatory indexes in the synovial tissue of rats with knee osteoarthritis,and meanwhile,it can delay the progression of knee osteoarthritis.But whether catalpol from Rehmannia glutinosa affects chondrocyte senescence and then delay the progression of knee osteoarthritis has not yet been clarified.OBJECTIVE:To investigate investigate whether catalpol from Rehmannia glutinosa could regulate ATDC5 chondrocyte senescence and the possible mechanisms.METHODS:ATDC5 chondrocytes were divided into blank group(0.1%bovine serum albumin),model group(0.1%bovine serum albumin+1µmol/L adriamycin),low-dose catalpol group(0.1%bovine serum albumin+1µmol/L adriamycin+20µmol/L catalpol from Rehmannia glutinosa)and high-dose catalpol group(0.1%bovine serum albumin+1µmol/L adriamycin+80µmol/L catalpol from Rehmannia glutinosa).Adriamycin-induced ATDC5 chondrocyte senescence model was constructed,and the corresponding treatments were given according to the above groups.Cell counting kit-8 assay was used to detect the effects of catalpol from Rehmannia glutinosa on ATDC5 chondrocyte viability,and to screen the optimal concentration of catalpol from Rehmannia glutinosa.The senescence of ATDC5 chondrocytes in each group was detected byβ-galactosidase staining after the corresponding treatments.Real-time fluorescence quantitative PCR and western blot were used to detect the mRNA and protein expression of P21,P53,type II collagen,matrix metalloproteinase 13,and interleukin-6.Immunofluorescence method was used to detect the expression of P21,P53 and type II collagen.Flow cytometry was used to detect apoptosis in each group.RESULTS AND CONCLUSION:ATDC5 chondrocytes were identified to be successfully induced and senescence model was induced.Catalpol from Rehmannia glutinosa at the concentrations of 0,20,40,and 80µmol/L showed no significant effects on the cell viability,suggesting that catalpol fr

关 键 词：地黄梓醇 ATDC5 软骨细胞 细胞衰老 骨关节炎 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R446