中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (34): 5467-5472.doi: 10.12307/2024.832

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

地黄梓醇调控ATDC5软骨细胞的衰老

贾瑞英1，梅  杰2，何  强2，李  丹1，孙  欣3，钱卫庆3，刘  振1   

  1. 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
  • 收稿日期:2023-12-13 接受日期:2024-01-25 出版日期:2024-12-08 发布日期:2024-03-14
  • 通讯作者: 刘振，副主任医师，菏泽市立医院，山东省菏泽市 274000 钱卫庆，博士后，主任医师，南京中医药大学附属南京中医院骨伤科，江苏省南京市 210022
  • 作者简介:贾瑞英，女，1980年生，2008年潍坊医学院毕业，助理研究员，主要从事卫生服务管理方面的研究。
  • 基金资助:
    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

Catalpol from Rehmannia glutinosa regulates senescence in ATDC5 chondrocytes

Jia Ruiying1, Mei Jie2, He Qiang2, Li Dan1, Sun Xin3, Qian Weiqing3, Liu Zhen1   

  1. 1Heze Municipal Hospital, Heze 274000, Shandong Province, China; 2Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, Shandong Province, China; 3Department of Orthopedics and Traumatology, Nanjing Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing 210022, Jiangsu Province, China
  • Received:2023-12-13 Accepted:2024-01-25 Online:2024-12-08 Published:2024-03-14
  • Contact: Liu Zhen, Master, Associate chief physician, Heze Municipal Hospital, Heze 274099, Shandong Province, China Qian Weiqing, MD, Chief physician, Department of Orthopedics and Traumatology, Nanjing Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing 210022, Jiangsu Province, China
  • About author:Jia Ruiying, Assistant researcher, Heze Municipal Hospital, Heze 274099, Shandong Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 3217100541 (to LD [project participant]); Social Development General Project of Jiangsu Provincial Department of Science and Technology, No. BE20211612 (to SX); Jiangsu Provincial Key Research and Development Program (Social Development), No. BE2020625 (to QWQ)

PDF

6

摘要：


文题释义：

地黄梓醇：熟地黄是中国“四大怀药”之一，记载可追溯至《神农本草经》。地黄是玄参科地黄的新鲜或干燥块根，梓醇是一种从熟地黄中分离得到的环烯醚萜葡萄糖苷，是熟地黄的主要活性成分，药理作用包括抗炎、抗氧化等作用。
细胞衰老：作为生物体所必然经历的自然过程，在整个生命周期中均可能发生，其突出的特点表现为细胞永久性的处于增殖停滞的状态，而衰老细胞的持续性存在和逐渐积累会分泌出大量的促炎性蛋白，进而会严重损害机体组织的功能。


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 +1 µmol/L阿霉素)、地黄梓醇低剂量组(0.1%牛血清白蛋白+1 µmol/L阿霉素+20 µmol/L地黄梓醇)及地黄梓醇高剂量组(0.1%牛血清白蛋白+1 µmol/L阿霉素+80 µmol/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0，20，40，80 µmol/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≥100 µmol/L时，细胞活力降低，提示可能存在毒性，故选择80 µmol/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调(P < 0.05)，高剂量组更为明显(P < 0.05)；⑤与模型组比较，地黄梓醇低、高剂量组的P21、P53荧光信号均显著减弱，而Ⅱ型胶原的荧光信号显著增强(P < 0.05)，高剂量组更为明显(P < 0.05)；⑥经Annexin V/PI法检测各组细胞凋亡情况，与空白组比较，模型组的凋亡情况无明显变化(P > 0.05)；与模型组比较，地黄梓醇低、高剂量组的凋亡指标均显著升高，且以高剂量组更为明显(P < 0.05)；⑦提示地黄梓醇能够通过促进衰老的ATDC5软骨细胞凋亡，进一步清除衰老的ATDC5软骨细胞，降低衰老相关表型进而延缓骨关节炎的进展。

https://orcid.org/0009-0008-9021-8156(贾瑞英)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 地黄梓醇, ATDC5, 软骨细胞, 细胞衰老, 骨关节炎

Abstract: 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+1 µmol/L adriamycin), low-dose catalpol group (0.1% bovine serum albumin+1 µmol/L adriamycin+20 µmol/L catalpol from Rehmannia glutinosa) and high-dose catalpol group (0.1% bovine serum albumin+1 µmol/L adriamycin+80 µmol/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 by β-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 80 µmol/L showed no significant effects on the cell viability, suggesting that catalpol from Rehmannia glutinosa is non-cytotoxic and can be used safely (P > 0.05); when the concentration was ≥ 100 µmol/L, the cell viability was reduced, suggesting that there may be cytotoxic. Therefore, 80 µmol/L was chosen as the high dose for subsequent experiments in this study. The percentage of positive cells in the model group was (86.93±2.18)%, which was significantly higher than that in the blank group [(17.32±0.72)%; P < 0.05]. Compared with the model group, the percentage of positive cells was significantly lower in the low- and high-dose catalpol groups [(57.28±1.73)% and (27.18±0.97)%, respectively; both P < 0.05]. Compared with the model group, the relative expression of P21, P53, matrix metalloproteinase 13, and interleukin-6 at mRNA and protein levels was significantly downregulated in the low- and high-dose catalpol groups, while the relative expression of type II collagen at mRNA and protein levels was significantly upregulated in both groups (P < 0.05), especially in the high-dose catalpol group (P < 0.05). Compared with the model group, the fluorescence intensities of P21 and P53 were significantly weakened in the low- and high-dose catalpol groups, while the fluorescence intensity of type II collagen was significantly enhanced in the low- and high-dose catalpol groups (P < 0.05), especially in the high-dose catalpol group (P < 0.05). The cell apoptosis detected by Annexin V/PI method showed that there was no significant difference between the model group and the blank group (P > 0.05); compared with the model group, the apoptotic index was significantly elevated in the low- and high-dose catalpol groups, especially in the high-dose catalpol group (P < 0.05). To conclude, catalpol from Rehmannia glutinosa can slow the progression of osteoarthritis by promoting apoptosis of senescent ATDC5 chondrocytes, further removing senescent ATDC5 chondrocytes, and decreasing the senescence-associated phenotypes.

Key words: catalpol from Rehmannia glutinosa, ATDC5, chondrocyte, cellular senescence, osteoarthritis

中图分类号: