Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (34): 5467-5472.doi: 10.12307/2024.832

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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)

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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

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