Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (23): 3636-3642.doi: 10.3969/j.issn.2095-4344.2645

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Inokosterone effects on proliferation and differentiation of osteoblasts from neonatal Sprague-Dawley rats

Jiang Tao1, 2, 3, Shao Min3, Chen Qingzhen1, 3, Ling Cuimin4, Shen Zhen1, 2, Wang Gang1, 2, Huo Shaochuan5, Lin Yanping1, 2, 3, Liu Haiquan3, Wang Qinsheng3, Zeng Zhenming3   

  1. 1Guangzhou University of Chinese Medicine; 2Laboratory of Orthopedics and Traumatology of Lingnan Medical Research Center, Guangzhou University of Chinese Medicine; 3The Third Affiliated Hospital of Guangzhou University of Chinese Medicine; 4Guangdong Second Traditional Chinese Medicine Hospital; 5Southern Medical University
  • Received:2019-08-08 Revised:2019-08-10 Accepted:2019-09-19 Online:2020-08-18 Published:2020-04-25
  • Contact: Chen Qingzhen, MD, Associate chief physician, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510240, Guangdong Province, China
  • About author:Jiang Tao, MD, Physician, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Supported by:
    Science and Technology Planning Project of Guangzhou City of China, No. 201707010463; National Natural Science Foundation of China, No. 81373654; Natural Science Foundation of Guangdong Province of China, No. 2015A030313351; Science and Technology Planning Project of Guangdong Province of China, No. 2014A020221052; Project of Administration of Traditional Chinese Medicine of Guangdong Province of China, No. 20164010

Abstract:

BACKGROUND: It is an effective strategy for the treatment of osteoporosis to promote the proliferation and differentiation of osteoblasts, but the influence of inokosterone on osteoblast proliferation and differentiation is rarely reported.

OBJECTIVE: To investigate the effects of inokosterone on the proliferation and differentiation of primary osteoblasts and the underlying molecular mechanism.

METHODS: Primary osteoblasts from neonatal Sprague-Dawley rats were isolated via the second enzyme digestion, and the cells were then cultured in osteogenic induction medium and identified. Cell counting kit 8 assay was performed to evaluate the effect of different concentrations of inokosterone (1, 5, 10 mg/L) on cell viability of primary osteoblasts. Early differentiation ability of osteoblasts was evaluated by alkaline phosphatase staining and alkaline phosphatase activity assay. To evaluate the mineralization ability of osteoblasts, alizarin red staining was performed to observe the number of calcium nodules. The expression level of osteogenic genes was detected by RT-qPCR at different time points. Furthermore, MDC staining was also used to observe the number of autophagosomes.

RESULTS AND CONCLUSION: Compared with the control group, inokosterone could inhibit the cell viability of primary osteoblast to some degree (P < 0.05) while significantly promoting alkaline phosphatase activity and mineralized nodules formation (P < 0.05). In addition, inokosterone upregulated the expression of osteogenic genes such as Collagen I, osteoprotegerin, osteopontin, osteocalcin and increased the number of autophagosomes. To conclude, inokosterone can promote osteogenic differentiation by upregulating osteogenic genes expression and activating autophagy of primary osteoblasts.

Key words: inokosterone, neonatal Sprague-Dawley rats, osteoblasts, osteogenic differentiation, autophagy, osteoporosis

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