| Objective To verify the regulation of Cyclin D1 and downstream signaling proteins on osteoblast proliferation cycle and explore the mechanism of Jiangu granule in promoting the proliferation of osteoblasts. Methods The osteoblast-like cell lines UMR-106 was knock-down of the Cyclin D1 gene by CRISPR/Cas9 gene editing and divided into 2 groups: gene knock-down + saline group (group 1) and gene knock-down + Jiangu granule group (group 2). The virus empty vector cell (group 3) was used as negative control. Cells without gene knock-down were divided into normal cells + saline group (group 4) and normal cells + Jiangu granule group (group 5). The cells were respectively treated by saline or Jiangu granule drug-containing serum for 24, 48 and 72 hours, and CCK8 method was used to detect cell proliferation. Gene expression of Cyclin D1, Cyclin E, CDK 2, CDK 4, Rb and E2F-1 were detected by Real time PCR. Results Western-bolt detection showed that the expression of Cyclin D1 protein decreased significantly in osteoblasts with gene knock-down. The results of CCK8 test shows that compared with normal cells + saline group, normal cells + Jiangu granule group’s multiplication rate was obviously increased (P < 0.05), negative control group had no significant difference (P > 0.05), while gene knock-down + saline group and gene knock-down + Jiangu granule group had much slower rate (P < 0.05 ). Compared with normal cells + Jiangu granule group, there was a significant decrease in gene knock-down + Jiangu granule group (P > 0.01), and there was no significant difference between gene knock-down + saline group and gene knock-down + Jiangu group (P > 0.05). Real time PCR demonstrated that compared with normal cells + saline group’s mRNA expression of Cyclin D1, CDK2, CDK4, Cyclin E, Rb and E2F-1, the expression in normal cells + Jiangu granule group was distinctly increased (P<0.05 or P<0.01), while negative control group had no significant difference (P > 0.05), and the expression in gene knock-down + saline group and gene knock-down + Jiangu granule groups were significantly reduced (P＜0.05 or P＜0.01). Compared with normal cells + Jiangu granule group’s, the gene knock-down + Jiangu granule group had decreased rate (P > 0.01), while there was no significant difference between gene knock-down + saline group and gene knock-down + Jiangu group (P > 0.05). Conclusion Cyclin D1 and its downstream signaling proteins are the key factors to regulate the proliferation of osteoblasts. The function of Jiangu granule in promoting osteoblast proliferation may be achieved by regulating Cyclin D1 and its downstream signaling proteins. |