Abstract. Duhuo Jisheng Decoction (DHJSD), a well known traditional Chinese folk medicine, is used for eliminating stagnation, removing blood stasis, promoting blood circulation and alleviating pain; it is commonly used for the treatment of various diseases, including osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effects of OA remain unclear. In the present study, the effects of DHJSD on the morphology of articular cartilage and the G1/S cell cycle progression in chondrocytes, as well as the underlying mechanisms, were investigated. A total of 27 two-month-old male Sprague Dawley rats were randomly divided into 3 groups: the control group (no papain-induced OA; received an equivalent amount of saline only), the model group (papain-induced OA; received an equivalent amount of saline only) and the DHJSD group [papain-induced OA; received a clinical oral dose of DHJSD (9.3 g/kg/day)]. After 8 consecutive weeks of treatment, the morphological changes in articular cartilage were observed under an optical microscope and by transmission electron microscopy (TEM) and the mRNA and protein expression levels of cyclin D1, CDK4, CDK6, retinoblastoma protein (Rb) and p16 were measured by RT-PCR and immunohistochemistry, respectively. Treatment with DHJSD significantly improved the arrangement of collagen fibers in the articular cartilage, as well as its structure and reduced cell degeneration compared with the model group. The mRNA and protein expression levels of cyclin D1, CDK4, CDK6 and Rb in the DHJSD-treated group were significantly increased compared with those in the model group, whereas p16 expression was significantly downregulated. Taken together, these results indicate that DHJSD treatment promotes chondrocyte proliferation by promoting the G1/S checkpoint transition in the cell cycle and by upregulating the expression of cyclin D1, CDK4, CDK6 and Rb and downregulating the expression of p16 and this may, in part, explain its clinical efficacy in the treatment of osteoarthritis.

Duhuo Jisheng Decoction (DHJSD) is a traditional Chinese herbal medicine that has multiple uses, including as a treatment for osteoarthritis (OA). However, the molecular mechanisms underlying the therapeutic effects of DHJSD on OA remain unknown. In the present study, a serum pharmacological method was applied to investigate the effects of DHJSD on the proliferation of chondrocytes treated with interleukin‑1β (IL‑1β) in vitro. This is a cell model commonly used to reproduce the mechanisms involved in degenerative arthropathies, including OA. The most effective intervention conditions of DHJSD serum were examined by MTT assay. The degenerative chondrocyte model was established by IL‑1β‑culture for 24 h, and was verified by optical microscopy and immunohistochemical analyses. Following the successful establishment of the degenerative chondrocyte model, the chondrocytes were subsequently randomly divided into two groups: The blank serum group and the DHJSD treatment group. Subsequent to treatment with the corresponding serum, cell proliferation was detected by MTT assay and DNA staining followed by FACS analysis, and the mRNA and protein expression levels of cyclin D1, cyclin‑dependent kinase 4 (CDK4), retinoblastoma tumor suppressor protein (Rb) and p16 were measured by reverse transcription polymerase chain reaction and western blotting, respectively. The results indicated that the most effective condition for the promotion of chondrocyte proliferation was 10% concentration of DHJSD 2‑h serum, and the degenerative chondrocyte model was successfully reproduced by IL‑1β‑treatment for 24 h. The mRNA and protein expression levels of cyclin D1, CDK4 and Rb in the DHJSD serum‑treated cells were significantly increased compared with those in the blank serum group, whereas p16 expression was significantly downregulated. These results indicate that treatment of cells with DHJSD‑containing serum is able to promote IL‑1β‑induced chondrocyte proliferation by promoting G1/S phase transition via modulating the expressions of cyclin D1, CDK4, Rb and p16, which contribute to the clinical efficacy of DHJSD in OA.

The aim of the present study was to investigate the effects of electroacupuncture (EA) on the proliferation of chondrocytes and the molecular mechanism(s) involved. Passage 2 chondrocytes were randomly divided into four groups and treated with EA or nocodazole. After treatment, cell proliferation was determined using an MTT assay and DNA staining followed by FACS. The mRNA expression levels of cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, phosphorylated retinoblastoma (pRb) and P16 were detected by RT-PCR, and the protein levels of cyclin D1, CDK4, CDK6, pRb and P16 were detected by western blotting. EA treatment significantly increased cell viability in a time-dependent manner and decreased the number of G0/G1 and G2/M phase chondrocytes and increased the number of S phase cells. The mRNA and protein levels of cyclin D1, CDK4, CDK6, (p)Rb and P16 consistently demonstrated a reverse trend with the levels in the chondrocytes treated with nocodazole. The expression levels of cyclin D1, CDK4, CDK6 and Rb were higher in chondrocytes receiving EA treatment when compared to levels in the untreated cells while expression of P16 was lower. In conclusion, EA treatment promotes chondrocyte proliferation via promotion of G1/S checkpoint transition in the cell cycle dependent on the activity of the P16-cyclin D1-CDK4/6-pRb pathway and this may, in part, explain its clinical effect in the treatment of osteoarthritis.