Electroacupuncture stimulation at CV4 prevents ovariectomy-induced osteoporosis in rats via Wnt-ß-catenin signaling.

The present study aimed to investigate the effect of electroacupuncture stimulation at CV4 (also termed Guanyuan) on femoral osteocalcin also termed bone gla protein (BGP), alkaline phosphatase (ALP), bone mineral density (BMD) and biomechanics, as well as the Wnt­ß­catenin signaling pathway in rats with postmenopausal osteoporosis. Female Sprague­Dawley rats (4.5­months old) were randomly divided into sham, Ovx, CV4 and mock groups (n=10/group). With the exception of those in the sham group, the rats were ovariectomized to induce postmenopausal osteoporosis. The rats in the CV4 and mock groups were given electroacupuncture at CV4 and non­acupoint, respectively. The rats in the Ovx model and sham groups underwent identical fixing procedures, but did not undergo electroacupuncture. Following treatment, hematoxylin and eosin staining was used to observe morphological changes in the left femoral trabecular bone, and a three­point­bending test was used to analyze femur biomechanics and determine the BMD. In addition, an enzyme­linked immunosorbent assay was used to measure the serum levels of ALP/BGP and reverse transcription­quantitative polymerase chain reaction was used detect the expression levels of Wnt3a, ß­catenin and Runx2. In the present study, it was demonstrated that electroacupuncture at CV4 significantly improved the osteoporotic morphological changes that occurred in the ovariectomized rats, increased serum ALP and BGP levels, enhanced the maximum and fracture loads, increased BMD (P<0.01), and activated the Wnt­ß­catenin signaling pathway. These findings demonstrated that electroacupuncture stimulation at CV4 affected bone formation and promoted bone metabolism in rats with postmenopausal osteoporosis, possibly by activating the Wnt­ß­catenin signaling pathway.

[Effect of electroacupuncture of "Guanyuan" (CV 4) on Wnt-ß-catenin signaling in osteoporosis rats].

OBJECTIVE: To observe the effect of electroacupuncture of "Guanyuan" (CV 4) on bone miner density, bone biomechanics, serum osteocalcin (BGP) and alkaline phosphatase (ALP) contents, and femoral osteoblastic Wnt-ß-catenin signaling in postmenopausal osteoporosis (PMOP) rats. METHODS: Forty female SD rats were randomly divided into sham operation (sham), model, EA-CV 4, and EA-non-acupoint (below the costal region) groups (n = 10 in each group). The PMOP model was established by performing an ovariectomy in the rats of the later 3 groups. EA (2 Hz, 1 mA) was applied to CV 4 for 20 min, once daily for one month, with one day's break between every 10 days. After the treatment, serum BGP and ALP contents were detected using ELISA, the right femoral bone miner density and biomechanics (maximum load, breakage load) were measured using a Dual Energy X-Ray Bone Densitometer and a Universal Material Testing Instrument, respectively. The expression levels of Wnt 3 a mRNA, ß-catenin mRNA and the bone-specific factors runt-related transcription factor 2 (Runx 2) mRNA of the femoral bone tissue were determined by real time RT-PCR. RESULTS: HE staining results suggested EA of "Guanyuan" (CV 4) can improve the morphological changes (trabeculae) of osteoporosis in ovariectomized rats. Compared with the control group, femoral maximum load and breakage load, bone density and serum BGP and ALP contents, femoral Wnt 3 a mRNA, ß-catenin and Runx 2 mRNA expression levels, and femoral Wnt 3 a.and ß-catenin immunoactivity were significantly down-regulated in the ovariectomized rats (model group) (P < 0.05). Following EA treatment, all the decreased levels of femoral maximum load and breakage load, bone density and serum BGP and ALP, femoral Wnt 3 a mRNA and protein, ß-catenin mRNA and protein and Runx 2 mRNA expression were obviously reversed in the EA-CV 4 group (P < 0.05) rather than in the non-acupoint group (P > 0.05). CONCLUSION: EA of CV 4 can improve the femoral biomechanics, increase bone density in OVX rats, which are associated to its effects in up-regulating levels of serum BGP and ALP, femoral Wnt 3 a mRNA and protein, ß-catenin mRNA and protein and Runx 2 mRNA expression, suggesting an involment of improved bone metabolism and activation of osteoblastic Wnt-ß-catenin signaling.

Duhuo Jisheng Decoction­containing serum promotes proliferation of interleukin­1ß­induced chondrocytes through the p16­cyclin D1/CDK4­Rb pathway.

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 interleukin­1ß (IL­1ß) 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 IL­1ß­culture 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, cyclin­dependent 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 2­h serum, and the degenerative chondrocyte model was successfully reproduced by IL­1ß­treatment for 24 h. The mRNA and protein expression levels of cyclin D1, CDK4 and Rb in the DHJSD serum­treated 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 DHJSD­containing serum is able to promote IL­1ß­induced 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.

[Effect of electroacupuncture of "Mingmen" (GV 4) on bone morphogenetic protein-2 expression in osteoporosis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Mingmen" (GV 4) on the bone morphogenetic protein-2 (BMP-2) content and biomechanics in osteoporosis rats so as to explore its mechanism underlying improvement of osteoporosis. METHODS: Fifty female SD rats were randomized into sham operation (sham), model, EA-GV 4, EA-non-acupoint (non-acupoint) and estrogen (medication) groups, with 10 rats in each group. Postmenopausal osteoporosis model was established by removing the rats' bilateral ovaries under anesthesia. EA (2 Hz/15 Hz, 1.0 mA) was applied to "Mingmen" (GV 4) or non-acupoint for 20 min, once daily for 30 times, with one day's interval between every two 10 times. Rats of the medication group were lavaged with Pentanoic Acid Estradiol (25 microg/mL, 2 mL/500 g), once every day (the dosage of estradiol was adjusted according to their body weight) continuously for 1 month. Rats of the model and sham groups experienced the fixing and fastening procedures as the other rats in the EA and medication groups. After intervention, the BMP-2 expression level of the femoral bone tissue, and bone biomechanical values were determined by immunohistochemistry and three-point bending tests, respectively. RESULTS: (1) In comparison with the sham operation group, the femoral biochemical maximum load and fracture load values were significantly decreased in the model group (P < 0.05). While compared with the model group, the biochemical maximum load and fracture load values were obviously increased in the EA-GV 4 and medication groups (P < 0.05), but not in the non-acupoint group (P > 0.05). (2) Compared with the sham group, the femoral BMP-2 expression of model group was significantly decreased (P < 0.05), compared with the model group, the expression of BMP-2 of GV 4 and medication groups significantly increased (P < 0.05). CONCLUSION: EA-GV 4 intervention can improve bone biomechanical changes in osteoporosis rats.

Duhuo Jisheng Decoction promotes chondrocyte proliferation through accelerated G1/S transition in osteoarthritis.

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 two­month­old 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 RT­PCR 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 DHJSD­treated 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.

Electroacupuncture promotes chondrocyte proliferation via accelerated G1/S transition in the cell cycle.

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.