Experiment Study On Effect Of Duhuo Jisheng Decoction On Osteoarthritis Chondrocyte Proliferation And Apoptosis

ObjectiveTo discuss the effect of Duhuo Jisheng Decoction (DHJSD) on osteoarthritis chondrocyte proliferation and apoptosis.Methods1. After one week of acclimation, healthy and clean, two-month-old Sprague Dawley rats of average gender (n=144) were randomly divided into blank group (n=36) and osteoarthritis model group (n=108). Osteoarthritis model was induced in double knees of rats by injecting0.2ml of4%papain solution on days1,4and7. After successfully establish the model, the animals were randomly divided into three groups:the model group (received an equivalent amount of saline only), the positive control group [received a clinical oral dose of Glucosamine Hydrochloride Capsules (0.15g/kg/day)] and the treatment group [received a clinical oral dose of DHJSD (9.3g/kg/day)]. The blank group also received an equivalent amount of saline only. Every treatment course includes two weeks. There are totally four courses.2. After every two courses, the animals were sacrificed and tibial plateau cartilage specimens were obtained. The morphological changes were observed under an optical microscope following staining with hematoxylin and eosin (H&E) and by transmission electron microscopy (TEM). The protein levels of Cyclin D1、CDK4、Rb、p16、Bcl-2、Bax、 Cytochrome c、Caspase-9and caspase-3were measured by immunohistochemistry. The mRNA levels of Cyclin D1、CDK4、 Rb and p16were measured by RT-PCR.3. A total of144two-month-old Sprague Dawley rats of average gender were randomly divided into two groups:the DHJSD group (n=108) treated with a dose of9.3g/kg/day DHJSD, which is the equivalent dosage for human in clinical use and the blank group (n=36) treated with an equivalent amount of saline only. All testing drugs and saline were administered via gastric gavage twice a day in the morning and afternoon for7consecutive days. The daily doses were given2h apart on the seventh day. Animals were anesthetized by intraperitoneal injection of2ml/kg2%pentobarbital sodium, and arterial blood in DHJSD group was collected from the abdominal aorta at1h,2h and3h after the final dose in DHJSD group, respectively and at2h in blank group.4. Cartilage was isolated from the knee joint of20～30health and clean, four-week-old SD rats, and used to establish a cultivation system of chondrocytes in vitro. The second-generation chondrocytes were seeded into96-well plates at a density of1.0x105cells/ml in0.1ml of medium. After synehronization, the cells were treated with10%、15%、20%、30%concentrations of different sampling time of DHJSD serum for24,36,48,60and72h, respectively. Cell viability was assessed by the MTT colorimetric assay.5. The degenerative chondrocyte model was established by exposing to10ng/ml IL-1β for24h, and verified by optical microscopy analyses and immunohistochemical staining of Collagen II analyses.6. After treated degenerative chondrocytes with the best intervention condition for24h,48h and72h, cell proliferation was detected by MTT assay and DNA staining followed by FACS analysis; cell apoptosis was detected by Flow Cytometry Analysis with Annexin V-FITC/PI Staining; mitochonrial membrane potential (ΔΨm) was measured by Flow Cytometry analysis with JC-1staining; Caspase-9,-3activation was analysesed by spectrophotometry; and the mRNA and protein expression levels of Cyclin D1, CDK4, Rb, p16, Bcl-2and Bax were measured by RT-PCR and Western Blot, respectively.Results1. The knee lateral X ray showed normal joint space, smooth articular surface, smooth lines dense shadow on joint edge and uniformity bone density in subchondral in normal group, while in model group, the joint space became narrow; the articular surface was not flat; on the edge of damage cartilage appeared bone hyperplasia and sclerosis; joint marginal appeared osteophyte formation. HE staining and transmission electron microscopy (TEM) results showed DHJSD and Glucosamine Hydrochloride Capsules could both promote OA cartilage repair and chondrocytes proliferation with no significant difference. DHJSD and Glucosamine Hydrochloride Capsules could both promote the mRNA and protein expression of Cyclin Dl, CDK4, Rb and inhibit p16, Cytochrome c, caspase-9, caspase-3mRNA and protein expression with no significant difference.2. Identification of the third generation chondrocytes with immunohistochemical staining of Collagen Ⅱ, the cytoplasm appeared brown and no coloration in the nucleus; with alcian blue staining, the nucleus were stained light blue, a lot of secretion particles and vesicles appeared in the cytoplasm. The best sampling time and concentration of DHJSD serum on promoting chondrocyte proliferation were2hour and10%, respectively. Compared with the normal chondrocytes,10ng/ml IL-1β induced chondrocytes became larger with some finger-like protrusions on the edge, and the cell membrane and cytoplasm was not clear. Meanwhile, the IL-1β-induced cells were polygonal changed, along with declining refraction. Besides, the immunohistochemical staining of Collagen II analyses showed that the brownish yellow staining in cytoplasm became shallow. After treated with DHJSD2h serum, the percentage of G0/G1phase cells was gradually decreased; however, the percentage of S phase cells and the proliferation index were increased. DHJSD2h serum treatment inhibited the apoptosis of degenerative chondrocytes, the loss of plasma membrane asymmetry (externalization of phosphatidylserine), collapse of mitochondrial membrane potential, and activation of caspase-9and caspase-3. DHJSD serum could promote the mRNA and protein expression of Cyclin D1, CDK4, Rb, Bcl-2and inhibit the mRNA and protein expression of p16, Bax.Conclusion1. DHJSD could promote osteoarthritis chondrocytes proliferation by promoting G1/S transition, in vivo and vitro.2. DHJSD could inhibit the mitochondrion-dependent pathway of apoptosis in osteoarthritis chondrocytes, in vivo and vitro.