The Expression Of P53, Caspase-3, Bcl-2 In Rats With Osteoarthritis

To detect the expression of p53, caspase - 3, bcl - 2 protein and mRNA in articular cartilage of knee joints in rats with experimental osteoarthritis ( OA) and to determine the functions of these three important apoptosis - related factors in OA.MethodsThe animal models of rat knee joint experimental osteoarthritis were established with Hulth method. After anesthesia with Petobarbital injected into abdominal cavity according to the weight, medial collateral ligament ,medial meniscus , anterior and posterior cruciate ligament of right knees were cut off by turns under the operating mgcroscop. Left knees were untreated as controls. All were divided into three groups randomly with 12 rats for each group,and raised for 4,8, 12 weeks. The articular cartilages of medial tibial plateau were harvested. Morphological changes were found through observing the appearance of cartilage and H - E stains. The expression of p53,caspase - 3 and bcl - 2 protein was detected through immunohistochemistry and ratios of positive cells were recorded. The expression of p53, caspase - 3 and bcl - 2 mRNA was detected through RT - PCR techniques and the average gray of each electrophoresis band was measured through computer software. The ratio to β - actin was defined as the relative value of mRNA. Data were analysed through CS2000 statistical software.ResultsThe cartilages of control knees in three groups were slightly white - blue, smooth and sheen. In OA models, cartilage tarnished in W4 group; tarnished ,became slightly white and rough in W8 group; fissured, rough and uneven in surface, slightly white - yellow in W12 group and even osteophyte occurred in medial side of tibial plateau (3/12). H - E stains showed that the chondrocytes were compact and well - distributed in controls, while in 0A, chondrocytes were less symmetrical and swollen in W4 group; chondrocytes were sparse, irregular, swollen and some assembled and more vacuolar cells occured in W8 group; chondrocytes became significantly sparse and some assembled into clusters, more vacancies and larger vacuoles were seen in W12 group. Immunohistochemistry showed the expression of p53, caspase - 3 , bcl - 2 protein in these three groups. p53 was mainly nucleolus -stained ;caspase -3 was mainly cytoplasm - stained; bcl - 2 was stained on both. There was no staining in the PBS treated in place of antibody. The ratios of p53, caspase - 3 , bcl - 2 positive cells statistically differed in OA models from that in controls in all groups (P < 0. 05 ) , which showed the increase of p53 and caspase - 3 protein expression and the decrease of bcl - 2 protein expression in OA. RT - PCR showed the expression of mRN A was consistent with that of protein. Moreover, there was no difference of p53, caspase - 3 , bcl - 2 mRN A expression in controls among these three groups. But in OA, p53 and caspase -3 mRNA expression was statistically different among W4,W8,W12 group,while bcl -2 mRNA expression showed no difference. This showed the increase of p53 and caspase - 3 mRNA expression which was direct correlated with time and the decrease of bcl - 2 mRNA expression without the correlation in OA.ConclusionThis study shows that the expression of p53, caspase - 3 , bcl - 2 protein and mRNA in articular cartilage alters from the early stage of OA. The up - expression of p53 and caspase - 3 mRNA is close correlated with OA process, while the down - expression of bcl - 2 mRNA is not. In conclusion, the balance between apoptosis and anti - apoptosis is disturbed in articular cartilage of OA with the dominance of apoptosis,which is a possible mechanism of OA.