RESUMO
Objective To analyze the effect of a Chinese medicine Zhengqingfengtongning on the expression of aggrecanase-1(ADAMTs-4)and aggrecanase-2(ADAMTs-5)in cartilage tissue of knee joint in rabbit models of osteoarthritis,and to study the mechanism of action of Zhengqingfengtongning in treatment for osteoarthritis. Methods 32 New Zealand rabbits were randomly divided into the observation group and the model group, and 16 healthy New Zealand rabbits were chosen as the blank group. The blank group did not receive the intervention treatment, the model group received physiological saline in gastric gavage, the observation group was given the Zhengqingfengtongning by gastric gavage. The soft tissue samples of knee joint in the three groups were taken at 4 weeks after drug intervention. The joint morphology,joint fluid and articular cartilage were observed by histopathology. The ADAMTs-4 and ADAMTs-5 mRNA were detected by RT-PCR. The data were then statistically analyzed. Results The grades of articular cartilage and Mankin's score of the model group were significantly higher than those of the observation group and blank group,the grades of articular cartilage and Mankin's score of the observation group were significantly higher than those of the blank group, and the difference between the two groups was statistically significant(P< 0.05). The ADAMTs-4 and ADAMTs-5 mRNA expressions of the model group were significantly higher than those of the observation group and blank group. The ADAMTs-4 and ADAMTs-5 mRNA expressions of the observation group were significantly higher than those of the blank group, and the difference between the two groups was statistically significant(P< 0.05). Conclusions The Zhengqingfengtongning can improve the degree of joint lesions in osteoarthritis animal models,which may be related to inhibiting the expression of ADAMTs-4 and ADAMTs-5.
RESUMO
OBJECTIVE: To observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA). METHODS: BMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups (n=3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×107 cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR. RESULTS: No significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group (P<0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group (P<0.05), but the indicators in 3 groups were significantly higher than those in the normal control group (P<0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower (P<0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group (P<0.05). CONCLUSIONS: Lentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.