RESUMO
OBJECTIVE To investigate the effects of β-sitosterol on the function of rheumatoid arthritis （RA） fibroblastic synoviocytes MH7A cells and its mechanism. METHODS Network pharmacology was adopted to screen the targets of β-sitosterol and the targets for the treatment of RA. After the intersection of them， topological analysis was performed to find the most critical target in the treatment of RA. MH7A cells were treated with different concentrations （0， 5， 10， 20， 40 μmol/L） of β-sitosterol， and CCK-8 was used to assay cell viability for screening the optimal concentration of β-sitosterol. MH7A cells were induced by 10 ng/mL TNF-α in vitro and treated with β-sitosterol （the optimum concentration）. CCK-8 and EdU were used to detect the ability of cell proliferation. Scratch experiment and Transwell invasion assay were used to analyze cell migration and invasion. The levels of interleukin-1β （IL-1β） and IL-6 in cell supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expressions of peroxisome proliferator-activated receptor α （PPARα） were measured by qRT-PCR and Western blot， respectively. The siRNA targeting PPARα was transfected into MH7A cells， and the effects of β-sitosterol on cell proliferation， migration， invasion， the secretion of inflammatory factors and the expression of PPARα after PPARα knockdown were detected by the above experimental methods. RESULTS PPARα was the most critical target of β-sitosterol in the treatment of RA. The optimal concentration of β-sitosterol was 20 μmol/L. Compared with model group， β-sitosterol decreased the viability of MH7A cells， and the number of proliferating cells also decreased significantly （P＜0.05）； the cell migration rate and the number of cell invasion decreased significantly （P＜0.05）. The levels of IL-1β and IL-6 were also significantly decreased （P＜0.05）， and the mRNA 15 and protein expression levels of PPARα were significantly increased （P＜0.05）. Compared with negative control small interfering RNA group， after PPARα knockdown， the cell viability increased by about 35.6% （P＜0.05）， the number of cell proliferation， the cell migration rate and the number of cell invasion increased significantly （P＜0.05）， and the levels of IL-1β and IL-6 also increased significantly （P＜0.05）. CONCLUSIONS β-sitosterol could effectively inhibit the proliferation， migration， invasion and secretion of inflammatory factors in MH7A cells， the mechanism of which may be associated with activating PPARα pathway.
RESUMO
BACKGROUND: Bone morphogenetic protein 2 is a most widely studied and osteogenic-inducing bone morphogenetic protein. However, simple bone morphogenetic protein can be diluted by tissue fluid and decomposed by protease after implantation, so it is difficult to maintain sustained drug concentration or play an effective role in bone induction. OBJECTIVE: To observe the effect of repairing osteoporotic fracture through calcium phosphate cement/fibrin glue composite as the carrier of bone morphogenetic protein 2. METHODS: Fifty-four female Sprague-Dawley rats were selected to remove bilateral ovaries for making osteoporosis models. Three months later, the middle femoral fracture models were made, and then randomized into three groups, with 18 rats in each group. Kirschner wire fixation group was injected nothing. The fracture end was injected with 0.5 mL calcium phosphate cement/fibrin glue (composite group) or calcium phosphate cement/fibrin glue loaded with recombinant human bone morphogenetic protein 2 (loaded group) . At 4 and 12 weeks after fracture, X-ray examination, micro-CT examination, biomechanical three-point bending test and pathological observation were performed. RESULTS AND CONCLUSION: (1) The fracture healing score in the loaded group was higher than that in the other two groups (P < 0.05) . (2) The bone volume fraction, trabecular thickness and number of trabeculae at 4 and 12 weeks in the loaded group were higher than those in the other two groups (P < 0.05) , and the trabecular segregation was lower than that in the other two groups (P < 0.05) . (3) The maximum load and stiffness at 4 and 12 weeks in the loaded group were higher than those in the other two groups (P < 0.05) , and the elastic modulus at 4 weeks after fracture was higher than that in the other two groups (P < 0.05) . (4) Fibrocartilage callus was mainly seen at 4 weeks after fracture in the Kirschner wire fixation group, and the callus was reconstructed into lamellar bone at 12 weeks after fracture with less callus content. Obvious fibrocartilage callus appeared at 4 weeks after fracture in the composite and loaded groups, and the callus was reconstituted into a plate-like bone at 12 weeks. (5) These results imply that the calcium phosphate cement/fibrin glue composite loaded with recombinant human bone morphogenetic protein 2 can promote the healing of osteoporotic fracture and improve bone strength.