ABSTRACT
Objective:To explore the effects of Alpha-2-macroglobulin-rich serum (A2MRS) on knee post-traumatic osteoarthritis (PTOA).Methods:The knee PTOA models were constructed by transection of the anterior cruciate ligament (ACL) in 80 SD male rats, aged 2 months and weighing from 250 to 300 g, which were randomized into 4 groups ( n=20): a high dose group (A2MRS containing 20 μg/μL A2M administered), a low dose group (A2MRS containing 10 μg/μL A2M administered), a positive control group (normal saline administered), and a blank control group (the knee joint cut pseudooperatively and normal saline administered). HE, toluidine blue staining, safranine O staining, modified Mankin scoring and Osteoarthritis Research Society International (OARSI) scoring were conducted to evaluate and compare the therapeutic effects of A2MRS on the knee PTOA among the 4 groups. Results:The rat cartilage was thinner with patchy and cracked surface, and the chondrocytes were reduced and distributed unevenly in the positive control group, compared with the blank control group. The modified Mankin score (3.89±0.93) and OARSI score (10.05±0.72) in the positive control group were significantly higher than those in the blank control group (0.67±0.07 and 3.10±0.29) ( P<0.05). The rat cartilage was thicker with basically complete and crack-free surface, and the chondrocytes were increased and distributed more evenly in the high dose group and the low dose group, compared with the positive control group. The modified Mankin scores (1.33±0.50 and 1.56±0.53) and OARSI scores (6.30±0.64 and 4.75±0.66) in the high dose group and the low dose group were significantly lower than those in the positive control group ( P<0.05). However, there were no such differences between the high dose group and the low dose group ( P>0.05). Conclusion:A2MRS effectively delays the pathological process of knee PTOA.
ABSTRACT
Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA.Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers.A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments.Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively.HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor.Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups.Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression.ResultsRestriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully.The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+mmu-miR-30b, pmirGLO+mmu-miR-30b inhibitor, pmirGLO-mFoxO3+mmu-miR-30b, pmirGLO-mFoxO3+mmu-miR-30b+mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively.Compared with pmirGLO+mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+mmu-miR-30b group (P0.05).In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05).Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05).Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b.There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA.