Study On The Repair Mechanism Of Sonic Hedgehog On Degenerative Knee Arthritis Via Up-regulation Of Timp-3 In Mice

Objective:Hedgehog(Hh)signaling pathway is extensive and critical roles in embryonic development and tissue homeostasis in vertebrates.It has been reported that Hh signaling is involved in the initiation and progress of osteoarthritis,however,the underlying mechanisms are still unclear.The purpose of our study is to investigate the role and mechanism of sonic hedgehog(Shh)factor in the model of degenerative knee arthritis induced by anterior cruciate ligament excision in mice.Methods:1.Four weeks after the degenerative knee osteoarthritis mouse model was established,Shh-N protein factor was injected into the knee joint cavity of the model group and sham-operated group.The levels of IL-1β and TNF-α in the synovial fluid were detected by ELISA,and the expression of Timp-3 in cartilage tissue was detected by qRT-PCR.2.The changes of spontaneous behavior(climbing time and long-distance movement ability)were recorded and analyzed by the behavior observation registration and analysis system of experimental animals.The behavior was measured before(time 0)and after operation.The behavior was measured every 4 weeks to 12 weeks.The analytical measurements were carried out between 4 p.m.and 8 a.m.on day 2.Since the mice needed to adapt to the test environment,the first two hours of data were excluded from the final analysis.Measure the weight before each assessment.At 13 weeks,the mice were killed and the knee joints were dissected.3.All data were expressed as mean ± standard deviation,and were statistically analyzed with graphpad prism 5.0.T-test was used for comparison between the two groups,and one-way ANOVA test was used for comparison between multiple groups.P < 0.05 indicated that the difference was statistically significant.Results:ELISA results indicated that compared with the sham-operated group,the levels of IL-1β and TNF-α in the osteoarthritis lumen of degenerative knee osteoarthritis model mice were significantly increased(24.5±5 vs 251±17.5 pg/mL,P <0.001;16.5±3.4 vs 197±15.6 pg/mL,P <0.001);compared with the PBS control group,the symptoms of osteoarthritis in Shh-N group were significantly alleviated,because the levels of inflammatory factors IL-1β and TNF-α in the synovial fluid were significantly lower than the PBS control group(116±8.7 vs 251±17.5 pg/m L,P <0.001;89±7.8 vs 197±15.6 pg/mL,P <0.001),the climbing time and long-distance movement ability of mice were significantly improved(1 700±185.6 vs 116±8.7 s,P <0.001;174±162.3 vs 132±34.5 m,P <0.001,respectively);the expression of metalloproteinase inhibitor 3(Timp-3)gene in cartilage tissue was significantly increased by qRT-PCR(5.02±0.34 vs 2.4±0.61 fold,P <0.001),and the A Disintegrin and Metalloprotease with Thrombospondin motifs 4(ADAMTS-4)level decreased significantly(P < 0.001).Conclusion:The study indicated that Shh factor can significantly improve the recovery of degenerative knee arthritis in mice.The mechanism may be through up-regulation of Timp-3 gene expression and degradation of endogenous polyproteoglycan enzymes.