The Regulation Function Of Pain Occurrence And The Formation Of Traumatic BME Of Knee Joint In HIF-1 α/COX-2 Signaling Pathway

Objective:By way of making rabbit knee traumatic bone marrow edema（BME）model to study the relationship between the expression of HIF-1α/COX-2 signal pathway,the degree of knee joint traumatic BME and the knee joint pains to explore the causes of forming of knee joint traumatic BME and pain,so as to provide guidance for clinical treatment of traumatic BME of knee joint.Methods:（1）The traumatic bone marrow edema model and non bone marrow edema model were made in 46 New Zealand white rabbits by free fall impact,The rabbits were divided into low-energy bone marrow edema group（12 rabbits）,medium-energy bone marrow edema group（12 rabbits）,high-energy bone marrow edema group（12 rabbits）and non bone marrow edema group（10 rabbits）.（2）On the 1st,7th,21st and 42nd day after modelling,MRI examination was performed on the knee joint of New Zealand white rabbits in different group to examined and measured the region of interest area（ROIA）which reflecting the severity of BME.Mechanical pain threshold measurement method was used to measure von Frey pain threshold for each group.（3）Some New Zealand white rabbits in each group were sacrificed on the 1st,7th,21st and 42nd day after modelling,and the subchondral bone tissue in BME knee area of the traumatic BME model was extracted.Western blot（WB）was used to detect HIF-1αin subchondral bone tissue in BME area and COX-2 protein expression.Enzyme-linked immune sorbent assay（ELISA）was used to detect the expression of tumour necrosis factor（TNF-α）in subchondral bone tissue of BME area.（4）The von Frey pain threshold,HIF-1α,COX-2 and TNF-αwere compared between traumatic BME of knee joint and non-BME group of knee joint soft tissue injury on the first day after modelling by independent sample test.（5）Different energy group of Traumatic BME of knee joint HIF-1α,COX-2 and the relative expression of TNF-α,von Frey pain threshold and ROIA were compared by one-way ANOVA,and LSD method was used for post-test.（6）Linear correlation analysis the relationship between von Frey pain threshold,HIF-1α,COX-2 and TNF-αrelative expression quantity and ROIA.（7）With von Frey pain thresholds dependent variable Y,HIF-1αcontent as independent variable X1,COX-2 content as X2,TNF-αcontent as X3,ROIA content as X4 were used for multiple linear regression analysis to analyze the influencing factors of HIF-1α/COX-2 signal pathway expression in BME pain.Results:（1）MRI showed high signal shadow of BME in all New Zealand rabbits with knee joint fat suppression T2WI model groups,and ROIA value was positively correlated with traumatic energy.There was no BME formed in the non-BME group of knee joint soft tissue injury.（2）ROIA of knee joint traumatic BME was measured according to Yadama method,and von Frey pain threshold of New Zealand white rabbits was measured by mechanical pain measurement method.（3）Knee joint traumatic BME group on the first day after modelling HIF-1α,COX-2,TNF-αwere compared with those in non-BME group the expression comparison was statistically significant（P<0.05）,von Frey pain threshold was not statistically significant between the two groups（P>0.05）.（4）Traumatic BME of knee joint on the first day after modelling,the differences of HIF-1α,COX-2,TNF-α,von Frey pain threshold and ROIA among the three groups were statistically significant（P<0.05）,on the 7th day after modelling,variance analysis of HIF-1α,COX-2,TNF-α,ROIA and von Frey the difference of pain threshold was statistically significant（P<0.05）.On the 21st day after modelling,COX-2,TNF-α,ROIA and von Frey the difference of pain threshold was statistically significant（P<0.05）,there was no significant difference in comparison of HIF-1α（P>0.05）.On the 42nd day after modelling,there were significant differences in TNF-α,ROIA and pain threshold among the three groups by analysis of variance（P<0.05）,HIF-1α,COX-2 had no significant difference（P>0.05）.（5）The von Frey pain threshold was negatively correlated with the expression of COX-2,HIF-1α,TNF-αin each group of knee joint traumatic BME the correlation coefficients were-0.819,-0.790,-0.814（P<0.05）,there was no correlation with ROIA of BME,and the correlation coefficient was-0.315（P>0.05）.BME ROIA the expression of HIF-1αwas positively correlated with COX-2,HIF-1αand TNF-α,the correlation coefficients were 0.455,0.423and 0.490 respectively（P<0.05）.The expression of HIF-1αwas positively correlated with the expression of COX-2 and TNF-α,the correlation coefficients were 0.746 and 0.754respectively（P<0.05）.（6）In the experimental group,von Frey pain threshold was used as the dependent variable Y and HIF-1αas the independent variable X₁the relative expression of COX-2 as the independent variable X₂the relative expression of TNF-αas independent variable X₃and ROIA in BME as independent variable X₄.The linear regression equation was Y=81.462-18.778X₁-24.552 X₂-0.153 X₃.According to the results of multiple linear regression analysis,it can be concluded that HIF-1α,COX-2 and TNF-αare the main factors affecting the pain of traumatic BME of knee joint.Conclusion:（1）The correlation between pain degree of traumatic bone marrow edema of knee joint and the severity of bone marrow edema is not high.（2）Pain caused by traumatic bone marrow edema of knee joint may be related to local hypoxia and the activation of HIF-1α/COX-2 signaling pathway.（3）Traumatic bone marrow edema of knee joint may activate HIF-1α/COX-2 signaling pathway and promote HIF-1α,COX-2 and TNF-αexpression.（4）Local hypoxic environment caused by bone marrow edema promotes HIF-1α,COX-2and TNF-α,and it may further mediate the development of bone marrow edema.