Effects Of Calcitonin Early Intervention On Cartilage And Subchondral Bone Of Unstable Knee In Ovariectomized Rats

Objective:The SD rats after bilateral anterior cruciate ligament transection(ACLT)combined with bilateral ovariectomy(OVX),that provides a model of osteoarthritis.To explore the effects of calcitonin early intervention on knee osteoarthritis cartilage and subchondral bone in rats with osteoporosis.Method: The random number table generated by the software of SPSS.30 SPF 3 months old female Sprague-Dawley(SD)rats were randomly divided into 3 groups(n = 10): sham operation(Sham)+ saline(S)control group,ACLT + OVX + elcatonin(E)treatment group,ACLT + OVX + saline(S)model group.ACLT combined with OVX to establish animal model of OA.Treatment group,treated with elcatonin injection(Elcatonin,1ml:10U/,ACLT+OVX),was given 10U/kg once a week,intramuscular injection on the right upper limb,and began to intervene 1 week after operation.The dose,frequency,injection mode and location of the saline intervention group were the same as that of the elcatonin intervention group.After intervention for 12 weeks,SD rats were given intraperitoneal injection of 10% chloral hydrate.After anesthesia,the blood of inner canthus was collected,and the biochemical markers(CTX-I and CTX-II)of bone metabolism were detected by ELISA.After opening bilateral knees,we observed the articular cartilage of each knee joint.After removing the soft tissue,the right distal femur was cut off and placed in liquid nitrogen.Real time PCR was used to detect mRNA expression of the MAPKs signaling pathway protein(ERK1,C-JUN,P38)and MMP-13 protein.The BMD,morphological structure and bone tissue morphometric parameters of the subchondral bone were detected by micro-CT.The left tibial plateau articular cartilage were stained with HE and sarranine.The full-thickness structure and cell morphology of cartilage were observed,and Mankin score was observed.Finally,rats in each group were sacrificed.Analysis: the above indexes were expressed as the mean with standard deviation.(x ±s),the differences between groups were analyzed with ANOVA,multiple factors were analyzed by logistic regression analysis and multiple stepwise regression analysis,the above statistics in the United States under the support of SPSS/PC+19.0.(P < 0.001,the difference is statistically significant)Results: 1.Observation of the gross specimens of the articular cartilage on the knee of the rats:In group Sham+S,the articular cartilage of the knee joint was smooth and intact.In group ACLT+OVX+S,the cartilage surface was thinner and had many small ulcers,and larger bony masses were seen around the joint.In group ACLT+OVX+E,the articular cartilage of the knee joint was slightly rough,and occasionally there was little destruction and there was less osteophyte around the joints.2.The morphological observation of the cartilaginous red and HE staining of the right tibial plateau joints:In group Sham+S,the articular cartilage was complete and the chondrocytes were basically normal.In group ACLT+OVX+S,the damage of cartilage surface was serious,and the chondrocytes were obviously less.In group ACLT+OVX+E,there were small cracks on the surface of cartilage,and articular cartilage became thinner.3.Modified Mankin score of articular cartilage:The modified Mankin score in group Sham+S was significantly lower than that in group ACLT+OVX+E,and the difference was statistically significant(P < 0.001).The modified Mankin score in group ACLT+OVX+S was significantly higher than that in group ACLT+OVX+E,and the difference was statistically significant(P < 0.001).4.The results of CTX-I and CTX-II were detected by ELISA in rat venous blood: CTX-I and CTX-II in group Sham+S were significantly lower than those in group ACLT+OVX+E(P < 0.001 and P < 0.001,respectively).The CTX-I and CTX-II in group ACLT+OVX+S were significantly higher than those in group ACLT+OVX+E,and the difference was statistically significant(P < 0.001,P < 0.001,respectively).5.BMD analysis of the subchondral bone of the left knee of SD rats: BMD in group Sham+S was significantly higher than that in group ACLT+OVX+E,the difference was statistically significant(P < 0.001);BMD in group ACLT+OVX+S was significantly lower than that in ACLT+OVX+E group(P < 0.001).6.The structural parameters of the subchondral bone of the tibia in the left knee joint of rats were found as follows:The values of BV/TV and Tb.N in group Sham+S were significantly higher than those in group ACLT+OVX+S,and the Tb.Sp value in group Sham+S was significantly lower than that in ACLT+OVX+S group.The values of BV/TV and Tb.N in group ACLT+OVX+E were significantly higher than those in group ACLT+OVX+S,and the Tb.Sp value in group ACLT+OVX+E was significantly lower than that in ACLT+OVX+S group.The difference was statistically significant(P < 0.001,P < 0.001,and P< 0.001,respectively).The difference between the Tb.Th values of the three groups was not significant,and the difference was not statistically significant.7.The relative mRNA expression of ERK1,C-JUN,P38 and MMP-13 protein in the femoral condyle of the right knee was detected by Real time PCR:the mRNA expression levels of ERK1,C-JUN,P38 and MMP-13 protein in group ACLT+OVX+E were significantly higher than those in Sham+S group(P < 0.001,P < 0.001,P < 0.001,P < 0.001,respectively).The mRNA expression levels of ERK1,C-JUN,P38 and MMP-13 protein in group ACLT+OVX+E were significantly lower than those in ACLT+OVX+S group(P < 0.001,P < 0.001,P < 0.001,P < 0.001,respectively).Conclusion: In the unstable knee joint of ovariectomized rats,the early application of elcatonin(10U/kg/ week)can inhibit the continuous destruction of the subchondral bone microstructures and protect the biomechanical properties of the subchondral bone and cartilage.At the same time,the expression of the MAPKs signal pathway may be inhibited and the expression of MMP-13 of the cartilaginous cell is downregulated and the degeneration of cartilage is relieved.Calcitonin has a potential therapeutic effect on OA.