Intraperitoneal Injection Of Thalidomide Alleviates Early Osteoarthritis Development By Suppressing Vascular Endothelial Growth Factor Expression In Mice

BackgroundOsteoarthritis(OA)is a joint disease that is mainly caused by the progressive changes of articular cartilage under the interaction of mechanical stress,biochemistry and genetics and so on.It occurs in the middle-aged and elderly people,involving multiple joints of the body.Weight bearing joints of knee and hip are more common.The clinical manifestations of OA are mainly manifested as joint pain and exercise restriction,pathological features of cartilage degeneration,synovitis,subchondral bone sclerosis and osteophyte formation.OA diagnosis depends mainly on clinical symptoms,signs and imaging data.Early OA mainly uses physical and medicine treatment in clinical.Middle and late stage of OA mainly use arthroscopic surgery and joint replacement surgery.The pathogenesis of OA is not yet fully understood.One of the more powerful doctrines suggests that OA was caused by a reduction in the number of chondrocytes and degradation of extracellular matrix(ECM)with variety of initiators.MMPs are one of the major protease families that degrade the structural fibrin component of articular cartilage,and matrix metalloproteinase-13(MMP-13)is one of the major enzymes which cause degradation of ECMs.Studies have shown that vascular endothelial growth factor(VEGF)was expressed in articular cartilage of OA.Increased levels of VEGF are associated with the progression of OA,and VEGF can increase the expression of MMPs.Therefore inhibiting the expression of VEGF can alleviate the development of OA.Thalidomide was initially used as a sedative and later found to be useful in the treatment of pregnant women's morning sickness.However,it was withdrawn from the global market due to its teratogenic effects on infants.It has been widely reported that thalidomide has anti-angiogenic properties and inhibits VEGF expression.Now it is mainly used in the field of anti-tumor and anti-angiogenic diseases.ObjectiveVEGF is expressed in articular cartilage and its levels increase along with the progression of OA.Thalidomide is a drug that has been reported to inhibit angiogenesis and reduce VEGF production by down-regulating its expression.Our objective in this study was to determine if intraperitoneal injection of thalidomide can attenuate early OA development in C57BL/6 mice.MethodsForty-eight mice of 10-week-old male C57BL/6 were randomly assigned into experimental group(Dmm+Th),control group(Dmm)and sham group(Sham)equally(n = 16 in each).The OA model was induced surgically in Dmm+Th and Dmm groups,and mice in the Dmm+Th group subsequently were treated with intraperitoneal injection of thalidomide(200 mg/kg/day).At 2 and 4 weeks after surgery,the pathological change of cartilage was assessed qualitatively by hematoxylin and eosin staining and Safranin O/Fast green staining,and quantitatively by the Osteoarthritis Research Society International score.The mRNA expression of MMP-13 and VEGF were measured by reverse transcription quantitative polymerase chain reaction(RT-qPCR).The protein expression of MMP-13 and VEGF were detected by immunofluorescence(IF)and immunohistochemistry(IHC)respectively.Production of VEGF in serum was evaluated by the Enzyme Linked Immunosorbent Assay(ELISA).Results1.The pathology results showed that compared with Sham group,the superficial fibrosis of articular cartilage and a small amount of proteoglycan were lost in Dmm group at 2 weeks after operation.Large amount of proteoglycan were lost at 4 weeks after operation,even with vertical cracks and erosion of calcification.Compared with Dmm group,the surface of articular cartilage in Dmm + Th group was slightly intact at 2 weeks after operation,proteoglycan and cartilage fibrosis decreased 4 weeks after operation,and the surface wear relatively reduced.The pathological scores of Dmm group and Dmm + Th group were significantly higher than those of Sham group(P<0.05).Nonetheless,pathological changes in the Dmm + Th group were significantly improved compared to the Dmm group(P<0.05).2.The results of RT-qPCR showed that the expression of VEGF and MMP-13 mRNA in articular cartilage of Dmm group increased with the development of early OA compared with Sham group.However,the expression of VEGF and MMP-13 mRNA in articular cartilage of Dmm + Th group decreased in different degrees when comparing with Dmm group,and the difference was statistically significant(P<0.05),3.The results of IHC showed that compared with Sham group,the number of VEGF-positive cells in articular cartilage of Dmm group increased with the degree of cartilage degeneration of early OA.Compared with Dmm group,the expression of VEGF in Dmm+Th group decreased in different degrees,and the difference between the two was statistically significant.4.The results of IF showed that compared with Sham group,the number of MMP-13 immunoreactive cells in articular cartilage of Dmm group and Dmm + Th group showed a trend of increase with the increase of early OA.The expression of MMP-13 in Dmm + Th group was significantly lower than that in Dmm group(P<0.05).5.The results of ELISA showed that compared with Sham group,the serum VEGF levels in Dmm group and Dmm + Th group increased to some extent with the development of early OA,and the difference was statistically significant(P<0.05).Compared with Dmm group,the serum VEGF concentration in Dmm + Th group was significantly lower(P<0.05).Conclusionl.The mouse OA model induced by DMM surgery showed early OA changes and progressive development.2.1n the mouse model of early OA,the mRNA and protein expression levels of VEGF and MMP-13 showed an increasing trend.3.Thalidomide could inhibit the expression of VEGF and MMP-13 and alleviate the development of early OA in mice,which may serve as a new drug for the treatment of OA.