Investigation On Potential Mechanism Of Genetic Deletion Of GIT2 Affecting Joints And Chondrocyte Differentiation In Rats With Rheumatoid Arthritis

Objective: This study was to investigate the effect of GIT2 gene deletion on joint function recovery and chondrocyte differentiation in rats with adjuvant arthritis.Methods:Healthy male Sprague Dawley SD rats were enrolled in the study.The rat signs were observed and the body weight of the rats was weighed.32 rats were randomly divided into normal group,rheumatoid group,gene knockout group and gene.Knockout plus rheumatoid group,8 in each group;Rat toe joint was collected after 24 d,and normal joint,rheumatoid group,gene knockout group,gene knockout plus rheumatoid group,rat joint tissue section HE staining,detection of Aggrecan,Sox9 expression by qRT-PCR and Western-blot and detection of type II collagen by immunohistochemistry;normal group,rheumatoid group,gene knockout group and gene knockout In the rheumatoid group,the volume of the right side(secondary side)of the rat was measured before and after the inflammation(0d),and on the 10 th,13th,17 th,21st and 24 th day after the inflammation.The degree of swelling(△ mL = volume after inflammation-volume before inflammation));expression of IL-1β,TNF-α,Aggrecan and Sox9 mRNA in toe articular cartilage after qRT-PCR detection;Western-blot detection of treated foot Protein expression of IL-1β,TNF-α,Aggrecan and Sox9 in the articular cartilage of the toe joint.Results:1.The RA model was established by CFA induction,and the models were weighed on days 0,10,13,17,21 and 24,and the body weight of each group was compared.The results are shown in Table 1.On the day of initial RA induction,there was no significant difference in body weight weighing between the groups(P>0.05).However,after the 10 th,the body weight of the model group was significantly lower than that of the model group and the GIT2-KO group,and the difference was statistically significant(P < 0.05).Compared with the normal group,the body weight of the GIT2-KO+ group decreased significantly,and the difference was statistically significant(P < 0.05).There was no significant change in body weight between the GIT2-KO group and the normal group.2.HE staining results showed that in addition to the thick synovial tissue,the synovial tissue of GIT2-KO group had different degrees of more severe RA manifestation,and the inflammatory cells also showed obvious infiltration compared with the normal group.The synovial tissue in the model GIT2-KO+ group was significantly thicker,infiltrated by more inflammatory cells,and showed greater severity in cartilage damage.3.According to the results of immunohistochemical analysis,type II collagen was highly expressed in the cartilage tissue of the normal group,but significantly decreased in the model group,the difference was statistically significant(P <0.05).Compared with the normal group,the expression of type II collagen in the cartilage tissue of the GIT2-KO group and the model GIT2-KO+ group decreased(P < 0.05).Compared with the model GIT2-KO+ group,the expression of type II collagen in the cartilage tissue of the model group and the GIT2-KO group was significantly increased,the difference was statistically significant(P <0.05).4.The degree of swelling of rat cartilage tissue showed that there was no significant difference in the degree of swelling of cartilage tissue between the groups at the initial stage of RA induction(P>0.05).However,after the 10 th day,the degree of swelling of cartilage tissue in the model group,GIT2-KO group and model GIT2-KO+ group was significantly higher than that in the normal group,the difference was statistically significant(P <0.05).Compared with the GIT2-KO group,the degree of swelling of cartilage tissue in the model GIT2-KO+ group was higher,and the difference was statistically significant(P <0.05).5.RT-PC analysis was used to detect the mRNA expression levels of IL-1β,TNF-α,Aggrecan and Sox9 in the cartilage tissues of each group.Western blotting was used to detect IL-1β in the cartilage tissue of each group.Protein expression levels of TNF-α,Aggrecan and Sox9.The results showed that RA induced initial(0 days),compared with the normal group,there was no significant difference in the expression levels of various factors between the groups(P>0.05).After the 10 th day,the mRNA expression levels of IL-1β,TNF-α,Aggrecan and Sox9 in each group gradually increased after the 10 th day,and were significantly higher than the normal group.The difference was statistically significant.Significance(P<0.05).Compared with the model GIT2-KO+ group,the mRNA and protein levels of IL-1β,TNF-α,Aggrecan and Sox9 mRNA expression levels in the rat cartilage tissues were decreased(P<0.05).Compared with the normal group,the mRNA expression levels and protein levels of IL-1β,TNF-α,Aggrecan and Sox9 in the cartilage tissues of GIT2-KO group were higher(P<0.05).Conclusion:1.On the 10,13,17,21,and24 days,the weight of rats in the model group surpassed those in the model + GIT2-KO group(all P < 0.05).The weight of rats in the GIT2-KO group was not significantly different compared to the normal group(P > 0.05).2.Compared with the normal group,the synovial tissues in the GIT2-KO group suffered from more severe RA in varying degrees,in addition to the appearance of thicker synovial tissues.3.Genetic deletion of GIT2 suppresses the expression of type II n rats with RA.4.Genetic deletion of GIT2 aggravated the degree of swelling in the cartilage.5.The mRNA and protein expressions of 1L-1,TNF-,Aggrecan,and Sox9 in rats of the model + GIT2-KO group were higher than in the model group.6.Genetic deletion of GIT2 might weaken chondrocyte differentiation in rats with RA,;the results of this study could be of great assistance and value in providing a stronger basis for potential mechanism of RA.