| Background Post-traumatic osteoarthritis(PTOA)is mainly characterized by articular cartilage destruction,synovial inflammation,osteophyte formation and subchondral bone degeneration.Appropriate intensity of exercise can protect the articular cartilage,but the mechanism is not clear.Mutations in the Sorting nexin 10(SNX10)regulate bone metabolism and inflammation processes which are potentially linked to the basic pathology of PTOA.Object Our preliminary experiment found that SNX10 gene expression was up-regulated in cartilage during high-intensity exercise,suggesting that SNX10 is involved in the regulation of chondrocyte function.In this study,gene knockout technology was used to systematically study the role of SNX10 in the exercise intervention of PTOA in animal experiments and cellular levels,revealing the importance of SNX10 in the function regulation of chondrocytes,and providing a theoretical basis for the exercise intervention of PTOA.Method Part 1 Effects of exercise on SNX10 in knee joint cartilage of PTOA mice1.45 20-week-old wild-type male mice are randomly divided into 5 groups: model control group(Nor,n=9),model group(Mod,n=9),control group(Con,n=9),model rest group(Res,n=9),and model rehabilitation group(Rh,n=9).2.Nor and Con group do not receive exercise intervention.Mice in Mod,Res and Rh group are subjected to high-intensity treadmill exercise for 5 weeks,with 5°slope,20m/min peak speed,5d/w,which resulted in PTOA animal model.1week rest after modeling,mice in the Rh group are given a low-intensity treadmill exercise for 4weeks with slope of 0°,8m/min constant speed,5d/w,for exercise rehabilitation intervention.Mice in Res group have a rest-cure for 5 weeks after modeling.3.Mice body weight is recorded every week.Mice in Nor and Mod group are sacrificed at the end of the fifth week of the experiment,and mice in Con,Res and Rh group are sacrificed at the end of the tenth week of the experiment.Sampling mice bilateral knee joint tissues of hind limbs and blood sample by eyeball extirpating.Micro-CT is used to detect related bone parameters.Mankin's score is detected by HE staining for paraffin sections,and OARSI score is detected by Safranin O-staining.The m RNA expression levels of SNX10,MMP9,?-catenin,COL-?,ADAMTS-5,TNF-? and IL-1? in cartilage are detected by RT-q PCR.The contents of IL-6,TNF-? and GAG in serum are determined by ELISA.The protein expression of SNX10 and MMP9 is detected by Western-blot.Part 2 Effects of SNX10 deficiency on knee cartilage injury in PTOA mice1.The genetic background is C57BL/6 male mice.18 Snx10-/-mice are randomly divided into two groups: model control group(KO-Nor,n=9)and model group(KO-Mod,n=9).18 homocluttering Snx10+/+ mice are randomly divided into model control group(WT-Nor,n=9)and model group(WT-Mod,n=9).2.KO-Nor and WT-Nor group have no exercise intervention.KO-Mod and WT-Mod group mice PTOA modeling methods are the same as in Part 1.3.Mice body weight is recorded every week and mice are sacrificed at the end of the fifth week.Sampling,Micro-CT,Mankin's score,OARSI score,RT-q PCR and ELISA are the same as those in Part 1.SNX10 m RNA is detected by RT-q PCR.The establishment of Snx10-/-mice is verified by Western-blot,as well as the protein expression changes of MMP9 and ?-catenin.Part 3 Effects of SNX10 deficiency on chondrocyte repair under mechanical stretch stimulation1.Wild type primary chondrocytes are divided into 4 groups according to the length of stretch: normal control group(CON),1-hour stretch group(1H),2-hour stretch group(2H),and 4-hour stretch group(4H).Mechanical stretch stimulation is performed with0.5Hz and 10% intensity.2.The m RNA expression levels of SNX10,MMP9,?-catenin,COL-?,TNF-? and IL-1?in chondrocytes are detected by RT-q PCR.Annexin V-FITC/PI assay kit is used to detect the apoptosis of chondrocytes in each group.Determine the suitable mechanical stretch scheme.3.Investigate the effects of different concentrations(2ng/ml,4ng/ml,6ng/ml,8ng/ml,10ng/ml)of IL-1? stimulator on the activity of wild-type primary chondrocytes.4.Snx10-/-and homocluttering Snx10+/+ primary chondrocytes are extracted.The single genotype is divided into 4 groups: control group(CON),stretch group for 1 hour(1H),IL-1? group(IL-1?),IL-1?+stretch group(S),a total of 8 groups with 2 genotypes.The CON group has no intervene.IL-1? and S group are treated with IL-1? stimulant.The 1H group is treated with 0.5Hz,10% intensity,1h mechanical stretch stimulation,and the S group was treated with 0.5Hz,10% intensity,1h mechanical stretch stimulation after the IL-1? stimulus.5.The m RNA expression levels of MMP9,COL-? and ?-catenin are detected by RT-q PCR.The protein expression of MMP9,COL-? and ?-catenin are detected by Western-blot and immunofluorescence.Results Part 1 Effects of exercise on SNX10 in knee joint cartilage of PTOA mice1.The body weight of mice decreased significantly in the Mod and Res group(P<0.05).2.The bone mineral density(BMD)and trabecular thickness(Tb.Th)of mice increased(P<0.05)and the connection density(Conn.D)decreased(P<0.05)in the Mod group.Bone mineral density increased in Rh group(P<0.05).3.The cartilage injury score(Mankin's)and calcification score(OARSI)were increased in Mod and Res groups(P<0.05)and the scores of Rh group were lower than Res group(P<0.05).4.Compared with Nor group,COL-? m RNA in the Mod group was significantly decreased after modeling(P<0.05),and m RNA of SNX10,MMP9,?-catenin,ADAMTS-5,TNF-? and IL-1? were significantly up-regulated(P<0.05).m RNA of ADAMTS-5 and IL-1? were significantly up-regulated in Res group compared with Con group(P<0.05).Compared with Res group,the COL-? m RNA in Rh group was significantly up-regulated(P<0.05),m RNA levels of ADAMTS-5,TNF-?,IL-1?,?-catenin,MMP9 and SNX10 were significantly down-regulated(P<0.05).5.Compared with Nor group and Con group,serum IL-6 and TNF-? contents in Mod group and Res group were significantly up-regulated(P<0.001),the content of GAG was significantly down-regulated(P<0.001).Compared with Res group,the contents of IL-6 and TNF-? in Rh group were significantly decreased(P<0.001),the content of GAG was significantly up-regulated(P<0.001).6.Compared with Con group,SNX10 protein expression in Res group was significantly up-regulated(P<0.05).Compared with the Res group,the protein expressions of SNX10 and MMP9 in the Rh group were significantly down-regulated(P<0.05).Part 2 Effects of SNX10 deficiency on knee cartilage injury in PTOA mice1.Compared with the Ko-Nor group,the body weight of mice in the KO-Mod group decreased significantly at 3-5 weeks(P<0.05).Mice in the KO-Mod group were heavier than those in the WT-Mod group at end of fifth week(P<0.05).2.Compared with other groups,the bone mineral density(BMD),connectivity density(Conn.D)and trabecular thickness(Tb.Th)of mice increased(P<0.05)in the KO-Mod group.3.Mankin's score and OARSI score were decreased significantly in KO-Mod than WT-Mod(P<0.05).4.Compared with WT-Mod group,the m RNA of COL-? in KO-Mod group was significantly up-regulated(P<0.05),m RNA levels of ADAMTS-5,TNF-?,IL-1?,MMP9 and ?-catenin were significantly down-regulated(P<0.05).5.Compared with WT-Mod group,serum IL-6 and TNF-? contents in KO-Mod group were significantly decreased(P<0.001),the content of GAG increased significantly(P<0.001).6.Western-blot results showed that Snx10-/-full-gene knockout mice model was successfully established.Gene knockout significantly down-regulated the expression of MMP9 and ?-catenin before and after modeling(P<0.05).Part 3 Effects of SNX10 deficiency on chondrocyte repair under mechanical stretch stimulation1.The m RNA levels of ?-catenin,TNF-? and IL-1? in chondrocytes were significantly down-regulated by 1h mechanical stretch(P<0.01),and the m RNA levels of COL-?were significantly up-regulated(P<0.05).The m RNA levels of SNX10,TNF-? and?-catenin were significantly up-regulated in 2H and 4H group(P<0.01),and the m RNA level of COL-? was significantly down-regulated(P<0.01).2.The apoptosis in 1H group was relatively mild,and the apoptosis in 4H group was significant.3.IL-1? at the concentration of 10ng/ml can significantly reduce the activity of chondrocytes.4.IL-1? could significantly up regulate MMP9 and ?-catenin m RNA(P<0.05),and down regulate COL-? m RNA(P<0.05).Combined with 1H mechanical stretch,KO group had more significant down-regulated effect of MMP9 and ?-catenin m RNA(P<0.01)than WT group,and COL-? m RNA was significantly up-regulated(P<0.05).5.IL-1? could significantly down-regulate the protein expression of COL-?(P<0.05),and up-regulate the protein expression of MMP9 and ?-catenin(P<0.05).Mechanical stretch could regulate the protein expression of COL-?,MMP9 and ?-catenin induced by IL-1?.Gene knockout combined with mechanical stretch could significantly down-regulate the protein expression of MMP9(P<0.05).Conclusions1.Low intensity treadmill shows regulatory effect on cartilage tissue in mice PTOA model.2.SNX10 gene deficiency can alleviate the degree of cartilage damage in the process of PTOA.3.SNX10 gene deficiency and appropriate mechanical stretch stimulation can regulate the expression of inflammatory factors,synthesis and degradation factors of chondrocytes,thereby protecting chondrocytes in PTOA.4.The appropriate intensity of exercise and mechanical stretch can promote cartilage tissue repair through SNX10 mediating MMP9 and ?-catenin. |
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