| Objestive:Section one,to investigate effect of diabetes on the Wnt/β-catenin signal pathway of rats with osteoarthritis.Section two,to investigate the effect and its mechanism of Wnt1,Wnt10b,β-catenin and classic Wnt/β-catenin signaling on occuranceand development of diabetic arthritis.Section three,to investigate the effect of regulation of Wnt/β-catenin signaling on the proliferation and apoptosis of human synovial cells with the interference of high glucose by regulating with Wnt/β-catenin signaling agonist LiCl and inhibitor DKK-1 based on the preliminary study.Methods:Section one,the 98 SD rats(body weight 180-200g)were chosen.The 20rats were randomly selected as Control group withnot any treatment.The 25 rats were randomly chosen to accept the single intraperitoneal injection of Streptozocin to build the 2-type diabete model and 22 rats were suceessfully built the model as Diabete group.The25SDratswereinjectedthe20μLmixtureliquid(4%nematolyt+0.03mol/L L-cysteine)in d 1,3 and 7 to build the ostearthris model.The last rats were built the combined group following the above methods.After 3 weeks of building models suceesfully,the CyclinD1,MMP-7,MMP-2 and MMP-9 expression levels in the knee joint cavity flushiing fluid of rats were detected by ELISA assay.The Wnt1,Wnt10b andβ-catenin gene and protein expression levels were checked by RT-PCR and Western blotting,respectively.The pathological change of knee joint were observed by HE staining assay.Section two,the human synovial cells model in vitro was built.The human synovial cells were cultured with the different concentrations of high level glucose,and the cell survival rates were meatured by MTT assay.The human synovial cells in logarithmic phase were taken and adjusted into 6×10~8/L.The human synovial cells were individed into the control group(normal RPMI1640 culture medium),20mmol/L high glucose group(20mmol/L glucose in the RPMI1640 culture medium),30mmol/L high glucose group(30mmol/L glucose in the RPMI1640 culture medium),40mmol/L high glucose group(40mmol/L glucose in the RPMI1640 culture medium)and mannitol group(19.5mmol/L mannitol in RPMI1640 culture medium).In different time points(24-96h),the cell survival rates in the groups were detected by MTT assay,the cell cycle distribution and the cell apoptosis was detected by the flow cytometry.The Wnt1,Wnt10b andβ-catenin gene and protein expression levels were checked by RT-PCR and Western blotting,respectively.The CyclinD1,MMP-7,MMP-2 and MMP-9 expression levels in cell supernate were detected by ELISA assay.Section three,the human synovial cells with the interference of high glucose(40mmol/L)in vitro were built.All cells were individed into 3groups:control group(40mmol/L high glucose interference without any treatment),DKK-1 group(40mmol/L high glucose interference without DKK-1treatment)and LiCl group(40mmol/L high glucose interference without LiCl treatment).In different time points(24-96h),the cell survival rates in the groups were detected by MTT assay,the cell cycle distribution and the cell apoptosis was detected by the flow cytometry.The Wnt1,Wnt10b andβ-catenin gene and protein expression levelswerecheckedbyRT-PCRandWesternblotting,respectively.The CyclinD1,MMP-7,MMP-2 and MMP-9 expression levels in cell supernate were detected by ELISA assay.Results:Section one,compared to the control group,the CyclinD1,MMP-7,MMP-2and MMP-9 expression levels in the knee joint cavity flushiing fluid and of rats Wnt1,Wnt10b andβ-catenin gene and protein expression levels in other 3 groups were significantly increaced and the expression levels in combined group were extremly significantly higher.In oestarthris and combined groups,there were the attenuated articular cartilage,modificated fibre and damaged matrix.These changes significantly happened in combined group.Section two,the high level glucose could improve the cell proliferation,reduce the cell apoptosis,have no obvious effect on the cell cycle.The effect to improve the cell proliferation had concentration dependent manner and time dependent manner.With the increase of glucose levels and delay of the treat time,the cell apoptosis inhibiting rates were generally improved,the CyclinD1,MMP-7,MMP-2 and MMP-9 expression levels were enhanced,the Wnt1,Wnt10b andβ-catenin mRNA and protein levels were increased.Section three,the LiCl interference could improve the cell proliferation,increase Wnt1,Wnt10b andβ-catenin gene and protein expression levels,enhance the CyclinD1、MMP-7、MMP-2and MMP-9 expression levels,and reduce the cell apoptosis,but have no obvious effect on the cell cycle.In DKK-1 group,the cell apoptosis rates were generally improved,the CyclinD1,MMP-7,MMP-2 and MMP-9 expression levels were reduced,the Wnt1,Wnt10b andβ-catenin mRNA and protein levels were decreased.The survival rate of human synovial cells in the experimental group were all increased with the prolongation of the culture time,with obvious time effect;compared with the control group,LiCl intervention can significantly improve the survival rate of human synovial cells,the survival rate of human synovial cells in DKK-1 group decreased significantly.In the control group,DKK-1 group and LiCl group,there was no significant difference in the percentage of G0/G1,G2/M and S cells at different time points of human synovial cells(24-96h).In the experimental group,the apoptosis rate of human synovial cells in the same culture time points,the apoptosis rate of LiCl group was significantly lower than that of the control group and DKK-1 group.Compared with the control group,DKK-1 intervention significantly increased the apoptosis rate of human synovial cells.With the extension of culture time,the apoptosis rate of human synovial cells in each experimental group decreased gradually.With the prolongation of cell culture time,the relative expression levels of Wnt1,Wnt10b andβ-catenin mRNA and protein in the cells increased significantly,and the difference was statistically significant.The expression levels of CyclinD1,MMP-7,MMP-2 and MMP-9 in the supernatant of cell culture medium of LiCl group were significantly higher than those of control group and DKK-1 group,but the expression level of four protein molecules in DKK-1 group was significantly lower than that in group.Show changes in different culture time points,with the level of cell culture time increased,the expression of CyclinD1,MMP-7,MMP-2 and MMP-9 in each experimental group has statistically significant.Conclusion:1、Highglucoseinternalenvirmentcouldimprovethe CyclinD1,MMP-7,MMP-2 and MMP-9 expression level.This effect was realized to aggravate the oestearthris by up-regulating Wnt1 and Wnt10b expression level and subsequently activating the Wnt/β-catenin signal pathway.2、The high glucose levels can improve the human synovial cells proliferation,inhibit the cell apoptosis,but have no significant effect.The CyclinD1,MMP-7,MMP-2 and MMP-9 expression levels in cell culture supernate were increased.Those effects had the concentration dependent manner and time dependent manner.The effects might be fulfilled by up-regulating Wnt1 and Wnt10b to activite the Wnt/β-catenin signaling.3、After DKK-1 intercepted Wnt/β-catenin signaling,the effects of high glucose interference on human synovial cells improving proliferation,inhibiting apoptosis and enhancing the CyclinD1、MMP-7、MMP-2 and MMP-9 were inhibited.AfterLiCl agitated Wnt/β-catenin signaling,the effects were improved. |
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