| [Objective]The impaired function of diabetic bone marrow mesenchymal cells(DM-BMSCs)is the main cause of diabetic bone regeneration deficiency.Currently,the specific mechanism of DM-BMSCs function impairment is still not fully elucidated.In this study,we firstly investigated the mechanism of Insulin-TGF-?1-SATB2 pathway regulation of autophagy affecting DM-BMSCs osteogenesis in vitro,and then studyed the effects of this pathway on the regeneration of type 2 diabetes mellitus in vivo.This study provides a potential therapitic strategy to promote diabetic bone regeneration.[Methods]Part 1.Effects of hyperglycemia and hyperinsulinemia on autophagy,aging and osteogenic differentiation of healthy bone marrow mesenchymal cells(1)Bone marrow mesenchymal cells cultured in low glucose(NG)and high glucose(HG)environment 3 days,the intracellular reactive oxygen species(ROS)was detected using the fluorescent probe DCFH-DA.(2)The autophagy proteins LC3-I and LC3-II were detected by Western Blot after 3 days of low glucose(NG)and high glucose(HG)culture,(rapamycin RA as a positive control),and semi-quantitative analysis of LC3-II/LC3-I ratio,immunofluorescence analysis of LC3 expression.(3)Low glucose(NG)and high glucose(HG)culture after insulin(final concentration 200uIU/ml)was added for 3 days,the expression of autophagy-related proteins LC3-I,LC3-II was analyzed by Western Blot,LC3-?/LC3-I ratio was semi-quantitatively analyzed.(4)Low glucose(NG)and high glucose(HG),at the same time,insulin(final concentration 200uIU/ml)was added for 3 days.The senescent phenotype of BMSCs was detected by SA-?-gal staining and the number of positive cells was quantified.(5)Low glucose(NG)and high glucose(HG)cultures were added at the same time.After 3 days addition of insulin(final concentration 200uIU/ml),the expression of autophagy-related proteins LC3-I,LC3-? was analyzed by Western Blot,LC3-?/LC3-I ratio was analyzed semi-quantitatively.(6)Low glucose(NG)and osteogenesis was initiated 3 days after the addition of insulin(final concentration 200uIU/ml)in the culture of glucose(HG),Osteogenesis was detected after 7 days of osteogenic induction.The amount of calcium nodules was analyzed by semiquantitative analysis.Real time PCR was used to detect osteogenic related genes.(7)Low-glucose(NG)and high-glucose(HG)cultures were added with insulin(final concentration 200uIU/ml)and cultured for 3 days to add recombinant human TGF-?1(final concentration was 5 ng/ml)(Rapamycin RA as a positive control),serum-free culture 6 hours after Western Blot analysis of autophagy-related protein LC3,P62 expression,semi-quantitative analysis of LC3-?/LC3-I ratio and P62 expression.(8)After low glucose(NG)and high glucose(HG)culture add insulin(final concentration 200uIU/ml)for 3 days,add TGF-?1 inhibitor(final concentration 5?M)and human recombinant cytokine TGF-?1(At the final concentration of 5 ng/ml),osteogenic induction was started.Real time PCR was used to detect the expression of osteogenic genes ALP,Runx2,Ocn and Opn 7 days after osteogenic induction.Part 2.Comparative analysis of autophagy,senescence,and osteogenic differentiation of bone marrow mesenchymal cells in patients with diabetes and healthy(1)Six hours after serum-free culture,Western Blot was used to detect the expression of autophagy protein LC3 and P62,and semi-quantitative analysis of LC3-?/LC3-?ratio,immunofluorescence analysis of LC3 expression.(2)SA-?-gal staining was used to detect the senescence phenotype of DM-BMSCs and BMSCs,quantitative analysis of the number of positive cells.(3)Alizarin red staining after 7 days of osteogenic induction was carried out and calcium nodule formation in DM-BMSCs and BMSCs was analyzed by semi-quantitative analysis.(4)Western Blot was used to detect the expression of TGF-?1,P-Smad3,Smad3 and SATB2 in DM-BMSCs and BMSCs.Part 3.Effects of Insulin-TGF-?1-SATB2 pathway on autophagy,senescence,and osteogenic differentiation of diabetic bone marrow mesenchymal cells(1)Insulin(final concentration 200uIU/ml)),TGF-?1 inhibitor(final concentration 5?M)and human recombinant cytokine TGF-?1(final concentration 5ng/ml)were added to high glucose medium,respectively.LC3 and P62 expression were detected after 6h serum-free culture using Western Blot,semi-quantitative analysis of LC3-?/LC3-? ratio and expression of P62.(2)Insulin(final concentration 200uIU/ml)),TGF-?1 inhibitor(final concentration 5?M)and human recombinant cytokine TGF-?1(final concentration 5ng/ml)were added to high glucose medium,respectively.SA-?-gal staining was used to detect senescent phenotype after 3 days of culture.(3)Insulin(final concentration 200uIU/ml)),TGF-?1 inhibitor(final concentration 5?M)and human recombinant cytokine TGF-?1(final concentration 5ng/ml)were added to high glucose medium,respectively.Osteogenic induction after 3 days of culture,expression of RUNX2,OSX and OPN were detceted by Western Blot after 7 days osteogenic induction.(4)Insulin(final concentration 200uIU/ml),TGF-?1 inhibitor(final concentration 5?M)and human recombinant cytokine TGF-?1(final concentration 5 ng/ml)were added to high glucose medium,respectively.Western Blot was used to detect TGF-?1 signaling pathway related protein and SATB2 after culture for 3 days.Part 4.Effect of TGF-?1 inhibitor on regeneration of jaw bone defect in type 2 diabetic rats(1)Type 2 diabetes mellitus rats were built in high-fat diet combined with intraperitoneal injection of STZ.(2)Mandibular defect were made and the expression of TGF-?1was detected in callus postoperative 3,7,14,21 days using Western Blot.(3)DM-BMSCs and gelatin sponge were transplanted into the critical bone defect area in mandible angle.TGF-?1 inhibitor was injected into defect area postoperative 1,3,5,7 days.New bone formation in the defective area was evaluated after 8 weeks by radiographic and histological analysis.[Results]Part 1:The effect of high glucose and high insulin environment on autophagy,aging and osteogenic differentiation of healthy bone marrow mesenchymal cells(1)High glucose promoted the expression of ROS in BMSCs.(2)High glucose promoted the aging of BMSCs.(3)High glucose promoted the autophagy of BMSCs.(4)High glucose and insulin exacerbates BMSCs senescence.(5)High glucose and insulin inhibits autophagy of BMSCs.(6)High glucose and insulin inhibits osteogenic differentiation of BMSCs.(7)TGF-?1 inhibits autophagic activation of BMSCs.(8)Inhibiting TGF-?1 signaling pathway promotes osteogenic differentiation of BMSCs.Part 2:Comparative analysis of autophagy,aging,and osteogenic differentiation of bone marrow mesenchymal cells in diabetes and healthy(1)Compared with BMSCs,the autophagic capacity of DM-BMSCs was decreased.(2)Compared with BMSCs,DM-BMSCs senescence was exacerbated.(3)Compared with BMSCs,the osteogenic differentiation of DM-BMSCs was attenuated.(4)Compared with BMSCs,TGF-?1 signal was highly expressed in DM-BMSCs,while SATB2 was lowly expressed.Part 3:Effects of Insulin-TGF-?1-SATB2 pathway on autophagy,senescence and osteogenic differentiation of diabetic bone marrow mesenchymal cells(1)Insulin inhibited autophagy activation of DM-BMSCs.Autophagy activation ability of DM-BMSCs increased when inhibiting TGF-?1 signaling pathway.(2)Insulin promoted DM-BMSCs senescence.DM-BMSCs aging was delayed when suppression TGF-?1 signaling pathway.(3)DM-BMSCs osteogenic differentiation was improved when restraining of TGF-?1 signaling pathway.(4)Insulin promoted TGF-?1 signaling pathway and the expression of T?R2.(5)TGF-?1 signaling pathway regulated the expression of SATB2 negatively.Part 4:Effect of TGF-?1 inhibitor on the regeneration of jaw bone defect in type 2 diabetic rats(1)High expression of TGF-?1 in the early stage of fracture healing of diabetes.(2)Local injection of TGF-?1 inhibitor promotion of bone regeneration of jaw bone of diabetes.[Conclusion]1.High glucose and insulin can inhibit BMSCs autophagy,promote aging,restrain osteogenic differentiation,and block TGF-?1 signaling pathway can increase the osteogenic differentiation of BMSCs in the high glucose and insulin environment.2.DM-BMSCs showed weak autophagy,increased the senescence,impotent osteogenic differentiation,and high expression of TGF-?1 signaling pathway.3.Insulin-TGF-?1-SATB2 pathway inhibited DM-BMSCs autophagy,promoted aging,and attenuated osteogenic differentiation.4.Inhibition of insulin-TGF-?1-SATB2 pathway can promote DM-BMSCs bone regeneration after transplantation. |
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