外源性硫化氢通过Wnt/β-catenin信号通路促进衰老BMSCs的成骨分化

Exogenous hydrogen sulfide promotes osteogenic differentiation of senescent BMSCs through Wnt/β-catenin signaling pathway

目的:探讨外源性硫化氢供体GYY4137对D-半乳糖(D-Gal)诱导的衰老骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨能力的影响及潜在机制。方法:应用不同浓度的D-Gal诱导BMSCs的衰老,CCK8法检测细胞活性及β-半乳糖苷酶染色检测细胞衰老的程度,确定D-Gal的最佳诱导浓度;应用不同浓度GYY4137进行干预后,检测GYY4137对正常BMSCs及衰老BMSCs细胞活性的影响,确定GYY4137最佳干预浓度。实验设CON组、GYY4137组、D-Gal组、D-Gal+GYY4137组,作相应处理后对各组BMSCs进行β-半乳糖苷酶染色,qRT-PCR法检测衰老相关基因(P16、P21、P53)和衰老相关炎症因子(IL-6、IL-8、TNF-α)的mRNA相对表达。对各组细胞进行成骨诱导分化,qRT-PCR法检测成骨分化相关因子碱性磷酸酶(ALP)、骨钙素(OCN)、Runt相关转录因子2(RUNX2)、人骨形态发生蛋白2(BMP2)的mRNA表达,通过茜素红染色法检测钙结节形成数量,ALP染色法检测ALP的生成。通过转录组测序检测D-Gal组、D-Gal+GYY4137组两组细胞的差异表达基因,根据测序结果,qRT-PCR对差异基因进行验证。应用Dickkopf相关蛋白1(dickkopf Wnt signaling pathway inhibitor 1,DKK1)抑制Wnt10b的表达后,检测各组衰老BMSCs的成骨分化能力。结果:D-Gal的最佳诱导浓度为30 g/L,GYY4137的最佳干预浓度为10μmol/L,D-Gal组β-半乳糖苷酶阳性细胞比例显著增加,衰老相关基因(P16、P21、P53)及衰老相关炎症因子(IL-6、IL-8、TNF-α)mRNA的表达量显著升高(P<0.05或P<0.01),应用GYY4137干预后,以上细胞衰老相关指标显著降低(P<0.05或P<0.01)。在成骨分化过程中,D-Gal+GYY4137组ALP、OCN、RUNX2、BMP2的mRNA表达,钙结节阳性比例显著高于D-Gal组(P<0.05或P<0.01);测序结果发现Wnt/β-catenin信号通路中的Wnt10b表达有显著差异,qRT-PCR结果显示GYY4137可增加衰老细胞Wnt10b及其下游基因β-catenin的表达(P<0.05或P<0.01),与测序结果一致。应用100μg/L DKK1抑制Wnt10b的表达后,GYY4137处理的衰老BMSCs成骨分化相关基因ALP、OCN、RUNX2、BMP2的mRNA表达明显降低(P<0.05或P<0.01)。结论:GYY4137可以缓解D-Gal诱导的BMSCs衰老进程,并且可能通过Wnt/β-cantenin信号通路促进衰老BMSCs的成骨分化。

Objective:To investigate the effect of exogenous hydrogen sulfide donor GYY4137 on the osteogenic ability of D-galactose(D-Gal)-induced senescent bone marrow mesenchymal stem cells(BMSCs)and its potential mechanism.Methods:BMSCs senescence was induced by different concentrations of D-Gal.The cell activity was detected by CCK8 method and the degree of cellular senescence was evaluated byβ-galactosidase staining to choose the optimal concentration of D-Gal.After treatment with various concentrations of GYY4137,the effects of GYY4137 on the activity of normal BMSCs and senescent BMSCs were detected,and the optimal concentration of GYY4137 was determined.CON group,GYY4137 group,D-Gal group and D-Gal+GYY4137 group were set up,andβ-galactosidase staining was performed on BMSCs in each group after corresponding treatment.The mRNA expression of cellular senescence-related genes(P16,P21,P53)and cellular senescence-related inflammatory factors(IL-6,IL-8,TNF-α)was detected by qRT-PCR.Osteoblastic induction differentiation was performed on cells in each group.mRNA expressions of osteogenic differentiation-related factors alkaline phosphatase(ALP),osteocalcin(OCN),Runt-related transcription factor 2(RUNX2)and human bone morphogenetic protein 2(BMP2)were detected by qRT-PCR.The number of calcium nodules formed was detected by alizarin red staining.ALP was detected by ALP staining.The differentially expressed genes of D-Gal group and D-Gal+GYY4137 group were detected by transcriptome sequencing.According to the sequencing results,the differentially expressed genes were verified by qRT-PCR.After the expression of Wnt10b was inhibited by DKK1,the osteogenic differentiation ability of senescent BMSCs in each group was detected.Results:The optimal induction concentration of D-Gal was 30 g/L,and the optimal intervention concentration of GYY4137 was 10μmol/L.Theβ-galactosidase content in D-Gal group was significantly increased.The mRNA expression levels of senescence related genes(P16,P21,P53)and senescence related inflammatory factors(IL-6,IL-8,TNF-α)were significantly increased(P<0.05 or P<0.01),and the above cell senescence related indicators were significantly decreased after GYY4137 intervention(P<0.05 or P<0.01).During osteogenic differentiation,the mRNA expressions of ALP,OCN,RUNX2 and BMP2 in D-Gal+GYY4137 group were significantly higher than those in D-Gal group(P<0.05 or P<0.01).The sequencing results showed that there were significant differences in Wnt10b expression in Wnt/β-catenin signaling pathway,and qRT-PCR results showed that GYY4137 increased the expression of Wnt10b and its downstream geneβ-catenin in senescent cells(P<0.05 or P<0.01),which was consistent with the sequencing results.The expression of Wnt10b was inhibited by 100μg/L DKK1,and the mRNA expressions of ALP,OCN,RUNX2 and BMP2 related to osteogenic differentiation of senescent BMSCs treated with GYY4137 were significantly decreased(P<0.05 or P<0.01).Conclusion:GYY4137 can alleviate the D-Gal-induced senescent process of BMSCs,and may promote osteogenic differentiation of senescent BMSCs through Wnt/β-cantenin signaling pathway.