Biomimetic electrical stimulation mediates 5-azacytidine-induced mesenchymal stem cell differentiation to skeletal muscle through activation of the CREB/SHP2/AKT signaling pathway and its effect on postnatal pelvic floor rehabil-itation
Han Xiaowen
Xi Yishu
Wang Ke
Guo Ya
Dong Li
Abstract：Objective To explore the role of CREB/SHP2/AKT signaling pathway in the differentiation of human bone marrow mesenchymal stem cells into skeletal muscle cells and the effect of this signaling pathway on postnatal pelvic floor rehabilitation.Methods The experiments were conducted from November 2022 to April 2023 in the Laboratory of the Third Hospital of Shijiazhuang.Using various analytical techniques on the human bone marrow stem cell osteogenic differenti-ation dataset,different gene expression patterns and regulatory relationships were revealed,shedding light on the underlying mechanisms of the disease.Experiments were performed using the human bone marrow MSC cell line CP-H166,and the cells were divided into a control group(NC group),a 5-azacytidine group,a 5-azacytidine+electrostimulation group,a 5-azacytidine+electrostimulation+CREB inhibitor group,and a 5-azacytidine+electrostimulation+CREB agonist group.The expression and distribution of relevant proteins and calcium ion concentration in cells of each group were detected by Western blot,Fura-2 calcium ion fluorescence staining and immunofluorescence staining.Results Western blot assay showed that 5-azacytidine and electrical stimulation treatments increased the expression levels of CREB,SHP2,AKT,MyoD,MyoG,and TPM1 proteins compared with the control group(P＜0.05).The addition of CREB inhibitor decreased the expression of SHP2 and AKT(P＜0.05),which in turn decreased the expression of MyoD,MyoG,and TPM1;whereas the addition of CREB agonist increased the level of the above-mentioned proteins(P＜0.05).The results of calcium fluorescence staining of Fura-2 showed that 5-aza-cytidine and electrical stimulation increased the calcium ion concentration(P＜0.05).Immunofluorescence staining showed that 5-azacytidine and electrical stimulation treatments increased the expression and distribution of p-CREB and MyoD(P＜0.05),and the addition of CREB agonists further increased the expression of these proteins(P＜0.05),whereas the addition of CREB inhibitors decreased their expression(P＜0.05).Conclusion The CREB/SHP2/AKT signaling pathway has an important role in the differentiation of BMSCs into skeletal muscle cells.Modulation of this signaling pathway can significantly affect the cell differentiation process and promote postnatal pelvic floor rehabilitation.
Keywords：Pelvic floor muscle functionBiomimetic electrical stimulationMesenchymal stem cellsSkeletal muscle cellsSHP-2CREB
Publication Date：2025-07-18
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 865-871 )