Neuregulin 4 promotes osteogenic differentiation of bone marrow mesenchymal stem cells in inflammatory microenvironment via autophagy activation

Background　 The inflammatory microenvironment inhibits osteogenic differentiation of bone-marrow mesenchymal stem cells (BMSCs). Neuregulin 4 (Nrg4) exerts anti-inflammatory effects on multiple organs, but whether Nrg4 can promote osteogenic differentiation of BMSCs in an inflammatory microenvironment and its underlying mechanism remain unclear. Objective　To investigate the effects of Nrg4 on osteogenic differentiation of BMSCs in the inflammatory microenvironment and related mechanisms.Methods　BMSCs were divided into four groups: control, TNF-α, Nrg4 (TNF-α+Nrg4), and CQ (TNF-α +Nrg4+CQ). ALP activity was measured using an alkaline phosphatase (ALP) assay kit, while the expression of osteogenic-related proteins ALP and osteocalcin (OCN), as well as autophagy-related proteins P62 and LC3B, was detected by Western blot. The number of mineralized nodules was assessed by alizarin red staining.Results　Compared with the control group, BMSCs in the inflammatory microenvironment exhibited reduced ALP activity (day 5, P＜0.001), decreased expression of ALP and OCN proteins (P＜0.05), upregulated P62 expression accompanied by downregulated LC3B-Ⅱ (P＜0.05), and fewer mineralized nodules (P＜ 0.001). Nrg4 treatment promoted ALP activity in BMSCs under inflammatory conditions (day 5, P＜0.001), increased the expression of ALP and OCN proteins (P＜0.05), downregulated P62 while upregulating LC3B-Ⅱ (P＜0.05), and enhanced the number of mineralized nodules (P＜0.001). However, when autophagy was inhibited by chloroquine, the pro-osteogenic effect of Nrg4 on BMSCs in the inflammatory microenvironment was significantly attenuated. Conclusion　Nrg4 promotes osteogenic differentiation of BMSCs in the inflammatory microenvironment, and this effect is associated with autophagy activation.