Peptide 11R‑VIVIT promotes fracture healing in osteoporotic rats

Abstract

Osteoporotic fracture healing is a complex clinical issue. The present study was conducted to investigate the repair properties of 11R‑VIVIT on osteoporotic fractures and to examine the potential effects of 11R‑VIVIT on osteoporotic bone marrow‑derived mesenchymal stem cells (BMSCs), A rat model of osteoporotic femoral fracture was established, and the effects of the daily local injection of 11R‑VIVIT or saline on fracture repairing were evaluated by micro‑CT scans and H&E staining. Moreover, BMSCs from osteoporotic rats were treated with 11R‑VIVIT, and the osteogenic and adipogenic differentiation of BMSCs was evaluated. The results revealed that 11R‑VIVIT promoted bone formation and increased fracture healing. In addition, 11R‑VIVIT promoted the differentiation of osteoporotic BMSCs into osteoblasts rather than adipocytes. Furthermore, mechanistic analysis revealed that 11R‑VIVIT promoted autophagy by blocking the protein kinase B (AKT)/nuclear factor of activated T‑cells (NFATc1) signaling pathway. Consistently, the activation and inhibition of autophagy using rapamycin and LY294002 confirmed the regulatory effects of 11R‑VIVIT on autophagy. On the whole, the findings of the present study demonstrate that 11R‑VIVIT promotes fracture healing in osteoporotic rats and enhances the osteogenic differentiation of osteoporotic BMSCs by dysregulating the AKT/NFATc1 signaling pathway.

Keywords: 11R‑VIVIT; BMSCs; NFATc1; osteoporotic fracture healing.

Figure 1

11R-VIVIT accelerates fracture healing in osteoporotic rats. (A) Bone mineral density. (B) Schematic timeline of the procedures during the study. (C and D) Micro-CT 3D reconstruction. (E) H&E staining of callus tissue at 6 weeks post-fracture (×200 magnification) and 16 weeks post-fracture (×100 magnification). ^*P<0.05, compared with baseline.

Figure 2

11R-VIVIT increases the osteogenic potential of osteoporotic BMSCs. (A) Flow cytometric analysis of BMSC surface antigens. (B) ALP staining results. (C) Alizarin Red S staining results. (D) Quantitative results of ALP staining. (E) Quantitative results of Alizarin Red S staining. (F) Semi-quantitative PCR detection of osteogenesis-related gens. ^*P<0.05 and ^**P<0.01, compared with the OVX group. BMSC, bone marrow-derived mesenchymal stem cell; OVX, ovariectomy; ALP, alkaline phosphatase.

Figure 3

11R-VIVIT impairs the adipogenic potential of osteoporotic BMSCs. (A) Oil Red O staining results. (B) Following adipogenic induction, the mRNA levels of PPARγ and LPL in BMSCs were detected by RT-qPCR. ^**P<0.01. BMSC, bone marrow-derived mesenchymal stem cell; OVX, ovariectomy; PPARγ, peroxisome proliferator-activated receptor γ; LPL, lipoprotein lipase.

Figure 4

11R-VIVIT alleviates the inflammatory levels of osteoporotic BMSCs and increased their viability. (A) Cell viability of BMSCs in the OVX group was decreased when compared with the normal group, and 11R-VIVIT treatment relieved the decreased cell viability caused by OVX. ^*P<0.05 and ^**P<0.01, normal group vs. OVX. ^##P<0.01, OVX vs. OVX + 11R-VIVIT. (B and C) Expression levels of the inflammatory factors, TNF-α and IL-1β, in BMSCs in the OVX group were increased, and 11R-VIVIT relieved the increase in the levels of TNF-α and IL-1β induced by OVX. ^**P<0.01. BMSC, bone marrow-derived mesenchymal stem cell; OVX, ovariectomy; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β.

Figure 5

11R-VIVIT activates autophagy by activating AKT/GSK-3β signaling. (A) Western blot analysis of the autophagy-related proteins, LC3-I/II and p62. The autophagy level in BMSCs from rats subjected to OVX was inhibited, and 11R-VIVIT promoted autophagy. (B) Western blot analysis of AKT and GSK-3β proteins. (C) Following osteogenic induction, the level of NFTAc1 in the nucleus of BMSCs in the OVX group was higher than that in the normal group. LY294002 and 11R-VIVIT inhibited the entry of NFTAc1 into the nucleus. ^*P<0.05 and ^**P<0.01, compared with BMSCs from rats subjected to OVX. BMSC, bone marrow-derived mesenchymal stem cell; OVX, ovariectomy; AKT, protein kinase B; GSK-3β, glycogen synthase kinase 3β; RAPA, rapamycin.

Figure 6

AKT/NFATc1 signaling pathway regulates the osteogenic differentiation of BSMCs. (A) ALP staining results. (B) Alizarin Red S staining results. (C) Quantitative results of ALP staining. (D) Quantitative results of Alizarin Red S staining. (E) Following osteogenic induction, the levels of osteogenic marker genes Runx2 and ALP of BMSCs in the OVX group were significantly reduced, while LY294002 and 11R-VIVIT promoted their expression. ^**P<0.01, compared with BMSCs from rats subjected to OVX. BMSC, bone marrow-derived mesenchymal stem cell; OVX, ovariectomy; ALP, alkaline phosphatase; AKT, protein kinase B; GSK-3β, glycogen synthase kinase 3β; NFAT, nuclear factor of activated T-cells; RAPA, rapamycin.