Shikonin intervention with bone marrow mesenchymal stem cells improves microstructure of femur in aged mice

doi:10.12307/2025.577

Abstract

Abstract: BACKGROUND: Shikonin, a purple naphthoquinone pigment extracted from Lithospermum erythrorhizon, exhibits diverse biological activities. Additionally, shikonin has been demonstrated to possess bone-protective effects. However, its action mechanisms remain unclear.
OBJECTIVE: To elucidate the molecular mechanisms by which shikonin promotes osteogenic differentiation in bone marrow mesenchymal stem cells, and its potential application in the treatment of osteoporosis.
METHODS: (1) Bone marrow mesenchymal stem cells were treated with varying concentrations (0.1, 0.2, and 0.4 µmol/L) of shikonin, and their proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. (2) Bone marrow mesenchymal stem cells were co-treated with different concentrations (0.1, 0.2, and 0.4 µmol/L) of shikonin and osteogenic induction medium. After osteogenesis induction for 7 and 14 days, alkaline phosphatase staining and alizarin red staining were performed. (3) Bone marrow mesenchymal stem cells were treated with different concentrations (0.2 and 0.4 µmol/L) of shikonin for 48 hours. RNA was extracted for real-time PCR analysis. (4) Bone marrow mesenchymal stem cells were treated with 0.2 µmol/L shikonin for 30, 60, and 120 minutes. Expression levels of p-P38, p-ERK, and p-JNK in the MAPK signaling pathway were detected by western blot assay. (5) Eighteen-month-old C57BL/6 mice were treated with shikonin for two months, followed by micro-CT scanning of rat femurs. The tibiae of mice were taken for osteocalcin immunofluorescence detection.
RESULTS AND CONCLUSION: (1) CCK-8 assay indicated that shikonin promoted the proliferation of bone marrow mesenchymal stem cells. (2) Alkaline phosphatase staining and alizarin red staining suggested that shikonin enhanced osteogenic differentiation of bone marrow mesenchymal stem cells. (3) RT-PCR results showed increased mRNA levels of osteogenic gene markers osteocalcin, alkaline phosphatase, bone morphogenetic protein 2, and Runt-related transcription factor 2. (4) Western blot assay revealed that shikonin induced phosphorylation of P38, ERK, and JNK in the MAPK signaling pathway of bone marrow mesenchymal stem cells. (5) Animal experiment findings indicate that shikonin can improve the bone microstructure of the femur of aged mice and increase the expression of osteocalcin in the tibia. The results show that shikonin promotes the osteogenic differentiation of bone marrow mesenchymal stem cells through the MAPK signaling pathway and improves the bone microstructure of osteoporotic mice.

Key words: bone marrow mesenchymal stem cell, shikonin, osteoporosis, MAPK signaling pathway, osteogenesis, engineered stem cell

CLC Number:

Hu Xiongke, Liu Shaohua, Tan Qian, Liu Kun, Zhu Guanghui. Shikonin intervention with bone marrow mesenchymal stem cells improves microstructure of femur in aged mice[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(7): 1609-1615.

Figures/Tables 2

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