Int J Stem Cells.  2022 Nov;15(4):405-414. 10.15283/ijsc21152.

Cannabidiol Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in the Inflammatory Microenvironment via the CB2-dependent p38 MAPK Signaling Pathway

• Li L
• Feng J
• Sun L
• Xuan Yw
• Wen L
• Li Yx
• Yang S
• Zhu B
• Tian Xy
• Li S
• Zhao Ls
• Dang Rj
• Jiao T
• Zhang Hs
• Wen N

• KMID: 2536210
• DOI: http://doi.org/10.15283/ijsc21152

Abstract

Background and Objectives
Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment.
Methods and Results
BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and pre-sented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs’ vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively.
Conclusions
CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.

Keyword

Cannabidiol (CBD); Osteogenic differentiation; Inflammatory microenvironment; Cannabinoid receptor 2 (CB2); p38 MAPK signaling pathway

Figure

• 

  Fig. 1 Morphology, phenotypic, and multidirectional differentiation characteristics of mouse BMSCs. (A) Re-presentative morphology image of passage 2 (P2) BMSCs was visual-ized by light microscopy (Scale bar, 100 μm). (B) P3 BMSCs surface markers were identified by flow cyto-metry. (C) Osteogenic differentiation capacities of isolated BMSCs were verified by alizarin red staining after incubation for 14 days (n=3; Scale bar, 100 μm). (D) Adipogenic differentiation capacities of isolated BMSCs were detected by oil red O staining after culture for 14 days (n=3; Scale bar, 100 μm). BMSCs: bone mesenchymal stem cells.

• 

  Fig. 2 CBD inhibits LPS-induced inflammatory response. (A) BMSCs’ viability was tested by CCK-8 assay after incubating with different concentrations of CBD for 72 hrs (n=8). (B) BMSCs were pretreated with LPS (10 μg/ml) for 12 hrs, and added different concentrations of CBD (0.5, 2.5, 5 μM) for 12 hrs. Then, BMSCs were harvested for detecting the mRNA expression levels of TNF-α and IL-6. β-actin was used as the internal control (n=5). CBD: cannabidiol, LPS: lipopolysaccharide, BMSCs: bone mesenchymal stem cells, CCK-8: cell counting kit-8, TNF-α: tumor necrosis factor-α, IL-6: interleukin-6, ns: no statistical significance. Data are presented as means±SD. *p<0.05 compared with the LPS group; #p<0.05 compared with the LPS plus 0.5 μM CBD group.

• 

  Fig. 3 CBD promotes osteogenic differentiation of BMSCs in the inflammatory microenvironment. (A) BMSCs were co-treated with LPS (10 μg/ml) and different concentrations of CBD (0.5, 2.5, 5 μM), as indicated for 1, 3, 5, and 7 days, then ALP activity was detected (n=5). (B) BMSCs were co-treated with LPS and CBD (0.5, 2.5, 5 μM), as indicated for 7 days. Western blot was performed for detecting Runx2, ALP, and OCN. β-actin was used as the internal control (n=3). (C) Quantitative analysis of (B). (D) BMSCs were co-treated with LPS and CBD (0.5, 2.5, 5 μM), as indicated for 7 days. Then, BMSCs were harvested for detecting the mRNA expression levels of Runx2, ALP, and OCN by qRT-PCR. β-actin was used as the internal control (n=5). CBD: cannabidiol, BMSCs: bone mesenchymal stem cells, Runx2: runt-related transcription factor 2, ALP: alkaline phosphatase, OCN: osteocalcin, qRT-PCR: quantitative real-time polymerase chain reaction. Data are represented as means±SD. *p<0.05 compared with the LPS group; #p<0.05 compared with the LPS plus 0.5 μM CBD group.

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  Fig. 4 CBD activates the CB2-dependent p38 MAPK pathway. (A) BMSCs were pretreated with LPS (10 μg/ml) for 12 hrs, then CBD (2.5 μM) were added and cultured for another 12 hrs. Western blots and quantitative analysis were performed for determining CB1 and CB2. β-actin was used as the internal control (n=3). (B) BMSCs were pretreated with LPS for 12 hrs, then CBD and AM630 (10 μM) were added as indicated and incubated for another 12 hrs. Western blots and quantitative analysis for p-p38 and p38 were performed. β-actin was used as the internal control (n=3). CB1: cannabinoid receptor 1, CB2: cannabinoid receptor 2, p-p38: phosphorylated p38. Data are presented as means±SD. *p<0.05 compared with the LPS group; #p<0.05 compared with the LPS plus CBD group.

• 

  Fig. 5 CB2-dependent p38 MAPK signaling pathway is required for CBD promoting osteogenic differentiation of BMSCs. BMSCs were pretreated with LPS (10 μg/ml) for 12 hrs. Then CBD (2.5 μM), AM630 (10 μM), and SB203580 (20 μM) were added as indicated and co-cultured with LPS for 7 days. (A) Western blots were performed for detecting the protein expression levels of Runx2, ALP, and OCN. β-actin was used as the internal control (n=3). (B) Quantitative analysis of (A). (C) BMSCs were harvested for measuring the mRNA expression levels of Runx2, ALP, and OCN using qRT-PCR. β-actin was used as the internal control (n=5). BMSCs were co-treated with these agents for 14 days and then alizarin red staining was performed. Representative images (D) and semiquantification (E) of alizarin red staining after incubation for 14 days (Scale bar, 100 μm). Data are presented as means±SD. *p<0.05 compared with the CBD plus LPS group. #p<0.05 compared with the CBD plus AM630 group.

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  Fig. 6 Schematic diagram of CBD intervention.

Reference

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