Berberine ameliorates the LPS-induced imbalance of osteogenic and adipogenic differentiation in rat bone marrow-derived mesenchymal stem cells

Abstract

Lipopolysaccharide (LPS) from oral pathogenic bacteria is an important factor leading to alveolar bone absorption and the implant failure. The present study aimed to evaluate the modulation of berberine hydrochloride (BBR) on the LPS-mediated osteogenesis and adipogenesis imbalance in rat bone marrow-derived mesenchymal stem cells (BMSCs). Cell viability, osteoblastic and adipogenic differentiation levels were measured using the Cell Counting Kit-8 assay, alkaline phosphatase (ALP) staining and content assay, and oil red O staining, respectively. Reverse transcription-quantitative PCR and immunoblotting were used to detect the related gene and protein expression levels. In undifferentiated cells, BBR increased the mRNA expression levels of the osteoblastic genes (Alp, RUNX family transcription factor 2, osteocalcin and secreted phosphoprotein 1) but not the adipogenic genes (fatty acid binding protein 4, Adipsin and peroxisome proliferator-activated receptorγ). LPS-induced osteoblastic gene downregulation, adipogenic gene enhancement and NF-κB activation were reversed by BBR treatment. In osteoblastic differentiated cells, decreased ALP production by LPS treatment was recovered with BBR co-incubation. In adipogenic differentiated cells, LPS-mediated lipid accumulation was decreased by BBR administration. The mRNA expression levels of the pro-inflammatory factors (MCP-1, TNF-α, IL-6 and IL-1β) were increased by LPS under both adipogenic and osteoblastic conditions, which were effectively ameliorated by BBR. The actions of BBR were attenuated by compound C, suggesting that the role of BBR may be partly due to AMP-activated protein kinase activation. The results demonstrated notable pro-osteogenic and anti-adipogenic actions of BBR in a LPS-stimulated inflammatory environment. This indicated a potential role of BBR for bacterial infected-related peri-implantitis medication.

Figure 1.

BBR alters osteogenic and adipogenic gene expression levels in undifferentiated bone marrow-derived mesenchymal stem cells. (A) Cells were incubated with DMSO or BBR (1, 5, 10, 50 or 100 µM) for 3 days, and then cell viability was determined using a Cell Counting Kit-8 assay. The cells were treated with BBR (1, 5 or 10 µM) alone or simultaneously with LPS (1 µg/ml), and then collected for reverse transcription-quantitative PCR analysis. The mRNA expression levels of the osteogenic genes (B) Runx2, (C) Spp1 and (D) Ocn, and the adipogenic genes (E) Fabp4, (F) Adipsin and (G) Pparγ were measured. Their relative changes were normalized to the GAPDH expression levels. The data are presented as the mean ± standard deviation of three independent experiments. ^#P<0.05, ^##P<0.01, ^###P<0.001, ^####P<0.0001 vs. control group; **P<0.01, ***P<0.001, ****P<0.0001 vs. LPS group. BBR, berberine hydrochloride; LPS, lipopolysaccharide; Runx2, RUNX family transcription factor 2; Ocn, osteocalcin; Spp1, secreted phosphoprotein 1; Fabp4, fatty acid binding protein 4; Pparγ, peroxisome proliferator-activated receptorγ.

Figure 2.

BBR promotes osteogenesis in bone marrow-derived mesenchymal stem cells. The cells were incubated with BBR (0, 1, 5 or 10 µM) alone or simultaneously with LPS (1 µg/ml) in osteogenic media for 7 days. Then, the cells were detected using ALP stain and reverse transcription-quantitative PCR analysis. (A) Images of the ALP stained cells were obtained using a camera. The mRNA expression levels of the (B) Alp, (C) Runx2, (D) Ocn and (E) Spp1 osteogenic genes were measured. Their relative changes were normalized to the GAPDH expression levels. The data are presented as the mean ± standard deviation of three independent experiments. ^##P<0.01, ^####P<0.0001 vs. control group; *P<0.05, **P<0.01 vs. LPS group. BBR, berberine hydrochloride; LPS, lipopolysaccharide; Runx2, RUNX family transcription factor 2; Ocn, osteocalcin; Spp1, secreted phosphoprotein 1; ALP, alkaline phosphatase.

Figure 3.

BBR inhibits adipogenesis in bone marrow-derived mesenchymal stem cells. The cells were incubated with BBR (0, 1, 5 or 10 µM) alone or simultaneously with LPS (1 µg/ml) in adipogenic media for 12 days. Then, the cells were detected using oil red O stain and reverse transcription-quantitative PCR analysis. (A) Images of the ALP stained cells were obtained using a microscope, at ×200 magnification. The mRNA expression levels of the (B) Fabp4, (C) Adipsin and (D) Pparγ adipogenic genes were measured. Their relative changes were normalized to the GAPDH expression levels. The data are presented as the mean ± standard deviation of three independent experiments. ^##P<0.01, ^####P<0.0001 vs. control group; ****P<0.0001 vs. LPS group. BBR, berberine hydrochloride; LPS, lipopolysaccharide; Fabp4, fatty acid binding protein 4; Pparγ, peroxisome proliferator-activated receptorγ; ALP, alkaline phosphatase.

Figure 4.

BBR protects bone marrow-derived mesenchymal stem cells from LPS-induced NF-κB activation under undifferentiated condition. Cells were incubated with DMSO (as the control), BBR (10 µM), LPS (1 µg/ml) or both BBR and LPS for 2h. Then, the cells were harvested for western blot analysis. (A) Bands of the p-and t- (B) NF-κB p65 subunit, (C) IκBα and GAPDH protein expression levels are shown and the results were semi-quantified. The data are presented as the mean ± standard deviation of three independent experiments. ^#P<0.05, ^####P<0.0001 vs. control group; **P<0.01 vs. LPS group. T, total, p, phosphorylated; BBR, berberine hydrochloride; LPS, lipopolysaccharide; p-, phosphorylated; t-, total.

Figure 5.

BBR alleviates LPS-induced inflammatory factor expression in both osteogenic and adipogenic differentiated bone marrow-derived mesenchymal stem cells. The cells were incubated with BBR (0, 1, 5 or 10 µM) or simultaneously with LPS (1 µg/ml) in osteogenic media for 7 days or in adipogenic media for 12 days. Then, the cells were harvested for reverse transcription-quantitative PCR analysis. The mRNA expression levels of the pro-inflammatory factors (A) MCP-1, (B) TNF-α, (C) Il-6 and (D) Il-1β were measured in differentiated osteoblastic cells. (E) MCP-1, (F) TNF-α, (G) Il-6 and (H) Il-1β expression levels were also measured in adipogenic differentiated cells. Their relative changes were normalized to GAPDH levels. The data are presented as the mean ± standard deviation of three independent experiments. ^#P<0.05, ^##P<0.01, ^###P<0.001, ^####P<0.0001 vs. control group; *P<0.05, ***P<0.001, ****P<0.0001 vs. LPS group. BBR, berberine hydrochloride; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1.

Figure 6.

BBR regulates osteogenic and adipogenic differentiation via AMPK activation. The cells were incubated with BBR (10 µM), C.C (1 µM) or both, in the presence or absence of LPS (1 µg/ml), in osteogenic media for 7 days or in adipogenic media for 12days. Then, the cells were collected for ALP staining, ALP content assay or oil red O staining. The images of the ALP stained cells were obtained using a (A) camera and (B) microscope at ×200 magnification. (C) Relative ALP content levels of osteogenic cells were measured and normalized to control levels. The images of the oil red O stained cells were obtained using a (D) camera and (E) microscope at ×200 magnification. The data are presented as the mean ± standard deviation of three independent experiments. ***P<0.001, ****P<0.0001. C.C, compound C; BBR, berberine hydrochloride; LPS, lipopolysaccharide; ALP, alkaline phosphatase.