CB1 enhanced the osteo/dentinogenic differentiation ability of periodontal ligament stem cells via p38 MAPK and JNK in an inflammatory environment

Abstract

Objectives: Periodontitis is an inflammatory immune disease that causes periodontal tissue loss. Inflammatory immunity and bone metabolism are closely related to periodontitis. The cannabinoid receptor I (CB1) is an important constituent of the endocannabinoid system and participates in bone metabolism and inflammation tissue healing. It is unclear whether CB1 affects the mesenchymal stem cell (MSC) function involved in periodontal tissue regeneration. In this study, we revealed the role and mechanism of CB1 in the osteo/dentinogenic differentiation of periodontal ligament stem cells (PDLSCs) in an inflammatory environment.

Materials and methods: Alkaline phosphatase (ALP) activity, Alizarin Red staining, quantitative calcium analysis and osteo/dentinogenic markers were used to assess osteo/dentinogenic differentiation. Real-time RT-PCR and Western blotting were employed to detect gene expression.

Results: CB1 overexpression or CB1 agonist (10 µM R-1 Meth) promoted the osteo/dentinogenic differentiation of PDLSCs. Deletion of CB1 or the application of CB1 antagonist (10 µM AM251) repressed the osteo/dentinogenic differentiation of PDLSCs. The activation of CB1 enhanced the TNF-α- and INF-γ-impaired osteo/dentinogenic differentiation potential in PDLSCs. Moreover, CB1 activated p38 MAPK and JNK signalling and repressed PPAR-γ and Erk1/2 signalling. Inhibition of JNK signalling could block CB1-activated JNK and p38 MAPK signalling, while CB1 could activate p38 MAPK and JNK signalling, which was inhibited by TNF-α and INF-γ stimulation.

Conclusions: CB1 was able to enhance the osteo/dentinogenic differentiation ability of PDLSCs via p38 MAPK and JNK signalling in an inflammatory environment, which might be a potential target for periodontitis treatment.

Keywords: CB1; MAPK signalling pathway; inflammation; osteo/dentinogenic differentiation; periodontal ligament stem cells (PDLSCs).

Figure 1

CB1 knock‐down inhibited the osteo/dentinogenic differentiation of PDLSCs. (A) Western blot results showed the knock‐down efficiency of CB1 shRNA in PDLSCs. β‐actin was used as an internal control. (B) ALP activity assay. (C) Alizarin Red staining. (D) Calcium quantitative analysis. (E‐H) Real‐time RT‐PCR results of the ON (E), DSPP (F), DMP1 (G) and BSP (H) expression levels during PDLSC osteo/dentinogenic differentiation. (I‐L) Real‐time RT‐PCR results of OSX (I), DLX2 (J), DLX3 (K) and DLX5 (L) expression levels in PDLSCs. GAPDH was used as an internal control. Student's t test was performed to determine statistical significance. Error bars represent the SD (n = 3). *P ≤ .05; **P ≤ .01

Figure 2

Overexpression of CB1 enhanced the osteo/dentinogenic differentiation of PDLSCs. (A) Western blot results showed the overexpression efficiency of HA‐CB1 in PDLSCs. β‐actin was used as an internal control. (B) ALP activity assay. (C) Alizarin Red staining. (D) Calcium quantitative analysis. (E‐H) Real‐time RT‐PCR results of the ON (E), DSPP (F), DMP1 (G) and BSP (H) expression levels during PDLSC osteo/dentinogenic differentiation. (I‐L) Real‐time RT‐PCR results of OSX (I), DLX2 (J), DLX3 (K) and DLX5 (L) expression levels in PDLSCs. GAPDH was used as an internal control. Student's t test was performed to determine statistical significance. Error bars represent the SD (n = 3). *P ≤ .05; **P ≤ .01

Figure 3

The function of R‐1 Meth and AM251 on osteo/dentinogenic differentiation in PDLSCs. A‐D, 10 µM R‐1 Meth was used to treat PDLSCs. A, Western blot results showed the expression of CB1 and CB2 in PDLSCs after treatment with 10 µM R‐1 Meth for 4 h. β‐actin was used as an internal control. B, The ALP activity assay. C, Alizarin Red staining. D, Calcium quantitative analysis. E‐H, 10 µM AM251 was used to treat the CB1‐overexpressing PDLSCs. E, Western blot results showed the expression of CB1 and CB2 in PDLSCs after treatment with 10 µM AM251 for 4 h. β‐actin was used as an internal control. F, ALP activity assay. G, Alizarin Red staining. H, Calcium quantitative analysis. Student's t test or one‐way ANOVA was performed to determine statistical significance. Error bars represent the SD (n = 3). **P ≤ .01

Figure 4

The effect of CB1 on MAPK signal pathways and PPAR‐γ in PDLSCs. A, Western blot results showed the expression of phosphorylated p38 MAPK, JNK, and Erk1/2, along with p38 MAPK, JNK, Erk1/2 and PPAR‐γ in the CB1‐overexpressing PDLSCs compared to the control group. B, Western blot results showed the expression of phosphorylated p38 MAPK, JNK and Erk1/2, along with p38 MAPK, JNK, Erk1/2 and PPAR‐γ in the CB1sh PDLSCs compared to the control group. C, 20 µM SB203580 or 20 µM SP600125 was used to treat the CB1‐overexpressing PDLSCs for 2 h. Western blot results showed the expression of phosphorylated p38 MAPK and JNK, along with p38 MAPK, JNK and PPAR‐γ in PDLSCs. β‐actin was used as an internal control

Figure 5

CB1 upregulated the osteo/dentinogenic differentiation potential of PDLSCs under TNF‐α and INF‐γ stimulation. A‐D, 10 ng/mL TNF‐α was used to treat PDLSCs. A, Real‐time RT‐PCR results showed the expression of CB1 at 1, 2, 4 and 8 h after 10 ng/mL TNF‐α treatment in PDLSCs. B, ALP activity assay. C, Alizarin Red staining. D, Calcium quantitative analysis. E‐H, 100 ng/mL INF‐γ was used to treat PDLSCs. E, Real‐time RT‐PCR results showed the expression of CB1 at 1, 2, 4 and 8 h after 100 ng/mL INF‐γ treatment in PDLSCs. F, ALP activity assay. G, Alizarin Red staining. H, Calcium quantitative analysis. GAPDH was used as an internal control. One‐way ANOVA was performed to determine statistical significance. Error bars represent the SD (n = 3). *P ≤ .05; **P ≤ .01

Figure 6

The function of R‐1 Meth on osteo/dentinogenic differentiation in PDLSCs under TNF‐α and INF‐γ stimulation. A‐C, 10 µM R‐1 Meth and 10 ng/mL TNF‐α were used to treat PDLSCs. A, ALP activity assay. B, Alizarin Red staining. C, Calcium quantitative analysis. D‐F, 10 µM R‐1 Meth and 100 ng/mL INF‐γ were used to treat PDLSCs. D, ALP activity assay. E, Alizarin Red staining. F, Calcium quantitative analysis. One‐way ANOVA was performed to determine statistical significance. Error bars represent the SD (n = 3). *P ≤ .05; **P ≤ .01

Figure 7

The effect of CB1 on MAPK signalling pathways and PPAR‐γ in PDLSCs under TNF‐α or INF‐γ stimulation. A, B, 10 ng/mL TNF‐α was used to treat PDLSCs for 4 h. A, Western blot showed the expression of phosphorylated p38 MAPK, JNK and Erk1/2, along with p38 MAPK, JNK, Erk1/2 and PPAR‐γ in PDLSCs. B, The quantitative analysis of phosphorylated p38 MAPK, JNK and Erk1/2 and PPAR‐γ. C, D, 100 ng/mL INF‐γ was used to treat PDLSCs for 4 h. C, Western blot showed the expression of phosphorylated p38 MAPK, JNK and Erk1/2, along with p38 MAPK, JNK, Erk1/2 and PPAR‐γ in PDLSCs. D, The quantitative analysis of phosphorylated p38 MAPK, JNK, and Erk1/2 and PPAR‐γ. Histone H3 was used as an internal control. Error bars represent the SD (n = 3). *P ≤ .05; **P ≤ .01