原著
ヒト歯肉線維芽細胞におけるカンナビノイド受容体GPR55の発現とGPR55を介したカンナビジオールの抗炎症作用の検討
2021 年 63 巻 1 号 p. 11-23
詳細
2021 年 63 巻 1 号 p. 11-23
Cannabidiol (CBD) in cannabis has an anti-inflammatory effect, and its receptor, G protein-coupled receptor 55 (GPR55), has been suggested to be involved in the regulation of inflammation. Here, we investigated GPR55 expression in human gingival fibroblasts (HGFs) and anti-inflammatory effects of CBD via GPR55.
GPR55 expression and localization were confirmed in control, LPS-stimulated (LPS), CBD-treated and LPS-stimulated (CBD+LPS), and CBD-treated (CBD) groups. We also compared interleukin (IL) -6 and IL-8 protein production in each group.
GPR55 expression levels were significantly reduced in the LPS and CBD+LPS groups (p<0.05) compared to control and CBD groups, and GPR55 protein was found diffusely in the cytoplasm of HGFs in all groups. In addition, IL-6 and IL-8 production was significantly reduced in non-knockdown HGFs in the CBD+LPS group compared to the LPS group (p<0.05). By contrast, in GPR55-knockdown HGFs, there was a significant increase in IL-6 and IL-8 production in the LPS and CBD+LPS groups compared with the control groups (p<0.05), but there was no significant difference in IL-6 and IL-8 production in GPR55-knockdown HGFs between the LPS and CBD+LPS groups.
In this study, we confirmed the homeostatic expression of GPR55 in HGFs and its tendency to decrease in expression during inflammation. It was also shown that part of an anti-inflammatory effect by CBD may be mediated by GPR55.
Periodontitis is an inflammatory disease of the periodontal tissue that is caused by dental plaque1). As the plaque matures, periodontal pathogens predominate. Periodontopathic bacteria are Gram-negative and their outer membrane contains lipopolysaccharide (LPS). LPS binds to Toll-like receptor 4 (TLR4) on the surface of periodontal tissue cells, including human gingival fibroblasts (HGFs), which constitute the connective tissue of the gingiva, and secretes proinflammatory cytokines through signal transduction2,3). In particular, interleukin (IL) -6 regulates the differentiation and proliferation of immune cells and also exacerbates periodontal disease by inducing bone resorption4). In addition, IL-8 induces the migration of inflammatory cells and osteoclasts in periodontal disease, and it is therefore thought to be involved in the destruction of periodontal tissues5). Mechanical plaque control for the removal of dental plaque, which is the source of infection, is an important part of periodontal treatment6). However, due to the limited effectiveness of mechanical plaque control7), chemical plaque control is being sought as an adjunctive therapy8). One of these adjunctive therapies is host modulation therapy, which is used to inhibit the secretion of inflammatory cytokines to prevent destruction of periodontal tissue9). In this study, we focused on cannabidiol (CBD), a major component of cannabinoids in cannabis that has anti-inflammatory properties.
CBD has no psychoactivity, unlike the other components of cannabis10), and has been widely studied for medical applications due to its beneficial antiepileptic effect11), pain relief ability12), anticancer activity13), and bone growth promoting activity14). CBD is a terpene phenol with the molecular formula C21H30O2 and a molecular weight of 314.47 g/mol. It is insoluble in water and soluble in organic solvents15). It has been clinically applied as a drug and is approved in some countries for advanced cancer pain and neuropathic pain relief and as epilepsy treatment16). In addition, the efficacy of CBD in the treatment of rheumatoid arthritis17) has led to the recent development of anti-inflammatory effects that are expected to be clinically applied18,19).
Two types of cannabinoid receptors, cannabinoid receptor 1 and 2 (CB1 and CB2), have been identified as receptors for CBD containing cannabinoids, which are G protein-coupled receptors (GPCRs) with seven transmembrane domains20). CB1 receptors are primarily distributed in the central nervous system and mediate most psychoactive effects, such as pain relief and muscle spasm relief through activation21). CB2 receptors are mainly expressed on peripheral and immune cells and mediate immunosuppressive effects such as anti-inflammatory and analgesic effects22). However, accumulating evidence from many studies has suggested the existence of new cannabinoid receptors that are distinct from CB1 and CB2 receptors.
In the mid-2000s, the orphan G protein-coupled receptor 55 (GPR55) was presented as a major candidate for a new cannabinoid receptor23). High GPR55 expression has been reported in malignant breast cancer cells and osteoclasts24,25). Therefore, there have been many studies on GPR55 and its therapeutics as targets for the treatment of cancer and bone metabolic diseases. Additionally, the relationship between GPR55 and inflammation has been reported18). For example, GPR55, when activated by the endogenous agonist lysophosphatidylinositol (LPI), is thought to play a role in exacerbating inflammation by activating transcriptional regulators such as nuclear factor-kappa B (NF-κB) through the activation of ERK and p38MAP kinases, thus promoting the production of proinflammatory cytokines26,27). Other reports showed increased GPR55 expression in intestinal tissues of rats with LPS-induced intestinal inflammation and suggested that CBD was effective in suppressing enteritis28). Furthermore, it has been reported that GPR55 is highly expressed in monocytes and natural killer cells, and GPR55 activation promotes the production of proinflammatory cytokines, which are conversely inhibited by CBD29). Thus, GPR55 has been suggested to be associated with inflammation in various tissues, but little has been clarified on its role in periodontal tissue. Because CB1 and CB2 expression in periodontal tissues and their association with inflammation have been reported30,31), it is likely that GPR55 is similarly relevant. The anti-inflammatory effect of CBD on rats induced by experimental periodontitis has been reported32), but there are few studies on CBD and periodontal tissues. Furthermore, the detailed mechanism underlying the anti-inflammatory effect of CBD and its association with GPR55 remain unclear. Therefore, we hypothesized that GPR55 is expressed in periodontal tissue and is associated with inflammation and CBD has an anti-inflammatory effect on periodontal tissue by acting as an antagonist of GPR55.
In the present study, we first confirmed the presence of GPR55 expression in HGFs and then observed changes in GPR55 expression in HGFs of inflammation induced by LPS stimulation. In addition, the relationship between anti-inflammatory effects of CBD and GPR55 was also investigated using HGFs with knockdown of GPR55 gene expression.
Immortalized HGFs (Applied Biological Materials Inc., BC, Canada) were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 μL/mL Antibiotic-Antimycotic (Thermo Fisher Scientific) and 10% fetal bovine serum (BioWest, Nuaillé, France) at 37°C in 5% CO2 atmosphere. Cells between passages 3 and 7 were used in experiments.
2, Effect of CBD and LPS on HGF cell proliferationEffects of CBD (Sigma-Aldrich, St. Louis, MO, USA) and LPS (from Escherichia coli O111: B4, Sigma-Aldrich) on HGF cell proliferation were assessed by MTT assay. Individual wells of 96-well microtiter tissue culture plates were seeded with 5.0×103 cells in 100 μL of complete medium. Cells were incubated overnight at 37°C in 5% CO2 atmosphere, the medium was replaced with reagent added medium adjusted to each concentration for 4 days. CBD concentrations evaluated were 0, 0.005, 0.05, 0.5, 5, and 10 μM, and LPS concentrations used were 0, 0.01, 0.1, 1, and 10 μg/mL. After 4 hours of incubation with the Cell Counting Kit-8 WST-8 (DOJINDO, Kumamoto, Japan), cell proliferation was measured using a spectrophotometer (SH-9000, Corona Electric, Hitachinaka, Japan) at 450 nm absorbance. Results are presented as optical density at 450 nm.
3, Experimental designTo investigate GPR55, CB1, and CB2 expression in HGFs, variations in expression upon LPS stimulation, and anti-inflammatory effects of CBD, the following conditions were established. Untreated cells were used as the control group, and cells stimulated by 0.1 μg/mL LPS for 24 h were included as the LPS group. Cells pre-treated with 0.5 μM CBD for 2 h and then cultured in 0.1 μg/mL LPS-supplemented medium for 24 h were used as the CBD+LPS group, and cells pre-treated with CBD for 2 h and then cultured in LPS-free medium for 24 h were included as the CBD group.
4, RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)Cells were seeded into 6-well plates at 2.5×105 cells/mL, incubated for 24 h, and then cultured for 24 h under the four conditions described above. Total RNA was extracted from HGFs using the Maxwell® RSC simplyRNA Cells Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. cDNA was then synthesized from total RNA using SuperScript® VILO Master Mix (Invitrogen, Thermo Fisher Scientific). β-Actin was used as the internal standard. Primer sequences used to detect each gene are as follows: CB1 (Hs01038522, Applied Biosystems, Thermo Fisher Scientific), CB2 (Hs00361490, Applied Biosystems, Thermo Fisher Scientific), GPR55 (Hs00271662, Applied Biosystems, Thermo Fisher Scientific), and β-Actin (NM_001101.2, Applied Biosystems, Thermo Fisher Scientific). Quantitative real-time PCR was performed using gene-specific primers and TaqMan Fast Advanced Master Mix (Invitrogen, Thermo Fisher Scientific), cDNA template (corresponding to 10 ng/μL cDNA), primers, and probes in a final reaction volume of 20 μL on a real-time PCR system. Cycling conditions for each gene were as follows: 95°C for 20 min, 40 cycles of 1 min at 95°C, and 60°C for 20 min. To compare gene expression levels between different samples, relative gene expression levels were calculated by the ΔΔCT method using StepOnePlus software (Applied Biosystems, Thermo Fisher Scientific).
5, Western blotting analysisCells were cultured under the same conditions as described above for qRT-PCR. Cells were then rinsed with cold PBS and divided into cytoplasmic and nuclear fractions. Samples were then electrophoresed in 10% resolving sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Then, membranes were reacted with specific antibodies against each target and internal standard and detected by the chemiluminescence method (ECL select, GE Healthcare, Chicago, IL, USA). β-Actin was used as the internal standard for cytoplasmic fractions and histone was included as the internal standard for nuclear fractions. Membranes were incubated at 4°C overnight with primary rabbit anti-GPR55 (ab3561, Abcam, Cambridge, UK), CB1 (ab23703, Abcam), and CB2 (ab3561, Abcam) serum. For internal standard, monoclonal anti-β-actin antibody produced in mouse clone (A5441, Sigma-Aldrich) and monoclonal anti-histone antibody (Histone H3 antibody, 9715S, Cell Signaling, Beverly, MA, USA) were used. Rabbit IgG HRP (NA934, GE Healthcare) and mouse IgG HRP (NXA931, GE Healthcare) were used as secondary antibodies. The ratio of protein expression to internal control was calculated using CS Analyzer Image Analysis Software (Atto Corporation, Tokyo, Japan).
6, Immunofluorescence assayHGFs were seeded onto 8-well culture slides at 1.0×104 cells/mL and incubated for 24 h, and then cells were cultured under the same conditions as described above for qRT-PCR. Cells were washed with 0.01 M PBS and fixed in 4% paraformaldehyde for 30 min. Cells were permeabilized for 20 min in 0.1% Triton X-100 in PBS, then treated with blocking buffer (1% BSA in PBS) for 1 h. The same primary antibodies used in western blotting analyses were used for staining of each receptor and the nucleus (DAPI stain), and cells were incubated at 4°C overnight. Cells were then washed with PBS and incubated with goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor 488 (A11008, Thermo Fisher Scientific) for 2 h. Thereafter, cells were contrast-stained by DAPI and sealed (Vector Laboratories, Burlingame, CA, US). Stained samples were evaluated under a confocal laser scanning microscope (LSM700, Carl Zeiss MicroImaging GmbH, Oberkochen, Germany).
7, Transfection with GPR55 siRNATo assess the presence of a GPR55-dependent inflammatory response, human GPR55 siRNA (Dharmacon, Horizon Discovery, Lafayette, CO, USA) and negative control siRNA (Dharmacon, Horizon Discovery) were used for transfection. Cells were transfected with 50 nM siRNA using DharmaFECT 1 Transfection Reagent (Dharmacon, Horizon Discovery) according to the manufacturer's instructions. Cells collected at 24-h post-transfection were used for enzyme-linked immunosorbent assays (ELISAs).
8, ELISAsTo evaluate the inhibitory effect of CBD on proinflammatory cytokine secretion, transfected HGFs were cultured in 24-well plates at 3.0×104 cells/mL and culture supernatants were collected. IL-6 and IL-8 protein expression in supernatants was quantified by ELISA (Quantikine ELISA Human IL-6, Human CXCL8/IL-8, R&D Systems, Minneapolis, MN, USA) and determined by a spectrophotometer (SH-9000, Corona Electric) used to measure the absorbance at 450 nm (reference wavelength of 570 nm). The concentration of each inflammatory cytokine was calculated according to a standard curve.
9, Statistical analysisAll data are presented as mean±standard deviation (SD) from five independent experiments. The normality of the distribution in measured data was confirmed by the Kolmogorov-Smirnov test. Subsequently, for qRT-PCR and Western blot data, the Tukey comparison test or the Games-Howell test was used as a one-way ANOVA and post-hoc test for comparisons between the four groups. Statistically significant differences between groups in the ELISA data were obtained using the Kruskal-Wallis test and the Steel-Dwass test as data showing a non-normal distribution. All statistical processing was performed using SPSS version 25 (IBM, Chicago, IL, USA). P values less than<0.05 were considered statistically significant.
To determine CBD and LPS concentrations used in the experiments, the concentration-dependent effects of each reagent on HGF cell proliferation were analyzed. CBD concentrations below 0.05 μM had no significant effect on cell proliferation after 1-4 days of exposure. Compared to the control group, cell proliferation in 5 and 10 μM CBD groups was significantly reduced after day 2 (p<0.05). After 4 days, there was a significant increase in the 0.5 μM CBD group compared to the control group (p<0.01) (Figure 1A). At LPS concentrations of 0.01 and 10 μg/mL, HGF cell proliferation was similar to that observed in the control group (Figure 1B). There was a tendency for HGF cell proliferation to decrease at LPS concentrations of 0.1 μg/mL and 1 μg/mL compared to the control group on days 3 and 4, but this decrease was not significant. Therefore, in subsequent experiments, the optimal concentration of CBD was determined to be 0.5 μM. The optimal concentration of LPS was determined to be 0.1 μg/mL, which has the highest production of pro-inflammatory cytokines in the range of 0-10 μg/mL.
Effects of A) CBD and B) LPS on HGF viability. Data represent mean±SD from five independent experiments. ＊p＜0.01, †p＜0.05, Tukey’s comparison test.
To assess the effect of LPS on mRNA expression of cannabinoid receptors GPR55, CB1, and CB2, changes in mRNA expression in HGFs at 1, 4, 12, and 24 h after 0.1 μg/mL LPS stimulation were analyzed using qRT-PCR. At 1 h after LPS stimulation, there was no significant difference between groups (Figure 2). In the LPS and CBD+LPS groups, there was a trend toward decreased GPR55 mRNA expression at 4-12 h after LPS stimulation, with a significant decrease in expression in both groups at 24 h (p<0.05). CB1 and CB2 mRNA expression was not observed in any group from the start of LPS stimulation to 24 h after treatment (data not shown).
GPR55 mRNA expression in HGFs. Cells were stimulated with 0.1 μg/mL LPS and GPR55 mRNA expression was analyzed by qRT-PCR. Changes in GPR55 gene expression levels were calculated by the 2－ΔΔCt method with non-stimulated cells set as reference (2－ΔΔCt＝1), which were normalized to mRNA expression of housekeeping gene β-Actin. Data represent mean±SD from five independent experiments. ＊p＜0.05, Tukey’s comparison test.
GPR55, CB1, and CB2 protein expression levels in each group were analyzed by western blotting 24 h after LPS stimulation. GPR55 protein was detected in the cytoplasmic fraction but not in the nuclear fraction. Furthermore, GPR55 protein expression in cytoplasmic fractions showed a significant downward trend in the LPS and CBD+LPS groups compared to the control group (p<0.05) (Figure 3). The CBD group showed the same expression trend as the control group. CB1 and CB2 protein expression was not observed in any group (data not shown).
GPR55 protein levels in HGFs were detected by Western blot, with β-Actin and histone levels included as controls. Histograms show mean±SD of relative quantification, which was normalized to the expression level in control cells. ＊p＜0.05, Games-Howell test.
The localization of each cannabinoid receptor in HGFs was analyzed by fluorescence immunostaining. In the control group, GPR55 expression was observed diffusely in the cytoplasm (Figure 4A). In the LPS, CBD+LPS, and CBD groups, GPR55 was also expressed in the cytoplasm as well as in the control group. CB1 was also expressed throughout HGFs in all conditions tested (Figure 4B). By contrast, CB2 expression was not observed (Figure 4C).
Localization of GPR55, CB1, and CB2 proteins in HGFs (control group, LPS group, CBD＋LPS group, and CBD group) evaluated by fluorescent microscopy. HGFs were pretreated with CBD (0.5 μM) for 2 h before LPS stimulation (0.1 μg/mL) for 24 h in the CBD＋LPS group. HGFs were then fixed and incubated with anti-GPR55, CB1, or CB2 antibody. (A) Distribution of GPR55 (green) and nucleus (DAPI, blue) in HGFs. (B) Distribution of CB1 (green) and nucleus (DAPI, blue) in HGFs. (C) Distribution of CB2 (green) and nucleus (DAPI, blue) in HGFs. Scale bar, 50 μm.
To compare changes in IL-6 and IL-8 production in HGFs with and without GPR55 (non-knockdown and GPR55-knockdown HGFs, respectively), we analyzed IL-6 and IL-8 production at 24 h after LPS stimulation by ELISA in each cell type. Similar to the control group, the CBD group of non-knockdown HGFs transfected with control siRNA showed little expression of inflammatory cytokines. There was a significant increase in the production of both IL-6 and IL-8 in the LPS and CBD+LPS groups compared to control and the CBD groups (p<0.05) (Figure 5A and C). However, in comparison between the LPS and CBD+LPS groups, IL-6 production in the CBD+LPS group was significantly suppressed by 28.2% (p<0.05). Similarly, IL-8 levels were reduced by 22.8% in the CBD+LPS group compared to the LPS group (p<0.05). Next, we analyzed IL-6 and IL-8 production in GPR55-knockdown HGFs transfected with GPR55 siRNA. GPR55 gene expression efficiency was determined by qRT-PCR by comparison with negative control siRNA, and the efficiency was stable and suppressed at 50%. In GPR55-knockdown HGFs, there was a significant increase in the production of both IL-6 and IL-8 in the LPS and CBD+LPS groups compared to the control and CBD groups (p<0.05). However, no significant change was observed between the LPS and CBD+LPS groups (Figure 5B and D). The production of proinflammatory cytokines in the LPS group was also significantly reduced in GPR55-knockdown HGFs (p<0.05) (Figure 5E and F). The significant difference between the LPS and CBD+LPS groups that was observed in non-knockdown HGFs was not observed in GPR55-knockdown HGFs.
Proinflammatory cytokine secretion was significantly upregulated by LPS stimulation and downregulated by CBD addition in HGFs. HGFs were treated with LPS in the presence or absence of CBD. Interleukin (IL)-6 and IL-8 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Data are presented as mean±SD from five independent experiments. Comparisons were analyzed by ANOVA. ＊p＜0.05, Kruskal-Wallis test with Steel-Dwass test.
Host modulation therapy has been investigated as a means of reducing inflammation in periodontal disease9). We have focused on CBD14,33) and its receptor, GPR55, as a means to achieve this. GPR55 has been reported to exacerbate inflammation by modulating transcription factors such as NF-kB through its activation26), but the expression and role of GPR55 in periodontal tissue remain unclear. Therefore, in the present study, we first confirmed the expression of GPR55 in HGFs and observed changes in its expression under LPS-induced inflammation. Then, we examined whether anti-inflammatory effects of CBD were affected by GPR55 knockdown. The results showed that GPR55 mRNA was homeostatically expressed in HGFs. Although GPR55 mRNA expression in immune tissues and the gastrointestinal tract has been previously reported34), it is not clear in periodontal tissues. To our best knowledge, this is the first report about GPR55 mRNA expression in periodontal tissues.
GPR55 mRNA expression showed a time-dependent decreasing trend in the presence of LPS. Lin et al.28) reported that GPR55 mRNA expression is increased in the intestines of rats treated with LPS for 1 h to induce enteritis. By contrast, it has been reported that GPR55 mRNA expression is reduced in glial cells after 8 h of LPS stimulation35). The results of the present study were consistent with the latter, but this may be due to the difference in the duration of action of LPS, in which GPR55 tended to increase after a short period of stimulation and then decrease. Evaluation of GPR55 protein expression indicated it is permanently present in the cytoplasmic fraction and was significantly reduced by LPS stimulation. These results are consistent with those for mRNA expression. By contrast, concerning CB1 and CB2, little or no expression of either mRNA or protein was observed in HGFs. It has been reported that CB1 and CB2 mRNA expression is increased in HGFs from patients with chronic periodontitis compared to healthy individuals36). These differences in mRNA and protein expression may be due to the time of exposure to inflammation and the interaction between multispecies cells. Thus, these findings must be investigated by examining the time of exposure to LPS, co-culturing epithelial cells and other periodontal tissue cells with HGFs, and using in vivo models. In the present study, GPR55 expression was reduced upon LPS stimulation. Miller et al. reported that cannabinoid receptors can increase or decrease their number depending on host conditions37). For example, decreased CB1 expression in human colorectal cancer38) and increased CB1 and CB2 expression in human prostate cancer have been reported39). These findings suggest that the expression of cannabinoid receptors varies according to tissue type, and elucidation of these mechanisms may lead to the development of new treatment methods for periodontal disease.
We also examined anti-inflammatory effects of CBD on HGFs and changes in proinflammatory cytokine production in HGFs by GPR55 knockdown. Anti-inflammatory effects of CBD have also been investigated in other tissues. For example, a study on the application of CBD in the treatment of rheumatoid arthritis, an inflammatory autoimmune disease, reported that CBD inhibits LPS-stimulated inflammatory cytokines in a murine model of collagen-induced arthritis40). Similarly, periodontal disease, an inflammatory disease, increases the production of proinflammatory cytokines such as IL-6 and IL-8, which are produced from HGFs by LPS stimulation41). IL-6 is involved in inflammatory cell migration and osteoclast formation42,43). The binding of IL-6 to HGFs promotes the production of tissue degrading enzymes. As a result, the periodontal tissue is destroyed by accelerated degradation of collagen fibers44). In addition, IL-8 similarly promotes periodontal tissue destruction by inducing neutrophil migration and local infiltration45). Thus, inhibition of IL-6 and IL-8 production is one of the most effective ways to prevent the worsening of periodontal disease. Therefore, we hypothesized that pre-administration of CBD, which is suggested to be a GPR55 antagonist, could inhibit IL-6 and IL-8 production by GPR55 activation. The results of the present study showed a rapid increase in IL-6 and IL-8 production in the LPS group compared to the control group at 24 h after stimulation, however, the production was significantly suppressed by the prior addition of CBD. By contrast, in GPR55-knockdown HGFs, the proinflammatory cytokine inhibitory effect of CBD could not be confirmed, because there was no significant difference between the LPS and CBD+LPS groups. It is possible that the action of CBD may be mediated by GPR55. However, IL-6 and IL-8 production in GPR55-knockdown HGFs was significantly lower than that in non-knockdown HGFs in the LPS group. In other words, in the knockdown cells, the effect of CBD would not have been observed in the knockdown cells, which would not have reflected the effect of increased proinflammatory cytokine production due to LPS exposure in the first place. It has been reported that activation of GPR55 leads to activation of transcription factors such as NF-κB through activation of RhoA and phosphorylation of ERK, ultimately leading to an increase in pro-inflammatory cytokines18). Assuming that CBD acts as an antagonist of GPR55, it may exert its anti-inflammatory effects by inhibiting such signaling. While a study suggested that CBD is a GPR55 antagonist46), some negative results have been reported and are inconclusive26,47). In addition to CB1 and CB2, CBD is associated with many other receptors, including GPR55, transient receptor potential channels, 5-hydroxytryptamine receptors, and peroxisome proliferator-activated receptor48). Therefore, additional studies on other receptors are needed. Furthermore, in the present study, knockdown of GPR55 mRNA suppressed IL-6 and IL-8 production in the LPS group. The fact that HGFs used in this study showed little CB1 and CB2 expression and altered GPR55 expression by LPS suggests that part of the inflammatory effect in HGFs may be mediated by GPR55. Schicho et al.49) reported that the inflammatory pathway is activated through GPR55 rather than CB1 or CB2 in a murine model of intestinal inflammation that knocked out GPR55. These results correspond with our opinion. It has been reported that HGFs express the TLR gene, which is widely known as a receptor for pathogens; TLR4 acts as a receptor for LPS and is involved in the expression of inflammatory cytokines50). Saliba et al.51) reported that GPR55 antagonists act on TLR4 to inhibit the production of prostaglandin E2 and other substances in TLR4-expressing rat microglial cells in a model of TLR4-induced neuroinflammation by LPS stimulation. GPR55 may be part of the inflammatory pathway in HGFs as an adjunct in addition to the TLR4-mediated pathway, considering the variation in expression under LPS stimulation. In summary, our results indicate that pretreatment of CBD inhibits inflammation in periodontal tissues and that GPR55 may be involved in the inflammatory pathway in periodontal tissues. More than 30% of the drugs currently on the market in Japan target GPCRs, and GPR55, a GPCR, is also considered a major target for drug discovery. Thus, the results suggest that GPR55 may be an effective new target for the treatment of periodontal disease and the possibility that CBD can be a tool for host modulation therapy.
The present study is the first to report the expression of GPR55 in HGFs, and the reduction of GPR55 expression under LPS stimulation. In addition, CBD was found to exert an anti-inflammatory effect by inhibiting the production of pro-inflammatory cytokines induced by LPS in HGFs. Further studies are needed to elucidate the role of GPR55 and the effect of CBD in periodontal tissue, including studies on its association with endogenous cannabinoids, its expression in other cells, the effects of CBD on downstream signaling, and its interaction with other receptors.
In this study, we investigated GPR55 expression in HGFs, its kinetics under LPS stimulation, and the involvement of GPR55 in the inflammatory response. GPR55 was permanently expressed in HGFs, and its expression tended to decrease upon LPS stimulation. Furthermore, we found that part of the inflammatory response by LPS may be mediated by GPR55 and the anti-inflammatory effect of CBD. These results suggest that GPR55 may be a potential therapeutic target in periodontitis and that CBD may be an effective therapeutic agent.
The authors extend their deepest gratitude to The Nippon Dental University, School of Life Dentistry, Department of Biochemistry and Department of Periodontology (Tokyo, Japan) for supporting this study.
Conflict of Interest: None.
