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. 2022 Apr 9;15(4):457.
doi: 10.3390/ph15040457.

Harpagophytum procumbens Root Extract Mediates Anti-Inflammatory Effects in Osteoarthritis Synoviocytes through CB2 Activation

Alessia Mariano  1 , Irene Bigioni  1 , Roberto Mattioli  1 , Antonella Di Sotto  2 , Martina Leopizzi  3 , Stefania Garzoli  4 , Pier Francesco Mariani  5 , Pietro Dalla Vedova  6 , Sergio Ammendola  7 , Anna Scotto d'Abusco  1
Affiliations

Harpagophytum procumbens Root Extract Mediates Anti-Inflammatory Effects in Osteoarthritis Synoviocytes through CB2 Activation

Alessia Mariano et al. Pharmaceuticals (Basel). .

Abstract

The endocannabinoid system is involved in the nociceptive and anti-inflammatory pathways, and a lowered expression of CB2 receptors has been associated with inflammatory conditions, such as osteoarthritis (OA). This suggests that CB2 modulators could be novel therapeutic tools to treat OA. In the present study, the involvement of Harpagophytum procumbens root extract, a common ingredient of nutraceuticals used to treat joint disorders, in CB2 modulation has been evaluated. Moreover, to clarify the effects of the pure single components, the bioactive constituent, harpagoside, and the main volatile compounds were studied alone or in a reconstituted mixture. Human fibroblast-like synoviocytes, extracted by joints of patients, who underwent a total knee replacement, were treated with an H. procumbens root extract dissolved in DMSO (HPEDMSO). The effectiveness of HPEDMSO to affect CB2 pathways was studied by analyzing the modulation of cAMP, the activation of PKA and ERK MAP kinase, and the modulation of MMP-13 production. HPEDMSO was able to inhibit the cAMP production and MAP kinase activation and to down-regulate the MMP-13 production. Pure compounds were less effective than the whole phytocomplex, thus suggesting the involvement of synergistic interactions. Present findings encourage further mechanistic studies and support the scientific basis of the use of H. procumbens in joint disorders.

Keywords: CB2; devil’s claw; endocannabinoid system; eugenol; fibroblast-like synoviocytes; harpagoside; osteoarthritis; volatile compounds; α-humulene; β-caryophyllene.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Determination of cAMP in cells treated with H. procumbens root extracted dissolved in DMSO (HPEDMSO). (A) Human primary synoviocytes (FLSs) were treated with 0.1 mg/mL of Harpagophytum procumbens root extract dissolved in DMSO (HPEDMSO), and after the indicated times, the amount of cAMP was determined by cAMP ELISA kit. (B) The amount of cAMP was determined in cells stimulated with forskolin for 20 min or in cells pre-treated with HPEDMSO for 1 h and then stimulated with forskolin for 20 min. * p < 0.05 vs. CTL.
Figure 2
Figure 2
Effects of HPEDMSO extract on PKA phosphorylation. Cells were treated with 0.1 mg/mL HPEDMSO for the reported times and then analyzed by immunofluorescence using anti-p-PKA primary antibody and Alexa Fluor 568 (red) secondary antibody. Nuclei were stained with DAPI (original magnification 40×). The bar graph represents the pixel intensities in the region of interest, obtained by ImageJ. * p < 0.05; ** p < 0.01 vs. CTL.
Figure 3
Figure 3
Effects of HPEDMSO extract on PKA phosphorylation after forskolin stimulation. Cells were treated with forskolin for 30 min or pre-treated with HPEDMSO for 1 h and then stimulated with forskolin for 30 min. Cells were analyzed by immunofluorescence using anti-p-PKA primary antibody and Alexa Fluor 568 (red) secondary antibody. Nuclei were stained with DAPI (original magnification 40×). The graph represents the pixel intensities in the region of interest, obtained by ImageJ. * p < 0.05 vs. CTL.
Figure 4
Figure 4
Effects of HPEDMSO on ERK phosphorylation. Cells were left untreated (CTL) or treated with 0.1 mg/mL of HPEDMSO. Cell extracts were analyzed at 5 min, 1 h, 4 h and 24 h after treatment by western blot, using anti-ERK/p-ERK and actin antibodies (upper side). The densitometric analysis (bottom side) was performed by ImageJ. Results are expressed as mean ± SEM of data obtained by three independent experiments. * p < 0.05; ** p < 0.01 vs. CTL.
Figure 5
Figure 5
Effects of HPEDMSO on c-Fos production and activation. (A) Cells were treated with 0.1 mg/mL HPEDMSO for 24 h and then the mRNA was extracted and analyzed by RT-PCR. c-Fos mRNA level was reported as relative mRNA expression level with respect to 18S mRNA (2−ΔΔCt method); (B) Cells were treated as described in A and then proteins were extracted and analyzed by western blot using anti-c-Fos/p-c-Fos and anti-actin antibodies; (C) Densitometric analysis of bands obtained in western blot experiment, performed by ImageJ. Results are expressed as mean ± SEM of data obtained by three independent experiments. * p < 0.05 vs. CTL.
Figure 6
Figure 6
Effects of HPEDMSO on MMP-13 production. Cells were treated with 0.1 mg/mL HPEDMSO for 24 h and then the mRNA was extracted and analyzed by RT-PCR. MMP-13 mRNA level was reported as relative mRNA expression level with respect to 18S mRNA (2−ΔΔCt method). The amount of MMP-13 produced was measured in the culture medium of cells and analyzed by ELISA. The results are reported as pg/mL. Results are expressed as mean ± SEM of data obtained by three independent experiments. *** p < 0.005 vs. CTL.
Figure 7
Figure 7
Effects of HPEDMSO on CB2, cAMP and MMP-13 production. Cells were treated with 0.1 mg/mL HPEDMSO, 8 ng/mL β-caryophyllene, 2 ng/mL α-humulene, 0.4 ng/mL eugenol and 0.4 μg/mL harpagoside and with the mixture of all four compounds (Mix) at same concentration, for 24 h. After treatment, the mRNA was extracted and analyzed by RT-PCR. CB2 (A) and MMP-13 (C) mRNA level was reported as relative mRNA expression level with respect to 18S mRNA (2−ΔΔCt method); The cAMP (B) amount was measured by cAMP ELISA kit. Results are expressed as mean ± SEM of data obtained by three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.005 vs. CTL.
Figure 8
Figure 8
Effects of HPEDMSO on intracellular Ca2+ concentration. Cells were treated with 0.1 mg/mL HPEDMSO for 20 min, 1 h and 24 h, then the amount of intracellular Ca2+ concentration was measured by Calcium Green-1 AM. The densitometric analysis (bottom) was performed by ImageJ. Results are expressed as mean ± SEM of data obtained by three independent experiments. * p < 0.05 vs. CTL.
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