Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes
Abstract
:1. Introduction
2. Results
2.1. Effects of pCBs on Viability, Apoptosis and Cell Cycle of Human HaCaT Cells
2.2. Effects of pCBs on Gene Expression of ECS Elements in HaCaT Cells
2.3. Effects of pCBs on Protein Expression of ECS Elements in HaCaT Cells
2.4. Effects of pCBs on Functional Activity of ECS Elements in HaCaT Cells
2.5. Endogenous Content of eCBs in HaCaT Cells Treated with pCBs
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Line and Treatment
4.3. MTT Cytotoxicity Assay
4.4. Determination of Apoptosis
4.5. Cell Cycle Analysis
4.6. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
4.7. Western Blotting
4.8. Receptor Binding Assay on Adherent Living Cells
4.9. TRPV1 Calcium Assay
4.10. Enzyme Assays
4.11. Quantitation of Endogenous Levels of eCBs and PEA
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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pCB | Time Point | IC50 (µM) |
---|---|---|
CBG | 6 h 12 h 24 h | 13.5 13.0 11.7 |
CBC | 6 h 12 h 24 h | 15.3 15.6 8.2 |
THCV | 6 h 12 h 24 h | 14.3 22.4 18.6 |
CBGA | 6 h 12 h 24 h | >25.0 24.5 25.3 |
pCBs | Gene Expression | Protein Expression | ||
---|---|---|---|---|
Receptors | Enzymes | Receptors | Enzymes | |
CBG | CB1 ↑++++ CB2 ↑+ | MAGL ↑+ | CB2 ↓+ TRPV1 ↑++++ PPARδ ↓+++ | NAPE-PLD ↑++++ MAGL ↑+++ |
CBC | CB1 ↓++++ CB2 ↓++ GPR55 ↓++++ PPARγ ↓++ | NAPE-PLD ↑+ | CB2 ↓+ TRPV1 ↑++++ PPARα ↓++ PPARδ ↓++++ | NAPE-PLD ↑++ MAGL ↑+++ FAAH ↑++ |
THCV | GPR55 ↑++++ PPARγ ↓++++ | CB1 ↓+ CB2 ↓+++ TRPV1 ↑++++ PPARα ↓++ PPARδ↓++++ | NAPE-PLD ↑++++ DAGLα ↑+++ DAGLβ ↓+++ MAGL ↑++++ | |
CBGA | GPR55 ↑+++ | FAAH ↓+++ | CB1 ↓++++ CB2 ↓++++ TRPV1 ↑++++ PPARα ↓++++ PPARδ ↓++++ | NAPE-PLD ↑++++ FAAH ↑++++ DAGLα ↑++++ MAGL ↑++++ |
HaCaT Cell Treatment | CB1/2 Binding Activity (pmol/mg of Protein) |
---|---|
Control | 4.033 ± 0.516 |
CBG | 8.027 ± 0.681 * |
CBC | 10.380 ± 0.750 ** |
THCV | 8.187 ± 0.302 * |
CBGA | 8.373 ± 0.410 * |
pCB | NAPE-PLD/FAAH Activity Ratio (Mean ± SEM) | DAGLα,β/MAGL Activity Ratio (Mean ± SEM) |
---|---|---|
Control | 1.00 ± 0.00 | 1.00 ± 0.00 |
CBG (6.0 µM) | 0.97 ± 0.36 | 1.33 ± 0.13 |
CBC (4.0 µM) | 0.41 ± 0.21 | 2.09 ± 0.23 *** |
THCV (9.0 µM) | 0.71 ± 0.30 | 1.56 ± 0.01 * |
CBGA (13.0 µM) | 2.51 ± 1.62 | 0.40 ± 0.06 * |
Gene | Forward Primer Sequence (5′→3′) | Reverse Primer Sequence (5′→3′) |
---|---|---|
cnr1 | CCTTTTGCTGCCTAAATCCAC | CCACTGCTCAAACATCTGAC |
cnr2 | TCAACCCTGTCATCTATGCTC | AGTCAGTCCCAACACTCATC |
gpr55 | ATCTACATGATCAACCTGGC | ATGAAGCAGATGGTGAAGACGC |
trpv1 | TCACCTACATCCTCCTGCTC | AAGTTCTTCCAGTGTCTGCC |
pparα | TGGGAAGGCAGCGTTGATTA | CTGTGTCCTTCCCACTCTCG |
pparγ | TGATGTCTTGACTCATGGGTGT | CACGGAGCTGATCCCAAAGT |
pparδ | AGGTTCCCCAAGAGGGAAGA | CAGGAGGAGACAGTTCCAACC |
napepld | TTGTGAATCCGTGGCCAACATGG | TACTGCCATGGTGAAGCACG |
Faah | CCCAATGGCTTAAAGGACTG | ATGAACCGCAGACACAAC |
daglα | AATGGCTATCATCTGGCTGAGC | TTCCGAGGGTGACATTCTTAGC |
daglβ | GCGCAAAGTAAACGGCAAGA | CTGCAGCTTGGGCTTTTCAT |
Mgll | ATGCAGAAAGACTACCCTGGGC | TTATTCCGAGAGAGCACGC |
Actb | TGACCCAGATCATGTTTGAG | TTAATGTCACGCACGATTTCC |
gapdh | CAGCCTCAAGATCATCAGCA | TGTGGTCATGAGTCCTTCCA |
Antibody | Diluition | Brand |
---|---|---|
CB1 receptor rabbit polyclonal | 1:200 | Cayman Chemical (Ann Arbor, MI, USA) |
CB2 receptor rabbit polyclonal | 1:200 | Cayman Chemical (Ann Arbor, MI, USA) |
GPR55 receptor rabbit polyclonal | 1:200 | Cayman Chemical (Ann Arbor, MI, USA) |
TRPV1 rabbit polyclonal | 1:1000 | OriGene (Rockville, MD, USA) |
PPAR α rabbit polyclonal | 1:1000 | Sigma-Aldrich (St. Louis, MO, USA) |
PPAR γ rabbit monoclonal | 1:1000 | Cell Signaling Technology (Danvers, MA, USA) |
PPAR δ rabbit polyclonal | 1:750 | Invitrogen (Waltham, MA, USA) |
NAPE-PLD rabbit polyclonal | 1:200 | Cayman Chemical (Ann Arbor, MI, USA) |
FAAH rabbit polyclonal | 1:200 | Cayman Chemical (Ann Arbor, MI, USA) |
DAGL-α rabbit polyclonal | 1:1000 | Invitrogen (Waltham, MA, USA) |
DAGL- β rabbit monoclonal | 1:1000 | Cell Signaling Technology (Danvers, MA, USA) |
MAGL rabbit polyclonal | 1:200 | Cayman Chemical (Ann Arbor, MI, USA) |
β-Actin rabbit monoclonal | 1:1000 | Cell Signaling Technology (Danvers, MA, USA) |
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Di Meo, C.; Tortolani, D.; Standoli, S.; Angelucci, C.B.; Fanti, F.; Leuti, A.; Sergi, M.; Kadhim, S.; Hsu, E.; Rapino, C.; et al. Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes. Int. J. Mol. Sci. 2022, 23, 5430. https://doi.org/10.3390/ijms23105430
Di Meo C, Tortolani D, Standoli S, Angelucci CB, Fanti F, Leuti A, Sergi M, Kadhim S, Hsu E, Rapino C, et al. Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes. International Journal of Molecular Sciences. 2022; 23(10):5430. https://doi.org/10.3390/ijms23105430
Chicago/Turabian StyleDi Meo, Camilla, Daniel Tortolani, Sara Standoli, Clotilde Beatrice Angelucci, Federico Fanti, Alessandro Leuti, Manuel Sergi, Salam Kadhim, Eric Hsu, Cinzia Rapino, and et al. 2022. "Effects of Rare Phytocannabinoids on the Endocannabinoid System of Human Keratinocytes" International Journal of Molecular Sciences 23, no. 10: 5430. https://doi.org/10.3390/ijms23105430