Carvacrol and TRPM2

Introduction

The TRPM2 structure comes from RCSB.org entry 6PUS. It features human TRPM2 bound to ADP-ribose, an NAD⁺ metabolite produced under oxidative stress and/or neuro degenerative conditions. The TRPV4 is from Xenopus. 6C8H The gadolinium inhibitor (arrow) is part of this structure. The TRPM2 mRNA and TRPV4 mRNA expression data are from Proteinatlas.org. The first study, out of Kyoto University in Japan, used the classic approach of comparing a wild type mouse model with a genetically manipulated version of the same strain in which the TRPM2 gene had been knocked out. This model allowed the comparison of the response to acute pain to pathological pain involving changes in neurons distal from the site of various injuries. The TRPV4 study used a pharmacological approach testing the the hypothesis that the mitogen activated protein kinase member p38 was somehow involved. The third study demonstrated that carvacrol inhibits TRPM2 and TRPV4 Ca²⁺ channel.

TRPM2 [1]

The Haraguchi study compared neuropathic pain in wild type and mice whose copies of the TRPM2 gene had been “knocked out.” Injuries were induced in the foot, sciatic nerve, and spinal cord.

Inflammatory pain via carrageenan injection

• The right hind paws were injected with 20µl of a 3% solution of carrageenan to induce inflammation associated pain. [1] The knockout mice had a diminished response
• Knock out mice and wild type mice showed the same response to mechanical and thermal stimuli.
• Mechanical allodynia was increased by almost 10x in the knock out (p<0.01) Allodynia is a condition in which mechanical stimuli that do not normally cause pain cause extreme pain.
• The latency to thermal hyperalgesia was increased by a small amount in the knock out mice.
• Paw edema was less in the knockout.
• TRPM2 mRNA was increased by 40x 8 hours after carrageenan injection in the wildtype mice.
• Some transient improvements in inflammatory cytokines were observed in the knockout mice. At 8 hours the myeloperoxidase was perhaps 25% less (p<0.01) in the knockout mice.

TRPM2 and macrophage

• Macrophage were isolated from the wildtype and TRPM2 mice and subjected to LPS/INFν stimulation.
• These agents increased TRPM2 mRNA in wildtype macrophage.
• Four hours after stimulation an increase was seen in secretion of the CXCL2.

The mice were subjected to partial sciatic nerve ligation.

• TRPM2 mRNA was increased at the ligation site in the wildtype mice.
• CXCL2, a chemokine secreted by monocytes and macrophage to attract leukocytes, did not experience its 24 hour spike in the knockout mice.
• TRPM2 knockout mice lacked the latent response.

Spinal cord injury pain

• For the spinal nerve transsection (SNT) model of pain the L4 spinal nerve was transected without damaging the L3 spinal nerve.
• By days 7 and 14 no change was seen in wild type or knockout mice in the 50% withdrawal reflex on the contralateral (opposite) side.
• By day 14 the 50% withdrawal was 100x less than the baseline in the wildtype and about one fourth baseline in the knockout on the ipsilateral (same) side.
• TRPM2 mRNA in spinal microglia almost double three days after spinal ligation.
• The microglia marker Iba-1 was 3x higher on day three in the wildtype mice.

TRPM2 in microglia

• Microglia were isolated, cultured, and treated with LPS/INFν
• CXCL2 mRNA and protein levels were increased in the wildtype compared to the knockout.
• iNOS mRNA and nitric oxide and oxidation products were reduced in the knockout relative to the wildtype.
• IL6 and IL1β and TNFα levels were unchanged. As a general note, the NRLP3 subunit of the inflammasome has reactive thiols. The inflammasome cleaves IL6 and IL1β pro-peptides into active cytokines,

TRPV4 [2]

This study out of China used a chronic compression of the dorsal root ganglion (CCD) model of neuropathic pain. [2] Haraguchi et al did an excellent job of demonstrating that TRPM2 was involved in neuropathic pain but did nothing to demonstrate how opening of the TRPM2 Ca²⁺ channel caused changes in mRNA transcription and so on. [1] Qu et al. addressed the hypothesis that the p38 kinase, a member of the MAPK cascade of kinases was some how involved in connecting TRPV4 conductivity with neuropathic pain in their CCD model of pain.

An image search was performed using terms “calcium and neuron and injury.” The role of Ca²⁺ in modulating adenylyl cyclase needs to be investigated further simply because that many cytokine receptors are G protein coupled receptors and because CB1 and CB2 activation might have synergistic affects with the TRPV4 activator carbacrol.

TRPV4 and p38 are tied together.

• TRPV4 inhibitor Ruthenium Red (RR), 10 nmol/L, injection resulted in a transient decrease in protein levels of TRPV4, p38, and phosphorylated p38. Protein levels at eight hours were indistinguishable from time 0. At 1 nM RR only p38 and p-38 were decreased relative to tubulin. RR is a salt of the heavy metal ruthenium.
• Injection with the TRPV4 agonist 4𝛼-PDD resulted in a transient increase in TRPV4 protein levels followed by a decrease in TRPV4 at eight hours. Protein levels of p38 also experienced a transient increase. Up to 100μM 4𝛼-PDD resulted more sustained increases by 8 hours.
• The p38 inhibitor SB203580 at 20 µM increased TRPV4 levels.
• The p38 activator anisomycin, 25 µg/mL, decreased TRPV4 protein levels.
• Ectopic dischargers were measured after CCD surgery. The frequency was not changed by inhibitors and agonists of p38 and TRPV4.
• The ectopic discharge amplitudes were increased by agonists and decreased by antagonists.
• Imunohistochemistry also showed that the number of TRPV4- or p38-positive small neurons changed significantly after CCD. Effectors of TRPV4 and p38 influenced this expression. [2] Qu et al speculated that the small neurons branching out into C fibers might be a source of neuropathic pain in their model.

Carvacrol modulates TRPM2 and TRPV4[3]

The studies of Haraguchi and Qu were presented in brief to illustrate the role of these two Ca²⁺ channels in neuropathic pain. Mustafa Nazıroğlu of Demiral University in Aspart Turkey published electrophysiology experiments in the SH-SY5Y neuronal cell line and HEK293 cells expressing TRPM2 and TRPV4. [3] Why carvacrol as a plant based inhibitor was not made clear. The world wide popularity of Turkish oregano oil and that this oil is 70% carvacrol is one explanation. Dr Nazıroğlu used two techniques. Fluo3-AM is a2+ sensitive dye that crosses the cell plasma membrane. Inside the cell, esterases cleave off the ester group that enabled the fluorophore part to cross the cell membrane. The other technique was whole cell patch clamping.

• Fluo-3 experiments
• H2O2, 1mM, is the TRPM2 activator.
• GSK, 100nM, is the TRPV4 activator
• Carvacrol, 100 µM, is a putative inhibitor and the test substance

SH-Sy5Y neuronal cell line

• The intracellular calcium indicator fluo3-AM was loaded into SH-SY5Y, a neuronal cell line.
• “a” p ≤ 0.05 vs. Cntrol.
• “b” p ≤ 0.05vs. H2O2 or GSK group. In the absence of extracellular ,Ca

Expressed individually in HEK293 cells

fluo-3 response to TRP agonists or of carvacrol (Fig 2 not shown.) GSK and 1% H2O2 had no effect on HEK293 cells not transfected with TRPM2 or TRPV4. (figure 3 not shown). In HEK293 cells expressing these channels, Carvacrol inhibited the increase in intracellular Ca²⁺.

Whole cell patch clamping

These sets of experiments involved more complicated sets of activators and inhibitors.  A lot of these small molecules involve cell survival. The reader is invited to read the rest of the study that is beyond the scope of this post to address the utility of carvacrol in treating neuropathic pain. Reversing neurodegenerative diseases is beyond the cope of this post.

• Poly ADP ribose polymerase (PARP) is a family of enzymes involved in DNA repair and cell death.
• ADP ribose
• Nmdg N-methyl-D-glucamine
• ACA a PARP inhibitor
• Ruthenium red TRPV4 inhibitor

The best oregano oil for pain

Turkish oregano oil contains very little β-caryophyllene. [4] The terpene content of oregano oils from around the world has been reviewed. [5] Could the optimal oregano oil for pain treatment also contain β-caryophyllene and other CB1/2 agonists?

References

1. Haraguchi, K., Kawamoto, A., Isami, K., Maeda, S., Kusano, A., Asakura, K., Shirakawa, H., Mori, Y., Nakagawa, T., & Kaneko, S. (2012). TRPM2 contributes to inflammatory and neuropathic pain through the aggravation of pronociceptive inflammatory responses in mice. The Journal of neuroscience : the official journal of the Society for Neuroscience, 32(11), 3931–3941. PMC free article
2. Qu, Y. J., Zhang, X., Fan, Z. Z., Huai, J., Teng, Y. B., Zhang, Y., & Yue, S. W. (2016). Effect of TRPV4-p38 MAPK Pathway on Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion. BioMed research international, 2016, 6978923. Free article
3. Nazıroğlu M. (2022)  A novel antagonist of TRPM2 and TRPV4 channels: Carvacrol. Metab Brain Dis. 2022 Jan 6:1–18. PMC free article
4. Tasdemir  D (2019) Antiprotozoal Activity of Turkish Origanum onites Essential Oil and Its Components Molecules 2019, 24, 4421 PMC free article
5. Leyva-López, N., Gutiérrez-Grijalva, E. P., Vazquez-Olivo, G., & Heredia, J. B. (2017). Essential Oils of Oregano: Biological Activity beyond Their Antimicrobial Properties. Molecules (Basel, Switzerland), 22(6), 989. PMC free article
6. van den Hoogen, N. J., Harding, E. K., Davidson, C., & Trang, T. (2022). Cannabinoids in Chronic Pain: Therapeutic Potential Through Microglia Modulation. Frontiers in neural circuits, 15, 816747. PMC free article