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. 2022 Mar;37(3):711-728.
doi: 10.1007/s11011-021-00887-1. Epub 2022 Jan 6.

A novel antagonist of TRPM2 and TRPV4 channels: Carvacrol

Mustafa Nazıroğlu  1   2
Affiliations

A novel antagonist of TRPM2 and TRPV4 channels: Carvacrol

Mustafa Nazıroğlu. Metab Brain Dis. 2022 Mar.

Abstract

The overload cytosolic free Ca2+ (cCa2+) influx-mediated excessive generation of oxidative stress in the pathophysiological conditions induces neuronal and cellular injury via the activation of cation channels. TRPM2 and TRPV4 channels are activated by oxidative stress, and their specific antagonists have not been discovered yet. The antioxidant and anti-Covid-19 properties of carvacrol (CARV) were recently reported. Hence, I suspected possible antagonist properties of CARV against oxidative stress (OS)/ADP-ribose (ADPR)-induced TRPM2 and GSK1016790A (GSK)-mediated TRPV4 activations in neuronal and kidney cells. I investigated the antagonist role of CARV on the activations of TRPM2 and TRPV4 in SH-SY5Y neuronal, BV-2 microglial, and HEK293 cells. The OS/ADPR and GSK in the cells caused to increase of TRPM2/TRPV4 current densities and overload cytosolic free Ca2+ (cCa2+) influx with an increase of mitochondrial membrane potential, cytosolic (cROS), and mitochondrial (mROS) ROS. The changes were not observed in the absence of TRPM2 and TRPV4 or the presence of Ca2+ free extracellular buffer and PARP-1 inhibitors (PJ34 and DPQ). When OS-induced TRPM2 and GSK-induced TRPV4 activations were inhibited by the treatment of CARV, the increase of cROS, mROS, lipid peroxidation, apoptosis, cell death, cCa2+ concentration, caspase -3, and caspase -9 levels were restored via upregulation of glutathione and glutathione peroxidase. In conclusion, the treatment of CARV modulated the TRPM2 and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting TRPM2 and TRPV4-mediated neurodegenerative diseases associated with the increase of mROS and cCa2+. The possible TRPM2 and TRPV4 blocker action of carvacrol (CARV) via the modulation oxidative stress and apoptosis in the SH-SY5Y neuronal cells. TRPM2 is activated by DNA damage-induced (via PARP-1 activation) ADP-ribose (ADPR) and reactive oxygen species (ROS) (H2O2), although it is inhibited by nonspecific inhibitors (ACA and 2-APB). TRPV4 is activated by the treatments of GSK1016790A (GSK), although it is inhibited by a nonspecific inhibitor (ruthenium red, RuRe). The treatment of GSK induces excessive generation of ROS. The accumulation of free cytosolic Ca2+ (cCa2+) via the activations of TRPM2 and TRPV4 in the mitochondria causes the increase of mitochondrial membrane depolarization (ΔΨm). In turn, the increase of ΔΨm causes the excessive generation of ROS. The TRPM2 and TRPV4-induced the excessive generations of ROS result in the increase of apoptosis and cell death via the activations of caspase -3 (Casp-3) and caspase -9 (Casp-9) in the neuronal cells, although their oxidant actions decrease the glutathione (GSH) and glutathione peroxidase (GSHPx) levels. The oxidant and apoptotic adverse actions of TRPM2 and TRPV4 are modulated by the treatment of CARV.

Keywords: Carvacrol; Glutathione; Neurodegeneration; Oxidative stress; TRPM2; TRPV4.

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Conflict of interest statement

The author has no conflicts of interest to declare.

Figures

Fig. 1
Fig. 1
The treatment of carvacrol (CARV) diminished H2O2 and GSK-mediated increase of cCa2+ concentration via inhibition of TRPM2 and TRPV4 in the SH-SY5Y cells. (Mean ± STD). The SH-SY5Y cells in the dishes of control (Cntr), H2O2, GSK, H2O2+CARV, and GSK+CARV groups were stained with the dye of Fluo 3-AM (1 µM for 60 min). After washing the cells, they were stimulated by H2O2 (1 mM) or GSK (100 nM), although they were inhibited by CARV (100 µM). The representative images groups on the increase of cCa2+ concentration through TRPM2 inhibition in the Cntr, H2O2, and H2O2+CARV in the confocal microscope with 40x oil objective were shown a, although the representative images on the cCa2+ concentration through TRPV4 inhibition in the groups of Cntr, GSK, and GSK+CARV groups in the LSCM-800 microscopee with 40x oil objective were shown d. The line (1b) and column (1c) mean fluorescence intensities of the Fluo 3-AM (150 s) are shown in the groups of Cntr, H2O2, and H2O2+CARV in the b and c, although the line (1e) and column (1f) mean fluorescence intensities of the Fluo 3-AM (150 s) are shown in the groups of Cntr, GSK, and GSK+CARV are shown in the e and f. The scale bar was kept as 10 μm. One example image of each figure was selected from 25-30 SH-SY5Y of 6 independent experiments for each condition (ap ≤ 0.05 vs. Cntr. bp ≤ 0.05 vs. H2O2 or GSK group)
Fig. 2
Fig. 2
There were no effects of H2O2 and GSK on the TRPM2 and TRPV4 activation in the absence of extracellular Ca2+(NoCa2+) and the treatments of CARV (24h) and PARP-1 inhibitors in the SH-SY5Y cells. (Mean ± STD). The SH-SY5Y cells in the CARV (24h), NoCa2+, DPQ (30 µM for 30 min) and PJ34 (1 μM for 30 min) groups were stained with Fluo 3-AM (1 µM for 60 min). After washing the cells, the stained cells were stimulated by H2O2(1 mM) or GSK(100 nM). Representative LSCM-800 (objective: 40x oil) images and mean colon values of Cntr, H2O2, and H2O2+CARV groups on the cCa2+ concentration through TRPM2 in the CARV (a and b), NoCa2+ (a and c), DPQ (a and d), and PJ34 (a and e) groups were shown in the Fig. 2. Effects of CARV treatments on the GSK-induced changes of cCa2+ intensity in the CARV (f and g) and NoCa2+ (f and h) groups were also shown by images (Fig. 2f) and columns (Figs. 2g and h). The scale bar: 10 μm
Fig. 3
Fig. 3
There was no effect of CARV on the increase of cCa2+ concentration in the absence of TRPM2 and TRPV4 in the HEK293 cells. (Mean ± STD). The HEK293 cells in the groups of control (Cntr), H2O2, GSK, H2O2+CARV, and GSK+CARV were stained with Fluo 3-AM (1 µM for 60 min). After washing the cells, the cells were stimulated by by H2O2 (1 mM) or GSK (100 nM), although they were inhibited by CARV (100 µM). The representative confocal microscope (objective: 40x oil) images, mean colon, and line values on the changes of cCa2+ concentration via the activation of TRPM2 in the HEK293 in the groups of Cntr, H2O2, and H2O2+CARV groups were shown in the a, b, and c, respectively. Effects of CARV treatments on the GSK-induced changes of cCa2+ concentration intensity in the HEK293 were also shown by images (d), lines (e), and columns (f). The scale bar: 10 μm. One example image of each figure was selected from 20-25 HEK293 of 6 independent experiments for each condition
Fig. 4
Fig. 4
The treatment of CARV modulated the increase of cCa2+ concentration via inhibition of TRPM2 and TRPV4 in the HEK293M2 and HEK293V4 cells. (Mean ± STD). The HEK293M2 and HEK293V4 cells in the in the control (Cntr), H2O2, GSK, H2O2+CARV, and GSK+CARV groups were stained with Fluo 3-AM (1 µM for 60 min). After washing the cells, the stained cells were stimulated by by H2O2 (1 mM) or GSK (100 nM), although they were inhibited by CARV (100 µM). Representative confocal microscope (objective: 40x oil) images, mean colon and line values of Cntr, H2O2, and H2O2+CARV groups on the cCa2+ concentration through TRPM2 in the HEK293M2 were shown a, b, and c, respectively. Effects of CARV treatments on the GSK-induced changes of cCa2+ intensity in the HEK293V4 were also shown by images (d), lines (e), and columns (f). The scale bar: 10 μm. One example image of each figure was selected from 20-25 HEK293 of 6 independent experiments for each condition
Fig. 5
Fig. 5
ADPR induced increase of TRPM2 current densities (pA/pF) in the SH-SY5Y cells but not in the HEK293 cells. The ADPR-induced increase of TRPM2 current density was diminished in the SH-SY5Y cells by the treatment of CARV. (Mean ± STD and n=4-6). The TRPM2 currents in the SH-SY5Y cells were induced by the stimulation of cytosolic ADPR (1 mM), but they were blocked by CARV (50 and 100 µM). W.C.: The record of whole cell. (a) Cntr (without ADPR). (b) Cntr (with ADPR and ACA). (c) ADPR+CARV (50 and 100 µM). (d) CARV (1 h)+ADPR. e. Mean currents densities of TRPM2 in the SH-SY5Y cells. f. HEK293 cells + ADPR (without TRPM2). g. HEK293M2 cells + ADPR (with TRPM2). h. HEK293M2 cells + CARV (1 h) + ADPR (with TRPM2). i. Mean currents densities of HEK293 and HEK293M2 after the treatments of ADPR, ACA, and CARV (ap ≤ 0.05 vs. Cntr. bp ≤ 0.05 vs. Cntr+ADPR group. Δp ≤ 0.05 vs. HEK293+ADPR group. ×p ≤ 0.05 vs. HEK293M2+ADPR group)
Fig. 6
Fig. 6
GSK induced increase of TRPV4 current densities (pA/pF) in the SH-SY5Y cells but not in the HEK293 cells. The modulator role of CARV. (Mean ± STD and n=3-6). The TRPM2 currents were induced in the SH-SY5Y cells by the stimulation of GSK (100 nM), but they were blocked by the treatment of CARV (100 µM). W.C.: The record of whole cell. a. Cntr (without GSK). b. Cntr (with GSK and RuRe). c. GSK+CARV (100 µM). d. GSK + RuRe and GSK + CARV (1 h). e. HEK293 cells + ADPR (without TRPM2). f. The mean currents densities of TRPM2 in the SH-SY5Y and HEK293 cells after the treatments of GSK, RuRe, and CARV (ͼp ≤ 0.05 vs. Cntr. ©p ≤ 0.05 vs. GSK)
Fig. 7
Fig. 7
The pretreatment of CARV (100 µM for 24 h) modulated GSK (100 nM for 60 min) and H2O2 (1 mM for 60 min)-mediated increase of cell viability, caspase -3 (CASP-3), caspase -9 (CASP-9), and apoptosis levels in the SH-SY5Y cells. (Mean ± STD and n=6). The CASP-3 (a), CASP-9 (b), apoptosis (c), and cell viability (d) levels were analyzed in the automatic microplate reader by Ac-DEVD-AMC substrate, Ac-LEHD-AMC substrate, commercial kit, and MTT, respectively. (*p ≤ 0.05 vs. control (Cntr) and MLT groups. ×p≤ 0.05 vs. H2O2 and GSK groups)
Fig. 8
Fig. 8
The pretreatment of CARV (100 µM for 24 h) modulated GSK (100 nM for 24 h) and H2O2 (1 mM for 24 h)-induced increase of SH-SY5Y cell death. (Mean ± STD and n=20-25). a. The images of bright field (BF, black-white). b. The images of PI (red and dead cells), Hoechst (blue and live cells), and merge images. c. The 2.5 D images of merges. d. The mean percentage of cell death (PI and Hoechst-positive) cell rates in the five groups. (Objective: 20×. Scala bar: 20 μm). (*p ≤ 0.05 vs. control (Cntr) and MLT groups. ×p≤ 0.05 vs. H2O2 and GSK groups)
Fig. 9
Fig. 9
The pretreatment of CARV (100 µM for 24 h) diminished GSK (100 nM for 1 h) and H2O2 (1 mM for 1 h)-induced increase of mROS, ΔΨm, and cROS in the SH-SY5Y cells. (Mean ± STD and n = 20-25). a. The images of mROS, ΔΨm, and cROS generation in the Cntr, H2O2, GSK, H2O2+ CARV, and GSK + CARV groups by using the stains of MitoTracker Red CM-H2Xros, JC-1, and DCFH-DA, respectively (Objective: 40×oil. Scala bar: 20 μm). b. The 2.5 images of mROS, ΔΨm, and cROS in the five group. Representative column of the fluorescence intensities of the mROS (c), (d), and DCFH-DA (e). (*p ≤ 0.05 vs. Cntr. ×p≤ 0.05 vs. H2O2 and GSK groups)
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