Korean J Physiol Pharmacol.  2017 May;21(3):309-316. 10.4196/kjpp.2017.21.3.309.

Activation of transient receptor potential vanilloid 3 by the methanolic extract of Schisandra chinensis fruit and its chemical constituent γ-schisandrin

• Nam Y
• Kim HJ
• Kim YM
• Chin YW
• Kim YK
• Bae HS
• Nam JH
• Kim WK

• KMID: 2376959
• DOI: http://doi.org/10.4196/kjpp.2017.21.3.309

Abstract

Transient receptor potential vanilloid 3 (TRPV3) is a non-selective cation channel with modest permeability to calcium ions. It is involved in intracellular calcium signaling and is therefore important in processes such as thermal sensation, skin barrier formation, and wound healing. TRPV3 was initially proposed as a warm temperature sensor. It is activated by synthetic small-molecule chemicals and plant-derived natural compounds such as camphor and eugenol. Schisandra chinensis (Turcz.) Baill (SC) has diverse pharmacological properties including antiallergic, anti-inflammatory, and wound healing activities. It is extensively used as an oriental herbal medicine for the treatment of various diseases. In this study, we investigated whether SC fruit extracts and seed oil, as well as four compounds isolated from the fruit can activate the TRPV3 channel. By performing whole-cell patch clamp recording in HEK293T cells overexpressing TRPV3, we found that the methanolic extract of SC fruit has an agonistic effect on the TRPV3 channel. Furthermore, electrophysiological analysis revealed that γ-schisandrin, one of the isolated compounds, activated TRPV3 at a concentration of 30 µM. In addition, γ-schisandrin (~100 µM) increased cytoplasmic Ca²âº concentrations by approximately 20% in response to TRPV3 activation. This is the first report to indicate that SC extract and γ-schisandrin can modulate the TRPV3 channel. This report also suggests a mechanism by which γ-schisandrin acts as a therapeutic agent against TRPV3-related diseases.

Keyword

Calcium channel; γ-schisandrin; Schisandra chinensis; TRPV3; 2-Aminoethyldiphenyl borate

MeSH Terms

Calcium
Calcium Channels
Calcium Signaling
Camphor
Cytoplasm
Eugenol
Fruit*
Herbal Medicine
Ions
Methanol*
Permeability
Schisandra*
Sensation
Skin
Wound Healing
Calcium
Calcium Channels
Camphor
Eugenol
Ions
Methanol

Figure

• 

  Fig. 1 Effect of methanolic extract of Schisandra chinensis (SCMeOH) on TRPV3 current (ITRPV3) induction in HEK293T cells overexpressing TRPV3 (hTRPV3-HEK293T).(A) Representative patch clamp recordings of ITRPV3 in response to continuous ramp-like voltage pulse in TRPV3-HEK293T cells. In the whole-cell patch clamp recording, ramp-like pulses from −100 to 100 mV (dV/dt=0.04 V/sec) were applied every 20 sec to determine the current-voltage relationship (I~V curves). The numbers in parentheses indicate the steady state ITRPV3 induced by 10 (1), 30 (2), or 100 (3) µg/mL SC fruit extract or 50 µM 2-APB (4), or inhibited by 10 µM ruthenium red (R.R) (5). (B) The corresponding I~V curves at each steady state ITRPV3. (C, D) Summary of normalized inward and outward currents at +100 mV (C) and -100 mV (D) induced by various concentrations of the SC extracts. The current densities were normalized to the maximum ITRPV3 with the 2-APB treatment and used at the end of the experiment. 2-APB, 2-aminoethyl diphenyl borate; SCMeOH, methanolic extract of dried Schisandra chinensis fruits; SCEtOH, ethanolic extract of dried Schisandra chinensis fruits; SCseed oil, Schisandra chinensis seed oil extract. *** indicates p<0.001 vs. the baseline current (control).

• 

  Fig. 2 Control experiments for TRPV3 currents (ITRPV3) in mock-transfected HEK293T cells.To confirm whether Schisandra chinensis (SC) extracts generate non-selective cationic currents, whole-cell patch clamp experiments were done in mock-transfected HEK293T cells (A~C). Representative patch clamp recording of mock-transfected HEK293T cells treated with the methanolic extract of dried Schisandra chinensis fruits (SCMeOH), ethanolic extract of dried SC fruits (SCEtOH), and SC seed oil (SCseed oil). In the case of SCMeOH, we treated the cells with 2-aminoethyl diphenyl borate (2-APB) to activate ITRPV3. We then treated the cells with the TRPV3 blocker ruthenium red (R.R) to inhibit TRPV3. It was observed that no current was generated from the SC extract or 2-APB treatment in the mock-transfected HEK293T cells.

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  Fig. 3 Structures of the four chemical compounds isolated from the Schisandra chinensis fruit extract.

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  Fig. 4 Effect of γ-schisandrin (γ-Sch) on TRPV3 activation in HEK293T cells overexpressing TRPV3.(A) Representative patch clamp recordings showing TRPV3 current (ITRPV3) activation by 30 µM γ-Sch. At the end of the experiment, 50 µM 2-APB was added to the bath solution in order to determine the maximum current. Following treatment with 10 µM ruthenium red (R.R), a TRPV3 inhibitor, ITRPV3 that was not due to a non-selective cationic current was determined. The numbers in parentheses indicate the corresponding current to voltage relations (I~V curves) depicted in Fig. 1B. (B) I~V relationship curves of steady state ITRPV3 due to 30 µM γ-Sch (1) and 50 µM 2-APB (2) treatments. The 10 µM R.R potently inhibited the inward current of ITRPV3; however, it had relatively no impact on the outward current at the same concentration. (C) Representative trace recordings of intracellular calcium measurements ([Ca2+]i), which were increased by 100 µM γ-Sch in HEK293T cells overexpressing TRPV3 (hTRPV3-HEK293T). (D) Comparison of peaks showing increases in [Ca2+]i induced by 100 µM γ-Sch or 100 µM 2-APB in the hTRPV3-HEK293T cells. The data was normalized by resetting the [Ca2+]i to 1. Data are presented as mean±standard error of the mean (n=8). **indicates p<0.01 and *** indicates p<0.001, each vs. the basal [Ca2+]i. 2-APB, 2-aminoethyl diphenyl borate.

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  Fig. 5 γ-schisandrin (γ-Sch) generated no additional currents in the mock-transfected HEK293T cells.(A) Representative patch clamp recordings after treatment of the cells with 30 µM γ-Sch.

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