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. 2017 May 31:8:335.
doi: 10.3389/fphys.2017.00335. eCollection 2017.

Inhibition of the TRPM2 and TRPV1 Channels through Hypericum perforatum in Sciatic Nerve Injury-induced Rats Demonstrates their Key Role in Apoptosis and Mitochondrial Oxidative Stress of Sciatic Nerve and Dorsal Root Ganglion

Fuat Uslusoy  1 , Mustafa Nazıroğlu  2   3   4 , Bilal Çiğ  3   4
Affiliations

Inhibition of the TRPM2 and TRPV1 Channels through Hypericum perforatum in Sciatic Nerve Injury-induced Rats Demonstrates their Key Role in Apoptosis and Mitochondrial Oxidative Stress of Sciatic Nerve and Dorsal Root Ganglion

Fuat Uslusoy et al. Front Physiol. .

Abstract

Sciatic nerve injury (SNI) results in neuropathic pain, which is characterized by the excessive Ca2+ entry, reactive oxygen species (ROS) and apoptosis processes although involvement of antioxidant Hypericum perforatum (HP) through TRPM2 and TRPV1 activation has not been clarified on the processes in SNI-induced rat, yet. We investigated the protective property of HP on the processes in the sciatic nerve and dorsal root ganglion neuron (DRGN) of SNI-induced rats. The rats were divided into five groups as control, sham, sham+HP, SNI, and SNI+HP. The HP groups received 30 mg/kg HP for 4 weeks after SNI induction. TRPM2 and TRPV1 channels were activated in the neurons by ADP-ribose or cumene peroxide and capsaicin, respectively. The SNI-induced TRPM2 and TRPV1 currents and intracellular free Ca2+ and ROS concentrations were reduced by HP, N-(p-amylcinnamoyl) anthranilic acid (ACA), and capsazepine (CapZ). SNI-induced increase in apoptosis and mitochondrial depolarization in sciatic nerve and DRGN of SNI group were decreased by HP, ACA, and CapZ treatments. PARP-1, caspase 3 and 9 expressions in the sciatic nerve, DRGN, skin, and musculus piriformis of SNI group were also attenuated by HP treatment. In conclusion, increase of mitochondrial ROS, apoptosis, and Ca2+ entry through inhibition of TRPM2 and TRPV1 in the sciatic nerve and DRGN neurons were decreased by HP treatment. The results may be relevant to the etiology and treatment of SNI by HP.

Keywords: Hypericum perforatum; TRPM2; TRPV1; apoptosis; mitochondrial oxidative stress; sciatic nerve injury.

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Figures

Graphical Abstract
Graphical Abstract
Poly (ADP-ribose) polymerase (PARP) catalyzes the transfer of ADP-ribose (ADPR) in nucleus during the DNA repair processes. TRPM2 channel is gated by ADPR and reactive oxygen species (ROS) through activation of ADP-ribose (ADPR) pyrophosphate enzyme in its nudix domain motif although it was inhibited by N-(p-amylcinnamoyl)anthranilic acid (ACA). TRPV1 channel is also activated by ROS and capsaicin (CAPS) but it is inhibited by capsazepine (CapZ).
Figure 1
Figure 1
Induction of sciatic nerve injury in right leg of the rats. After anesthesia (A), the common sciatic nerve of the right hind paw was exposed the middle of thigh by blunt dissection through the biceps femoris (B). For sciatic nerve crush, a hemostatic sterile clamp was used. The sciatic nerve was crushed for a total of 30 s. Then, the wound was closed with a 2.0 suture and rats were allowed to recover in the postoperative room. In sham-operated rats, the same surgical procedure was followed, the connective tissue was freed, and no ligatures were applied.
Figure 2
Figure 2
Effect of Hypericum perforatum (HP) treatment on [Ca2+]i concentration and TRPM2 in sciatic nerve (A) and dorsal root ganglion neurons (DRGNs) (B) of control, sham and SNI-induced rats. (n = 8 and mean ± SD). The animals received oral HP for 4 weeks. Then, these dissected neurons of control, sham and SNI were further in vitro treated with CPx (0.1 mM) and ACA (0.025 mM) before loading Fura-2 for 125 s. ap ≤ 0.05 and bp ≤ 0.001 vs. control and sham groups. cp ≤ 0.05 and dp ≤ 0.001 vs. sham+ACA and sham+HP groups. ep ≤ 0.001 vs. sham+HP+ACA group. fp ≤ 0.001 vs. SNI group. gp ≤ 0.05 and hp ≤ 0.001 vs. SNI+ACA group. kp ≤ 0.05 vs. SNI+HP group (C).
Figure 3
Figure 3
Effect of Hypericum perforatum (HP) treatment on [Ca2+]i concentration and TRPV1 in sciatic nerve (A) and dorsal root ganglion neuron (DRGN) (B) of control, sham and SNI-induced rats. (n = 8 and mean ± SD). The animals received oral HP for 4 weeks. Then, these dissected neurons of control, sham and SNI were further in vitro treated with CAPS (0.01 mM) and CapZ (0.1 mM) before loading Fura-2 for 125 s. ap ≤ 0.05 and bp ≤ 0.001 vs. control and sham groups. cp ≤ 0.05 and dp ≤ 0.001 vs. sham+CapZ and sham+HP groups. ep ≤ 0.001 vs. sham+HP+CapZ group. fp ≤ 0.001 vs. SNI group. gp ≤ 0.05 and hp ≤ 0.001 vs. SNI+CapZ group. kp ≤ 0.05 vs. SNI+HP group (C).
Figure 4
Figure 4
Effects of HP on TRPM2 channel activation in dorsal root ganglion neuron (DRGN) of control and SNI-induced rat. The TRPM2 currents in DRGN were stimulated by intracellular ADPR (1 mM in patch pipette) but they were inhibited by extracellular ACA (0.025 mM) in the patch-chamber. W.C.: Whole cell. Control (without SCI induction and stimulation): Original recordings from control neuron (A). (B). Control+ADPR group (without SCI induction). (C). SNI group (with SCI induction). (D). SCI+HP group: The rats received HP after SCI induction. (E). HP group: The rats received HP without SCI induction. (F). TRPM2 channel current densities in the DRGN. The numbers in parentheses indicated n numbers of groups were indicated by numbers in parentheses. (ap ≤ 0.001 vs. control. bp ≤ 0.001 vs. control+ADPR group. cp ≤ 0.001 vs. control+ADPR+ACA group. dp ≤ 0.001 vs. SNI+ADPR group. ep ≤ 0.001 vs. SNI+ADPR+ACA group).
Figure 5
Figure 5
Effects of HP on TRPV1 channel activation in dorsal root ganglion neuron (DRGN) of control and SNI-induced rat. The TRPV1 currents in DRGN were stimulated by extracelular capsaicin (CAPS and 0.01 mM in patch chamber) but they were inhibited by extracellular CaPZ (0.1 mM) in the patch-chamber. W.C.: Whole cell. Control (without SCI induction and stimulation): Original recordings from control neuron (A). (B). Control+CAPS group (without SCI induction). (C). SNI group (with SCI induction). (D). SCI+HP group: The rats received HP after SCI induction. (E). HP group: The rats received HP without SCI induction. (F). TRPV1 channel current densities in the DRGN. The numbers in parentheses indicated n numbers of groups were indicated by numbers in parentheses. (ap ≤ 0.001 vs. control. bp ≤ 0.001 vs. control+CAPS group. cp ≤ 0.001 vs. control+CAPS+CapZ group. dp ≤ 0.001 vs. SNI+ADPR group. ep ≤ 0.001 vs. SNI+ADPR+ACA group).
Figure 6
Figure 6
Effects of Hypericum perforatum (HP) on the apoptosis and cell viability (MTT) levels through TRPM2 (A) and TRPV1 (B) in sciatic nerve of SNI-induced rats (mean ± SD and n = 3). Apoptosis level was measured by using a commercial kit. Values expressed as fold increase (experimental/control). These neurons were dissected from control, SNI and treated animals. The animals were received HP via gastric gavage. The neurons in TRPM2 and TRPV1 experiments were stimulated with cumene hydroperoxide (CPx and 0.1 mM) capsaicin (CAPS and 0.01 mM) although they were inhibited by ACA (0.025 mM) and CapZ (0.1 mM), respectively. (ap ≤ 0.001 and ep ≤ 0.05 vs. control, sham, sham+HP, sham+HP+ACA and sham+HP+CapZ groups. bp ≤ 0.001 and cp ≤ 0.05 vs. SNI group. dp ≤ 0.05 and fp ≤ 0.001 vs. SNI+ACA and SNI+CapZ groups. gp ≤ 0.05 vs. SNI+HP group).
Figure 7
Figure 7
Effects of Hypericum perforatum (HP) on the intracellular ROS production and cell mitochondrial membrane depolarization (JC-1) levels through TRPM2 (A,C) and TRPV1 (B,D) in sciatic nerve of SNI-induced rats (mean ± SD and n = 3). Values expressed as fold increase (experimental/control). Sciatic neurons were dissected from control, SNI and HP treated animals. The neurons in TRPM2 and TRPV1 experiments were stimulated with cumene hydroperoxide (CPx and 0.1 mM) capsaicin (CAPS and 0.01 mM) although they were inhibited by ACA (0.025 mM) and CapZ (0.1 mM), respectively. (ap ≤ 0.05 and cp ≤ 0.001 vs. control, sham, sham+HP, sham+HP+ACA and sham+HP+CapZ groups. bp ≤ 0.05 and dp ≤ 0.001 vs. SNI group. ep ≤ 0.05 vs. SNI+ACA group).
Figure 8
Figure 8
Effects of Hypericum perforatum (HP) on the PARP-1, caspase 3 and 9 expression levels in sciatic nerve (A,C), DRGN (B,C), skin (D,F) and muscle (Musculus piriformis) (E,F) and skin (F) of rats with SNI (mean ± SD and n = 3). Anti-β-actin was used as an internal control for the concentration of PARP1, caspase 3 and 9. Values expressed as fold increase (experimental/control). Sciatic nerve DRGN neurons were dissected from control, SNI and HP treated animals. (ap ≤ 0.05 vs. control, sham, sham+HP groups. bp ≤ 0.05 and cp ≤ 0.001 vs. SNI group).
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