Inhibition of apoptosis signal-regulating kinase by paeoniflorin attenuates neuroinflammation and ameliorates neuropathic pain

Adult male Sprague-Dawley rats (180–200 g) were provided by the Experimental Animal Center at Nanjing Medical University, Nanjing, China. The animals were housed five to six per cage under pathogen-free conditions with soft bedding under controlled temperature (22 ± 2 °C) and photoperiods (12:12-h light-dark cycle). They were allowed to acclimate to these conditions for at least 2 days before inclusion in experiments. For each group of experiments, the animals were matched by age and body weight.

CCI surgery was performed according to our previous study [24]. Rats were anesthetized with 4% pentobarbital sodium, and a 7-mm segment of the right common sciatic nerve was exposed at the mid-thigh level. Four ligatures (4-0 chromic catgut) thread at four sites with approximately 1-mm intervals were loosely tied around the nerve. The animals in the control group received identical surgery but without nerve injury.

NQDI-1 was purchased from Selleck Chemical Inc. (Houston, TX). Antibodies for p-p38 (Tyr182) (1:800, #9211S), ASK1 (1:1000, #8662S), pERK1/2 (Thr202/Tyr204) (1:1000, #4370), and p-JNK (Thr183/Tyr185) (1:1000, #9255S) and CGRP antibody (1:100, #14959) were purchased from Cell Signaling Technology (Beverly, MA). Antibody for GAPDH (1:5000, G9545) was purchased from Sigma-Aldrich Inc. (St. Louis, USA). Antibody for p-ASK1 (Thr845) (1:1000, bs-3031R) was purchased from Bioss (Woburn, MA). Antibodies for IBA-1 (1:100, ab178847), GFAP (1:100, ab7260), IL-1β (1:1000, ab200478), and TNF-α (1:1000, ab6671) were purchased from Abcam (Cambridge MA). Anti-mouse IgG, HRP-linked Antibody (1:3000, #7076) and Anti-rabbit IgG, HRP-linked Antibody (1:3000, #7074) were purchased from Cell Signaling Technology (Beverly, MA). All other chemicals were purchased from Sigma Chemical Co (St. Louis, MO).

Rats were performed according to our previous study [24]. The animals were placed in the testing environment daily for at least 2 days before baseline testing for acclimatization. Mechanical sensitivity was detected by Von Frey hairs (Woodland Hills, Los Angeles, CA) test. The animals were placed in boxes with elevated metal mesh floor for 30 min before testing. A series of Von Frey hairs with logarithmically incrementing stiffness were used to stimulate the plantar surface of each hind paw perpendicularly. Each rat was tested for three times, and the averages of the threshold were measured. For testing thermal hyperalgesia, rats’ foot withdrawal latency to heat stimulation was measured. An analgesia meter (UGO Basile, Italy) was used to provide a heat source. The animals were placed in boxes with a smooth and temperature-controlled glass floor. The heat source was focused on a portion of the hind paw, which was flushed toward the glass, so that a radiant thermal stimulus was delivered to that site. The stimulus shuts off when the hind paw withdrew (or the stimulus was removed after 20 s to prevent tissue damage). The intensity of the heat stimulus was maintained constant throughout all experiments. The elicited paw movement occurred at latency between 9 and 14 s in the control animals. Thermal stimuli were delivered three times to each hind paw at 5- to 6-min intervals. Behavioral tests were performed blindly.

The three-dimensional (3D) structure of the Ask1 protein was retrieved from the RCSB Protein Data Bank database (http://​www.​rcsb.​org/​), and the PDB ID was 4BF2. Chemical structure of paeoniflorin (CAS NO. 23180-57-6) and the recognized Ask1 inhibitor NQDI-1 (CAS NO. 175026-96-7) were retrieved from PubChem database (https://​pubchem.​ncbi.​nlm.​nih.​gov/​), and the PubChem CID of the two ligands were 442534 and 5522952, respectively. PDBQT files of ligands (paeoniflorin and NQDI-1) and protein (Ask1) were prepared as described in the protocol [25]. Then, the protein-ligand docking studies were performed using the AutoDock Vina program [26], which is one of the widely used methods for protein-ligand docking. AutoDock Vina significantly improves the average accuracy of predictions. Here, PDBQT file of paeoniflorin and NQDI-1 was taken as ligand and PDBQT file of Ask1 was taken as protein. These ligands were docked with ASK1 around its important binding sites as described [27]. The prediction results and optimum ligand-bound conformations were performed with AutoDockTools [28, 29].

To identify temporal expression or the phosphorylated levels of proteins, whole protein samples were analyzed. In brief, samples (cells or spinal cord segments at L1–L6) were collected and washed with ice-cold phosphate-buffered saline before being lysed in a radioimmunoprecipitation assay lysis buffer. Then, whole sample lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Bedford, MA). The membranes were blocked with 5% bovine serum albumin for 1 h at room temperature, probed with antibodies overnight at 4 °C with the primary antibodies, and then incubated with HRP-coupled secondary antibodies. Data were analyzed with the Molecular Imager (Gel DocTM XR, 170-8170) and the associated software Quantity One-4.6.5 (Bio-Rad Laboratories, Berkeley, CA).

Under deep anesthesia, the animals were transcardially perfused with PBS followed by 4% paraformaldehyde, and L4 and/or L5 lumbar segment was dissected out and post-fixed in the same fixative. The embedded blocks were sectioned into 20-μm thick. The sections from each group (five mice in each group) were incubated with anti-CGRP antibody (1:100, #14959), anti-IBA-1 antibody (1:100, ab178847), and anti-GFAP antibody (1:100, ab7260). Then, the free-floating sections were washed with PBS and incubated with the secondary antibody (Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG, 1:300, #711-545-152, Jackson Laboratories, USA) for 2 h. After washing out three times with PBS, the samples were studied under a confocal microscope (Leica TCS SP2, Leica Biosystems, Wetzlar, Germany) for morphologic details of the immunofluorescence staining. Images were randomly coded and transferred to a computer for further analysis; the analyzed images were 8-bit grayscale.

Among several models of painful neuropathy have been developed in rats, CCI is the most commonly used neuropathic pain model to study the mechanism and to assess the effects of various treatments. As shown in Fig. 1a and b, the mechanical and thermal thresholds of rats, measured using the Von Frey test, were minimal at day 14 and last up to day 21 after CCI surgery. Mitogen-activated protein kinases (MAPKs: p38, JNK, and ERK MAPK) are critical for intracellular signal transduction and participate in regulating neural plasticity and inflammatory responses. Inhibition of MAPK pathways has been proved to attenuate inflammatory and neuropathic pain. ASK1, a member of the MAP3K family, could activate p38 MAPK and JNK pathway. To explore whether ASK1 play an important role in the process of neuropathy, we measured the phosphorylation of ASK1 using western blot. As shown in Fig. 1c, CCI increased the expression of p-ASK1 compared to the control group in the spinal cord of rats but did not affect the of ASK1 expression. These data suggested that CCI surgery could successfully decrease the mechanical and thermal thresholds of rats and the phosphorylation of ASK1.

Considering the significant changes of ASK1 in the neuropathic pain process, and lack of reports about the effect of ASK1 on the mechanical allodynia and thermal hyperalgesia, we measured the mechanical and thermal thresholds after ASK1 inhibitor NQDI1 administration. As shown in Fig. 2a and b, compared with the CCI group, ASK1 inhibitor NQDI1 (4 μg/20 μl, i.t.) significantly increased the mechanical and thermal thresholds in the ipsilateral paws of rats at 2, 4, and 8 h (P < 0.001) after a single administration of NQDI1. We then measured the analgesic effect of consecutive administration of NQDI1. As shown in Fig. 2c and d, ASK1 inhibitor NQDI1 (4 μg/20 μl, i.t. once daily for 3 days) markedly increased the mechanical and thermal thresholds in the ipsilateral paws of rats.

We further verified the effect of ASK1 inhibitor on MAPK kinases. As shown in Fig. 2e and f, compared with the control group, CCI increased the phosphorylation of p38, JNK, and ERK MAPK in the spinal cord of rats, and ASK1 inhibitor NQDI1 (4 μg/20 μl, i.t.) selectively alleviated the phosphorylation of p38 and JNK (P < 0.05), but did not affect the expression of p-ERK.

These data suggested that ASK1 inhibitor NQDI1 could attenuate neuropathic pain and decreased the phosphorylation of p38 and JNK.

Currently, there is no effective medication for treating neuropathic pain via inhibiting ASK 1. Thus, we are trying to find drugs that are clinically used to inhibit ASK 1 phosphorylation. As shown in Fig. 3a, the results of AutoDock suggested that paeoniflorin, a monoterpene glucoside which is the principal bioactive component purified and extracted from the root of Paeonia lactiflora pall, tightly bound to ASK1, which is equivalent to the ASK1 inhibitor, NQDI1. We subsequently measured the effect of paeoniflorin on ASK1 phosphorylation. As shown in Fig. 3b, different doses of paeoniflorin (20, 40, and 60 mg/kg, i.p.) significantly decreased the phosphorylation of ASK1 at 2 h after administration. These data suggested that paeoniflorin may mimic the ASK1 inhibitor NQDI1 and inhibit ASK1 phosphorylation.

In consideration of the effect on ASK1 phosphorylation, we measured whether paeoniflorin could alleviate neuropathic pain like ASK1 inhibitor NQDI1. We measured the mechanical and thermal thresholds after paeoniflorin administration. As shown in Fig. 4a and b, compared to the CCI group, paeoniflorin (40 and 60 mg/kg, i.p.) increased the mechanical and thermal thresholds in the ipsilateral paws of rats at 2, 4, and 8 h after a single administration in a dose-dependent manner.

We then measured the analgesic effects of consecutive administration of paeoniflorin on 14 days after CCI surgery. As shown in Fig. 4c and d, paeoniflorin (40 mg/kg, i.p.) significantly increased the mechanical and thermal thresholds in the ipsilateral paws up to day 21 after five times of administration. We also found that paeoniflorin and NQDI1 have no effect on the mechanical and thermal thresholds in naive rats (Fig. 4i).

To explore the effect of paeoniflorin on the development of pain, paeoniflorin was administrated immediately after CCI surgery. As shown in Fig. 4e and f, paeoniflorin (40 mg/kg, i.p.) significantly delayed the development of neuropathic pain and the analgesic effect lasted until day 27.

Subsequently, we also measured the effects of paeoniflorin on MAPK kinases. As shown in Fig. 4g and h, paeoniflorin (40 and 60 mg/kg, i.p.) significantly decreased the phosphorylation of p38 and JNK induced by CCI surgery.

These data suggested that paeoniflorin could decrease the phosphorylation of p38 and JNK, delay the progress of neuropathic pain, and attenuate neuropathic pain, which is consistent with the experimental results of ASK1 inhibitor NQDI1.

Microglia and astrocytes have recently emerged as key contributors to the pathological of neuropathic pain. To test the response of glial cells, GFAP and IBA-1 expression have been used as markers of astrocytes and microglia activity, respectively. As shown in Fig. 5a and b, compared with the CCI group, paeoniflorin (40 mg/kg) significantly decreased the expression of GFAP and IBA-1 induced by CCI. Chronic pain significantly induced the response of glial cells. Activated glia release a variety of neuroexcitatory substances that potentiate neurotransmission, especially proinflammatory cytokines (TNF-α and IL-1β). Compared with the CCI group, paeoniflorin (40 and 60 mg/kg, i.p.) significantly reduced the expression of IL-1β and TNF-α in the spinal cord induced by CCI (Fig. 5c).

Nitzan-Luques found the significance of calcitonin gene-related peptide (CGRP) in Aβ-touch afferents in the development of neuropathic pain. Intrathecal injection of CGRP could induce mechanical allodynia in naïve rats [30]. Moreover, administration of CGRP-P inhibitor almost reversed the allodynia in the model of spinal nerve ligation (SNL) [31]. Interestingly, we also found that paeoniflorin administration for 5 days distinctly suppressed the overexpression of CGRP in the spinal cord (Fig. 5d).

These data suggested that paeoniflorin could reduce the response of astrocytes and microglia, decrease the expression of IL-1β and TNF-α, and downregulate the expression of CGRP induced by CCI.