Celastrol ameliorates inflammatory pain and modulates HMGB1/NF-κB signaling pathway in dorsal root ganglion

Xiumei Zhang a, Wenpin Zhao a, Xingfang Liu c, Zhihua Huang b,d, Reai Shan c,d, Cheng Huang b,d*

Summary

Tripterygium wilfordii Hook, possesses anti-inflammatory activity, but the underlying mechanism remains to be fully clarified. We aim to investigate whether HMGB1 in dorsal root ganglion (DRG) participates in the effect of celastrol on inflammatory pain. Complete Freund’s adjuvant (CFA)-induced inflammatory pain rat model was used. Paw withdrawal latency (PWL) was detected to evaluate the effects of celastrol on CFA-evoked inflammatory pain. After application results in increasing NF-κB production, which subsequently drives the production of pro-inflammatory cytokines and chemokines, contributing to the initiation and maintenance of pain hypersensitivity [3-5]. It is evident that HMGB1 is activated under pathological conditions and involved in the pathogenesis of inflammatory pain.
Previous studies have demonstrated that pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α ) contribute to the pathogenesis of inflammatory pain [6,7]. These cytokines activate the NF-κB IL-1β through activating NF-κB and up-regulating inflammatory mediators in CFA rats, we hypothesized that the HMGB1/NF-κB signaling pathway was involved in the anti-inflammatory effect of celastrol on the DRG of CFA rats. In the present study, we explore whether celastrol regulated HMGB1 and NF-κB expression in the DRG of rats with CFA-induced inflammatory pain, and subsequently influenced the expression levels of IL-1β, IL-6, TNF-α, COX-2 and MCP-1. Additionally, we further investigated normal saline to the hind paw, while the other groups received CFA injection to the hind paw. The rats in the CFA+CEL group were treated with celastrol (CEL, Shanghai Tianxi Chemical Co., Ltd. with a purity of 99.99%, 1mg/kg, i.p.) at 10 hours post-CFA injection and once a day in the next 14 days. The rats in the Ctrl and CFA+VEH group were treated with normal saline and DMSO (0.1ml/100g, i.p.), respectively. PWL of the ipsilateral hind paw was measured following administration of normal saline, DMSO and celastrol on day 1, 3, 7, and 14 post-CFA injection, plantar surface of the hind paw. To avoid tissue injury, light intensity was set to obtain a baseline latency of approximately 20 s. Three PWLs were recorded with an interval of 5 minutes between stimulations of each hind paw. The mean PWL of the three trials was used for statistical analysis. Rats not exhibiting pain hypersensitivity after injecting CFA were discarded.

2. Materials and Methods

2.1. Quantitative RT-PCR analysis

Total RNA was extracted from the DRG of L4-L6 segments and prepared for quantitative DRG of L4-L6 segments. Thirty micrograms of protein per sample were denatured and then separated with 10% SDS-PAGE and western-blotted on a PVDF (Millipore, CA) membrane using a minigel and mini transblot apparatus (Bio-Rad, Hercules, CA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 for 60 minutes at room temperature. Subsequently the membranes were incubated in the primary antibody solutions respectively, for overnight at 4◦C. The antibodies were rabbit anti-HMGB1 (1:2000, ab79823, statistically significant.

3. Results

3.1. Celastrol alleviates CFA-induced thermal hyperalgesia

To explore the analgesic effects of celastrol treatment, celastrol (CEL, 1mg/kg) or vehicle (VEH) was intraperitoneally injected once a day for 14 consecutive days. PWL was measured on day 1, 3, 7, and 14 post-CFA injection. As shown in Fig. 2, PWL was significantly decreased in the

3.2. Celastrol inhibited CFA-induced IL-1β, IL-6, IL-17, TNF-α and MCP-1 up-regulation in the DRG

To confirm the inhibitory effect of celastrol on the expression of IL-1β, IL-6, IL-17, TNF-α and MCP-1 in the DRG of CFA rats. IL-1β expression levels were checked on day1, 3, 7, and 14 post-CFA injection. As shown in Fig. 4 A and B, the levels of IL-1β mRNA and protein in the CFA+VEH group were significantly increased on day 3 (P < 0.05), 7 (P < 0.05), and 14 (P < 0.01), the Ctrl group on day 1 (P < 0.05), 3 (P < 0.05), and 7 (P < 0.01), respectively. As expected, the level of COX-2 protein in the CFA+CEL group was significantly lower than that of the CFA+VEH group on day 1 (P < 0.001), 3 (P < 0.01), and 7 (P < 0.05), respectively.

3.3. Celastrol inhibited CFA-induced GFAP and CD11b mRNA up-regulation in the DRG

To assess whether satellite glial cell and immune cell are involved in the inhibitory effect of celastrol on CFA-induced pain hypersensitivity in the DRG. The levels of mRNA of GFAP, an activation marker of satellite glial cell, and CD11b, an activation marker of macrophages, were determined on day 1, 3, 7, and 14 post-CFA injection. As shown in Fig. 6, the level of GFAP mRNA in the CFA+VEH group was significantly increased compared to that of the Ctrl group (P < 0.05) on day 3, 7, and 14, respectively. Whereas, the level of GFAP mRNA in the CFA+CEL group was significantly lower than that of the CFA+VEH group on day 3 (P < 0.01), 7 (P < 0.001), and 14 (P < 0.001), respectively. Similarly, the level of CD11b mRNA in the CFA+VEH group was significantly increased on day 3 (P < 0.01), 7 (P < 0.05), and 14 (P < 0.01), respectively, compared with that of the Ctrl group. It was markedly reduced in the CFA+CEL group compared to that of the CFA+VEH group on day 3 (P < 0.01), 7 (P < 0.05), and 14 (P < 0.01), respectively.

3.4. Celastrol suppressed CFA-induced NF-κB activation in the DRG

To examine whether celastrol inhibits NF-κB activation induced by CFA in the DRG, the levels of NF-κB-p65 mRNA and protein were tested. As shown in Fig. 7, the level of NF-κB-p65 mRNA was significantly increased on day 3 (P < 0.05), 7 (P < 0.01), and 14 (P < 0.01) after CFA injection. Additionally, Western blot results showed that p-NF-κB-p65 expression was markedly increased on day 1 (P < 0.05), 3 (P < 0.01), 7 (P < 0.05), and 14 (P < 0.05) post- CFA injection. Celastrol significantly suppressed the up-regulation of NF-κB-p65 mRNA due to CFA injection on day 3 (P < 0.01), 7 (P < 0.01), and 14 (P < 0.001). Moreover, celastrol significantly inhibited the up-regulation of p-NF-κB-p65 on day1 (P < 0.05), 3 (P < 0.001), 7 (P < 0.001), and 14 (P < 0.001) post-CFA injection in the DRG.

4. Discussion

In the present study, we found that celastrol suppressed up-regulation of HMGB1 and NF-κB mRNA and protein up-regulation in the DRG by CFA, and subsequently attenuated thermal releasing pro-inflammatory molecules [26]. In peripheral nerve injury and painful diabetic neuropathy conditions, the up-regulation of HMGB1 expression in the spinal cord and the DRG was also observed [22,24,27]. An immunohistochemical study also revealed that HMGB1 was increased in satellite cells and neurons of the DRG [21]. Consistent with previous results, we found that CFA injection led to up-regulation of HMGB1 mRNA and protein in the DRG, which was suppressed by celastrol, suggesting that celastrol exerts anti-inflammatory effect through GFAP protein were up-regulated in the spinal cord of mice with inflammatory pain [22,28]. These results suggest that the activation of spinal glial cells are involved in chronic inflammatory pain.
The DRG was reported to connect the peripheral and central nervous systems and play a critical role in pain transmission. Since the GFAP for an activation marker of satellite glial cell and CD11b for an activation marker of macrophages in the DRG. We further explored the effects of celastrol on CFA-induced chronic inflammatory pain and found that celastrol attenuated the hypersensitivity partially through suppressing the HMGB1/NF-κB signaling pathway, and the production of pro-inflammatory cytokines, inflammatory mediators as well as the activation of satellite glial cell and immune cell in the DRG. Our results suggest that HMGB1 is a new potential target for celastrol to relieve CFA-induced chronic inflammatory pain.

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