Lidocaine Potentiates SOCS3 to Attenuate Inflammation in Microglia and Suppress Neuropathic Pain

Abstract

Lidocaine is one of the typical local anesthetics that are frequently used in the peripheral nerve blocks and pain management. Emerging evidence have shown that lidocaine may exert anti-inflammatory effect involving neuropathic pain. However, the effect and underlying mechanism of lidocaine in suppressing neuroinflammation in neuropathic pain are incompletely revealed. In this study, effects of lidocaine on the suppressors of cytokine-signaling protein 3 (SOCS3) in microglia are investigated in chronic constriction injury (CCI) rat model and lipopolysaccharide (LPS)-stimulated BV-2 cells. It was shown that intrathecal injection of lidocaine substantially alleviated CCI-induced neuropathic pain, as reflected by the decreased thermal latency and mechanical threshold. Lidocaine reduced the CCI-evoked spinal injury and cell apoptosis. CCI induced an significant increase of IBA1⁺ microglia accompanied by the increase of inflammatory cytokines IL-6 and IL-1β, which were suppressed after lidocaine administration. SOCS3 expression in IBA1⁺ microglia was notably upregulated in response to lidocaine injection, which presented in a similar pattern in LPS-activated BV-2 cells. Furthermore, lidocaine upregulated SOCS3 expression dependent of pCREB, and CREB silencing greatly discounted this effect. The intrathecal injection of lentiviral vectors LV-SOCS3 efficiently alleviated CCI-evoked neuropathic pain and reduced spinal IBA1⁺ microglia. SOCS3 overexpression contributed to the inhibition of neuroinflammation by decreasing the expression and activation of p38 MAPK and NF-κB stimulated by LPS. Collectively, lidocaine promoted the SOCS3 expression in microglia, in turn leading to suppression of IBA1⁺ microglia accumulation and p38 MAPK and NF-κB, which may expand our understanding on lidocaine in suppressing neuroinflammation and neuropathic pain.

Keywords: Lidocaine; Microglia; Neuroinflammation; Neuropathic pain; SOCS3.

Fig. 1

Effect of intrathecal lidocaine administration on behavioral performance and spinal injury in CCI rats. CCI and sham-operated rats were used to evaluate effects of lidocaine administration on neuropathic pain. Behavioral tests were performed 3 days before CCI as baseline, and 1, 4, 7, 10 and 14 days after CCI. Lidocaine and saline were intrathecally injected into CCI or sham rats 6 days post-surgeries. Histological examinations were performed 6 h after the last behavioral test. Changes of mechanical allodynia (a) and thermal hyperalgesia (b) in sham and CCI rats at indicated time points in response to Lidocaine injection or not. The differences between groups were analyzed by repeated measures ANOVA followed by LSD tests. **p < 0.01, *p < 0.05 versus Sham + saline; ^#p < 0.05 versus CCI + saline. N = 12 rats/group; c Diagram of the target areas of interest (rectangle box) for histological and immunological studies; d TUNEL staining of the spinal dorsal horn in each group of rats; Scale bar: 100 μm e Quantification of the ratio of TUNEL stained areas in each group of animals. N = 6 rats/group

Fig. 2

Inhibitory effect of lidocaine on microglia in CCI rats and LPS-stimulated BV-2 cells. a Representative immunohistochemical staining of hyperactive microglia using IBA1 antibody in spinal dorsal horn of CCI or sham rats 14 days postoperatively, scale bar: 100 μm; b Quantitative analysis of IBA1 antibody stained areas in spinal section in these animals; c, d IL-6 and IL-1β mRNA levels in spinal dorsal horn of CCI or sham rats receiving lidocaine or saline. N = 6 rats/group, *p < 0.05, **p < 0.01 versus Sham + saline; ^#p < 0.05, ^##p < 0.01 versus CCI + saline. N = 6 rats/group; e, f BV-2 cell were preconditioned with 10 μg/mL of lidocaine for 1 h and then exposed to 1.0 μg/mL LPS for 24 h. Western blot detection and quantification of IBA1 expression in BV-2 cells were performed to evaluate the effect of lidocaine. g, h L-6 and IL-1β mRNA levels in BV-2 cells. *p < 0.05, **p < 0.01 versus Control; ^#p < 0.05, ^##p < 0.01 versus LPS

Fig. 3

Lidocaine potentiated the expression of SOCS3 in microglia of CCI rats and activated BV-2 cells in a CREB-dependent manner. a Double immunofluorescence staining of the co-localization of SOCS3 expressing cells with IBA1⁺ microglia in response to CCI and lidocaine exposure. Scale bar: 200 μm N = 6 rats/group; *p < 0.05 versus Sham + saline; ^#p < 0.05 versus CCI + saline; b Quantification of the ratio of SOCS3⁺IBA1⁺ cells relative to IBA1⁺ microglia in each group of animals; c, d Western blot analysis and quantification of the effects of lidocaine treatment on CREB phosphorylation and SOCS3 expression as well as the regulatory effect of CREB on SOCS3. *p < 0.05, **p < 0.01, versus control; ^#p < 0.05 versus LPS; ^&p < 0.05, ^&&p < 0.01 versus LPS + Lido + NC

Fig. 4

SOCS3 overexpression alleviated neuropathic pain in CCI rats and microglia activation. CCI animals were intrathecally injected with LV-SOCS3 or LV-Ctrl vectors 3 days prior to the CCI, then behavioral tests and histological examinations were performed after the injections. a Evaluation of spinal SOCS3 mRNA levels in rats 48 h after the injection. b Double immunofluorescence staining of SOCS3⁺ with IBA1⁺ microglia to examine the transfection efficiency of SOCS3 to microglia in CCI rats; Scale bar: 200 μm c Quantification of the ratio of SOCS3⁺IBA1⁺ cells relative to IBA1⁺ microglia in each group of animals, N = 6 rats/group; d The effects of SOCS3 overexpression on mechanical allodynia (paw withdrawal thresholds) and thermal hyperalgesia (paw withdrawal lantency) of CCI rats. N = 6 rats/group. e, f Effect of SOCS3 overexpression on the presentation of IBA1⁺ spinal microglia in CCI rats. Scale bar: 100 μm; N = 6 rats/group. The differences between groups were analyzed by t test or repeated ANOVA followed by LSD tests. *p < 0.05, **p < 0.01 versus CCI + LV-Ctrl. g BV-2 cells were transfected with SOCS3 overexpressing vectors or control for 48 h and incubated with LPS for another 24 h, then the IBA1 expression was determined using western blot. **p < 0.01 versus LPS + LV-Ctrl

Fig. 5

SOCS3 overexpression suppressed neuroinflammation by inhibiting NF-κB and p38-MAPK signaling in BV-2 cells. BV-2 cells were transfected with SOCS3 overexpressing vectors or the matched control vectors, and were pretreated using lidocaine followed by the addition of LPS. a–c After 24 h of incubation, supernatant IL-1β, IL-6 and TNF-α levels in each culture were evaluated by ELISA. d Activation of NF-κB and p38-MAPK signaling cascades in BV-2 cells in response to various treatments were determined by western blot. **p < 0.01 versus Control; ^#p < 0.05 versus LPS; ^&p < 0.05 versus LPS + LV-Ctrl