Bupivacaine effectively relieves inflammation-induced pain by suppressing activation of the NF-κB signalling pathway and inhibiting the activation of spinal microglia and astrocytes

Abstract

The pain induced by local acute inflammation results in mild to severe discomfort, in addition to the possibility of physiological dysfunction and psychiatric disorders, such as sleep disorders and depression. However, the pathogenesis of pain is yet to be fully elucidated. In the present study, the effects of bupivacaine were explored in rat models inflammatory pain in order to investigate the anti-pain mechanism of bupivacaine. Complete Freund's adjuvant (CFA) was injected into the right rear foot of the rats to establish a model of transient inflammation-induced pain. Rats were randomly divided into four groups (n=8): CFA, CFA plus bupivacaine, CFA plus saline and untreated. The mechanical withdrawal threshold (MWT) of the rats was detected prior to and following CFA injection, and the results demonstrated that the MWT in the right rear foot significantly decreased from the 1st day of CFA injection (P<0.01; n=8), as compared with the untreated controls. Bupivacaine treatment was demonstrated to significantly increase the MWT of rats treated with CFA stimulation, as compared with the CFA group (P<0.01). Rotarod testing was performed to assess the motor activity of the rats, and the results demonstrated no significant differences among the four groups (P>0.05). Furthermore, the respective body weights of the rats were determined every two days before and after CFA injection, and no significant differences were detected among the four groups (P>0.05). Western blot analysis was performed to analyze expression levels of IκB and nuclear factor (NF)-κB, and the results demonstrated that bupivacaine increased the expression of IκB and decreased the expression levels of NF-κB, as compared with the rats with CFA-induced inflammatory responses, suggesting that bupivacaine inhibited NF-κB activation in the dorsal horn of the lumbar spinal cord of the model rats. Furthermore, reverse transcription-quantitative polymerase chain reaction analysis was performed to analyze the expression levels of inflammatory cytokines, which demonstrated that bupivacaine significantly inhibited the expression of TNF-α, IL-1β and IL-6, as compared with the untreated group (P<0.01). Moreover, bupivacaine treatment significantly decreased the expression of spinal microglial marker OX42 and astrocyte marker-glial fibrillary acidic protein, as compared with the rats in the CFA group (P<0.01). The present findings demonstrated that treatment with bupivacaine significantly decreased the activation of microglia and astrocytes in rat models of inflammatory pain. Therefore, the present results may provide clarification of the pathogenesis and mechanism of inflammation-induced pain and may provide novel therapeutic strategies for the clinical treatment of pain.

Keywords: bupivacaine; glial fibrillary acidic protein; inflammatory cytokines; inflammatory pain; nuclear factor-κB; ox42.

Figure 1.

Mechanical withdrawal threshold was detected in rats prior to and following CFA injection. The right hind paw of the rat was sterilized with iodophor after anesthesia and CFA (1 µl/g) was injected into the rear portion of the plantar site. Untreated rats were used as the controls. Mechanical withdrawal threshold was detected prior to CFA injection (day 0) and on days 1, 3, 5 and 7 post-injection (n=8; **P<0.01). Data are presented as the mean ± standard error of the mean. CFA, complete Freund's adjuvant.

Figure 2.

Body weight. To construct the rat model of inflammation-induced pain, 100 µl CFA was subcutaneously injected into the right hind foot. Body weight of the rats was assessed prior to CFA injection and every two days post-injection (n=8). Untreated rats were used as the controls. Data are presented as the mean ± standard error of the mean. CFA, complete Freund's adjuvant.

Figure 3.

Bupivacaine increases the mechanical withdrawal threshold of rats treated with CFA stimulation. Rats were randomly divided into four groups (n=8): CFA, CFA plus bupivacaine, CFA plus saline and untreated. The right hind paw of the rat was sterilized with iodophor following anesthesia and CFA (1 µl/g) was injected into the rear portion of the plantar site. Mechanical withdrawal threshold in rats was detected prior to (day 0) and on days 1, 3, 5 and 7 post-injection (n=8, **P<0.01 vs. the CFA group). Untreated rats were used as the controls. Data are presented as the mean ± standard error of the mean. CFA, complete Freund's adjuvant.

Figure 4.

Motor activity was not obviously altered in the different groups. Data from the four different groups were recorded prior to (day 0) and on days 1, 3, 5 and 7 post-CFA injection. Untreated rats were used as the controls. Rats were placed on the round bar and the instrument of rotating rods were set in on Uniformly Accelerating mode. Data are presented as the mean ± standard error of the mean. CFA, complete Freund's adjuvant.

Figure 5.

Bupivacaine inhibits NF-κB activation in the lumbar spinal dorsal horn of rat models of CFA-induced pain. Spinal dorsal horn tissues of lumbar segments were harvested from the different groups of rats and total cell lysates were used to detect the expression of IκB and nuclear NF-κB by western blotting analysis. (A) Samples in the CFA group at 6, 24 and 48 h post-CFA injection. (B) Expression of IκB and NF-κB in four different groups were determined by western blotting analysis. CFA, complete Freund's adjuvant; Ctrl, control; Bup, bupivacaine; SS, saline solution; NF, nuclear factor.

Figure 6.

Bupivacaine inhibits the secretion of inflammatory cytokines. Total RNA samples were extracted from four different groups and transcribed into cDNA to assess the expression levels of (A) TNF-α, (B) IL-1β and (C) IL-6 inflammatory cytokines via quantitative polymerase chain reaction analysis. Data are presented as the mean + standard error of the mean. TNF-α, tumor necrosis factor-α; IL, interleukin; CFA, complete Freund's adjuvant. **P<0.01 vs. the untreated group; ^##P<0.01 vs. the CFA group.

Figure 7.

Treatment with bupivacaine decreases the expression levels of OX42 and GFAP. (A) Spinal dorsal horn tissues of lumbar segments were harvested from rats in the different groups and total cell lysates were used to detect the expression levels of OX42 and GFAP via western blot analysis. Histograms of the expression of (B) OX42 and (C) GFAP relative to the untreated Ctrl in each group. Data are presented as the mean + standard error of the mean. **P<0.01 vs. the untreated group; ^##P<0.01 vs. the CFA group. CFA, complete Freund's adjuvant; GFAP, glial fibrillary acidic protein; Ctrl, control.