Nalbuphine alleviates inflammation by down-regulating NF-κB in an acute inflammatory visceral pain rat model

Abstract

Introduction: Nalbuphine can relieve patients' inflammation response after surgery compared to other opioid drugs. However, its molecular mechanism has not been clear. Activation of NF-κB signaling pathway under oxidative stress and inflammation can maintain pain escalation.

Methods: We firstly investigated the effect of nalbuphine on writhing test and mechanical allodynia using a rat model of inflammatory visceral pain (acetic acid (AA) administrated). Cytokines (including tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-2, and IL-6 in plasma were tested with ELISA technology. Expression levels of TNF-α, IκBα and p-NF-κB p65 at the spinal cord (L3-5) were measured by western blot or RT-qPCR.

Results: We found that the paw withdrawal threshold (PWT) values of rats were reduced in the model group, while the numbers of writhing, levels of IL-1β, IL-2, IL-6, and TNF-α in plasma, and p-NF-κB protein and its gene expressions in the lumbar spinal cord were up-regulated. Subcutaneously injection of nalbuphine (10 μg/kg) or PDTC (NF-κB inhibitor) attenuated acetic acid-induced inflammatory pain, and this was associated with reversal of up-regulated IL-1β, IL-2, IL-6, and TNF-α in both plasma and spinal cord. Furthermore, acetic acid increased p-NF-κB and TNF-α protein levels in the white matter of the spinal cord, which was attenuated by nalbuphine. These results suggested that nalbuphine can significantly ameliorate inflammatory pain via modulating the expression of NF-κB p65 as well as inflammation factors level in the spinal cord.

Conclusion: In conclusion, nalbuphine inhibits inflammation through down-regulating NF-κB pathway at the spinal cord in a rat model of inflammatory visceral pain.

Keywords: Inflammation; NF-κB; Nalbuphine; Visceral pain.

Fig. 1

The effects of acetic acid, nalbuphine, and PDTC on the writhing test and paw withdrawal threshold (PWT). a The number of writhes for 30 minutes in each group. b Mechanical paw withdraw threshold in each group (data were the mean ± SEM, n = 12). *p < 0.05, AA vs control; ^#p < 0.05, AA+nalbuphine vs AA, ^&p < 0.05; AA+PDTC vs AA

Fig. 2

Results of plasma levels of several inflammatory factors. The expressions of IL-1β, IL-2, IL-6, and TNF-α were compared between groups (data were the mean ± SEM, n = 8). *p < 0.05, AA vs control; ^#p < 0.05, AA+nalbuphine vs AA, ^&p < 0.05; AA+PDTC vs AA.

Fig. 3

Levels of IκBα and p-NF-κB p65 (Ser536) in four groups. a The protein levels of IκBα and p-NF-κB p65 (Ser536) in the spinal cord were detected via WB. GAPDH was used as a loading control. b The mRNA expression of NF-κB p65 in the spinal was detected via RT-qPCR. (Data are the mean ± SEM, n = 4). *p < 0.05, AA vs control; ^#p < 0.05, AA+nalbuphine vs AA, ^&p < 0.05; AA+PDTC vs AA

Fig. 4

The mRNA and protein levels of IL-1β, TNF-α, and IL-6 in the lumbar spinal cord of rats. a-c The mRNA expressions of IL-1β, TNF-α, and IL-6 in the spinal cord were detected via RT-qPCR. d The protein levels of IL-1β, TNF-α, and IL-6 in the spinal cord were detected via western blotting. GAPDH was used as a loading control. (Data were the mean ± SEM, n = 4). *p < 0.05, AA vs control; ^#p < 0.05, AA+nalbuphine vs AA, ^&p < 0.05; AA+PDTC vs AA

Fig. 5

The expressions of p-NF-κB and TNF-α in the spinal were observed by IHC. a The number of positive cells at representative locations. b Representative images of p-NF-κB and TNF-α after immunohistochemical staining (SLIDEVIEW VS200; × 20, × 200). (Data are the mean ± SEM, n = 4). *p < 0.05, AA vs control; ^#p < 0.05, AA+nalbuphine vs AA, ^&p < 0.05; AA+PDTC vs AA