Analgesic Effects Of Aloperine Against Neuropathic Pain Induced By Chronic Constriction Injury Via Regulation Of TLR4/NF-κB Signaling Pathway In Mice

Objective The present study aimed at investigating the analgesic effects of Aloperine(ALO)on neuropathic pain induced by chronic constriction injury on sciatic nerve and exploring the potential relationship with TLR4/NF-κB signal pathway.Methods1.To detect the analgesic effects of aloperine on CCI-induced neuropathic pain in mice.Mice were randomly divided into sham group,CCI group,CCI+ALO(20,40,and 80mg/kg)group and CCI+ pregabalin(10 mg/kg)group;the sciatic nerve of Sham group mice was exposed only,whereas the sciatic nerves of other group mice suffered CCI surgery to induced neuropathic pain.Saline,ALO(20,40 and 80 mg/kg)and pregabalin(10 mg/kg)were injected intraperitoneally for 8 consecutive days from the 7th day post-surgery.1 The von Frey filaments test was performed to measure the paw withdrawal threshold(PWT)and radiant heat test was performed to detect the paw withdrawal latency(PWL);(2)Electrophysiological examinations were measured the sensory nerve action potential(SNAP)amplitude and sensory nerve conduction velocity(SNCV)of CCI mice;(3)Hematoxylin-Eosin(H&E)and Toluidine Blue staining were used to observe the histopathological impairments in sciatic nerve;Transmission electron microscopy was used to evaluated the ultrastructural changes of sciatic nerve in mice.2.To explore the analgesic mechanism of aloperine on neuropathic pain induced byCCI via TLR4/NF-κB pathway.Mice were randomly divided into sham group,CCI group and CCI+ALO(80 mg/kg)group;mice in the sham group,the sciatic nerve was exposed only;mice in other groups,the sciatic nerves suffered CCI surgery to induced neuropathic pain.Saline and ALO(80 mg/kg)were injected intraperitoneally for 8 consecutive days from the 7th day post-surgery.(1)Immunofluorescence: to detected the effects of ALO on the activation of microglia in the spinal cord of mice with neuropathic pain caused by CCI.(2)ELISA method: to detect the effects of ALO on the expression of IL-6 and TNF-α in spinal cord of CCI-mice.(3)Western blot analysis: to examine the effects of ALO on the expression of TLR4 and NF-κB in the spinal cord.(4)The von Frey filaments test was performed to detect the effects of LPS(10μg/10μl)on the PWT of CCI+ALO(80 mg/kg).BV2 microglia were randomly divided into control group,LPS(1 μg/ml)group and LPS+ALO treated groups.(1)CCK-8: to detect the cytotoxicity of each dose of ALO on normal cells and LPS-treated cells.(2)ELISA method: to detect the IL-6 and TNF-α expression levels in cell culture supernatants.(3)Immunofluorescence: to observe the effects of ALO on the activation of BV2 microglia induced by LPS.(4)Western Blot assay: to detect the effects of ALO on the expression levels of TLR4,p-NF-κB p65/NF-κB and p-IκBα/IκBα protein in LPS-treated BV2 cells.(5)Reverse transcriptase–polymerase chain reaction(RT-PCR)method was used to quantify the mRNA level of TNF-α and IL-6 in each group of cells.Results1.The analgesic effects of aloperine on CCI-induced neuropathic pain.Compared with the CCI-group,administration of ALO(80mg/kg)(1)obviously raise the PWT in the von Frey filaments test(P<0.01)and prolong the PWL in the radiant heat test(P<0.01).(2)obviously accelerate the SNCV and increase the SNAP amplitude(P<0.01).(3)partially recover the damaged histopathology in sciatic nerve.2.Aloperine ameliorated the CCI-induced neuropathic pain in mice through TLR4/NF-κB pathway.Compared to the CCI group,treatment with ALO(80mg/kg)(1)significantly inhibited the over-activation of microglia induced by CCI(P<0.01).(2)markedly down-regulated the CCI-induced high expression levels of TNF-α and IL-6 proteins in the spinal cord(P<0.01).(3)significantly decreased the expression of TLR4 and NF-κB in the spinal cord of mice with neuropathic pain induced by CCI(P<0.01).(4)intrathecal injected with LPS(10 μg/10 μl),the PWT of mice in CCI+ALO(80mg/kg)group was significantly decreased.Compared with the LPS model group,LPS+ ALO(100 μM)group:(1)significantly reduced the expression levels of TNF-α and IL-6 proteins in LPS-treated BV2 cells(P<0.01).(2)significantly inhibited the activation of BV2 cells cultured with LPS(P<0.01).(3)significantly decreased the expression of TLR4,p-NF-κB p65/NF-κB and p-IκBα/IκBαproteins in LPS-treated BV2 cells(P<0.01).(4)significantly reduced the mRNA expression of TNF-α and IL-6 in LPS-treated BV2 cells(P<0.01).Conclusions Aloperine exerts analgesic and neuroprotective effects against CCI-induced neuropathic pain in mice via anti-inflammatory effect mediated by inhibiting the activation of TLR4/NF-κB pathway,which can contribute to provide additional options for safe and effective neuropathic pain remedies.