MAPK/ERK Pathway Attenuates Oxaliplatin-induced Neuropathic Pain Through Nav1.6

Background and purposesOxaliplatin(OXA),a third-generation platinum-based chemotherapy drug,has achieved good efficacy in the treatment of solid tumors such as colorectal cancer and ovarian cancer.The use of OXA often cause acute or chronic peripheral neurotoxicity,which is named Oxaliplatin-induced peripheral neurotoxicity(OIPN).Acute OIPN is a transient peripheral neuropathy,mainly associated with cold stimulation.It is usually mild to moderate.The onset of acute OIPN is rapid and recovery occurs within a week.Chronic OIPN,however,is one of the most common a dose-limiting toxic effect characterized by bilateral symmetrical paresthesia,disturbance,or pain with a "glovelike" distribution at the ends of the hands or feet.OIPN severely limits the quality of chemotherapy patients’ life,and at the same time severely reduces the clinical use of OXA.Therefore,it is of great significance to strengthen the exploration of its pathogenesis inducement,in-depth study of its molecular mechanism to reduce the severe neurotoxic effects brought by OXA and to research and develop drugs for clinical use.Studies have shown that the pathogenesis of peripheral neuropathic pain induced by OXA is closely related to voltage-gated ion channels,MAPK family and oxidative stress.The voltage-gated sodium channel subtype Nav1.6 has been clearly implicated in OXA-induced neuropathic pain.However,the mechanism of its action remains unclear.The MAPK/ERK pathway can regulate the expression of ion channels and participate in a variety of neurological diseases.Whether this pathway regulates the expression of Nav1.6 in OXA-induced neuropathic pain has not been reported.Therefore,based on the OXA-induced neuropathic pain model in rats,this study explored the regulatory relationship and mechanism between MAPK/ERK pathway and Nav1.6 in the peripheral nervous system.It will provide a research basis for the pathogenesis of OXA-induced neuropathic pain and suggest a new direction for drug research and development.Methods(1)A rat model of OXA-induced peripheral neuropathic pain was established.Rats were randomly divided into 5 groups: na(?)ve group,vehicle group,and three OXA groups with different time points(7,14 and 21 days).Na(?)ve group was normal rats without treatment.OXA group was rats intraperitoneally injected with 6mg/kg OXA on day 1,3,5,7.Vehicle group was rats intraperitoneally injected with 5%glucose at the same dose as OXA group.The paw withdrawal threshold was measured by Von-Frey filaments to reflect mechanical allodynia.The cold hyperalgesia and thermal hyperalgesia were evaluated by the paw-withdrawal latency.The acetone score was also used to detect cold hyperalgesia.All these were used to determine whether the OXA-induced neuropathic pain model had been successfully established.(2)The expressions of p-Raf1,Raf1,p-ERK,t-ERK,p-CREB,CREB in MAPK/ERK pathway and Nav1.6 were detected in Dorsal Root Ganglion(DRG)of OXA rats.We used double-immunofluorescent staining to detected the co-expression of pCREB and Nav1.6 with CGRP,NF200,IB4,Neu N and GS in rats DRGs.The changes in the fluorescence intensity of single and co-expression of Nav1.6 and p-CREB in DRG of OXA and vehicle rats were also detected.The protein expressions of Nav1.6,p-Raf1,Raf1,p-ERK,t-ERK,p-CREB and CREB in DRG of na(?)ve,vehicle and OXA rats were detected by Western-Blot at 7,14 and 21 days,respectively.RT-q PCR was used to detected the m RNA expression of Nav1.6 in DRGs of vehicle and OXA rats at different time points.(3)Effective siRNA was screened in PC12 cells and the regulatory relationship of Raf1,ERK,CREB and Nav1.6 were verified in vitro.The most effective siRNAs for Raf1 and CREB were screened by transfection of Raf1-siRNA and CREB-siRNA(siRaf1 and siCREB)in PC12 cells respectively.Their effects on the expressions of Raf1,t-ERK,p-ERK,p-CREB,CREB and Nav1.6 were detected by Western Blot.(4)The pain behavior and related molecular changes of rats were detected after intrathecal injection of siRaf1,U0126(ERK inhibitor)and siCREB respectively.The effect of intrathecal injection of siRaf1 on the pain behavior of OXA rats and the expressions of t-ERK,p-ERK,CREB,p-CREB,Nav1.6 were detected.Different doses of ERK inhibitor U0126(5 μg/10μl,10 μg/10μl and 20 μg/10μl)were administered intrathecally to detect its influence on pain behavior and the expressions of p-ERK,t-ERK,p-CREB,CREB and Nav1.6 proteins and Nav1.6 m RNA.The effect of siCREB on pain behavior and the protein expression of p-CREB,CREB and Nav1.6were detected.Results(1)OXA-induced neuropathic pain model in rats was successfully established.The results showed that at 7,14 and 21 days after the first injection of OXA,the mechanical paw-withdrawal threshold and cold paw-withdrawal latency of rats significantly decreased,and the acetone score significantly increased,suggesting that the model established was successful.The thermal paw-withdrawal latency was not changed.(2)The expressions of p-Raf1,p-ERK,p-CREB,CREB and Nav1.6 were upregulated in DRG of OXA-induced neuropathic pain rats,and p-CREB was coexpressed with Nav1.6.Nav1.6 was co-expressed with CGRP,NF200,IB4 and Neu N,but not with GS in DRG,indicating that Nav1.6 was expressed in small,medium and large diameter DRG neurons,but not in satellite glial cells.P-CREB was co-expressed with CGRP,Neu N,IB4 and NF200,indicating that p-CREB was expressed in small,medium and large diameter neurons.P-CREB and Nav1.6 are co-expressed in both vehicle and OXA rats DRGs.The expressions of Nav1.6,p-Raf1,p-ERK,CREB and p-CREB in DRG were significantly increased when compared with the vehicle group at 7,14 and 21 days after the injection of OXA.While the expressions of t-ERK and Raf1 were not changed.The m RNA expression of Nav1.6 was up-regulated in OXA rats.(3)Knockdown of Raf1 or CREB both induced the down-regulation of Nav1.6 in vitro experiments.The effective siRaf1 was screened in PC12 cells.Inhibition of Raf1 significantly reduced the expression of p-ERK and Nav1.6,but had no effect on t-ERK.Knocking down CREB by siCREB also caused down-regulation of Nav1.6.(4)In vivo experiments showed that inhibition of Raf1,p-ERK and CREB alleviated OXA-induced neuropathic pain and inhibited the upregulation of Nav1.6.After the establishment of OXA-induced neuropathic pain model in rats,inhibition of Raf1 by intrathecal administration of siRaf1 resulted in an increase in mechanical paw-withdrawal threshold,a decrease in acetone score and the reversal of the up-regulation of p-ERK,Nav1.6 induced by OXA.Intrathecal injection of ERK inhibitor U0126(5 μg/10μl,10 μg/10μl and 20 μg/10μl)in rats injected with OXA increased the mechanical paw-withdrawal threshold and decreased the acetone score to different degrees.It suggested that U0126 alleviated the OXA-induced pain behavior in a dose-dependent manner.At the same time,administration of U0126(20 μg/10μl)also reversed the increase of p-CREB and Nav1.6 expression induced by OXA.Similarly,both mechanical allodynia and cold hyperalgesia were significantly alleviated by intrathecal administration of siCREB in OXA model rats,accompanied by down-regulation of m RNA and protein expression of Nav1.6.ConclusionThe MAPK/ERK pathway regulates Nav1.6 in DRG and participates in OXAinduced neuropathic pain by regulating the expression of transcription factor CREB.