The Role Of Hippocampal MiR-181c In Neuropathic Pain Of CCI Rats And Its Preliminary Regulatory Mechanism

Pain is an unpleasant feeling and emotional experience associated with actual or potential tissue damage.It is a complex physiological and psychological activity.Recent studies have shown that hippocampus is involved in pain perception after peripheral nerve injury.MicroRNA(miRNA)is a non-coding RNA in eukaryotes,which can regulate gene expression by targeting specific mRNAs for degradation or inhibiting translation.Recent studies have shown that miR-181 c is highly expressed in the nervous system.Administration of exogenous miR-181 c agomir can reduce hippocampal neuron apoptosis.The mechanism may be due to the inhibition of activation of TLR4 signaling pathway which resulted in the decreased expression of IL-1β and TNF-α.Further studies showed that TNF-α and TLR4 are direct downstream targets of miR-181 c.Recent studies have shown that upregulation of TNF-α and TLR4 expression in the hippocampus is involved in pain perception,pain behavior,and anxiety and depression-like behavior.Therefore,it can be speculated that that intra-hippocampal injection of miR-181 c may alleviate neuropathic pain in CCI rats by down-regulating the expression of TLR4 and TNF-α.Objectives:1.To explore whether hippocampal miR-181 c can alleviate neuralgia in CCI rats by down-regulating TNF-α and TLR4 expression.2.To explore whether intra-hippocampal CA1 injection of PDTC may produce an analgesic effect through down-regulating the expression of NF-κB and up-regulating the expression of the transcription factor CTCF,thereby promoting the production of miR-181 c in CCI rats.Methods:1.SD male rats with 5~6 weeks old were randomized into three groups(n=5): Sham group,CCI3 d group,CCI7 d group.Thermal withdrawal latency(TWL)was evaluated before the surgey and on 1,3,5,7d after surgery.The expression of miR-181 c,TLR4 and TNF-α mRNA in hippocampus was detected by RT-qPCR at 7 post-operative days and the expression of TLR4 and TNF-α was detected by western blot.2.SD male rats were randomized into six groups(n=5): Sham group,CCI goup(CCI+intra-hippocampal CA1 injection of saline),CCI+miR-181 c agomir negative control group,CCI+miR-181 c antagomir negative control group,CCI+miR-181 c agomir group,CCI+miR-181 c antagomir group.For the rats after ligation,agomir NC,antagomir NC,agomir,antagomir(20 nM),saline(0.5 ul)were intra-hippocampally administrated twice a day for 7 days.TWL were evaluated before the surgery and on the 1st,3rd,5th and 7th day after surgery.The expression of miR-181 c,TLR4 and TNF-α mRNA in hippocampus was detected by RT-qPCR.The expression of CTCF,Nrf2,TLR4 and TNF-α was detected by WB.3.All rats were randomized into four groups(n=4): sham group(sham operation),PDTC-treated sham group,vehicle-treated CCI group,PDTC-treated group.For the rats after ligation,PDTC(0.5 μg/μl,0.5 μl),saline(0.5 μl)were intra-hippocampally administrated twice a day for 7 days consecutively.TWL were evaluated before the surgery and on the 1st,3rd,5th and 7th day after surgery.The expression of miR-181 c in hippocampus was detected by RT-qPCR.The expression of CTCF,NF-κB p65 and NF-κB p50 was detected by WB.Results:1.Decreased TWL was exhibited in the CCI rats on the 1st day after nerve injury compared to sham rats,and the allodynia was sustained for 7 days.The CCI rats presented increased expression of TLR4 and TNF-α(at the mRNA and protein level)and the decreased expression of miR-181 c than the sham group.2.The CCI group displayed significantly decreased thermal withdrawal latency on days 1,3,5,and 7 compared with the sham rats,which were markedly increased by miR-181 c agomir adiministration.Compared with that in the vehicle-treated CCI,miR-181 c agomir administration significantly suppressed the increased expression of TLR4 and TNF-α(at the mRNA and protein level).At the same time,the expression of miR-181 c in the hippocampal tissue was significantly increased after miR-181 c agomir administration in nervr injury-induced neuropathic pain rats.On the contrast,miR-181 c antagomir treatment suppressed the expression of miR-181 c but signifcantlly increased the expression of TLR4 and TNF-α.Compare to sham group,the expression of CTCF in the cytosol and nucleus of hippocampal tissue were significantly decreased after nerve injury,and the expression of Nrf2 in the nucleus of hippocampal tissue was significantly increased.Compared to CCI group,miR-181 c agomir treatment increased the expression of CTCF in the cytosol and nucleus of hippocampal tissue but markedly decreased the expression of Nrf2 in the nucleus.3.Repeated administration of PDTC markedly suppressed CCI-induced the decreased.Compared to CCI group,PDTC treatment significantlly reversed CCI-induced the decreased expression of miR-181 c.In addition,compared to CCI group,PDTC treatment significantlly increased the expression of CTCFand the level of NF-κB p50,p65 in the cytosol of hippocampal tissue.Intra-hippocampal injection of PDTC significant reversed the CCI-induced nuclear translocation of p50 and p65 subunits of NF-κB in hippocampal of rats.Conclusions: 1.Intra-hippocampus administration of miR-181 c agomir significantly attenuated CCI-induced thermal hyperalgesia and the increased expression of TLR4 and TNF-α in hippocampus of CCI rats.2.Intra-hippocampus administration of PDTC significantly attenuated CCI-induced the thermal hyperalgesia,NF-κB activation and the decreased expression of CTCF,which leads to the increased expression of miR-181 c.