Study On LncRNA BC168687 Involving In P2X₇ Receptors-mediated Diabetic Neuropathic Pain In Rats

Background:Long non-coding RNA?lncRNA?,known as non-protein-coding RNA transcripts longer than 200 nucleotides?nt?,is located in the nucleus or cytoplasm.Although lnc RNA has no protein coding function,it could regulate the expression of gene in various levels with the form of RNA,thus involving in numerous physiological and pathological processes.Transient receptor potential channels?TRPs?are a class of non-selective cation channels that include at least 28different gene-encoded products,and TRPs are primarily responsible for the introduction of sensations such as chemical,thermal and mechanical stimulus.TRPV1 receptor is one of the most widely studied non-selective cation channel proteins that could be activated by capsaicin,heat,acidosis,and some endogenous substances.Moreover,TRPV1 is mainly distributed in small and medium-sized neurons of dorsal root ganglia?DRG?and trigeminal ganglia?TG?in peripheral nervous system?PNS?.Several studies have showed that TRPV1 receptor may be considered as a pre-painful factor in acute inflammatory pain models,and it plays a vital role in the generation of pain and the enhancement of hyperalgesia.P2X₇receptor,a kind of adenosine 5'-triphosphate?ATP?-gated ion channel,could be expressed on satellite glial cells?SGCs?and involves in numerous inflammatory and neurological diseases.Activation of P2X₇ receptor by ATP could leads to the influx of Na⁺and efflux of Ca²⁺and K⁺,which can transmit extracellular signals and cause biological effects.The release of ATP from damaged neuronal vesicles during acute injury in PNS could activate the P2X₇ receptor on adjacent glial cells,allowing the surrounding glial cells to release ATP in autocrine or paracrine form.Then the ATP signal will be further amplified by this cascade reaction,thus promoting inflammation.Furthermore,activation of P2X₇ receptor increases the release of inflammatory cytokines such as tumor necrosis factor alpha?TNF-??and interleukin 1 beta?IL-1??in glial cells and then trigger chronic pain.Therefore,we concluded that inhibition of the upregulation of P2X₇ and TRPV1 receptors may have great significance of alleviating diabetic neuropathic pain?DNP?,and lncRNA may be involved in this pathophysiological process.Objective:In this study,high glucose and high free fatty acids?HGHF?-induced SGCs injury model and streptozocin?STZ?-induced model rats were established and then treated with lncRNA BC168687 siRNA.This study was aimed to explore the role of lncRNA BC168687 in the physiological and pathological processes of DNP mediated by P2X₇ and TRPV1 receptors.Methods:1.Cellular experiment.In this research,we established HGHF induced primary cultured SGCs injury model and then detected the relative indicators after transfection of lncRNA BC168687 siRNA.Experimental Groups:Control group?Control?,HGHF model group?HGHF?,HGHF treated with lncRNA BC168687siRNA group?HGHF+BCsi?,and HGHF treated with scrambled siRNA negative group?HGHF+NCsi?.Experimental Contents:?1?The screening of siRNA.?2?The level of P2X₇ mRNA in each group was determined by quantitative real-time PCR?qPCR?.?3?The expression of P2X₇ protein in each group was measured by Western Blot?WB?.?4?The fluorescent density of P2X₇ receptor and glial fibrillary acidic protein?GFAP?in each group was observed by double-labelling immunofluorescence.?5?The level of ATP in each group was analyzed by luciferin-luciferase method.?6?The concentration of nitric oxide?NO?in each group was assayed by Nitrate reductase method.?7?The level of reactive oxygen species?ROS?in each group was inspected by ROS Assay Kit.?8?The expression level of phosphorylation?p?-ERK in each group was measured by WB.2.Animal experiment.In this study,we firstly established the model of DNP rats by STZ,and then detected the relative indicators in rats after intrathecal injection with lncRNA BC168687 siRNA.Experimental Groups:Control group?Control?,DNP model group?DNP?,DNP treated with lncRNA BC168687 siRNA group?DNP+BCsi?,and DNP treated with scrambled siRNA negative group?DNP+NCsi?.Experimental Contents:?1?The measurement of blood glucose.?2?The observation of animal behavior.?3?The level of P2X₇ and TRPV1 mRNA in DRG of rats in each group was determined by qPCR.?4?The expression of P2X₇ and TRPV1 protein in DRG of rats in each group was measured by WB.?5?The relative level of TRPV1receptor in DRG of rats in each group was detected by immunohistochemistry?IHC?.?6?The fluorescent density of P2X₇ receptor and GFAP in DRG of rats in each group was observed by double-labelling immunofluorescence.?7?The inflammatory cytokine of TNF-?and IL-1?in serum of rats in each group was analyzed by enzyme-linked immunosorbent assay?ELISA?.?8?The phosphorylation level of ERK and p38 signaling pathway in DRG of rats in each group was measured by WB.Result:1.Cellular experiment.?1?The sequence of BC168687-2400 siRNA was selected as the most effective siRNA of lncRNA BC168687?2?The results of qPCR and WB showed levels of P2X₇ mRNA and protein in cell of HGHF group were obviously higher than that in Control group?P<0.01?;and the level of P2X₇ mRNA and protein in HGHF+BCsi group were obviously lower than those in HGHF group?P<0.01?.No significant differences appeared between the HGHF and the HGHF+NCsi group?P>0.05?.?3?Results of double-labelling immunofluorescence showed that the fluorescent density of P2X₇ and GFAP in the HGHF group increased significantly compared to Control group?P<0.01?;and fluorescent density of P2X₇and GFAP in the HGHF+BCsi group were lower than those in HGHF group?P<0.01?,but no significant differences were found between the HGHF and the HGHF+NCsi group?P>0.05?.?4?The measurement of ATP,NO,and ROS showed that the level of ATP,NO,and ROS in HGHF group were significantly higher than that in Control group?P<0.01?;the level ATP,NO,and ROS of HGHF+BCsi group were significantly lower than those in HGHF group?P<0.01?.There were no significant differences between the HGHF and the HGHF+NCsi group?P>0.05?.?5?The results of WB displayed that relative level of pERK in cell of HGHF group were obviously higher than that in Control group?P<0.01?;and HGHF+BCsi group were obviously lower than those in HGHF group?P<0.01?.No significant differences appeared between the HGHF and the HGHF+NCsi group?P>0.05?.2.Animal experiment.?1?There was no significant difference in postprandial blood glucose?PBG?levels of rats with normal diet?P>0.05?.After high glucose and high fat diet for four weeks and then intraperitoneal injection with STZ to induce DNP model,the PBG of DNP group were obviously higher than that in Control group?P<0.01?.?2?Animal behavior test showed that the mechanical withdrawal threshold?MWT?and thermal withdrawal latency?TWL?of DNP group were decreased significantly compared with Control group?P<0.01?;and the MWT and TWL of DNP+BCsi group were obviously lower than those in DNP group?P<0.01?.There were no significant difference between DNP group and DNP+NCsi group?P>0.05?.?3?The results of qPCR,WB,IHC and double-labelling immunofluorescence showed that compared to the Control group,the level of P2X₇ and TRPV1 mRNA and protein in DRG of rats in the DNP group increased significantly?P<0.01?.And the level of P2X₇ and TRPV1 mRNA and protein in the DNP+BCsi group were lower than those in the DNP group?P<0.01?.No significant difference appeared between the DNP and the DNP+NCsi group?P>0.05?.?4?The results of ELISA showed the level of TNF-?and IL-1?in serum of rats in DNP group were significantly higher than that in Control group?P<0.01?;and the level of TNF-?and IL-1?in DNP+BCsi group were significantly lower than those in DNP group?P<0.01?.No significant differences were observed between the DNP and the DNP+NCsi group?P>0.05?.?5?The results of WB displayed that pERK and p-p38 in DRG of rats of DNP group were obviously higher than that in Control group?P<0.01?;and the relative level of pERK and p-p38in DNP+BCsi group were obviously lower than those in DNP group?P<0.01?,while no significant differences were found between the DNP and the DNP+NCsi group?P>0.05?.Conclusion:LncRNA BC168687 is involved in the generation of diabetic neuropathic pain,and lnc RNA BC168687 siRNA could alleviate diabetic neuropathic pain mediated by P2X₇ and TRPV1 receptors.