The Effects And Possible Mechanism Of LncRNA-UC.25+in The Dorsal Root Ganglion P2Y₁₄ Receptor-mediated Diabetic Neuropathic Pain In Rats

Background:Diabetes mellitus（DM）is a common multiple metabolic disease worldwide,diabetic neuropathic pain（DNP）caused by neuropathy is one of the most serious complications,involving progressive neuronal damage and dysfunction.Neurons in the dorsal root ganglia（DRG）are responsible for the transmission of nociceptive information,and changes of molecules or receptors function in the DRG could cause pain.The development of neuropathic pain is related to abnormal signal transmission between neurons.Satellite glial cells（SGCs）tightly surround the cell body of DRG neurons.SGCs play an important role in nociceptive signal communication.Activated and damaged cells release large amounts of adenosine triphosphate（ATP）outside the cell,thereby activating two types of P2 purinergic receptors,including P2X and P2Y receptors.Studies have shown that P2Y receptors were involved in diabetic neuropathic pain,but whether P2Y₁₄ receptor was involved in DNP,and the process and mechanism of P2Y₁₄ receptor-mediated DNP were still not clear.Long noncodingRNA（long noncodingRNA,lncRNA）is a type ofRNA molecule that does not encode proteins.It can participate in various biological processes such as maintaining cell and tissue homeostasis.Although the function of most lncRNAs is unclear,lncRNAs are closely related to the occurrence and development of many diseases.In this study,we established a type 2 diabetic neuropathic pain rat model to explore the role and mechanism of lncRNA-UC.25+in dorsal root ganglion P2Y₁₄ receptor-mediated diabetic neuropathic pain in rats.Objective:1.To establish a rat model of DNP and investigate the role of dorsal root ganglion P2Y₁₄ receptors in diabetic neuropathic pain.2.To observe the role of lncRNA-UC.25+on the diabetic neuropathic pain mediated by P2Y₁₄ receptor in DNP rats DRG satellite glial cells.3.To investigate the possible mechanism of lncRNA-UC.25+regulating P2Y₁₄receptor-mediated neuropathic pain in type 2 diabetes through cell experiments.Methods:We established a rat model of DNP and randomly divided the rats into5 groups:normal control group（Control group）,Diabetic neuropathic pain model group（DNP group）,DNP combined with P2Y₁₄ short hairpinRNA（shRNA）treatment group(DNP+P2Y₁₄ shRNA group),DNP combined with lncRNA-UC.25+shRNA processing group（DNP+lncRNA-UC.25+shRNA group）and DNP combined with scramble shRNA negative control group（DNP+NC shRNA）.Contents of the experiment:（1）Measurement of the mechanical withdrawal threshold and thermal withdrawal latency;（2）Real-time PCR（Q-PCR）and Western blot were used to detect the expression level of P2Y₁₄ receptor and lncRNA-UC.25+in the dorsal root ganglia（DRG）of each group rats;（3）Co-expression of P2Y₁₄ receptor and satellite glial cell marker（glial fibrillary acidic protein,GFAP）in DRG was detected by immunofluorescence double-labeled method;（4）The protein expression level of interleukin 1β（IL-1β）and tumor necrosis factor（TNF-α）were detected by Western blot;（5）The changes of expression and phosphorylation level of p38 MAPK pathway were tested by Western blot;（6）The SOD activity,GSH and MDA contents in serum and DRG of rats in each group were detected by the kit;（7）Using bioinformatics to predict P2Y₁₄ transcription factors;（8）Co-immunoprecipitation assay（RIP）was used to identify the protein that interact with lncRNA-UC.25+,HUVECs were cultured and divided into two groups:co-transfected with pc DNA3.0-UC.25+-12×MS2bs and pc DNA3.0-Flag-2×MS2 plasmid experiment group,co-transfected with pc DNA3.0-12×MS2bs and pc DNA3.0-Flag-2×MS2 plasmid control groups.And then observed the protein expression changes in STAT1 by Western blot;（9）Cultured Human Umbilical Vein Endothelial Cells（HUVEC）and transfected lncRNA-UC.25+plasmid and STAT1 plasmid,observed the mRNA and protein expression level of STAT1 and P2Y₁₄ receptor by Q-PCR and Western blot.Result:（1）Compared with Control group,the mechanical withdrawal threshold and thermal withdrawal latency were significantly reduced in DNP group（p<0.01）.The mechanical withdrawal threshold and thermal withdrawal latency of DNP+P2Y₁₄shRNA group and DNP+UC.25+shRNA group were significantly higher than DNP group rats（p<0.01）.There was no significant difference between DNP group and DNP+NC shRNA group（p>0.05）;（2）The results of Real-time PCR and Western blot indicated that compared with the Control group,the expression of UC.25+in the dorsal root ganglion of the DNP group was significantly increased（p<0.01）,and the mRNA and protein expression levels of the P2Y₁₄ receptor were significantly increased（p<0.01）,compared with the DNP group,the expression levels of UC.25+in the DNP+P2Y₁₄shRNA group and DNP+UC.25+shRNA group were reduced（p<0.01）,and the mRNA and protein expression levels of the P2Y₁₄ receptor were significantly reduced（p<0.05）.There was no significant difference between DNP group and DNP+NC shRNA group（p>0.05）;（3）The results of immunofluorescence double-labeled showed that P2Y₁₄ receptor co-expressed with satellite glial cell marker glial fibrillary acidic protein（GFAP）,and the fluorescence intensity of DNP group and DNP+NC shRNA group was significantly higher than Control group（p<0.05）,the fluorescence intensity of DNP+P2Y₁₄ shRNA group and DNP+UC.25+shRNA group was lower than DNP group（p<0.05）;（4）Western blot detected the changes of IL-1βand TNF-αexpression in the dorsal root ganglia of rats in each group.Compared with the Control group,the protein expressions levels of IL-1βand TNF-αin the DNP group were significantly increased（p<0.01）.Compared with DNP group,the protein expressions levels of IL-1βand TNF-αin DNP+P2Y₁₄ shRNA group and DNP+UC.25+shRNA group were significantly reduced（p<0.01）.There was no significant difference between DNP group and DNP+NC shRNA group（p>0.05）;（5）The results of Western blot showed that there was no significant difference in the expression of p38 in DRG among the five groups（p>0.05）.However,the phosphorylation of p38 protein in the DNP group was significantly higher than Control group（p<0.01）.The p38phosphorylation level of DNP+P2Y₁₄ shRNA group and UC.25+shRNA group were lower than DNP group（p<0.01）.There was no significant difference between DNP group and DNP+NC shRNA group（p>0.05）;（6）The oxidative stress test results showed that compared with the Control group,the SOD activity and GSH content in the serum and DRG of DNP rats were decreased（p<0.01）,the MDA content was increased significantly（p<0.01）.After DNP rats treating with P2Y₁₄ shRNA or UC.25+shRNA,compared with DNP group,SOD activity and GSH content in rat serum and DRG increased significantly（p<0.01）,the MDA content was decreased（p<0.01）.There was no significant difference between DNP group and DNP+NC shRNA group（p>0.05）;（7）The results of bioinformatics indicated that STAT1 is a transcription factor for P2Y₁₄;（8）The results of co-immunoprecipitation experiment（RIP）showed that compared with co-transfection of pc DNA3.0-12×MS2bs and pc DNA3.0-Flag-2×MS2 plasmid control group,the protein expression levels of STAT1 in co-transfection of pc DNA3.0-UC.25+-12×MS2bs and pc DNA3.0-Flag-2×MS2 plasmid experiment group was significantly increased（p<0.001）;（9）After lncRNA-UC.25+plasmid transfected in HUVEC cells,the protein expression of P2Y₁₄receptor and STAT1 were significantly increased（p<0.01）.After STAT1 plasmid transfected,the protein expression of P2Y₁₄ receptor was significantly increased（p<0.01）.Conclusion:1.The P2Y₁₄ receptor in DRG participated in the DNP process of type 2 diabetic rats.P2Y₁₄ shRNA can relieve DNP in type 2 diabetic rats.2.LncRNA-UC.25+shRNA can alleviate DNP mediated by P2Y₁₄ receptors,lncRNA-UC.25+may mediate DNP by directly or indirectly regulating P2Y₁₄ receptors in satellite glial cells of DRG.3.LncRNA-UC.25+may regulate P2Y₁₄ receptor by transcription factor STAT1to alleviate DNP.