The Effects And Mechanism Of LncRNA-UC.25+ In The Spinal Cord P2Y₁₄ Receptor-mediated Diabetic Neuropathic Pain In Rats

Background:Diabetic neuropathic pain is a common complication of diabetes mellitus,and its complicated pathogenesis and clinical manifestations have brought great troubles to its treatment.As an important part of the central nervous system,the spinal cord is an important part to regulate the occurrence and development of diabetic neuropathic pain.Spinal microglia can regulate the activity of spinal neurons and play a role in the regulation of chronic pain.In the event of nerve injury or diabetes,microglia are greatly activated,promoting the release of various inflammatory cytokines and chemokines,enhancing neuronal excitability and promoting pain transmission.At the same time,the activated microglia trigger the release of ATP extracellular,which activates the purinergic P2 receptor.Studies have shown that P2Y₁₂ receptors are involved in diabetic neuropathic pain,and P2Y₁₂ and P2Y₁₄ receptors have a high degree of sequence homology,but there is no report that P2Y₁₄ receptors are involved in diabetic neuropathic pain.Lnc RNAs do not have the function of protein coding,and only regulate gene expression level by RNA state.More and more studies have confirmed that the dysregulation of lnc RNAs is closely related to many human diseases and plays a role in promoting or inhibiting the occurrence and development of diseases.In this study,we established an animal model to explore whether P2Y₁₄ receptor was involved in the process of diabetic neuropathic pain,and whether lnc RNA-UC.25+affected diabetic neuropathic pain by affecting the expression of P2Y₁₄ receptor and its possible mechanism.Objective:1.To investigate the role of spinal cord P2Y₁₄ receptor in neuropathic pain in type 2 diabetes.2.To explore the role and possible mechanism of lnc RNA-UC.25+in rat spinal cord P2Y₁₄ receptor mediated diabetic neuropathic pain.Methods:1.The model of neuropathic pain in type 2 diabetes mellitus was established,and SD rats were randomly divided into four groups:control group（Control）,model group（DNP）,model combined with P2Y₁₄ sh RNA treatment group(DNP+P2Y₁₄ sh RNA),and model combined with scramble sh RNA negative control group（DNP+NC sh RNA）.The mechanical withdrawal threshold and thermal withdrawal latency of each group were measured respectively.The m RNA and protein expression of P2Y₁₄ receptor in rat spinal cord were detected by quantitative real-time PCR and Western blot.The expression of interleukin-1β（IL-1β）and tumor necrosis factor-αreceptor（TNF-α）and the phosphorylation level of p38 MAPK in rat spinal cord were detected by Western blot.The co-expression of P2Y₁₄ receptor and microglial marker OX42 in rat spinal cord was detected by immunofluorescence.2.On the basis of determining the involvement of P2Y₁₄ receptor in diabetic neuropathic pain,the animal model was established again,and SD rats were randomly divided into four groups:control group（Control）,model group（DNP）,model combined with UC.25+sh RNA treatment group（DNP+UC.25+sh RNA）,and model combined with scramble sh RNA negative control group（DNP+NC sh RNA）.The mechanical withdrawal threshold and thermal withdrawal latency of each group were measured respectively.Real-time PCR and Western blot were used to detect the expressions of lnc RNA-UC.25+and P2Y₁₄ receptor m RNA and protein in rat spinal cord.The expression of IL-1βand TNF-αand the phosphorylation of p38 MAPK in rat spinal cord were detected by Western blot.The co-expression of P2Y₁₄ receptor and microglial marker OX42 in rat spinal cord was detected by immunofluorescence.RNA binding protein immunoprecipitation assay（RIP）combined with Western blot were used to detect the transcription factor STAT1 interacting with lnc RNA-UC.25+.Chromatin immunoprecipitation（Ch IP）was used to detect the interaction between transcription factor STAT1 and promoter of P2Y₁₄ gene.Human umbilical vein endothelial cells（HUVEC）were cultured and transfected with lnc RNA-UC.25+overexpression and low expression plasmids and STAT1 overexpression and low expression plasmids respectively.The expression of STAT1 and P2Y14 receptors were detected by real-time PCR and Western blot.Results:1.Before the start of the experiment,there was no difference in various indexes between the rats in each group（p>0.05）.Compared with the Control group,the mechanical withdrawal threshold and the thermal withdrawal latency in DNP group were significantly decreased（p<0.01）;The m RNA and protein expression of P2Y₁₄ were significantly increased（p<0.01）;Phosphorylation of inflammatory cytokines IL-1βand TNF-αand signaling pathway p38 MAPK were significantly increased（p<0.01）;P2Y₁₄ was co-localized with microglial marker OX42 and its co-expression was increased（p<0.01）.Compared with DNP group,the mechanical withdrawal threshold and thermal withdrawal latency were significantly increased in DNP+P2Y₁₄ sh RNA group（p<0.01）;the m RNA and protein expression of P2Y₁₄were significantly decreased（p<0.01）;Phosphorylation of inflammatory factors and signaling pathway p38 MAPK were significantly reduced（p<0.01）;The co-expression of P2Y₁₄and OX42 was decreased（p<0.01）.There was no significant difference between the DNP+NC sh RNA group and DNP group（p>0.05）.2.Compared with the Control group,the mechanical withdrawal threshold and thermal withdrawal latency of DNP group were significantly reduced（p<0.01）;The expression of P2Y₁₄ m RNA and protein and lnc RNA-UC.25+were significantly increased（p<0.01）;IL-1β,TNF-αand p38 MAPK phosphorylation were also significantly increased（p<0.01）;The co-expression of P2Y₁₄ and OX42 in the DNP group was significantly higher than Control group（p<0.01）.After UC.25+sh RNA treatment,compared with DNP group,the mechanical withdrawal threshold and thermal withdrawal latency of DNP+UC.25+sh RNA group were significantly increased（p<0.01）;The expression of P2Y₁₄ m RNA and protein and lnc RNA-UC.25+were significantly reduced（p<0.01）;IL-1β,TNF-αand p38 MAPK phosphorylation were also significantly reduced（p<0.01）;The co-expression of P2Y₁₄ and OX42 was reduced（p<0.01）.There was no significant difference between the DNP+NC sh RNA group and DNP group（p>0.05）.RIP assay showed that lnc RNA-UC.25+could bind to STAT1.The results of Ch IP assay showed that STAT1 could specifically bind to the DNA fragment of P2Y₁₄ gene promoter,and there was an interaction between the two.Cell experiments showed that lnc RNA-UC.25+was overexpressed in normal HUVEC cells,and the protein expression of STAT1 was significantly increased（p<0.01）;the expression of lnc RNA-UC.25+was low in HUVEC cells with high glucose,and the protein expression level of STAT1 was significantly decreased（p<0.01）,indicating that lnc RNA-UC.25+could regulate the expression of STAT1;Similarly,when STAT1 was overexpressed in normal HUVEC cells,the expression level of P2Y14 protein was significantly increased（p<0.01）,while STAT1 was low in high glucose HUVEC cells,and the expression level of P2Y14 protein was significantly reduced（p<0.01）,indicating that STAT1 can regulate P2Y14 receptor expression.Conclusion:1.P2Y₁₄ sh RNA can reduce the expression of P2Y₁₄ receptor in the spinal cord of rats with type 2 diabetic neuropathic pain,thereby inhibiting the activation of microglia;reducing the expression of inflammatory factors IL-1βand TNF-αand the phosphorylation level of p38 MAPK,it shows that the spinal cord P2Y₁₄ receptor is involved in the DNP process.2.LncRNA-UC.25+shRNA can down-regulate the expression of P2Y₁₄receptors,reduce the release of inflammatory factors and the level of p38 MAPK phosphorylation,indicating that lnc RNA-UC.25+can relieve the spinal cord P2Y₁₄receptor-mediated diabetic neuropathic pain.The underlying mechanism may be that lnc RNA-UC.25+regulates the expression of P2Y₁₄ receptor through the transcription factor STAT1,thereby alleviating DNP mediated by the P2Y₁₄ receptor.