| Background and Objective:Diabetes mellitus is a common disease.Diabetic neuropathic pain in clinical practice is the common complication of diabetes mellitus.It was reported that the expression of P2X3 receptor in dorsal root ganglia was significantly increased in the neuropathic pain of diabetes mellitus,the hyperalg-esiahe in the neuropathic pain of diabetic rats was relieved by P2X3 receptor antagonists.Thereby P2X3 receptor in dorsal root ganglia is involved in the pathological changes of neuropathic pain in diabetic rats,which provide a new target of prevention and cure for the intractable neuropathic pain of diabetes mellitus.Calcitonin gene-related peptide(CGRP)synthesized by dorsal root ganglia is a major sensory neurotransmitter.CGRP is also involved in diabetic neuropathic pain.It was reported that the expression change of mRNA-like non-coding RNA(mlncRNA)and the factors affecting the expression change of mlncRNA were related to neurological diseases.According to the preliminary studies in the application of SOLiD high-throughput transcriptome and sequence screening,we found that there was upregulated expression of mlncRNA2 in the dorsal root ganglia of type 2 diabetic rats.By testing the effect of mlncRNA2 siRNA on the neuropathic pain of type 2 diabetic rats,this study might provide an experimental basis for the prevention and treatment of diabetic neuropathic pain.Methods:Type 2 diabetic rats were used as the experimental model of diabetic neuropathic pain.The rats were randomly divided into:normal saline control group(Ctrl + NS),diabetes mellitus group + saline control group(DM + NS),diabetes mellitus group + mlncRNA2 siRNA treatment group(DM + mlnc2 si),diabetes mellitus group + scrambled siRNA negative control group(DM + SC si).The studies were made as following:1.During the experiment,the value changes of fasting plasma glucose(FPG)and postprandial blood glucose(PBG)were measured.2.Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were tested.3.The rats tail sensory nerve conduction velocity were tested.4.Serum lipids and fasting insulin were measured.5.Immunohistochemistry and Western blot were used to oberserve the expression changes of P2X3 receptor and CGRP in rat dorsal root ganglia.6.RT-PCR were used to test the expression changes of mlncRNA2,P2X3 receptor and CGRP in rat dorsal root ganglia on RNA levels.7.Serum TNF-a level were measured.8.Serum oxidative stress indicators-catalase and superoxide dismutase activity were measured.Results:1.In order to enhancing the weight of model rats,rats were given high-sugar high-fat diet for 4 weeks.After 5 weeks,the weight in model rats was higher than that in the control rats(p<0.01,F3,28=36.85).After 7 weeks,type 2 diabetic model was established.The values of fasting plasma glucose(FPG)and postprandial blood glucose(PBG)in type 2 diabetic rats were higher than the diabetic diagnosis level in the normal range.After 8 weeks,the gain of weight in type 2 diabetic rats was lower than that in the control rats(p<0.05,F3,28=3.81).After type 2 diabetic rats given mlncRNA2 siRNA,the FPG and PBG in DM + mlnc2 si group were significantly decreased compared with those in DM + NS group and DM + SC si group(p<0.01)FPG-F2,21=11.44,PBG-F2,21=15.28).There was no significantly difference between DM + NS group and DM + SC si group(p>0.05).2.During the establishment processing of type 2 diabetic rat model,the MWT and TWL in model rats were gradually decreased.After the establishment of type 2 diabetic model,the MWT and TWL values in DM + NS group were significantly lower than those in the Ctrl + NS group(p<0.01,MWT-F3,28=21.87,TWL-F3,28=13.32).After given mlncRNA2 siRNA in type 2 diabetic rats,the MWT and TWL values in DM + mlnc2 si group were significantly increased compared with those in DM + NS group and DM + SC si group(p<0.01,MWT-F2,21=13.54,TWL-F2,21=12.36).There was no significantly difference between DM + NS group and DM + SC si group(p>0.05).3.During the establishment processing of diabetes mellitus model,the sensory conduction velocity(SCV)of rat tail nerve was gradually decreased.After the establishment of type 2 diabetic model,the SCV in DM + NS group had a significant difference compared with that in the Ctrl + NS group(p<0.01,F3,28=21.37).After given mlncRNA2 siRNA in type 2 diabetic rats,the SCV in DM+mlnc2 si group was significantly increased compared with that in DM + NS group and DM + SC si group(p<0.01,F2,21=32.56).There was no significantly difference between DM + NS group and DM + SC si group(p>0.05).4.The serum levels of total cholesterol(TC),triglyceride(TG)and low density lipoprotein(LDL)in DM + NS group were significantly increased compared with those in Ctrl + NS group(p<0.01).The serum levels of TC,TG and LDL in the DM + mlnc2 si group were significantly lower than those in the DM + NS group and DM + SC si group(p<0.01,TC-F2,21=23.46,TG-F2,21=28.88,LDL-F2,21=41.09).There was no significantly difference between DM +NS group and DM + SC si group(p>0.05).High-density lipoprotein(HDL)level in DM + NS group was significantly decreased compared with that in Ctrl + NS group(p<0.05).The level of HDL in DM + mlnc2 si group was significantly higher than that in DM + NS group and DM + SC si group(p<0.05,F2,21=6.39).There was no significantly difference between DM + NS group and DM + SC si group(p>0.05).The values of fasting insulin(FINS)in DM + NS group,DM + mlnc2 si group and DM + SC si group were significantly increased(p<0.05,F3,28=4.01)compared with those in Ctrl + NS group.There was no statistically difference for the FINS among DM + NS group,DM + mlnc2 si group and DM + SC si group(p>0.05,F2,21=2.88).The IRI of the diabetic rats was significantly lower than that in the normal rats(p<0.05).5.RT-PCR results showed that the mRNA expression of mlncRNA2,P2X3 receptor and CGRP of dorsal root ganglia in DM + NS group was significantly increased compared with that in Ctrl + NS group(p<0.01).The expression levels of mlncRNA2,P2X3 receptor and CGRP in the DM + mlnc2 si group were significantly lower than those in the DM + NS group and DM + SC si group(p<0.01,mlncRNA2-F2,27=43.21,P2X3-F2,27=34.14,CGRP-F2,27=23.33).There was no significantly difference between DM + NS group and DM + SC si group(p>0.05).6.Immunohistochemistry and western blot results showed that the expression levels of P2X3 receptor and CGRP of dorsal root ganglia in DM + NS group were significantly increased compared with those in Ctrl+NS group(p<0.01).The expression of P2X3 receptor in the DM + mlnc2 si group was significantly lower than that in the DM +NS group and DM + SC si group(p<0.01,IHC:P2X3-F2,27=28.12,CGRP-F2,27=21.97,WB:P2X3-F2,27=13.32,CGRP-F2,27=10.52).There was no significantly difference between DM + NS group and DM + SC si group(p>0.05).7.Immunofluorescence double-label results showed that the expression levels of P2X3 receptor and CGRP in DRG neurons in type 2 diabetic rats were increased,which were inhibited by mlncRNA2 siRNA.8.Serum levels of TNF-a in DM + NS group were significantly increased compared with those in Ctrl + NS group(p<0.01).The TNF-a levels in DM+ mlnc2 si group was significantly lower than that in DM + NS group and DM + SC si group(p<0.01,F2,21=45.34).There was no significantly difference between DM +NS group and DM + SC si group(p>0.05).9.Oxidative stress results showed that the serum CAT and SOD activity in DM + NS group was significantly lower than that in the Ctrl + NS group(p<0.01).The serum CAT and SOD activity in the DM + mlnc2 si group was significantly higher than that in DM + NS group and DM + SC si group(p<0.01,CAT-F2,21=11.33,SOD-F2,21=13.46).There was no significantly difference between DM + NS group and DM + SC si group(p>0.05).Conclusion:MlncRNA2 siRNA has a inhibitive effect on the diabetic neurop-athic pain which mediated by P2X3 receptor and CGRP in dorsal root ganglia. |
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