The Effect And Mechanism Of MiRNA-9-5p In Hypothalamic Paraventricular Nucleus On Sympathetic Nerve Activity In Diabetes

Objective:One of the important pathological features of Type 2 diabetes mellitus（T2D）is sympathetic hyperexcitability.This persistent hyperexcitability further worsens T2D,and the mechanism of causing parasympathetic hyperactivity in the Paraventricular nucleus（PVN）in T2D rats is currently unclear.This study aims to investigate the effects of miRNA-9-5p（miR-9-5p）and small-conductance calcium Ca²⁺-activated K⁺channel（SK）in PVN of T2D rats on sympathetic nerve activity and its internal mechanism,and to clarify the effect of SK3signaling pathway mediated by the change of miR-9-5p level in PVN on sympathetic nerve activity.Methods:In this study,Diabetes mellitus（DM）rat model was induced by High fat diet（HFD）combined with 30 mg/kg Streptozocin（STZ）.Healthy SD rats fed with normal diet were used as DM control group（DC）.The changes of Renal sympathetic nerve discharge（RSND）,plasma Norepinephrine（NE）,Mean arterial pressure（MAP）,Heart rate（HR）and fasting blood glucose indexes were observed after 12 weeks of parallel rearing in the DC and DM groups.Enzyme linked immunosorbent assay（ELISA）was used to determine the levels of insulin,leptin and triglyceride in rats of the two groups.Real-time PCR was used to determine the levels of KCNN3 mRNA and miR-9-5p in PVN of rats in both groups.Western blot was used to detect SK3 protein expression in PVN and Supraoptic nucleus（SON）of DC and DM rats.Microinjection of artificial cerebrospinal fluid and Recombinate adeno-associated virus（rAAV）（rAAV-miR-9-5p）and/or anti-9-5p recombinant adeno-associated virus（rAAV-anti-9-5p）into PVN was performed in the two groups,respectively.Blood glucose and sympathetic driving indexes of rats were obtained and compared.The number of Fos B and SK3 positive cells in PVN of each group was detected by immunofluorescence assay.Microinjection of artificial cerebrospinal fluid and recombinant adeno-associated virus of KCNN3（rAAV-KCNN3）and/or recombinant adeno-associated virus of siKCNN3（rAAV-siKCNN3）into PVN of rats in the two groups was performed to obtain and compare the blood glucose and sympathetic driving indexes of rats.The number of FOSB and SK3 positive cells in PVN of each group was detected by immunofluorescence assay.MiR-9-5p and/or anti-9-5p and KCNN3 and/or siKCNN3 were transfected into primary brain neurons and NG108 cells,respectively,to detect the expression of SK3 protein in the cells after intervention.Dual luciferase reporter gene assay was used to detect the targeting relationship between miR-9-5p and KCNN3.Results:1.Compared with DC rats,miR-9-5p gene expression in paraventricular nucleus of DM rats was significantly up-regulated（2.22±0.11 vs 1.00±0.00,P<0.05）,KCNN3mRNA expression had no significant difference（1.04±0.05 vs 1.00±0.00,P=0.44）,while SK3protein expression was significantly down-regulated（0.61±0.05 vs 1.00±0.00,P<0.05）.2.PVN overexpression of miR-9-5p induced RSND in DC and DM rats[DC:（40.67±2.29 vs 60.00±2.67）%of Max,DM:（68.33±2.64 vs 91.17±1.66）%of Max,P<0.05],plasma NE[DC（232.83±8.41 vs 325.83±12.04）pg/mL,DM（372.83±13.99 vs 489.83±18.02）pg/mL,P<0.05],blood glucose level[DC（4.85±0.33 vs 8.53±0.52）mM,DM（13.27±0.37 vs18.67±0.56）mM,P<0.05]and Fos B positive neurons[DC（4.50±0.56 vs 8.33±0.61）cells/4×10⁴μm²,DM（8.00±0.58 vs 13.83±0.60）cells/4×10⁴μm²,P<0.05].In contrast,miR-9-5p knockdown in the paraventricular nucleus resulted in RSND in DC and DM rats[DC:（40.67±2.29 vs 29.33±1.02）%of Max,DM:（68.33±2.64 vs 51.17±2.50）%of Max,P<0.05],plasma NE[DC（232.83±8.41 vs 171.17±10.54）pg/mL,DM（372.83±13.99 vs283.67±9.19）pg/mL,P<0.05],blood glucose level[DC（4.85±0.33 vs 2.88±0.518）mM,DM（13.27±0.37 vs 8.58±0.56）mM,P<0.05],and the number of Fos B-positive neurons[DC（4.50±0.56 vs 2.17±0.31）cells/4×10⁴μm²,DM（8.00±0.58 vs 4.17±0.31）cells/4×10⁴μm²,P<0.05]were significantly decreased.3.PVN overexpression of KCNN3 induced RSND in DC and DM rats[DC（40.00±1.71vs 30.00±1.41）%of Max,DM（65.83±2.98 vs 47.50±2.93）%of Max,P<0.05],Plasma NE[DC（232.67±6.38 vs 187.00±6.51）pg/mL,DM（346.00±11.43 vs 257.50±6.60）pg/mL,P<0.05]and blood glucose[DC（5.28±0.32 vs 2.98±0.19）mM,DM（12.88±0.54 vs 8.38±0.68）mM,P<0.05]were significantly decreased.In contrast,KCNN3 knockdown in the paraventricular nucleus induced RSND[DC（40.00±1.71 vs 59.83±2.57）%of Max,DM（65.83±2.98 vs 87.50±2.47）%of Max,P<0.05],Plasma NE[DC（232.67±6.38 vs328.00±14.79）pg/mL,DM（346.00±11.43 vs 461.17±13.22）pg/mL,P<0.05]and blood glucose levels[DC（5.28±0.32 vs 9.23±0.60）mM,DM（12.88±0.54 vs 18.72±0.84）mM,P<0.05]increased significantly.4.Overexpression of miR-9-5p in the paraventricular nucleus resulted in a significant down-regulation of the number of SK3 positive neurons in DC and DM rats[DC（11.50±0.67vs 7.33±0.56）cells/4×10⁴μm²,DM（7.17±0.48 vs 2.67±0.42）cells/4×10⁴μm²,P<0.05].Cell transfection with miR-9-5p can significantly down-regulate SK3 protein levels in primary brain nerve cells（1.00±0.00 vs 0.50±0.03,P<0.05）and NG108 cells（1.00±0.00 vs 0.53±0.04,P<0.05）;Transfection with anti-9-5p can significantly increase the level of SK3 protein in primary brain nerve cells（1.00±0.00 vs 0.97±0.04,P<0.05）and NG108 cells（1.00±0.00 vs0.94±0.05,P<0.05）.Targetscan predicted that miR-9-5p and the 3’untranslated region（3’UTR）of KCNN3 mRNA had sequence binding,and the dual luciferase reporter gene confirmed that miR-9-5p targets the regulation of KCNN3 gene（100.00±0.00 vs 49.58±3.42,P<0.05）.Conclusions:The up-regulated miR-9-5p in PVN of T2D rats may mediate sympathetic hyperexcitability through the targeted inhibition of SK3 protein expression.