| Background:Non-coding RNA,in a form of RNA,plays an extremely important role in the regulation of genes expression.Messenger-like long noncoding RNAs(mlncRNAs)exist in cells.MlncRNAs are spliced similarly to mRNA and also have poly A tail at 3,end,but have no typical open reading frame.Data have shown that both the expression of mlncRNAs and the factors influencing mlncRNAs expression are associated with the onset of nervous system's diseases.However the mechanisms underlying are not clear.Diabetes mellitus is one of metabolic syndromes,characterized as high blood sugar and high blood lipid.Diabetic cardiac autonomic neuropathy results in the dysfunction of heart or even sudden cardiac death and is harmful to the health.The postganglionic nerve fibers of superior cervical ganglia(SCG)distribute to cardiac plexus and regulate the activity of the heart.Recently,glucotoxicity and lipotoxicity-mediated nervous damage is emerged as one of the hotspots in the research of diabetic neuropathy.Chronic inflammation,increased high free fatty acids(FFAs)and oxidative stress associated with glucotoxicity and lipotoxicity can directly and indirectly activate P38 mitogen activated protein kinase(P38 MAPK)signal pathway and other stress-related signal pathways,induce the transcription of non-coding RNA in the neuronal cells,regulate the expression of target genes related to nervous cellular function,and be involved a series responses of neuron to the glucotoxicity and lipotoxicity.Our pervious studies have shown the high expression of mlncRNA2 in SCG of diabetic rats through the validation of SOLiD database and the prediction of sequence.In this investigation,we will inhibit the high expression of SCG mlncRNA2 by transfection with mlncRNA2-small interfering RNA(siRNA)on both integral and cellular levels,and then explore the effect of SCG mlncRNA2-siRNA in the regulation of diabetic cardiac autonomic neuropathy and its mechanism by the technologies of integral,cellular and molecular levels.Our results will provide new targets for the prevention and treatment of diabetic cardiac autonomic neuropathy.Objective:1?After the inhibition of high expression of mlncRNA2 in SCG by transfection with mlncRNA2-siRNA on integral level,we screened the mlncRNA2-mediated genes related to diabetic cardiac autonomic neuropathy in SCG by high-throughput microarray.Further we performed the functional verification of the SCG mlncRNA2-targeted significant genes selected by microarray and explored the role of SCG mlncRNA2 in regulating diabetic cardiac autonomic neuropathy.2.We established sympathetic PC 12 cellular model with high expression of mlncRNA2 induced by combined high D-glucose and high FFAs,and then explored the role of P38 MAPK signal pathway in the high expression of mlncRNA2 induced by combined high D-glucose and high FFAs in PC 12 cells.3?Through the cellular levels' transfection of mlncRNA2-siRNA on the model of sympathetic PC12 cells with high expression of mlncRNA2 induced by both combined high D-glucose and high FFAs,we verified the changes of SCG mlncRNA2-targeted significant genes selected by our integral level's study,observed the expression of insulin receptor substrate 1(IRS1),combined with the differences in PC 12 cells neurites outgrowth,and analyzed the effect of mlncRNA2 in IRS 1-mediated inhibitory neurites outgrowth of PC 12 cells cultured with combined high D-glucose and high FFAs and its mechanism;This will be helpful for our verification of the target genes significantly associated with mlncRNA2 selected by our integral level's study and deep exploration mlncRNA2-siRNA-mediated diabetic cardiac autonomic neuropathy on the cellular level.4.Through the cellular levels' transfection of mlncRNA2-siRNA on the model of sympathetic PC 12 cells with the high expression of mlncRNA2 induced by combined high D-glucose and high FFAs,we observed the expression of P2X7,combined with changes of[Ca2+]i and the levels of interleukin-6(IL-6)and tumor necrosis factor ?(TNF-?)in PC 12 cells,and investigated the effect of mlncRNA2-siRNA in P2X7-mediated inflammatory injury of PC12 cells and the mechanism underlying.5.We analyzed P3 8 MAPK signal pathway in high FFA-induced P2X7 expression and subsequent secretion of IL-6 on the model of sympathetic PC 12 cells.Method:1.After the inhibition of high expression of SCG mlncRNA2 by transfection with mlncRNA2-siRNA on intergal level,we screened the mlncRNA2-mediated genes in SCG related to diabetic cardiac autonomic neuropathy by high-throughput microarray.Further we performed the functional verification of SCG mlncRNA2-targeted significant genes selected by high-throughput microarray,observed the levels of fasting and postprandial blood glucose,IL-6,TNF-a,norepinephrine(NE)and oxidative stress markers,combined with the changes of cardiac autonomic function such as ECG,blood pressure,heart rate,heart rate variability and baroreflex sensitivity,and further evaluated the effect of SCG mlncRNA2-siRNA in the mediation of diabetic cardiac autonomic neuropathy and the mechanism underlying.2.We used PC 12 cells with combined high D-glucose and high FFAs as a model of diabetic nervous cellular damage,observed the dose-effect and time-effect of high D-glucose or high FFAs on the expression of mlncRNA2 by the method of real-time PCR,determined the best time and concentration of high D-glucose or high FFAs,which can induce high expression of mlncRNA2 in PC 12 cells,further compared the difference of mlncRNA2 expression in PC 12 cells cultured with high D-glucose or high FFAs or combined high D-glucose and high FFAs,and established sympathetic PC 12 cells model with high expression of PC 12 mlncRNA2 induced by combined high D-glucose and high FFAs.Moreover,after the inhibition of P38 MAPK signal pathway by the P38 MAPK specific inhibitor SB-203580,we further analyzed mlncRNA2 expression of PC 12 cells by the technology of real-time PCR,and elucidated the role of P38 MAPK signal pathway in combined high D-glucose and high FFA-induced mlncRNA2 expression.3.Through the cellular levels' transfection of mlncRNA2-siRNA in the sympathetic PC 12 cells with high expression of mlncRNA2 induced by combined high D-glucose and high FFAs,we analyzed IRS1 mRNA and protein expression,and the level of IRS1 serine 302 phosphorylation in PC 12 cells by the technologies of real-time PCR and western blot,combined with the change of neurites outgrowth in PC 12 cells,and explored the role of mlncRNA2 in regulating PC 12 cells neurites outgrowth.Moreover,after the inhibition of P38 MAPK signal pathway by the P38 MAPK specific inhibitor SB-203580,we further analyzed IRS1 mRNA and protein expression and the level of IRS1 serine 302 phosphorylation in PC 12 cells by the technologies of real-time PCR and western blot,combined with the changes of neurites outgrowth in PC 12 cells,and elucidated the role of mlncRNA2-siRNA in the inhibition of PC 12 cells neurites outgrowth induced by combined high D-glucose and high FFAs and the possible mechanism underlying.4.Through the cellular levels' transfection of mlncRNA2-siRNA in the sympathetic PC 12 cells with high expression of mlncRNA2 induced by both combined high D-glucose and high FFAs,we analyzed P2X7 mRNA and protein expression of PC 12 cells by the technologies of real-time PCR and western blot,combined with the changes of[Ca2+]i and the levels of IL-6 and TNF-a in PC12 cells,and explored the role of mlncRNA2 in the regulating inflammatory reaction of PC 12 cells.Moreover,after the inhibition of P38 MAPK signal pathway by the P38 MAPK specific inhibitor SB-203580,we further analyzed P2X7 mRNA and protein expression of PC 12 cells by the technologies of real-time PCR and western blot,combined with the changes of[Ca2+]i and the levels of IL-6 and TNF-a in PC 12 cells,and elucidated the influence of mlncRNA2-siRNA on the inflammatory reaction and functional damage of PC 12 cells induced by combined high D-glucose and high FFAs and the possible mechanism underlying.5?Using a model of sympathetic PC 12 cell treated with FFAs,we analyzed P2X7 mRNA and protein expression of PC 12 cells by the technologies of immunofl-uorescence,real-time PCR and western blot,combined with the changes of[Ca2+]i and the levels of IL-6 in PC12 cells,and explored the effect of high FFAs in P2X7 expression and subsequent secretion of IL-6.Moreover,after the inhibition of P38 MAPK signal pathway by the P38 MAPK specific inhibitor SB-203580,we further analyzed P2X7 mRNA and protein expression of PC 12 cells by the technologies of immunofluorescence,real-time PCR and western blot,combined with the changes of[Ca2+]i and the level of IL-6 in PC12 cells,and elucidated the role of P38 MAPK signal pathway in the high FFA-induced P2X7 expression and subsequent secretion of IL-6.Result:1?Diabetic rats exhibit accelerated heart rate,prolongation of QT interval,high level of blood pressure,decreased total power spectrum(TP),extremely low frequency(VLF),low frequency(LF)and high frequency(HF),increased ratio of LF/HF,down-regulation of baroreflex sensitivity,rise in fasting and postprandial blood glucose,increased levels of IL-6,TNF-a,NE and MDA in blood serum,and decreased activities of GSH-PX and T-NOS in blood serum.These results indicated prompted inflammatory reaction and oxidative stress in SCG,and the abnormal cardiac autonomic functional changes in diabetic rats.These abnormal changes were alleviated after the inhibition of SCG high expression of mlncRNA2 by transfection with mlncRNA2-siRNA.Microarray results showed there were 6 up-regulated and 8 down-regulated genes related to the knockdown of mlncRNA2 expression by transfection with mlncRNA2-siRNA.The Pannexin-1 and IRS1 are the two genes associated with nervous cellular growth and functional injury,which were down-regulated and up-regulated by 7.4618 and 10.1036 times,respectively,after the decreased expression of mlncRNA2 by transfection with mlncRNA2-siRNA.Immunofluorescence results showed Pannexin-1 and IRS1 were mainly co-expressed with neurons of the SCG.Inhibition of the expression of mlncRNA2 by transfection of mlncRNA2-siRNA can significantly suppress the up-regulation of Pannexin-1 mRNA and protein,inhibit abnormal phosphorylation of IRS 1 at the site of serine 302,and alleviate the down-regulation of IRS1 mRNA and protein expression of SCG in diabetic rats by the testes of real-time PCR and western blot.Results of western blot showed increased p-P38 levels in SCG of diabetic rats,which was inhibited after the decreased expression of mlncRNA2 by transfection with mlncRNA2-siRNA.2.Both 75mM D-glucose and 0.75mM FFAs can induce mlncRNA2 expression in PC 12 cells cultured for 3 days.But mannitol,which imitated hypertonic environment,did not stimulate PC12 mlncRNA2 expression.mlncRNA2 expression in PC 12 cells cultured with combined 75mM D-glucose and 0.75mM FFAs after 3 days are 1.91 times and 1.93 times of its expression in PC12 cells cultured with 75mM D-glucose or 0.75mM FFAs for 3 days,respectively.Inhibition of P38 MAPK pathway by specific P38 MAPK inhibitor SB-203580 can inhibit the up-regulation of the expression of PC12 mlncRNA2 induced by combined high D-glucose and high FFAs,suggesting that P38 MAPK signal pathway may be involved in the increased expression of mlncRNA2 of PC 12 cells cultured with combined high D-glucose and high FFAs.3.Treatment with combined high D-glucose and high FFAs inhibited PC 12 cells neurites outgrowth.Inhibition the mlncRNA2 expression induced by combined high D-glucose and high FFAs by transfection of mlncRNA2-siRNA alleviated the inhibitory neurites outgrowth of PC 12 cells cultured with combined high D-glucose and high FFAs.The expression of IRS1 mRNA and protein was decreased,while the level of IRS 1 serine phosphorylation was increased in PCl2 cells treated with combined high D-glucose and high FFAs,which was suppressed after the inhibition of mlncRNA2 expression by transfection of mlncRNA2-siRNA in PC 12 cells cultured with combined high D-glucose and high FFAs.P38 MAPK inhibitor SB-203580 can inhibit the increased expression of mlncRNA2 and the abnormality level of IRS 1 serine phosphorylation,alleviated the down-regulation of IRS1,and promoted PC 12 cells neurites outgrowth.4.Treatment with combined high D-glucose and high FFAs induced the increased levels of IL-6 and TNF-?,and ATP or BzATP-induced Ca2+ signals in PC12 cells.The Inhibition of mlncRNA2 expression induced by combined high D-glucose and high FFAs by transfection of mlncRNA2-siRNA suppressed the increased levels of IL-6 and TNF-a,and ATP or BzATP-induced Ca2+ signals in PC 12 cells cultured with combined high D-glucose and high FFAs.The expression of P2X7 mRNA and protein was increased in PC 12 cells treated with combined high D-glucose and high FFAs,which was suppressed after the inhibition of the mlncRNA2 expression by transfection of mlncRNA2-siRNA in PC 12 cells cultured with combined high D-glucose and high FFAs.The inhibition of P38 MAPK by specific P38 inhibitor SB-203580 can inhibit the increased expression of mlncRNA2,the up-regulation of P2X7,the increased[Ca2+]i evoked by ATP or BzATP,and the high levels of IL-6 and TNF-a in PC 12 cells cultured with combined high D-glucose and high FFAs.5?High FFAs induced P2X7 expression and IL-6 release significantly in PC 12 cells.Moreover,high FFAs enhanced ATP or BzATP-induced Ca2+ signals in PC 12 cells.Inhibition of P2X7 by transfection with P2X7-siRNA or co-culture with BBG(a specific P2X7 inhibitor)at high concentrations of FFAs decreased ATP or BzATP-promoted Ca2+ signals and IL-6 release in PC12 cells.High FFAs induced the phosphorylation of P38 in PC12 cells.Blockade of P38 pathways by SB-203580 inhibited P2X7 up-expression,ATP or BzATP-evoked[Ca2+]i rises as well as IL-6 release in PC 12 cells exposed to high FFAs.Conclusion:1.Chronic inflammation,high FFAs and oxidative stress associated with glucotoxicity and lipotoxicity directly and indirectly activated stress-related P38 MAPK signal pathway,induced the expression of SCG mlncRNA2-mediated Pannexin-1 and IRS1,participated in inflammatory reaction and functional injury of SCG,and was involved in the pathogenesis of SCG mlncRNA2-mediated diabetic cardiac autonomic neuropathy.2.Combined high D-glucose and high FFAs can induce the up-regulation of mlncRNA2 in PC 12 cells;P38 MAPK signal pathway may be involved in the increased expression of mlncRNA2 of PC 12 cells cultured with combined high D-glucose and high FFAs.3.Glucotoxicity and lipotoxicity directly and indirectly activated P38 MAPK signal pathway,regulated the expression of mlncRNA2-mediated P2X7 and IRS1 in PC 12 cells,inhibited the neurites outgrowth of PC 12 cells,induced inflammatory reaction of PC 12 cells,and thus related to mlncRNA2-mediated functional injury of PC 12 cells treated with combined high D-glucose and high FFAs.4?High FFAs induced P2X7 expression and IL-6 release in PC 12 cells via P38 MAPK signal pathway.Therefore,Pannexin-1,IRS1 and P2X7 genes related to SCG/PC12 mlncRNA2 up-regulation induced by glucotoxicity and lipotoxicity may be the potential new targets for the prevention and treatment of diabetic cardiac autonomic neuropathy. |
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