Effects And Mechanism Of Lentivirus-mediated Silence Or Overexpression Of Spinal NGF Gene On Myocardial Protective Effect Of Intrathecal Morphine Preconditioning

Objective Ischemic heart disease is one of the diseases with the highest morbidity and mortality in China.And the main treatment is to restore the ischemic myocardial blood flow reperfusion as early as possible.After revascularization of ischemic myocardium,excessive generation of oxygen radicals,Ca²⁺ and inflammatory factors further aggravates myocardial injury.Substances produced locally during myocardial ischemia can stimulate the activation of TRPV1,which is highly expressed on cardiac sensory afferent neurons.Nociceptive stimulation signals are transmitted along sensory afferent fibers through dorsal root ganglia to the dorsal horn of the spinal cord at the corresponding segment,and finally uploaded to the corresponding central and cerebral cortex.What's more,nerve growth factor?NGF?can maintain the growth and function of sensory nerve and sympathetic nerve,enhance the sensitivity of primary afferent neurons to nociceptive stimulation,and play an important role in the process of nociceptive stimulation of sensory neurons.It has been confirmed that NGF is involved in the neural stress response following myocardial Ischemia reperfusion?IR?.Evidences prompted that intrathecal morphine preconditioning?ITMP?reduces the cardiac injury of IR via the central nervous system.However,the molecular mechanism is not fully understood and little is known about the significance of NGF in myocardial injury of IR and intrathecal morphine-induced cardioprotection.This study was designed to investigate the relationship and mechanism between the myocardial protective effect of ITMP and the expression of NGF in spinal cord by specific silencing or overexpression of NGF gene in spinal cord.Methods NGF overexpressed lentivirus?LV?vector?LV-NGF?was designed and constructed;Three short hairpin RNA?sh RNA?targeting NGF genes were synthesized,and lentiviral vectors were constructed for NGF silencing: LV-NGF sh RNA1,LV-NGF sh RNA2,and LV-NGF sh RNA3.NGF overexpression efficiency was verified in vivo?SD rats?,and gene silencing efficiency of NGF sh RNA was screened and verified.Then we selected the NGF sh RNA with the best silencing efficiency.The infection efficiency was determined by fluorescent microscope.Besides,we detected the level of NGF m RNA and expression of NGF protein by quantitative reverse transcription polymerase chain reaction?q RT-PCR?and western blotting,respectively.Intrathecal catheter placement models were established in healthy male Sprague-Dawley?SD?rats?weighting 260 ± 20 g?.The whole study consisted of two parts.In the first experiment,we investigated the effect of NGF gene overexpression in spinal cord on myocardial protection of intrathecal morphine preconditioning.And the rats were randomly divided into the following groups?n=6?: Sham group,ischemia/reperfusion group?I/R?without or with lentivirus-mediated nerve growth factor?LV-NGF?infection?I/R and LV-NGF+I/R group?,and intrathecal morphine preconditioning group?ITMP?group without or with LV-NGF infection?ITMP+I/R and LV-NGF+ITMP+I/R group?,as well as the empty viral infected group [LV-CON?NGF?+ITMP+I/R].In the second experiment,We investigated the effect of spinal cord NGF gene silencing on myocardial protection of ITMP.The rats were randomly assigned to the following groups?n = 6?: Sham group,I/R,ITMP without or with lentivirus-mediated NGF sh RNA infection?ITMP+I/R and LV-NGF sh RNA+ITMP+I/R group?,as well as other empty viruses infected group [LV-CON?NGF sh RNA?+ITMP+I/R].During the operation,the ECG was monitored and the number and duration of arrhythmias were counted.At the end of the reperfusion,the hearts of rats were obtained and the area at risk?AAR?,infarct size?IS?and IS/ARR were calculated;We detected the expression of c Tn I in serum;The T2-6 segments of the spinal cords were extracted for determination of the level of NGF m RNA and TRPV1 m RNA by q RT-PCR;Then the spinal cord and DRG in T2-6 were separated for detection of the expression of NGF/Trka,TRPV1/p-TRPV1 by western blot.In addition,the expression of SP and CGRP in spinal cord and DRG were examined by immuno-fluorescence.Results 1.The NGF gene overexpression effects and NGF sh RNA gene silencing effects were examined in vivo.Spinal cord tissue sections were observed by fluorescence microscopy,and the lentivirus could infect T2-6 spinal cord segments successfully after injection.The overexpression effect of LV-NGF gene was perfectly,which the expression levels of NGF m RNA and protein were significantly increased compared with Sham group.In the three constructed NGF sh RNAs,the LV sh NGF3 had the best gene silencing effects.Compared with the ham group,the level of NGF m RNA and NGF protein was effectively inhibited in the LV sh NGF3 group,inferring that the LV-NGF and LV-NGF sh RNA3 could make NGF gene overexpression or silenced successfully.2.Overexpression of NGF gene in spinal cord alleviated myocardial protective effect of intrathecal morphine preconditioning.Compared with the I/R group,the area of myocardial infarction in the ITMP+I/R group was significantly reduced,but infarct size?IS?could be increased by intrathecal LV-NGF injection in advance.The level of NGF m RNA in spinal cord and the expression of NGF/Trka protein in spinal cord and DRG increased,which suggested that the overexpression of NGF gene in spinal cord could alleviate the myocardial protective effect of ITMP.3.Silencing of NGF gene in spinal cord enhanced the myocardial protective effect of ITMP.Intrathecal injection of LV-NGF sh RNA reduced myocardial IS.Additionally,the level of NGF m RNA in spinal cord and expression of NGF/Trka protein in spinal cord and DRG were decreased significantly,suggesting that silencing of NGF gene enhanced the myocardial protective effect of morphine.4.TRPV1 modification was induced by spinal NGF gene and SP and CGRP released.The expression of TRPV1/ p-TRPV1 protein in the spinal cord and DRG were decreased in the ITMP group compared with the I/R group while they were elevated with the infection of LV-NGF in spinal cord and DRG.However,after transfected NGF sh RNA,the expression of TRPV1/p-TRPV1 decreased significantly.Compared with IR group,immunofluorescence results showed that the expression of SP and CGRP in spinal cord and in DRG descended obviously in ITMP group;Overexpression of NGF gene significantly enhanced SP and CGRP expression in spinal dorsal horn and DRG.Conversely,silencing of spinal NGF gene significantly reduced the expression of SP and CGRP in spinal dorsal horn and DRG.Conclusion The cardiac protective effects of intrathecal morphine preconditioning may implement through mediating NGF expression in the spinal cord,and may involve TRPV1.