Electroacupunture Zusanli Point Protect Myocardium Against Ischemia-reperfusion Injury In Rats

Background:Ischemia-reperfusion injury is a complex pathogenetic process involves multiple signal transductions and inflammatory mediators.At present,it is thought that both systemic and regional inflammatory reaction participat in ischemia-reperfusion injury,leucocyte (neutrophil primarily),cytokine and chemokine as well as complement is activated during myocardial ischemia and significantly aggravated at reperfusion period,then initiate myocardial inflammatory cascade reaction.Research find that stimulating efferent vagus nervus and applicating neurotransmitter acetylcholine or nicotine can inhibite endotoxemia leading to systemic inflammatory response syndrome,and depress significantly cytokine releasing such as:tumor necrosis factor-α,interleukin-1β,interleukin-6,interleukin-8 meanwhile unaffect of the level of anti-inflammatory factor interleukin-10,which is named "the cholinergic anti-inflammatory pathway"The response of the myocardium to reperfusion is similar to inflammatory responses induced by sepsis.It is a pathogenetic process including some mediators of inflammation participating and excessive inflammatory reaction out of control. Therefore base of above-mentioned finding that electric stimulating efferent vagus nervus can inhibite excessive inflammatory and protect organs in vivo,we hypothesis that any method that can electric stimulating efferent vagus nerve possibly regulate myocardial ischemia- reperfusion injury at direct.Our study is to aim mainly at whether if how to antagonise the injury caused by inflammation overexpression in myocardium,then provide a new and available method for protecting myocardium against ischemia-reperfusion injury.Objective:To study the protection of myocardium against ischemia-reperfusion injury via activating the cholinergic anti- inflammatory pathway by electric stimulating vagus nerve or electric acupuncture at Zusanli point in rats.Method:1.100 Sprague-Dawley male rat were randomly divided in 5 groups:①sham-operation control group(SHAM group)②myocardial ischemia-reperfusion group(IR group)③vagus nerve stimulation group(STM group)④electroacupuncture Zusanli point group(ZSL group)⑤electroacupuncture non-acupoints group(FJFX group).Except for SHAM group,each group was subjected to 30 min of myocardial ischemia followed by 2h of reperfusion.In addition,in STM group, electrode continued stimulating left cervical vagus nerve at the intensity of 5 V,2ms,1HZ before and after 10min of reperfusion;in ZSL group, electric acupuncture continued stimulating Zusanli point before and after 10min of reperfusion;in FJFX group,electric acupuncture continued stimulating non-acupoints to exclude the effect of electric stimulus to myocardium injury.To record the incidence rate of arhythmia,and the change of heart rate,mean arterial pressure;sample was collected after 120min of reperfusion.2.Myocardium inflammation were dectected by HE staining and examined under the light microscope;myocardial area at risk and infart region was determined by Evan's blue dye perfusion and triphenyl tetrazolium chloride(TTC) staining;the level of plasma troponinIin was detected by immunoturbidimetry;the level of tissue TNF-αand IL-6 in myocardium and plasm was detected by Elisa;The activity of myeloperoxidase(MPO) in myocardium was detected by chromatometry;the formation of malondialdehyde(MDA) in myocardium was detected by thio-barbituric acid method;the expression of nuclear factor-κB p65(NF-κBp65) in myocardium was analyzed by immunohistochemical technique;myocardial apoptotic cells were examined by the TUNEL assay. Results:1.Vital signs and arhythmia:compared with SHAM group,MAP decreased in IR group,ZSL group,STM group and FJFX group.there is no difference in these groups.HR in these group decreased during reperfusion period and STM group dereased more rapidly at the sitmulation period.When stimulation stopped,HR recovered.The incidence rate of arhythmia in STM group and ZSL group decreased more significantly than IR group(P＜0.05).2.Histodiagnosis by light microscope:myocardium structure is normal and cardiocyte rank tightly,no edma and inflammtory cell infiltrate in SHAM group.IR group and FJFX group have more significantly change than STM group and ZSL group.Extensive cloudy swelling,lamellar necrosis,many inflammatory cells infiltration existed in IR group.3.Area at risk and infarct size were detected by EB/TTC staining.There is a significant difference between IR group and STM group in%infart size.the levels of cTnI in each group before ischmia are similar.after 120min of reperfusion,there is a increase in each group,compared with SHAM group(P＜0.05),the level of cTnI in STM group and ZSL group are lower than IR group(P＜0.05);there is no difference in ST group and ZSL group(P＜0.05).4.Injury caused by inflammation and oxidation:MPO activity and MDA formation in other 4 groups increase more significantly than SHAM group.The MPO activity and MDA formation in IR group are higher than STM group and ZSL group,compared to SHAM group,TNF and IL-6 concentration in plasma and myocardium are higher in IR group,STM group,ZSL group and FJFX group(P＜0.05).IR group caused a farther increase of TNF an IL-6 compared to ZSL group.There is no difference between STM group and ZSL group.The immuno-histochemical stain of NF-κB demonstrated that compared with SHAM group,the colour in cytoplasma was obviously deeper in IR group.there is positive staining in cytoplasma and nuclei,indicating that NF-κB was activated from cytoplasma into nuclei;vagus nerve stimulation(VNS) and eclectroacupuncture Zusanli point can reduce its expression,however which was still higher than SHAM group.5.Cardiocyte apoptosis index:There were hardly any TUNEL-positive nuclei in myocardium of SHAM group.numerous TUNEL-positive nuclei in the myocardium were observed in the IR group. Electric vagus nerve stimulation and electroacupuncture Zusanli reduced the amout of TUNEL-positive cells as AI(apoptosis index ) are lower in STM group and ZSL group than in IR group.(P＜0.05).Conclusion:Electroacupuncture Zusanli point can inhibite the inflammtion induced by myocardial I/R injury and protect myocardium without vagus nerve dissection;The mechanism is invovled about activation of cholinergic anti-inflammatory pathway,blunting NF-κB p65 expression,down-regulation TNF and IL-6 release,inhibiting apoptosis.