| ObjectiveBy observing the effect of intradermal auricular electroacupuncture stimulation(iaES)on lipopolysaccharide(LPS)induced endotoxemia rats’ survival rate and serum pro-inflammatory cytokines(TNF-α,IL-1β,IL-6),the influence of antagonists(atropine,propranolol)blocking receptors with drugs(mAChRs,β-ARs)and neurotomy(cervical vagotomy,splanchnicotomy)respectively from the central and peripheral nervous system disproving the different neural anti-inflammatory pathways.To illustrate differences between central and peripheral autonomic nervous anti-inflammatory pathways of iaES.MethodsExperiment 1:Effect of iaES on the survival rate of lethal endotoxemia rats40 SPF male Sprague-Dawley male rats(230-250 g)were randomly divided into model group and iaES group(n=20).All the rats were under anesthesia of urethane to accomplish all the interventions.The rats of iaES group received iaES intervention 30 minutes before and after LPS(10 mg/kg),1 h in total,but the model group did not receive the corresponding iaES intervention,just as model control.The survival of the rats of 7 days was observed and recorded daily.Experiment 2:Mechanism of the vagus pathway in the anti-inflammatory effect of iaESExperiment 2.1:Central mAChRs mechanism of vagus nerve anti-inflammatory effect of iaES42 SPF male Sprague-Dawley rats(230-250 g)were randomly divided into 6 groups(n=7),normal group,model group,iaES group,saline(i.c.v.)group,atropine methyl nitrate(i.c.v.)group,and atropine methyl nitrate(i.c.v.)only group.All the rats were under anesthesia of urethane to accomplish all the interventions.Normal group of rats received saline(6 mg/kg,i.p.)without iaES as blank control only.Model group of rats received LPS(6 mg/kg,i.p.)made of endotoxemia model without iaES as model control only.Rats of iaES group received iaES half an hour before and after LPS(6 mg/kg,i.p.)injection,1 h in total.Saline(i.c.v.)group of rats were injected with normal saline(5 μL)through lateral ventricle,and atropine methyl nitrate(i.c.v.)group of rats were injected with atropine methyl nitrite(5 μg/kg in 5 μL)through the lateral ventricle.Both two groups of rats received lateral ventricle injection 30 min before iaES intervention.And then received iaES half an hour before and after LPS(6 mg/kg,i.p.)injection,1h in total.The rats of atropine methyl nitrate(i.c.v.)only group were injected with atropine methyl nitrite(5 μg/kg in 5μL)through the lateral ventricle,but without iaES,and then received LPS(6 mg/kg,i.p.)at 1 h after lateral ventricle injection.Serum levels of pro-inflammatory cytokines of TNF-α,IL-1β and IL-6 were determined by ELISA at 3 h after LPS or normal saline injection.Experiment 2.2:Peripheral nerve mechanism of vagus nerve anti-inflammatory effect of iaES14 SPF male Sprague-Dawley rats(230-250 g)were randomly divided into 2 groups(n=7),cervical vagotomy group,sham cervical vagotomy group.All the rats were under anesthesia of urethane to accomplish all the interventions.The rats in the cervical vagotomy group received bilateral cervical vagotomy,while the sham cervical vagotomy group of rats only exposed bilateral cervical vagus nerves without separation or nerve resection.All neurotomy operations of both neurotomy groups and sham groups were completed 30 min before iaES.And then received iaES half an hour before and after LPS(6 mg/kg,i.p.)injection,1 h in total.Serum levels of pro-inflammatory cytokines of TNF-,IL-1β and IL-6 were determined by ELISA at 3 h after LPS injection.Experiment 2.3:Peripheral mAChRs mechanism of vagus nerve anti-inflammatory effect of iaES21 SPF male Sprague-Dawley rats(230-250 g)were randomly divided into 3 groups(n=7),atropine methyl nitrate(i.p.)group,atropine sulfate(i.p.)group,atropine sulfate(i.p.)only group.All the rats were under anesthesia of urethane to accomplish all the interventions.Atropine methyl nitrate(i.p.)group of rats were intraperitoneally injected with atropine methyl nitrate(4 mg/kg).Rats of atropine sulfate(i.p.)group were intraperitoneally injected with atropine sulfate(4 mg/kg).Both two groups of rats were injected 15 min before iaES intervention.And then received iaES half an hour before and after LPS(6 mg/kg,i.p.)injection,1 h in total.The rats of atropine sulfate(i.p.)only group rats were injected with atropine sulfate(4 mg/kg,i.p.).This group of rats didn’t receive iaES intervention.And then received LPS(6 mg/kg,i.p.)at 45 min after injection.Serum levels of pro-inflammatory cytokines of TNF-α,IL-1β and IL-6 were determined by ELISA at 3 h after LPS injection.Experiment 3:Mechanism of peripheral sympathetic pathway in the anti-inflammatory effect of iaESExperiment 3.1:Peripheral nerve mechanism of sympathetic anti-inflammatory effect of iaES14 SPF male Sprague-Dawley rats(230-250 g)were randomly divided into 2 groups(n=7),splanchnicotomy group,and sham splanchnicotomy group.All the rats were under anesthesia of urethane to accomplish all the interventions.The splanchnicotomy group of rats received splanchnic great nerve resection,while the rats in the sham splanchnicotomy group only exposed the splanchnic great nerve without separation or nerve resection.All neurotomy operations of both neurotomy groups and sham groups were completed 30 min before iaES.And then received iaES half an hour before and after LPS(6 mg/kg,i.p.)injection,1 h in total.Serum levels of pro-inflammatory cytokines of TNF-α,IL-1β and IL-6 were determined by ELISA at 3 h after LPS injection.Experiment 3.2:Peripheral β-ARs mechanism of sympathetic anti-inflammatory effect of iaES14 SPF male Sprague-Dawley rats(230-250 g)were randomly divided into 2 groups(n=7),propranolol(i.p.)group,propranolol(i.p.)only group.All the rats were under anesthesia of urethane to accomplish all the interventions.Propranolol(i.p.)group rats were intraperitoneally injected with propranolol(4 mg/kg).And the rats were injected 15 min before iaES intervention.And then received iaES half an hour before and after LPS(6 mg/kg,i.p.)injection,1 h in total.The rats of propranolol(i.p.)only group were injected with propranolol(4 mg/kg,i.p.).The rats didn’t receive iaES intervention.And then received LPS(6 mg/kg,i.p.)at 45 min after injection.Serum levels of pro-inflammatory cytokines of TNF-α,IL-1 β and IL-6 were determined by ELISA at 3 h after LPS injection.iaES method:Two disposable sterile acupuncture needles(0.18*10 mm)were taken and pierced parallelly into the central area of cymba concha and cavum concha respectively along the skin.The depth of the needles was 6 mm,and the electroacupuncture apparatus(HANS-200A)was connected to two needles respectively.The parameters of iaES were as follows:current intensity of 4 mA,frequency of 30 Hz,wave width of 0.2 ms ±30%,time of 1 h separated in 30 min before and after LPS.Experiment 2,3 shared same batch of rats,so the results of Experiment2,3 were compared with the normal group,model group and iaES group.ResultsThe results showed that iaES could significantly improve the survival rate of rats with lethal dose of endotoxemia,increase the survival rate from 25%to 50%(P<0.05).Moreover,iaES can reduce the systemic inflammatory response induced by LPS,and reduce the levels of serum pro-inflammatory cytokines(TNF-α,IL-1β and IL-6).Compared with the normal group,TNF-α,IL-1β and IL-6 of model group were significantly increased(P<0.0001,P<0.0001,P<0.0001).Compared with model group,TNF-α,IL-1β and IL-6 of iaES group were significantly decreased(P<0.0001,P<0.0001,P<0.0001).Intracerebroventricular injection of atropine methyl nitrate could reverse the anti-inflammatory effect of iaES,the differences were significant compared with intracerebroventricular injection of saline group(P<0.0001,P<0.01,P<0.0001).While intracerebroventricular injection of saline had no effect on the anti-inflammatory effect,no significant differences were observed compared with the iaES group(P>0.05).And intracerebroventricular injection of atropine methyl nitrate only had no effect on inflammation,differences were not significant compare with the model group(P>0.05).Cervical vagotomy group affected the anti-inflammatory effect of iaES,compared with iaES group(P<0.01,P<0.05,P<0.01).While sham cervical vagotomy had no impact on iaES inhibiting serum pro-inflammatory cytokines compared with iaES group(P>0.05).Peripheral injections of atropine methyl nitrate can sharply inhibit inflammatory reaction,decrease the levels of serum cytokines(P<0.0001,P<0.01,P<0.0001).Peripheral injections of atropine sulfate can also reduce the serum cytokines in combination with iaES or with drug only(P<0.0001,P<0.01,P<0.0001).And the anti-inflammatory effect was better than that of iaES only,but there was no statistical significance between iaES and drug combination group and drug only group(P>0.05).Splanchnicotomy affected the anti-inflammatory effect of iaES compared with iaES group(P<0.0001,P<0.05,P<0.0001).But no effect was observed in sham splanchnicotomy group compared with iaES group(P>0.05).Peripheral injections of propranolol can exert anti-inflammatory effects in combination with iaES or with drugs only(P<0.0001,P<0.0001,P<0.0001).,The anti-inflammatory effect was better than that of iaES only(P<0.0001,P<0.0001,P<0.0001),but there was no statistical significance between acupuncture and drug combination group and drug only group(P>0.05).ConclusioniaES can improve the survival rate of endotoxemia rats,and to reduce the serum pro-inflammatory cytokines(TNF-α,IL-1β,and IL-6).The central mAChRs anti-inflammatory mechanism is by iaES activating the central mAChRs.For the peripheral autonomic nerve anti-inflammatory mechanism,in addition to activating vagus nerve mediated cholinergic anti-inflammatory pathway(CAP),and requiring the activation of greater splanchnic nerve(GSN)as well.While the anti-inflammatory effect of iaES seems to be independent on the peripheral mAChRs and β-ARs,we cannot draw a conclusion of combination of iaES with antagonists. |
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