The Role Of Electroacupuncture In Mitigating Sepsis-associated Encephalopathy Based On Nrf-2/HO-1/Ca²⁺Signaling Pathway

Objective Sepsis-associated encephalopathy is a common and serious complication of sepsis,characterized by cognitive impairment,which may lead to poor prognosis,increased mortality and poor quality of life.The traditional Chinese electroacupuncture（EA）has a complementary and alternative therapeutic effect on many central nervous system diseases.Studies have shown electroacupuncture can play an anti-inflammatory and antioxidant stress effect by activating the nuclear factor erythroid-2-related factor-2（Nrf-2）/heme oxygenase-1（HO-1）signaling pathway,thus protecting against sepsis-related organ damage;HO-1 can regulate cellular function by regulating intracellular Ca²⁺signaling pathway.Nrf-2/HO-1signaling pathway is one of the most important endogenous protective systems in the body that is widely involved in anti-inflammation and anti-oxidative stress.Free calcium ion is an important second messenger in cells and plays an important role in regulating almost all physiological activities of cells and the body.In this study,we investigated the possible protective effect of electroacupuncture on neuronal,synaptic and cognitive impairments to mitigate sepsis-associated encephalopathy,and the role of Nrf-2/HO-1/Ca²⁺signaling pathway involved.Methods PartⅠ:One Sepsis-associated encephalopathy model was establisheded by cecal ligation and puncture（CLP）.The Sprague Dawley（SD）rats were randomly divided into four groups:Control group（rats underwent a sham surgical procedure）,CLP group（rats underwent a CLP surgical procedure）,CLP+EA group（rats underwent a CLP surgical procedure and treated with electroacupuncture）,and CLP+sham EA（rats underwent a CLP surgical procedure and treated with sham electroacupuncture）.EA at Baihui（DU20）,Quchi（LI11）and Zusanli（ST36）or sham EA were performed 30 minutes once a day for 10 consecutive days starting from 2days before CLP.Started from day 9 after CLP,cognitive function was examined by Morris water maze test for 6 days.On day 14 after CLP,the 14-day survival rate was observed,the rats were euthanized,and then the hippocampal neuron count,the dendrite spine density,the synaptic proteins,the oxidative stress markers such asmalondialdehyde（MDA）and superoxide dismutase（SOD）,and the expression of Nrf-2 and HO-1were detected.PartⅡ:Health male C57BL/6J mice were randomly divided into 4 groups:Control group（rats underwent a sham surgical procedure）,CLP group（rats underwent a CLP surgical procedure）,CLP+EA group（rats underwent a CLP surgical procedure and treated with electroacupuncture）,and CLP+EA+ZnPP group（rats underwent a CLP surgical procedure and treated with electroacupuncture and zinc protoporphyria）.All mice were injected with virus carrying calcium ion fluorescent probes and implanted with optical fiber into the hippocampal CA1 area,so the real-time calcium fluorescence signals in targeted brain area during behavior tests were recorded to reflect the changes of neuronal activities.Three weeks after administration,when the calcium signals were fully expressed,sepsis-associated encephalopathy model was establisheded by CLP.EA at DU20、LI11 and ST36 acupoints were conducted 30 min a day for 10 consecutive days starting from 2 days before CLP.Mice in CLP+EA+ZnPP group were intraperitoneally injected with ZnPP 50μmol/kg 12 h before each electroacupuncture administration.Open field and novel object recognition tests were conducted 12～14 d after CLP respectively to record the number of rearing,recognition index and related calcium signals to reflect the changes of cognitive function and hippocampal neuron activity in mice.After the last tests,mice were sacrificed and brains were collected,the neuron number in hippocampal CA1 area was caculated by Nissl staining,and the expression of Nrf-2mRNA and the activity of its downstream HO-1 were detected.Results PartⅠ:Hippocampal neuronal injury,synaptic deficit,oxidative stress response,high mortality rate and cognitive dysfunction were shown in CLP rats comparing with those in Control rats.However,electroacupuncture treatment suppressed oxidative stress,hippocampal neuronal injury and synaptic impairement.The survival rate and spatial learning and memory abilities were also improved after electroacupuncture treatment.Furthermore,electroacupuncture enhanced the expression of Nrf-2 mRNA and HO-1 protein in hippocampus,but sham EA didn’t have the same effect.Electroacupuncture may inhibit hippocampal oxidative stress response by upregulating the Nrf-2/HO-1 signaling pathway,thereby alleviating neuronal and synaptic impairements,cognitive dysfunction and sepsis-associated encephalopathy.PartⅡ:Comparing with the Control group,the neuron count in hippocampal CA1 area were reduced,the levels of Nrf-2 and HO-1 were decresed,the number of rearing and recognition index of mice were decreased,and the amplitudes of related calcium signals were declined in CLP group.Comparing with the CLP group,the neuron count and the levels of Nrf-2 and HO-1 in hippocampal area were increased,the number of rearing and recognition index of mice were elevated,and the amplitudes of related calcium signals were enhanced in the CLP+EA group.No statistical differences in the parameters mentioned above were found between the CLP+EA+ZnPP and CLP groups,the HO-1 inhibitor reversed the protective effect of electroacupuncture.Calcium ion is an important second messenger in cells,which regulates almost all physiological activities of cells and the body.Electroacupuncture may function through upregulating the Nrf-2/HO-1/Ca²⁺signaling pathway to increase the level of free calcium ions in hippocampal neurons,thereby enhances neuronal activity and function,alleviates cognitive dysfunction and improves sepsis-associated encephalopathy.Conclusion Taken together,the results suggested that electroacupuncture might alleviate neuronal loss and synaptic injury,enhance neuronal function,improve cognitive function and exert neuroprotection on sepsis-associated encephalopathy.The mechanism may be related to the upregulation of the Nrf-2/HO-1/Ca²⁺signaling pathway,which reduces the oxidative stress response in the hippocampus and increases the level of free calcium ions in hippocampal neurons.