[Mechanism of electroacupuncture combined with antibiotics targeting dorsal motor nucleus of vagus nerve to activate vagus-adrenal axis, alleviate inflammatory response and reduce mortality in septic mice].

OBJECTIVES: To explore the mechanism by which electroacupuncture (EA) combined with antibiotics regulates the vagus-adrenal axis via the dorsal motor nucleus of vagus (DMV), promotes catecholamine secretion, attenuates inflammatory storm, and improves survival rate in septic mice.

METHODS: Experiment 1:fifty C57BL/6 mice were randomly divided into sham operation group, model group, antibiotic group, EA group, and EA + antibiotic group, with 10 mice in each group. Cecal ligation and puncture (CLP) was performed to establish the sepsis model. Mice in the EA group received 15 min EA (10 Hz, 0.5 mA) at bilateral "Zusanli" (ST36), once daily for 3 consecutive days. Mice in the antibiotic group were intraperitoneally injected with imipenem-cilastatin sodium (30 mg/kg) every 12 h for 3 consecutive days. Mice in the EA+antibiotic group received EA and antibiotic treatment the same as the above. The 3-day survival rate and body weight changes of mice in each group were recorded. ELISA was adopted to detect serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). HE staining was used to observe pathological changes and the number of inflammatory cells in cecal tissues. Whole-brain optical clearing was performed to detect the expression of proto-oncogene c-Fos protein (c-Fos) in the DMV region. Experiment 2:fifty Chat-Cre mice were randomly assigned to sham operation + empty virus group, model + empty virus group, EA-antibiotic combination + empty virus group, EA-antibiotic combination + inhibitory virus group, and antibiotic + activating virus group, with 10 mice per group. Corresponding viruses were injected into the DMV 3 weeks before model establishment. CLP operation was conducted 3 weeks after virus injection, followed by daily intraperitoneal injection of clozapine N-oxide (CNO, 3 mg/kg), combined with antibiotics or EA treatment (the same interventions and parameters as Experiment 1). Serum IL-6 and TNF-α levels were measured by ELISA;cecal inflammatory infiltration was evaluated via HE staining. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was applied to detect serum catecholamines including dopamine (DA), epinephrine (E), norepinephrine (NE), as well as their metabolites 3-methoxytyramine (3-MT), normetanephrine (NMN), and metanephrine (MN) levels. Immunofluorescence staining was used to quantify the counts of forkhead box protein P3 positive / cluster of differentiation 4 positive (FOXP3+/CD4+) cells in cecal tissues.

RESULTS: Experiment 1:compared with the sham operation group, the model group exhibited markedly decreased 3-day survival rate and body weight (P<0.000 1), elevated serum IL-6 and TNF-α levels (P<0.000 1), and increased inflammatory cell infiltration in cecum (P<0.000 1). Compared with the model group, the EA + antibiotic group showed improved survival rate (P<0.05), alleviated body weight loss (P<0.000 1), decreased serum IL-6 and TNF-α levels (P<0.000 1), reduced inflammatory cell infiltration (P<0.000 1), and elevated c-Fos positive cell density in DMV (P<0.000 1). Moreover, all above indicators were superior to those in the single EA or single antibiotic group to varying degrees. Experiment 2:in comparison with the sham operation + empty virus group, the model + empty virus group had higher serum inflammatory factor levels and more cecal inflammatory infiltrates (P<0.0001), and lower levels of catecholamines and their metabolites, as well as fewer FOXP3+/CD4+ cells in cecal tissues (P<0.000 1). Compared with the model + empty virus group, the EA-antibiotic combination + empty virus group, EA-antibiotic combination + inhibitory virus group, and antibiotic + activating virus group displayed reduced serum inflammatory factors and relieved cecal inflammatory infiltration (P<0.000 1, P<0.01, P<0.05), accompanied by increased levels of catecholamines and metabolites, and more FOXP3+/CD4+ cells in cecum (P<0.01, P<0.05, P<0.001, P<0.000 1). Relative to the EA-antibiotic combination + empty virus group, all the above indicators were reversed in the EA-antibiotic combination + inhibitory virus group (P<0.01, P<0.001, P<0.05, except for 3-MT).

CONCLUSIONS: EA at ST36 combined with antibiotics exerts optimal therapeutic effects on CLP-induced septic mice. The underlying mechanism may involve activating cholinergic neurons in DMV, regulating the vagus-adrenal axis to facilitate catecholamine secretion, suppressing excessive inflammatory response, and consequently elevating survival rate.