Background The incidence, mortality and disability rates of ischemic stroke are high, and the reperfusion injury after thrombolytic therapy has a great impact on patients. Acupuncture is a characteristic therapy for the treatment of the disease, but the action mechanism remains unclear.
Objective To observe the effect of Tongdu Tiaoshen electroacupuncture (EA) pretreatment on miR-124-3p/glycogen synthase kinase β (GSK-3β) /cyclin D (Cyp-D) signaling pathway and mitochondrial permeability transition pore (MPTP) of cerebral ischemia-reperfusion injury (CIRI) rats, and explore its possible mechanism of prevention and control of CIRI.
Methods From June to August 2022, a total of 100 clean SD rats were randomly divided into the sham operation group, model group, EA group, agonist group and EA + inhibitor group, with 20 rats in each group. For 7 d of intervention before modeling, in the EA group and EA + inhibitor group, "Baihui" (GV 20) , "Fengfu" (GV 16) and "Dazhui" (GV 14) were selected to perform electroacupuncture 1 time a day for 7 days. For 24 h before modeling, miR-124 agonist and inhibitor (5 nmol) were injected into the lateral ventricles in the EA + agonist group and inhibitor group. Except for the sham operation group, the right cerebral ischemia-reperfusion model of rats was prepared by the modified suture method in the rest groups. The right cerebral cortex of rats was taken, the degree of neurological impairment in each group was observed using mNSS scale and TTC staining, the nerve cell injury was observed by TUNEL staining and transmission electron microscopy. The expressions of GSK-3β, p-GSK-3β, Cyp-D, Cyt-C and Caspase-3 in cerebral cortex of each group were detected by immunofluorescence staining, Western blot and real-time quantitative PCR. The degree of MPTP openness was detected by flow cytometry, the higher rate of MPTP-positive cells indicated greater degress of MPTP openness.
Results Except for the sham operation group, all rest groups of rats were successfully modeled. Compared with the sham operation group, mNSS score and infarct volume of brain tissue were increased, mitochondrial structure was seriously damaged, cell apoptosis index was increased, p-GSK-3β positive expression was reduced, Cyp-D positive expression was enhanced, miR-124-3p, Cyp-D, Cyt-C and Caspase-3 mRNA expressions were increased, p-GSK-3β/ GSK-3β ratio was decreased, relative Cyp-D, Cyt-C, and Caspase-3 protein expressions were increased, and MPTP openness degree was increased in the model group (P<0.05) . Compared with the model group, mNSS score and relative infarct volume were decreased, mitochondrial structure destruction was relieved, p-GSK-3β/GSK-3β ratio was increased, and relative expression of Caspase-3 protein was decreased in the electroacupuncture group and inhibitor group; the mRNA expression of GSK-3βwas increased in electroacupuncture + agonist group (P<0.05) ; apoptosis index was decreased, positive expression of p-GSK-3β was reduced and positive expression of Cyp-D was enhanced respectively, miR-124-3p, Cyp-D, Cyt-C and Caspase-3 mRNA expressions were decreased, Cyp-D and Cyt-C protein expressions were decreased, the degree of MPTP openness was decreased in the electroacupuncture group, inhibitor group and electroacupuncture + agonist group (P<0.05) . Compared with the electroacupuncture group, p-GSK-3β positive expression was enhanced, Cyp-D positive expression was reduced, miR-124-3p and Cyp-D, Cyt-C, Caspase-3 mRNA expressions were decreased, GSK-3β mRNA expression was increased, Cyp-D and Cyt-C protein expressions were decreased, and MPTP openness degree was reduced in the agonist group (P<0.05) ; the apoptosis index was increased, the positive expression of P-GSK-3β was reduced, the positive expression of Cyp-D was enhanced, the expressions of miR-124-3p, Cyt-C and Caspase-3 mRNA were increased, the expressions of Cyp-D and Cyt-C protein were increased, and the openness degree of MPTP was increased in the electroacupuncture + agonist group (P<0.05) .
Conclusion Tongdu Tiaoshen electroacupuncture pretreatment can alleviate neurological impairment in cerebral ischemia-reperfusion rats, the mechanism may be related to mediating miR-124-3p/GSK-3β/Cyp-D signaling pathway, inhibiting MPTP openness and thus reducing cell injury. The results of this study further verified the therapeutic effect of Tongdu Tiaoshen electroacupuncture in the treatment of CIRI from the mechanism, providing a new scientific basis for preventive treatment of disease of TCM and promote its clinical application.
