Study On The Protective Effect And Mechanism Of Xinbao Pills In Brain Injury After Cardiopulmonary Resuscitation Based On Mitochondrial Quality Control

ObjectiveBrain injury after cardiopulmonary resuscitation is essentially cerebral ischemiareperfusion injury,which is the main cause of death and long-term disability in patients with cardiac arrest(CA),and there is no treatment as so far.Therefore,it is urgent to develop new drugs or therapies for the prevention and treatment of brain injury after cardiopulmonary resuscitation.A variety of constituent herbs or active ingredients of Xinbao Pills have been proved to perform neuroprotective effects,but there is no reported literature on the neuroprotective effect and mechanism of Xinbao Pills.Hence,the neuroprotective effect and mechanism of Xinbao Pills were studied in vitro and in vivo by establishing the oxygen and glucose deprivation and reoxygenation(OGD/RO)model as well as post-cardiopulmonary resuscitation brain injury(PBI)model in this paper.Methods1.The components of Xinbao Pills Lyophilized Powder and Containing Serum were analyzed and compared by widely-targeted metabolic analysis.Subsequently,PC 12 cells were intervened by oxygen-glucose deprivation for 2 hours in a three gas incubator and reoxygenation to establish the ischemia-reperfusion injury model of nerve cells.Based on this model,the efficacy of Xinbao Pills Lyophilized Powder and Containing Serum were compared by the cell viability with MTT test,the cell morphology observing with microscope and the LDH level detection with kit,and then the dosage form with the best efficacy was selected to be detected its effect on the pH value and osmotic pressure of DMEM culture medium and carried out for further experiments.2.PC 12 cells were intervened by oxygen-glucose deprivation for 2 hours in a three gas incubator and reoxygenation to establish the ischemia-reperfusion injury model of nerve cells.The experiment was divided into following groups:Control group(routine culture,Control),Model group(oxygen and glucose deprivation and reoxygenation,OGD/RO),Lowdose-Xinbao Pills experimental group(OGD/RO+XBP-L),Medium-dose-Xinbao Pills experimental group(OGD/RO+XBP-M)and High-dose-Xinbao Pills experimental group(OGD/RO+XBP-H).And then,the ATP level of cells was detected by kit,the change of mitochondrial membrane potential was observed under fluorescence microscope by TMRE,the morphology of mitochondria was observed under confocal microscope by Mito-Tracker Red CMXRos,and the expression level of the mitochondrial quality control related proteins includingPGC-1α、NRF1、TFAM、Drp1、MFF、FIS1、OPA1、MFN1、MFN2、p62、PINK1、Parkin、LC3 and other protein including AMPK、p-AMPK were investigated by Western blotting,and the content of mtDNA as well as the expression of AMPK gene were detected by PCR.Finally,by adding AMPK inhibitor Compound C,MTT assay and Western blotting were used to explore whether Xinbao Pills mediated the regulation of mitochondrial quality control through AMPK target.3.The post-cardiopulmonary resuscitation brain injury model of rats was established by clamping the trachea and the cardiopulmonary resuscitation was carried out 5 minutes after cardiac arrest.Male SD rats of 300-350g were randomly divided into Sham group(Sham),Model group(PBI),Low-dose-Xinbao Pills experimental group(PBI+XBP-L)and Highdose-Xinbao Pills experimental group(PBI+XBP-H).The experimental groups were given Xinbao Pills and the Sham group as well as PBI group were given the same volume of saline.The neurological deficit score(NDS)of each group was evaluated,the morphology of neurons was observed by HE staining,and the contents of neuron-specific enolase(NSE)in serum as well as the mitochondrial respiratory chain complex Ⅰ-Ⅳ and Succinate dehydrogenase(SDH)in cortex were detected by ELISA kit,the expression level of the mitochondrial quality control related proteins including PGC-1α、NRF1、TFAM、Drp1、MFF、FIS1、OPA1、MFN1、MFN2、p62、PINK 1、Parkin、LC3 and other proteins including AMPK、p-AMPK were investigated by Western blotting,the content of mtDNA as well as the expression of AMPK gene were detected by PCR,and the colocalization expression of mitochondrial marker ATPB and autophagy marker LC3 was detected by immunofluorescence staining.ResultsExperiment 1:(1)There were significant differences in the components between Xinbao Pills Lyophilized Powder and Containing Serum,and the content of most components in Lyophilized Powder was higher than that in Containing Serum.(2)Xinbao Pills Lyophilized Powder has better protective effect in PC 12 cells induced by OGD/RO.(3)Xinbao Pills Lyophilized Powder had no significant effect on the pH value and osmotic pressure of DMEM culture medium.Experiment 2:(1)Xinbao Pills ameliorated the mitochondrial damage in PC 12 cells induced by OGD/RO.(2)Xinbao Pills not only promoted mitochondrial biogenesis and mitochondrial fusion but also inhibited mitochondrial fission and over-activated mitophagy in PC 12 cells induced by OGD/RO.(3)Xinbao Pills mediated the regulation of mitochondrial quality control by promoting the phosphorylation of AMPK.Experiment 3:(1)Xinbao Pills ameliorated the neurological function in rats with brain injury after cardiopulmonary resuscitation.(2)Xinbao Pills ameliorated the mitochondrial damage in rats with brain injury after cardiopulmonary resuscitation.(3)Xinbao Pills not only promoted mitochondrial biogenesis and mitochondrial fusion but also inhibited mitochondrial fission and over-activated mitophagy as well as prevented the transfer of LC3 to mitochondria in rats with brain injury after cardiopulmonary resuscitation.(4)Xinbao Pills promoting the phosphorylation of AMPK in rats with brain injury after cardiopulmonary resuscitation.ConclusionXinbao Pills performed the protective effect against brain injury after cardiopulmonary resuscitation by ameliorating mitochondrial injury and its mechanism can be ascribed to the mediation of AMPK on mitochondrial quality control.