Base On The C-Myc/CLIC4 Pathway To Explore The Pathogenesis Of Hippocampal Injury Induced By Lipopolysaccharide In Rat

In the veterinary field,endotoxemia is mainly secondary to animal pyomesia,pyoderma and severe surgical infections.Endotoxemia often leads to hippocampal injury,and its clinical incidence is about 70%,which can cause brain atrophy,cognitive impairment and even death.Hippocampal injury induced by endotoxemia is associated with poor prognosis and high mortality.However,the occurrence,development and molecular mechanism of hippocampal injury induced by endotoxemia are still unclear,resulting in the lack of corresponding diagnosis and treatment methods and effective treatment drugs in clinical practice.Therefore,the study of the pathogenesis of hippocampal injury induced by endotoxemia has been called the hot spot and difficulty of veterinary clinic.In this study,SD rats were used to establish endotoxemia model,and the pathogenesis of hippocampal injury induced by LPS was explored from three levels: tissue level,cell level and molecular level.The mechanism of c-Myc/CLIC4 pathway in LPS-induced PC12 cell apoptosis was verified by genetic editing of PC12 cells using CRISPR/Cas9 gene editing technology.This study aims to provide a new theoretical basis for the pathogenesis of hippocampal injury induced by endotoxemia,and provide theoretical basis and experimental basis for future treatment.Thirty-six healthy male Sprague-Dawley rats were randomly divided into CON group and LPS group.The CON group was injected with 1 m L/kg normal saline,and the LPS group was intraperitoneally injected with 10 mg/kg LPS.After 4 hours,the blood was collected from heart and the rat was sacrificed,and then,the hippocampus taken for futher experiments.Through detecting inflammatory factors in rat serum,hippocampal histopathological and hippocampal transmission electron microscopy,detecting mitochondrial apoptosis-related pathway protein,and the m RNA and protein expression changes of c-Myc/CLIC4 pathway,this study aims to clarify the pathogenesis of mitochondrial apoptosis pathway in the occurrence and development of hippocampal injury induced by LPS,and explore whether the c-Myc/CLIC4 pathway is involved in the regulation of mitochondrial apoptosis pathway.In the in vitro,PC12 cells were selected and divided into 5 groups,CON group,LPS group,sgc-Myc group,sg CLIC4 group,and sg CON group.The c-Myc gene and the CLIC4 gene was knocked out by CRISPR/Cas9.The following treatment was performed: the CON group was replaced with a new complete medium;the other groups were replaced with a complete culture containing 200 ?g/m L LPS.Flow cytometry,Annexin V-FITC and Western blot were used to detect the change of apoptosis level after knocking out c-Myc/CLIC4 pathway,thus confirming the regulation of c-Myc/CLIC4 pathway on mitochondrial apoptosis pathway.The results of inflammatory factors showed that the levels of IL-1?,IL-6,TNF-? and IL-18 in the LPS group were significantly increased than those in the CON group(p < 0.01),which proved that the endotoxemia model was successfully established.Histopathological results showed that the hippocampal neurons in the CON group were closely arranged,and cellular integrity.The number of hippocampal neurons in the LPS group was significantly reduced,the surrounding between the glial cells was significantly widened.Ultrastructure observation of hippocampus showed that the nuclear membrane of CON group was clear and the mitochondrial structure was complete.In the LPS group,the nuclear membrane was damaged,the mitochondria was swollen,and the mitochondrial crete was ruptured,which proved that the activation of mitochondrial apoptosis pathway is one of the mechanisms of hippocampal injury induced by LPS.Western blot analysis showed that compared with the CON group,the expression of cleaved caspase-3,cleaved caspase-9,Cyt C and Bax/Bcl-2 in LPS group increased significantly(p < 0.01).Real-time quantitative PCR and Western blot showed that the expression of c-Myc and CLIC4 m RNA and protein in LPS group were significantly increased(p < 0.01),while p53 m RNA and protein levels were not significantly different from those in CON group(p > 0.05),demonstrating that regulation of the mitochondrial apoptotic pathway is associated with activation of the c-Myc/CLIC4 pathway.In vitro,flow cytometry and Annexin V-FITC showed that the apoptosis rate of LPS group and sg CON group was significantly increased than that of CON group(p < 0.01),while compared with the CON group,was no significant difference(p > 0.05),in the sgc-Myc group and the sg CLIC4 group.Western blot results showed that expression of mitochondrial apoptosis pathway-related protein in LPS group and sg CON group increased significantly(p < 0.01),while the expression of protein in sg CLIC4 group sgc-Myc group and was not significantly(p > 0.05),which proved that c-Myc/CLIC4 pathway regulates mitochondrial apoptosis pathway and mediates hippocampal neuronal injury.The results of in vivo experiments confirmed that the increase of neuronal apoptosis level during hippocampal injury induced by endotoxemia was related to the activation of mitochondrial apoptosis pathway by c-Myc/CLIC4 pathway.In vitro experiments have demonstrated that activation of the c-Myc/CLIC4 pathway is an important factor in lps-induced neuronal apoptosis,and c-Myc,as the upstream regulatory factor of CLIC4,plays an important role in the occurrence and development of hippocampal injury induced by endotoxemia.Combined with the results of this study,it is suggested that the c-Myc/CLIC4 pathway may be a key clinical target for the prevention and treatment of hippocampal injury induced by endotoxemia.