The Study On The Molecular Mechanism Of EGCG Against Mitochondrial Dysfunction After Experiment Subarachnoid Hemorrhage

Background Subarachnoid hemorrhage (SAH) is a common disease induced by the rupture of aneurysm and cerebral vascular malformation, with the characteristics of high death rate, high disability rate. Mitochondrial dysfunction, which are related to the death of neurons, has been observed in Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic lateral sclerosis (ALS) diseases. Epigallocatechin gallate (EGCG) is a main component of green tea polyphenols. In recent years, many researchers focus on the protective effects of EGCG on the disease of cancer, inflammatory, cardiovascular, and neurodegenerative diseases. In this study, PC 12 cell was used to establish the in vitro SAH model. Therefore, the mechanisms of mitochondrial quality control was used to assay the EGCG neuro-protective effects based on autophagy, mitochondrial membrane potential, and ROS alteration.Method 1.To selected the best protective concentration of EGCG on PC 12 SAH model by MTT assay.2. The cells were randomly divided into three groups:a:control group; b:SAH group; c:EGCG (50 μmol/L) pre-treatment group. The level of △Ψm (mitochondrial membrane potential) and ROS (reactive oxygen species) were investigated by mitochondrial membrane potential detection kit and active oxygen detection kit.3. Detecting the expression of mitochondrial fission and fusion genes:FIS1, MFN1,OPA1, PINK1, and PARKIN gene.4. The expression of autophgosome in every group was observed through transmission electron microscope. The expressions of ATG5, BECLIN1, LC3-Ⅱ gene in all groups were investigated by qRT-PCR.Results 1.The cells proliferation ability was significantly increased after SAH (P< 0.01), whereas after EGCG pre-treatment, cell viability decreased to normal levels (P< 0.05), especially at 50 umol/L.2.Compared with the control group, the red fluorescence intensity decreased significantly in SAH group. The fluorescence intensity obvious increased compared with SAH group, which retained to control group. Quantitative analysis found that depolarization of the mitochondrial membrane potential in SAH group changed obviously, with statistical significance compared with the control group (P< 0.05); after pre-treatment with EGCG, the mitochondrial membrane potential increased significantly (P< 0.01 vs SAH, P< 0.001 vs Con). The expression of ROS in SAH group also significantly increased when compared with the control group (P< 0.01). After EGCG pre-treatment, the level of ROS were significant decreased (P< 0.01 vs SAH, P< 0.001 vs Con).3. Effect of EGCG on mitochondrial quality control. The expression of FIS1, MFN1, OPA1 increased in the SAH group, but the expression of FIS1 were significant differences compared with the blank control group (P< 0.05). However, after EGCG pre-treatment, the expression of FIS1、MFN1、OPA1 decreased compared with SAH group. The expression of PINK1 and Parkin increased in the SAH group, showing significant difference compared with the blank control group (P< 0.01). The expression of PINK1 decreased in EGCG group, which were significant difference with SAH group and control group; the expression of PARKIN has no significant change in SAH group.4. Effect of EGCG on autophagy. The expression of autophgosome in every group was observed by using transmission electron microscope. Compared with the SAH group, the number of autophagic bodies decrease in EGCG pre-treatment group. Compared with the normal group, the expression of LC3-II and BECLIN1 increased, but the expression of ATG5 gene decreased in SAH group. After EGCG pre-treatment, the expression of autophagy related genes were all decreased (P< 0.05).Conclusion The neuro-protection effects of EGCG was relied on mtichondrial quality control system, through which autophagy was activated to eliminate the damaged mitochondria. Therefore, after EGCG pre-treatment, mitochondrial membrain poteintial and ROS retained to normal levels after SAH.