The Mechanism Study Of THC Induced Autophagy Through Modulation PI3K/AKT/mTOR Pathway In The Role Of Apoptosis Following Traumatic Brain Injury In Rats

Part 1Title:Tetrahydrocurcumin induced autophagy and provided neuroprotection in rats after traumatic brain injuryObjective:The aim of this study is to explore whether Tetrahydrocurcumin(THC)could investigate the expression of autophagy and has neuroprotection after traumatic brain injury(TBI).Methods:Rats were randomly divided into five groups:Sham,TBI,TBI+vehicle,TBI+THC(four subgroups:5mg/kg,25mg/kg,50mg/kg,100mg/kg)(each n=30).THC with purity more than 96%was dissolved in the vehicle(saline containing 1%dimethyl sulfoxide).Sixty minutes after TBI,THC or equivalent volumes of vehicle were administered via intraperitoneal injection.We used the Western blot analysis to detect the expression of autophagy related proteins LC3 and Beclin-1 according to different timepoints and THC concentrations.Rats were assessed on 24 h after TBI,beam-walking tests for neurological deficits were performed.The lower score on behalf of the more serious damage.Harvested the cortical tissue,we weight each sample recorded as wet weight,dried at 80℃ for 72 h,then weight.According to the formula,we calculated the brain water content.For Nissl staining,we stained the sections of paraffin-embedded brain tissue(5-μm thick)by cresyl violet according to the directions.Results:The expression of LC3-Ⅱ and Beclin-1 increased in the TBI group when compared with the sham group(p<0.01).Administration of THC increased LC3-Ⅱand Beclin-1 expression and as the THC concentration administered increased to 50 mg/kg and 100 mg/kg,the expression LC3-Ⅱ and Beclin-1 gradually declined relative to the 25 mg/kg group(p<0.001),but remained higher than in the TBI group.Administration of THC after TBI increased the expression of LC3-Ⅱ and Beclin-1 with time passing and reached its highest level at 24 h.We used the beam-walking test scores to evaluate the motor function of the rats after TBI at 24.The motor scores of the TBI and TBI+vehicle groups were lower than the scores of the Sham group(p<0.001).The THC-treated group exhibited reduced neurological deficits compared with the TBI group(p<0.01).The results showed that TBI and TBI+vehicle groups had significantly increased the brain water contents compared with the Sham group(p<0.001).Brain edema was attenuated in THC-treated groups.The results of Nissl staining showed that few Nissl bodies were found in TBI group(p<0.001),THC treatment significantly decreased the number of damaged neuronal cells compared with the TBI+vehicle group(p<0.05).Conclusion:Administration of THC improved the expression of autophagy and attenuated brain edema and improved neurobehavioral performance after traumatic brain injury in rats.Part 2Title:Tetrahydrocurcumin reduces apoptosis via the mitochondrial apoptotic pathway by modulating autophagy in rats after traumatic brain injuryObjective:The aim of this study is to investigate the function of autophagy and the mechanism of THC regulation of apoptosis after TBI in rats.Methods:MDA and GPx levels reflect lipid peroxidation and antioxidant levels,respectively.Tissue samples were homogenized in 2 ml phosphate buffer saline(PBS,pH 7.4)according to the manufacturer’s instructions of the commercial kits for MDA and GPx detection.We used the Western blot analysis to detect the expression of mitochondrial apoptosis related proteins Bax,Bcl-2,Cytochrome-c and Cleaved caspase-3.The brain tissue sections were examined for apoptotic cells using a terminal deoxynucleotidyl transferase dUTP nick end-labeling(TUNEL)detection kit following the manufacturer’s instructions.The sections were incubated with the TUNEL reagent.Fluorescence was imaged on an Olympus IX71 inverted microscope system.Results:MDA levels were higher and GPx activities were reduced in the TBI and TBI+vehicle groups relative to the sham group(p<0.01).Treatment with THC reduced MDA levels(p<0.05)and rescued GPx activity(p<0.01).We used Western blot analysis to examine expression of cleaved caspase-3,an indicator of apoptosis.Cleaved caspase-3 expression was higher at 24 h post-TBI relative to the sham group(p<0.001).Additionally,treatment with THC reduced the cleaved caspase-3 level relative to the TBI+vehicle group(p<0.05).Only a few TUNEL-positive cells were found in the cortex of the sham group,the apoptotic index of the cortex increased after TBI(p<0.001).THC treatment reversed this post-TBI apoptotic index.Then we examined expression of biological markers of the mitochondrial apoptotic pathway,namely Bax,Bcl-2,and Cytochrome-c upon TBI.When mitochondria were injured by TBI,the expression of Bax was increased(p<0.001),the Bcl-2 and the Cytochrome-c were decreased when compared with the sham group(p<0.001).Treatment with THC at 24 h post-TBI reversed the expression levels of mitochondrial Bax,Bcl-2 and Cytochrome-c relative to the TBI+vehicle group(p<0.05,p<0.01,p<0.01).In cytoplasmic protein levels.The level of cytoplasmic Bax protein decreased,whereas the levels of cytoplasmic Bcl-2 and cytochrome c proteins increased relative to the sham group(p<0.001).After treatment with THC,the level of cytoplasmic Bax protein increased,whereas the levels of cytoplasmic Bcl-2 and cytochrome c proteins decreased when compared with the TBI+vehicle group(p<0.01).To determine whether this autophagy activation is associated with the mitochondrial apoptotic pathway,we used the autophagy inhibitor 3-MA and examined the expression of mitochondrial apoptosis-associated proteins by western blot analysis.Expression of two autophagy markers,Beclin-1 and LC3-Ⅱ,were reduced in the TBI+THC+3-MA group when compared with the TBI+THC group(p<0.001).Similarly,treatment with THC+3-MA reduced expression of Bax,a protein associated with the mitochondrial apoptotic pathway,relative to the group treated with THC alone(p<0.01).Treatment with THC+3-MA increased the level of cleaved caspase-3 relative to treatment with THC alone(p<0.05).Conclusion:Administration of THC alleviates oxidative stress and enhances antioxidant enzyme activity after TBI.THC suppresses the mitochondrial apoptotic pathway by activating autophagy after TBI.Part 3Title:Tetrahydrocurcumin induced autophagy might via modulation of PI3K/AKT/mTOR pathway in rats after traumatic brain injuryObjective:The aim of this study is to investigate the mechanism of THC induced autophagy after TBI in rats.Methods:We used the Western blot analysis to detect the expression of PI3K/AKT/mTOR pathway related proteins AKT,p-AKT,mTOR and p-mTOR and the autophagy related proteins LC3 and Beclin-1.Results:We examined the expression of p-AKT and p-mTOR after TBI.The expression of p-AKT and p-mTOR were increased after TBI,compared with the sham group(p<0.01).Treatment with THC resulted in a significant reduced in p-AKT and p-mTOR activity compared to the TBI group(p<0.01).The expression of p-AKT and p-mTOR in the group preteated with LY294002(PI3K kinase inhibitor)were further decreased(p<0.01),while the expression of LC3-II and Beclin-1 were further increased compare with the THC treated group(p<0.01,p<0.05).Conclusion:Administration of THC could inhibite the activation of PI3K/AKT/mTOR pathway and THC might induce autophagy through modulation of PI3K/AKT/mTOR pathway.