Effect And Mechanism Of Green Tea Polyphenols On Secondary Brain Injury After Intracerebral Hemorrhage

Objective Intracerebral hemorrhage(ICH)is a global cerebrovascular disease with high prevalence and high disability rate.At present,there is no effective agents and therapy for ICH clinical treatment,which seriously affects the quality of life and brings a heavy burden to the society and the families of patients.Recent studies have found that oxidative stress plays an important role in Secondary brain injury(SBI)after ICH,which can cause many pathophysiological changes and injuries at the brain.Green tea polyphenol(GTP)could penetrate the blood-brain barrier,and show strong antioxidant capacity and less toxic effects,so it may play an important role in cerebrovascular and nervous system diseases.However,the effect of GTP on cerebral hemorrhage injury and its mechanism are still unclear and need to be further studied.Therefore,this study intends to establish a rat model of cerebral hemorrhage and an in vitro oxidative damage model with primary neurons to study the effects of GTP on secondary brain injury caused by cerebral hemorrhage,and to further explore the molecular target and mechanism of GTP on oxidative injury after cerebral hemorrhage..Methods Male SD rats were selected to establish the rat model of acute intracerebral hemorrhage with collagenase and heparin injection.Rats were ramdonly divided as sham operation group,model control group and GTP group,and each group was further divided into 3 d and 7 d,respectively.The rats in the GTP group were intraperitoneally injected with 60mg/kg green tea polyphenol once a day,and the rats in the model control group and the sham operation group were intraperitoneally injected with the same volume of normal saline.After neurobehavioral scores were performed at the corresponding time points,brain tissue was collected to determine Malondialdehyde(MDA).MDA content,Superoxide dismutase(SOD),Glutathione peroxidase(GSH-PX)activity,and hematoxylin eosin(H&E)staining were used to evaluate the lesions in cortex and hippocampus of rats.To observe the protective effect of GTP on secondary cerebral hemorrhage injury in rats,the apoptosis of neurons in cortex and hippocampus was detected by TUNEL staining mediated by deoxyribonucleotide terminal transferase.Neonatal cortical neurons of SD rats were isolated andcultured,followed by stimulating with hydrogen peroxide(H2O2)for 24 hours,to simulate the peroxidation injury after intracerebral hemorrhage in neuron cell culture model.Control group(CTRL),solvent(dimethyl sulfoxide,DMSO)group,epigallocatechin-3-gallate(EGCG,the main active monomer of green tea polyphenols)group,and N-acetyl-L-cysteine group(NAC,the antioxidant used as positive control)were designed for the cell culture study.Firstly,the contents of Reactive oxygen species(ROS)and MDA in each group were detected,and the activities of SOD and GSH-PX were determined.Secondly,the apoptosis of neurons in each group was detected by TUNEL staining.Lastly,the p62-KEAP1Nrf2 pathway protein and other indicators were detected by Western blot to further explore the molecular mechanism of green tea polyphenols in improving oxidative damage after intracerebral hemorrhage.1.Evaluation of neurofunctional behavioral effects in rats:After the establishment of cerebral hemorrhage rat model by collagenase and heparin injection,the neurobehavioral score was conducted to observe the protective effect of GTP on the neurobehavioral injury after cerebral hemorrhage.2.Detection of antioxidant capacity of rat brain tissue:The rats in each group were sacrificed,and the corresponding brain tissues were collected to determine MDA content by Thibabituric Acid(TBA)method;SOD activity was determined by xanthine oxidase method;the activity of GSHPx was determined directly by 5’,5-dithio-p-nitrobenzoic acid(DTNB)method to detect the effect of GTP on the antioxidant capacity of brain tissue after cerebral hemorrhage in rats.3.Detection of pathological injury and neuronal apoptosis in rat brain tissue:After HE staining,the pathological changes of cortical and hippocampus of rats after cerebral hemorrhage were observed under 40x light microscope.After TUNEL staining,the effects of GTP on neuronal apoptosis in cortex and hippocampus of rats after cerebral hemorrhage were also observed and analyzed under 40x light microscope.4.Detection of oxidative stress injury in primary neurons of rats:Newborn cortical neurons of SD rats were isolated andcultured,followed by stimulating with hydrogen peroxide(H2O2)for 24 hours,to simulate the peroxidation injury after intracerebral hemorrhage in neuron cell culture model.H2DCFDA(DCF)staining was used to detect the production of ROS;thiobarbituric acid(TBA)method was used to determine the content of MDA;water-soluble tetrazole-8(WST-8)colorimetric method was used to determine the activity of SOD;and nicotinamide adenine dinucleotide phosphate(NADPH)method was used to detect the activity of GSH-PX.5.Detection of apoptosis of primary neurons after oxidative stress induction:The TUNEL staining of cells was performed by immunohistochemical technique,and the effects of H2O2 and EGCG on the apoptosis of primary nerve cells were observed and captured under 40X light microscope.6.Investigation of the p62-Keapl-Nrf2 molecular pathway:Western blot was used to detect the expression levels of Nrf2 and its upstream proteins p62 and Keap1,to explore whether EGCG can alleviate the oxidative injury of neurons via p62-Keapl-Nrf2 pathway.Results 1.Compared with rats of sham operation group,the neurobehavioral scores of rats in the model group were significantly decreased.After 3 d administration of GTP,there was no statistical difference in the neurobehavioral scores between the GTP group and the model group.However,after 7 d treatment of GTP,the neurobehavioral scores of rats in the GTP-treated group improved significantly compared with the model group,indicating that GTP can effectively improve the neurobehavioral damage after cerebral hemorrhage in rats.2.The detection of antioxidant indexes in brain tissue of rats in each group showed that compared with the sham operation group,the content of MDA in brain tissue of rats in model group and GTP group increased,while the activities of SOD and GSH-Px decreased.After 3 d of GTP,there was no statistical difference in the detection indexes between the green tea polyphenols group and the model group.However,after 7 d of GTP,the content of MDA in brain tissue of green tea polyphenols group decreased while the activities of SOD and GSHPx increased,compared with model group.This set of results indicate that GTP treatment can effectively enhance the antioxidant capacity of brain tissue after cerebral hemorrhage in rats.3.Studies on the staining of brain tissue sections of rats showed that the administration of GTP for 7 d could significantly relieve the pathological damage of cerebral hemorrhage at cortex and hippocampus tissue in rats,and TUNEL staining showed that the apoptosis of nerve cells in cortex and hippocampus after cerebral hemorrhage was significantly reduced by GTP treatment.These results indicate that GTP can effectively improve the pathological damage of cerebral hemorrhage in brain tissue and significantly inhibit the apoptosis of nerve cells.4.The results of cellular experiments showed that the intracellular ROS aggregation in primary neurons was significantly increased after exposure to H2O2,compared with the control group;EGCG or ROS inhibitor N-acetylcysteine treatment can effectively prevent intracellular ROS accumulation.At the same time,the MDA content of primary neurons increased after H2O2 treatment,while the activities of SOD and GSH-PX decreased.Treatment with EGCG or ROS inhibitor N-acetylcysteine could effectively reduce the MDA content,and significantly increase the activities of SOD and GSH-PX.These results suggested that EGCG could exert the same antioxidant effect as NAC,and thus effectively inhibit the oxidative damage of primary neurons in rats.5.TUNEL staining showed that the TUNEL positive cells increased after H2O2 treatment,indicating that H2O2 induced neuronal apoptosis;either EGCG or ROS inhibitor NAC can effectively block the increase of TUNEL positive cells,suggesting EGCG decreased neuronal apoptosis.Experimental data from primary cell culture is consistent with the results of animal experiments,which further suggested that GTP can effectively reverse the oxidative damage induced by intracerebral hemorrhage at brain tissue,and inhibit the neuronal apoptosis caused by oxidative damage.6.Western blotting showed that the expression of p62 and Nrf2 protein decreased and the expression of Keap1 increased in the primary neurons after treatment with H2O2.However,both EGCG and NAC could effective enhance the expression of p62 and Nrf2,accompanied by the decrease of Keap1 expression,indicating that the p62-Keapl-Nrf2 pathway may be involved in the neuroprotective effect mediated by EGCG.These results suggest that GTP can reverse the oxidative damage caused by brain hemorrhage by regulation on p62-Keap1Nrf2 signaling pathway.Conclusions 1.The experimental results of cerebral hemorrhage model rats showed that green tea polyphenols could enhance the antioxidant capacity of brain tissue,reduce the apoptosis of nerve cells,and effectively improve the pathological damage of brain tissue after cerebral hemorrhage in rats.2.The results of cell experiments showed that green tea polyphenols could effectively improve the antioxidant capacity and inhibit cell apoptosis after oxidative damage of primary neurons in rats,and the antioxidant effect was related to the activation of p62-Keapl-Nrf2 signaling pathway.3.GTP can improve oxidative stress injury and neuronal apoptosis after intracerebral hemorrhage,and then play a protective role in the secondary injury of intracerebral hemorrhage.