Antioxidant Effect Of Persimmon Leaf Extract To Cell Models Of Alzheimer’s Disease And The Effect To Nrf2/HO-1 Pathway

BackgroundNowadays,with the increasing problems of aging population,increasingly importance has been attached to senile disease and neurodegenerative diseases.The incidence of neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease is increasing,which will lead to increasingly serious social problems.Alzheimer’s disease is a progressive disease of the central nervous system,which is characterized by mental decline and personality change.The pathogenesis of Alzheimer’s disease is not very clear,and there is no specific drug.Therefore,it is of great significance to study the mechanism of neurodegenerative diseases and to develop drugs that can effectively delay the degeneration of the disease.Amyloid cascade is an important hypothesis in the pathogenesis of AD,and the amyloid fibrils formed by beta amyloid protein aggregation are neurotoxic,which are also the major component of senile plaques.Aβ aggregation can induce oxidative stress,increase ROS and damage mitochondria function.Oxidative stress can promote the aggregation of Aβ and the phosphorylation of microtubule associated proteins TAU,and aggravate the imbalance of redox reaction in AD brain.Oxidative stress is a common key factor associated with various pathogenic mechanisms of AD,suggesting the complexity of the disease.Therefore,it is an effective method to prevent and cure AD by searching for suitable antioxidants and multi-target therapy.Nrf2lHO-1 is considered to be the most important endogenous antioxidant signaling pathway.Nrf2 is an important transcription factor in regulating oxidative stress,Under the condition of oxidative stress,Nrf2 enters into the nucleus,and enhance the expression of antioxidant enzyme gene,and then promove the resistance of cells to oxidative stress.HO-1 is one of the antioxidant enzymes downstream of Nrf2,protecting neurons by restoring mitochondrial function.Nrf2/HO-1 is an important protective system against oxidative stress.Recent research found that Persimmon leaves have antioxidant,hypolipidemic,hypoglycemic,antibacterial,hemostatic efficacy.The clinical found that the curative effect of persimmon leaf extract is better than that of Ginkgo biloba extract in circulation ischemia.The Persimmon leaf extract of Naoxinqing can protect the ischemia reperfusion induced brain injury,prevent neuronal excitotoxicity.Persimmon leaf extract has been used to control a variety of cerebrovascular disease.Our previous study found,PLE can improve the learning and memory ability of aging mice induced by D-galactose,improve the activity of antioxidant enzymes SOD,GSH-Px,So does it have the same effect on the nerve cells,especially in the AD cell model.Whether it can play a neuroprotective role by reducing the aggregation of A beta,reducing the activity of ROS and regulating the expression of Nrf2/HO-1?Based on the above background,we investigated whether PLE has antioxidant effect on AD cell model,and whether Nrf2 and HO-1 are involved in antioxidant regulation in this study.To provide the basis for drug development of AD.ObjectiveHEK293 cells(20E2)carrying the Sweden mutant APP gene were used as AD cell model.By detecting the level of A beta 1-42 in cell supernatant,the fluorescence intensity of intracellular ROS and the mitochondrial membrane potential to investigate the effect of PLE on oxidative stress.And study the action mechanism though Nrf2/HO-1 signaling pathway.Methods:1.We detecte the expression of APP protein by Western Blotting and the extracellular level of Aβ1-42,by ELISA kit in 20E2 group and NC group,to determine whether the cell model is successful.2.The effect of different concentrations of PLE on the Cell Viability was measured by CCK-8.And then select the best concentration.3.Set groups:SH-SY5Y cell as normal control group(NC group),20E2 as the model group(20E2 group),20E2 treated with PLE as treated group(20E2+PLE group).4.Reactive oxygen species(ROS)levels of each group were detected with DCFH-DA fluorescent probe.5.Mitochondrial membrane potential was detected by JC-16.The extracellular level of Aβ1-42 were detected by ELISA kit7.Cytoplasmic Nrf2,Nuclear Nrf2,Whole-cell HO-1 were detected by Western blotting.Rusult:1 The expression of APP protein and Aβ1-40:The expression levels of APP protein and Aβ1-40 were detected with a higher expression in model group compared with those in NC group2 The effect of different concentrations of PLE on the Cell Viability:PLE could increase the activity of SH-SY5Y cells at 1.5-6 g/mL(P<0.05),and increase the activity of 20E2 in 3-6 g/mL(P<0.05),PLE also inhibited cell activity after more than 6 g/mL,In order to reduce the toxicity of drugs,the final selection of 3 g/mLfor drug intervention concentration.3 Reactive oxygen species(ROS)levels:Compared with NC group,the intracellular ROS fluorescence signal in the 20E2 group was significantly enhanced(P<0.05),the intracellular oxidative damage was obvious.After PLE intervention,the fluorescence signal of ROS in 20E2+PLE group was decreased(P<0.05)4 Mitochondrial membrane potential was detected by JC-1:Compared with group NC cells,20E2 group display weak red fluorescence(P<0.05)and strong green fluorescence(P<0.05).Mitochondrial membrane potential was decreased.With the intervention of PLE,20E2+PLE group red fluorescence intensity was enhanced(P<0.05)and green fluorescence was weak(P<0.05),mitochondrial membrane potential rise.5 The extracellular level of Aβ1-42 by ELISA kit.Compared with the NC group,the extracellular level of Aβ1-42 in the 20E2 group was significantly increased(P<0.05).After PLE intervention,the extracellular level of Aβ1-42 was significantly reduced in the 20E2+PLE group(P<0.05).6 the expression of Cytoplasmic Nrf2,Nuclear Nrf2,Whole-cell HO-1:Compared with NC group,the expression of Nrf2 protein and HO-1 protein increased in 20E2 group(P<0.05);Compared with 20E2 group,the expressions of Nuclear Nrf2 and Whole-cell HO-1 were up-regulated and the Cytoplasmic Nrf2 were down-redulated in 20E2+PLE group(P<0.05).Conclusion:PLE can reduce the level of oxidative stress of model group effectively,it possibly reduce the aggregation of AP and prevent oxidizing via activating Nrf2/HO-1 pathway.