Studies On Pharmacodynamics And Mechanism Of Areca Catechu L Extracts Against High-altitude Hypoxia

High-altitude hypoxia will lead to metabolic changes and increase ROS synthesis in vivo.Hypoxia induced ROS will lead to oxidative damage to the body,including protein carbonylation,DNA damage,lipid peroxidation and so on.High-altitude hypoxia can also cause massive release of inflammatory factors.Recent studies have shown that Areca catechu L extracts have various biological activities such as anti-oxidation and anti-inflammation.However,the effects of Areca catechu L extract on the oxidative damage and inflammatory response induced by hypoxia were not reported.We will study from the following aspects:First,the Areca catechu L was screened for its anti-hypoxic potential by using the simulated hypoxic rat model.The dose of 250 mg/kg of arecoline,anhydrous ethanol extract of Areca catechu L,50%ethanol extract were tested for blood gas,histopathological changes and oxidative stress.The result of the experiment showed that the hypoxemia can be improved by anhydrous ethanol extract of Areca catechu L and 50%ethanol extract.The PaO₂ and SatO₂ in hypoxic rats were increased respectively by 12.99%,and 2.46%（P<0.05）by anhydrous ethanol extract,and20.00%、5.79%（P<0.01）by 50%ethanol extract.The result of pathology indicated that the inflammatory injury of myocardial tissue and the infiltration of inflammatory cells can be reduced by the anhydrous ethanol extract of Areca catechu L and 50%ethanol extract,as well as the damage of brain tissue and the nuclear condensation of neuronal cells can be improved.The oxidative stress data showed that the activity of antioxidant enzymes was increased and the lipid peroxidation injury was reduced by the anhydrous ethanol extract of Areca catechu L and 50%ethanol extract.Thereinto,in the anhydrous ethanol extract of Areca catechu L group,the activity of SOD in myocardium and brain tissue was respectively increased by 15.15%and 7.82%（P<0.05）,the content of MDA in brain tissue decreased by 28.20%（P<0.05）.In 50%ethanol extract group,the SOD activity in myocardium and brain tissue was increased significantly by16.79%and 7.89%（P<0.05）.The content of NO in brain tissue was increased significantly by29.03%（P<0.05）.At the same time,the arecoline had no effect on blood gas,histopathological changes and oxidative stress of hypoxic rat.Second,the anti-plateau anoxic pharmacodynamics of anhydrous ethanol extract and 50%alcohol extract of Areca catechu L was studied by using the plateau hypoxia-induced rat model.Three dose of anhydrous ethanol extract of Areca catechu L（250 mg/kg,500 mg/kg,and 750mg/kg）and 50%ethanol extract were performed for blood gas,histopathological changes,oxidative stress of myocardium and brain tissue and serum inflammatory factors.The results of acute hypoxia in plateau showed that the hypoxemia can be improved by the low and medium dosage of Areca catechu L anhydrous alcohol extract.PaO₂ and SatO₂ in low dose group were respectively increased by 16.78%and 6.29%（P<0.05）,and in the middle-dose group which were increased by 13.40%and 5.13%respectively（P<0.05）.The result of pathology indicated that the hyperchromatic cytoplasm of myocardial tissue and the nuclear condensation of neuronal cells can be improved by anhydrous ethanol extract of Areca catechu L and 50%ethanol extract.,as well as the damage of brain tissue and the infiltration of inflammatory cells can be reduced,the nuclear condensation of neuronal cells can be improved.At the same time,both anhydrous ethanol extracts and 50%ethanol extracts could release the oxidative stress in hypoxic rats.In the medium and high dose groups of Areca catechu L anhydrous alcohol extract,the content of MDA in myocardial tissue of the rats was significantly decreased（P<0.05）and the activity of GSH was significantly increased,in the high dose groups,the activity of SOD and the content of NO were significantly increased（P<0.05）.;as for the brain tissue,in the medium and high dose groups of Areca catechu L anhydrous alcohol extract,the activity of SOD and GSH was significantly increased（P<0.05 or P<0.01）,in the high dose group,the content of NO was significantly increased（P<0.05）.The activity of SOD in myocardial tissue was significantly increased in the medium and high dose groups of the Areca catechu L 50%ethanol extract,the content of NO was significantly increased in the high dose groups;as for the brain tissue,the activity of GSH was significantly increased in the high dose groups,the content of NO was all significantly increased（P<0.05）in the low,middle and high dose groups.In addition,the release of pro-inflammatory cytokines MCP-1,IL-6,TNF-αcan be inhibited by the anhydrous alcohol extract and 50%ethanol extract.The expression of MCP-1 in the serum of each drug group was significantly decreased（the fold difference was less than or equal to 0.85）.The expression of IL-6 in medium and high doses of 50%ethanol extract were decreased respectively by 0.85 and 0.79 times.The expression of TNF-αrespectively decreased by 0.60,0.66,and 0.73 fold in medium and high dose of anhydrous ethanol extracts and high dose of 50%alcohol extracts.Third,the anhydrous ethanol extract of Areca catechu L was further explored for the anti-hypoxia mechanism by establishing an in vitro hypoxia model of H9C2 cardiomyocytes.Three dose of Areca catechu L anhydrous alcohol extract（5,10,and 20μg/mL）was investigated for the effects on H9C2 cell survival rate.The 20μg/mL Areca catechu L alcohol extracts was tested for the the effects on the intracellular MDA content,SOD,GSH activity,Nrf2 and Caspase-3 mRNA expression levels.The results showed that the survival rate of H9C2 cells（P<0.01）and the activities of intracellular antioxidant enzymes SOD and GSH could be significantly increased（respectively by 14.90%and 28.94%,P<0.05）,the relative expression of Nrf2 mRNA was decreased by 0.66-fold（P<0.05）by the anhydrous ethanol extract of Areca catechu L.The following conclusions can be drawn from the above study:1)anhydrous ethanol extracts and 50%ethanol extracts of Areca catechu L are effective components to against hypoxia.2)anti-hypoxia effect of Areca catechu L extract is related to its antioxidant capacity,excess oxygen free radicals clearance,oxidative stress damage reduction and inflammatory response inhibition.3)Areca catechu L alcohol extract can enhance hypoxia tolerance of H9C2 cell and increase the activity of antioxidant enzyme,so that mRNA expression of Nrf2 in hypoxic cells tend to normoxia level,which protects cells from oxidative stress producing the anti-hypoxia effect.