Effects Of Sappan Extract On Autophagy Mediated Via PI3K/Akt/mTOR Signaling Pathway

Objective:The effects of sappan extract on PI3K/Akt/mTOR signaling pathway and autophagy mediated by ApoE^-/-mice AS model induced by high fat diet and HUVECs injury model induced by ox-LDL were observed,and the mechanism of sappan extract regulating autophagy was further explored.Method:1.Experiment 1:40 ApoE^-/-mice were randomly divided into blank group?model group?sappan extract group and atorvastatin group.Except blank group,the other three groups were fed with high fat diet for 8 weeks to construct AS mice model.From the first day of model replication,the blank group and the model group were given the same volume of sodium carboxymethyl cellulose suspension?0.1ml/10g?intragastric administration.The sappan extract suspension?0.1ml/10g?and atorvastatin suspension?0.1ml/10g?were given intragastrically once a day in the sappan extract group and the atorvastatin group,respectively.The general status and weight changes of mice in each group were observed;HE staining to detect the pathological morphology of aorta in mice;Oil red O staining was used to detect the lipid level of aortic sinus;TUNEL was used to detect the apoptotic level of endothelial cells;ELISA was used to detect blood lipid?TC?TG?LDL?HDL?and inflammatory factors?IL-6 and IL-8?;The expression levels of PI3K?Akt?p-Akt?mTOR and p-mTOR in arterial tissues were detected by immunohistochemistry;Western blot was used to detect Beclin-1?LC3I?LC3II?p62?PI3K?Akt?p-Akt?mTOR and p-mTOR in arterial tissue.The expression levels of PI3K?Akt and mTOR proteins in arterial tissues were detected by RT-PCR.2.Experiment 2:HUVECs were divided into blank group?model group?sappan extract group?inhibitor group and agonist group.Except blank group,the other four groups were induced by ox-LDL solution 100mg/L for 12 hours to construct HUVECs injury model.Before replication,the sappan extract group?inhibitor group and agonist group were given corresponding drug pretreatment for 6 hours.The specific methods were as follows:sappan extract group was given 80 micromol/L suspension of sappan extract,inhibitor group was given LY294002?PI3K inhibitor?10microM,agonist group was given Insulin?PI3K/Akt/mTOR signaling pathway agonist?200nM,while blank group and model group were not pretreated.After the model was replicated?ox-LDL solution induced for 12 hours?,cell proliferation level was detected by MTT method and cell viability was calculated;apoptotic level was detected by flow cytometry;the morphological changes of endothelial cells were observed by inverted phase contrast microscopy;and the level of autophagic flow was observed by transmission electron microscopy;detection of inflammatory factors IL-6 and IL-8,SOD and MDA related to oxidative stress and NO and eNOS related to cell function by ELISA;apoptotic proteins?Bax?Caspase-3 and Bcl-2??adhesion factors?ICAM-1 and VCAM-1??autophagic proteins?Beclin-1?LC3I?LC3II?p62?and?PI3K?Akt?p-Akt?mTOR?p-mTOR?were detected by Western blot.Result:Experiment 1:1.The weight of mice in each group increased gradually after feeding,but there was no significant difference between the groups?P>0.05?.2.The sappan extract group could significantly improve the pathological changes of aorta in AS model mice induced by high fat diet,and significantly inhibit the formation of plaque.Compared with the model group,the difference was statistically significant?P<0.01?.3.Sappan extract can significantly reduce the levels of TC?TG?LDL?IL-6 and IL-8,and increase the level of HDL.Compared with the model group,the difference is significant?P<0.01?.4.Sappan extract can significantly inhibit the apoptosis of endothelial cells,compared with the model group,the difference has significant statistical significance?P<0.01?.5.Sappan extract can down-regulate the expression of PI3K?p-Akt and p-mTOR proteins in arteries.Compared with the model group,the difference is significant?P<0.01?,but has no significant effect on the expression of Akt and mTOR proteins.Compared with the model group,there is no significant difference?P>0.05?.6.Sappan extract could down-regulate the expression of PI3K?Akt and mTOR in arteries,which had significant statistical significance compared with the model group?P<0.01?.7.Sappan extract could up-regulate the expression of Beclin-1 and LC3II/LC3I,down-regulate the expression of p62 protein.Compared with the model group,the difference was significant?P<0.01?.Experiment 2:1.The pretreatment of sappan extract could significantly increase the cell viability of HUVECs,which had significant statistical significance compared with the model group?P<0.01?.2.The pretreatment of sappan extract could significantly reduce the total apoptotic level of HUVECs,and the difference was statistically significant compared with the model group?P<0.01?.3.Sappan extract could significantly inhibit the expression of Bax and Caspase-3 protein,and up-regulate the expression of Bcl-2 protein.Compared with the model group,the difference was statistically significant?P<0.01?.4.Sappan extract could significantly inhibit the expression of PI3K,p-Akt and p-mTOR proteins in HUVECs.Compared with the model group,the difference was statistically significant?P<0.01?,but had no significant effect on the expression of Akt and mTOR proteins.There was no significant difference between the two groups?P>0.05?.5.Sappan extract could significantly up-regulate the expression of Beclin-1 and LC3II/LC3I in HUVECs,and down-regulate the expression of p62 protein.Compared with the model group,the difference was statistically significant?P<0.01?.6.Sappan extract can significantly increase the content of SOD and decrease the content of MDA in HUVECs.Compared with the model group,the difference is significant?P<0.01?.7.Sappan extract can significantly reduce the content of IL-6 and IL-8 in HUVECs,and the difference is significant compared with the model group?P<0.01?.8.Sappan extract could down-regulate the expression of ICAM-1 and VCAM-1 in HUVECs,and the difference was statistically significant compared with the model group?P<0.05?.9.Sappan extract can significantly increase the content of NO and eNOS in HUVECs,compared with the model group,the difference has significant statistical significance?P<0.01?.10.Sappan extract can significantly improve morphological changes caused by ox-LDL induced cell damage and dysfunction,promote the synthesis of autophagic lysosomes and promote the degradation of organelles.Conclusion:1.Sappan extract can inhibit the accumulation of lipid and the formation of atherosclerotic plaque in the arteries.2.Sappan extract can regulate the balance of proliferation and apoptosis of endothelial cells.3.Sappan extract can alleviate oxidative stress and inflammation and improve endothelial cell function.4.Sappan extract can inhibit the activation of PI3K/Akt/mTOR signaling pathway,and then promote the autophagy level of endothelial cells.