Effects Of Synthetic Peptide,AREGEM On Proliferation,Apoptosis And Oxidant Of Vascular Smooth Muscle Cells Incubated With High Glucose

Many studies have shown that oxidative stress is closely related to the occurrence development and complications of various chronic diseases including cardiovascular disease,cancer and diabetes.Antioxidant therapy can reverse the damage of oxidative stress to tissue,thus preventing or delaying the occurrence and progress of many diseases.In this study,we applied high glucose-incubated vascular smooth muscle cell(VSMCs)model and used the selected highly active peptides to further study the biological antioxidant activity of peptides at the cellular level and its effects of peptides on proliferation and apoptosis of VSMCs.The main research contents and results are as follows:1.The antioxidant activity of peptide AREGEM was evaluated in vitro.Based on the screening of the wheat germ antioxidant peptide sequence AREGETVVPG,we obtained the sequences of MAREGETVVPG,MAREGE and AREGEM by modifying the peptide sequences.The antioxidant activity of these synthetic peptides were evaluated by in vitro chemical method based on HAT and HET mechanisms.Highly active antioxidant peptide sequence AREGEM was screened.2.Biological antioxidative activities of peptide AREGEM at the cellular level was further studied.The activities of total antioxidation capacity(T-AOC),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)and malondialdehyde(MDA)were measured by enzyme-linked immunosorbent assay.The results show that the antioxidative enzyme(T-AOC,GSH-Px and SOD)activities in VSMCs exposed to high glucose were significantly increased in the presence of AOP(p<0.01),whereas the MDA level was decreased(p<0.01).In addition,the 2',7'-dichlorofluorescein diacetate(DCFH-DA)fluorescent probe assay further demonstrated that AREGEM reduced intracellular ROS accumulation in VSMCs.These findings indicated that the peptide exhibited a protective role against high glucose-induced oxidative stress in VSMCs.3.The protection effects of AREGEM on the VSMCs proliferation induced by high glucose was investigated in different perspectives including cell proliferation and cell cycle.MTT assay results showed that AREGEM significantly attenuated high glucose-induced VSMCs proliferation.For example,the cell proliferation was from 138.9 ± 5.1%to 104.4 ± 4.4%of the normal glucose control value when the peptide concentration was increased from 7.5 ?M to 45 ?M,which is comparable to that of a normal glucose condition(p>0.05,NS).As expected,AREGEM had neither toxicity(p>0.05)to the cells nor promotion effect in the growth within the concentration range of 7.5 ?M to 60 ?M.Propidium iodide(PI)single-staining method demonstrated that the high glucose environment could decrease the number of VSMCs in G0/G1 phase and increase the number of cells in S phase.After treatment with AREGEM at different concentrations(15 ?M and 45 ?M),the number of cells arrested in G0/G1 phase increased while the number of cells in S phase decreased and the proliferation index decreased significantly.The results showed that the peptide AREGEM mainly inhibited the transition of cells in G0/G1 phase into S phase,thus inhibiting cell proliferation.4.The effects of AREGEM on the apoptosis of VSMCs in high glucose was investigated.The apoptotic cells were detected by fluorescence microscopy combined with modem apoptosis detection technique.The expression of relevant proteins involved in apoptosis,such as Bcl-2 and Bax,were investigated by western blot analysis.Both Hoechst 33258 staining method and Annexin V-FITC/PI double staining showed that glucose at high concentrations could inhibit the apoptosis of smooth muscle cells.However,this apoptotic inhibition of high glucose was significantly attenuated at the presence of AREGEM.Thus,our results demonstrated that high glucose levels prevent serum deprivation-induced apoptosis,whereas this effect could be abolished by AREGEM treatment.However,AREGEM had no effect on the serum deprivation cell apoptosis level in normal glucose condition.The mechanism of AREGEM attenuating the inhibitory effects of high glucose on cellular aptoposis may be associated with its capacity of modulating caspase-3 activity and the ratio of Bcl-2/Bax protein within the cells.In addition,the level of intracellular ROS was increased in VSMCs treated with high glucose,resulting in an increase in the phosphorylation level of AKT at the Ser473 site,whereas addition of AREGEM reduced the AKT phosphorylation in VSMCs cultured with high glucose.