Nirmala Koju, Abdoh Taleb, , Zhou Jifang, , Lv Ge, and Ding Qilong
Background: Oxidative stress is associated with endothelial dysfunction, the initial step in the pathogenesis of various cardiovascular disorders.
Methods: HUVECs were divided into 3 groups including control group, model group (cells treated with 10-6 M Ang II for 24 hr) and treatment groups (cells treated with 0.1 mM, 0.25 mM, 0.5 mM and 1 mM ALA for 30 minutes and further incubated with 10-6 M Ang II for 24 hrs). The cytoprotective effect of ALA against Ang II was tested by MTT assay, ROS generation was evaluated using Di-chloroflourescein (DCFH) and Mitosox Red, NO, ET-1 and antioxidant enzymes (SOD, GPx, CAT) and IDH) activity were analyzed by their respective kits. Furthermore, Western blot was used to detect the protein expression of Akt, p-Akt, eNOS, p-eNOS, Nrf2, PGC1-α, Sirt3 and Nox4. RT-PCR was used to detect the gene expression of Nox4 and eNOS.
Results: The results indicated that ALA in a dose-dependent manner lowered Ang II-induced loss in endothelial cell viability, reactive oxygen species and ET-1 production. Furthermore, ALA pre-treatment increased NO Level and antioxidant enzyme activity which is crucial for ROS elimination. The western blot results showed that Ang II markedly decreased Akt, eNOS, Nrf2, PGC1-α, Sirt3 protein expression which was enhanced with the pre-treatment of ALA that was further inhibited by a Wortmannin and L-NAME.
Conclusion: Our findings demonstrated that ALA possesses antioxidant activity against Ang II-induced oxidative stress partly by antagonizing AT1 receptor, suppressing Ang II-induced NADPH oxidase, increasing antioxidant enzyme activity and up-regulating PI3K/Akt/eNOS/NO dependent signaling pathway.
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