| Objective:A series of novel halophenol compounds previously prepared by our group exhibited significant protective activities against H2O2-induced injury on HUVECs, among which 5, 2’-dibromo-2,4’,5’- trihydroxy-diphenyl-methanone(LM49), 2,3-dibromo- 4,5-dihydroxydiphenyl-methanone(LM48) and 3’-chloro-3,4-dihydroxy-diphenyl- methanone(LM46) showed high protective effects. These compounds also have significant anti-atherosclerotic effects and protective effects against myocardial ischemia-reperfusion injury in rats. These protective activities indicated that these compounds may have potential in cardiovascular disease through inhibition of oxidative stress. However, their action mechanisms are still unclear. This study aimed to search for the possible target and mechanism, through which halophenols compounds exhibited cytoprotective effects, and to provide the basis for the design and further structural optimization of new halophenols compounds with high activity. Methods:The heme oxygenase-1(HO-1) expression induced by LM49, LM48 and LM46 was first investigated by Western blot. Meanwhile, pretreatment with Zn PP, an inhibitor of HO-1, the protective effect of these compounds were detected by MTT. In addition, the further research confirmed that the levels of reactive oxygen species(ROS) and the expression of tumor necrosis factor-α(TNF-α) in H2O2-induced EA.hy926 by flow cytometry and ELISA. Meanwhile, Zn PP also weakened their inhibitory effects of the three compounds on ROS and TNF-α expression. Moreover, six other halophenols derivatives(LM50, LM51, LM52, LM53, LM54 and LM55) with different cytoprotective activities were detected for their induction on HO-1 expression. Further test revealed that LM49 induced HO-1, Nrf2, p38, JNK, Erk1/2 and AKT, PKC-δ. Results:The results indicated that LM49, LM48 and LM46 showed obvious induction on HO-1 expression. The order of HO-1 expression was LM49>LM48>LM46, which was in accordance with the their cytoprotective activities. In addition, the further research confirmed that these compounds could significantly reduce the levels of ROS and inhibited the expression of TNF-α in H2O2-induced EA.hy926 in the following order: LM49>LM48>LM46. Meanwhile, Zn PP also weakened their inhibitory effects of the three compounds on ROS and TNF-α expression. Meanwhile, pretreatment with Zn PP, could result in an obvious decrease of their protective effect. Moreover, six other halophenols derivatives with different cytoprotective activities were detected for their induction on HO-1 expression. The results showed that the HO-1 expression levels of the six compounds were also consistent with the cytoprotective activities. However, the action mechanisms involved HO-1 are still unclear. Further test revealed that LM49 induced HO-1 and Nrf2 expression in a dose-dependent manner and activated the phosphorylation of Erk1/2 and AKT instead of p38, JNK and PKC-δ. The inhibitor of Erk and PI3 K, PD98059 and Wortmannin were used to detect the expression of HO-1. Conclusions:(1) HO-1 protein played an important role in antioxidative and cytoprotective effects of halophenol compounds. LM49,LM48 and LM46 induced the expression of HO-1 protein, which futher suppressed the expression of TNF-α and reduced the level of ROS to exhibited cell protection activity.(2) Halophenol compounds with high cytoprotective activitives induced transcription activation of Nrf2 and regulated expression of HO-1 protein mediated by Erk1/2 and PI3K/AKT. |
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