Oridonin Attenuates Low Shear Stress-induced Endothelial Dysfunction And Oxidative Stress By Activating NRE2 Pathway

Atherosclerosis occurs widely in the population,leads to many clinical problems including ischemic heart disease,stroke,ischemia of lower extremity arteries,and make a great burden on medical care worldwide.It is widely realized that atherosclerosis always begins with dysfunction of vascular endothelial cells,and the low shear stress produced by disturbed blood flow is the main cause of endothelial dysfunction.Oridonin,the major active ingredient of the traditional Chinese medicinal herb Rabdosia rubescens,has been used to treat cancer and inflammation because of its apparent antioxidative activity.However,the effect of oridonin about anti-atherosclerosis is rarely reported.We screen out some potential targets of oridonin by network pharmacology,and four of them are highly related,which are adenylate cyclase 1(ADCY1),adenylate cyclase 2(ADCY2),Nuclear factor erythroid 2-related factor 2(NRF2),and NROB1.NRF2 is reported to be a mechanosensitive transcription factor sensing blood flow and transfer the mechanical signal to biological signal.The NRF2 is downregulated by low shear stress,which promote the occurrence of endothelial dysfunction and oxidative stress.That’s why the mechanosensitive transcription factors such as NRF2 is regarded as the potential target for the prevention and treatment of atherosclerosis.In this study,we explored whether oridonin can improve endothelial cell dysfunction caused by low stress and the correlation between the effect and NRF2 in vitro experiments.Also,We used zebrafish as experimental animals to observe the drug effects of oridonin on lipid-lowering,antioxidant and anti-inflammatory.Objective:To investigate whether oridonin can improve low-shear-induced vascular endothelial cell dysfunction and whether oridonin has the potential to treat atherosclerosis.Methods:We predicted the potential targets of oridonin to treat cardiovascular disease by text mining with chemometric and chemogenomic methods.After three times verification,we found that ADCY1,ADCY2,NRF2,and NROB1 are highly related with the effect of oridonin for cardiovascular disease,and we chose NRF2 for further investigation.The EA.hy926 cells were treated with DMSO(0.1%),oridonin(100μg/m L)or oridonin(100μg/m L)+NRF2 si RNA(0.1μmol/L)for 24 h,and then placed on the lower plate of the chamber under low shear stress(3 dyn/cm~2)induced by continuous fluid flow.The NO level,ET-1 m RNA expression,e NOS m RNA expression,and PGIS m RNA expression in EA.hy926 cells were analyzed to determine EC dysfunction.Meanwhile,the EA.hy926 cells were also collected to measure secreted SOD activity,MDA content,GSH content and GSSG content using the ELISA after the LSS experiment.we fed zebrafish larvae with a high-cholesterol diet(HCD)to establish a zebrafish AS model.5 dpf Tg(fli1:EGFP)zebrafish larvae,constitutively expressing GFP in ECs,were fed for 10 days with the HCD enriched with 4%cholesterol and supplemented with 10μg/g of fluorescent cholesteryl ester analog.After anesthesia,we observed the zebrafish with a fluorescence microscope.We used Nile red staining to evaluate whether oridonin could reduce lipid accumulation.We also used Tg(mpx:EGFP)zebrafish(neutrophils are specifically labeled with green fluorescent protein)to evaluate the anti-inflammation effect of oridonin.We used DCFH-DA to detect ROS expression in HCD zebrafish and evaluate the anti-oxidative effect of oridonin.Results:Oridonin significantly increased the m RNA expression of NRF2,HMOX1,and NQO1 in EA.hy926 cells.Low shear stress(LSS)significantly reduces NO activity and the m RNA of e NOS and PGIS,100μg/m L oridonin significantly increase NO activity,gene silencing of NRF2 counteracted the effects of oridonin on intracellular NO activity.LSS significantly induced ROS activity,which was significantly inhibited by 100μg/m L oridonin,gene silencing of NRF2 counteracted the effects of oridonin on the activity of ROS.Compared with the AS group,treatment of 50μg/m L and 100μg/m L oridonin significantly decreased lipid accumulation around the blood vessel of zebrafish.The Nile red staining also showed that oridonin reduced lipid level in HCD zebrafish.The treatment of 50μg/m L and 100μg/m L oridonin significantly decreased oxidative stress and inflammation in HCD zebrafish.Conclusion:Oridonin effectively improved endothelial dysfunction and oxidative stress induced by low shear stress,and we thought that the atherosclerotic protection of oridonin is based on the NRF2 pathway.Also,oridonin reduced lipid level and significantly decreased oxidative stress and inflammation in HCD zebrafish.