Tanshinone ?A Protects Endothelial Cells From H₂O₂-Induced Injuries Via PXR Activation

Objective:The pregnane X receptor?PXR?is one of the nuclear receptor superfamilies,widely distributed in the liver,intestine,kidney and cardiovascular system,involved in the body of many drug metabolism enzymes and transporters regulation,can regulates all stages of xenobiotic metabolism and transport and is responsible for important inductive drug interactions,such as drug absorption,distribution,metabolism and excretion.Tanshinone ?A?Tan ?A?is a pharmacologically active substance extracted from the rhizome of Salvia miltiorrhiza Bunge?also known as the Chinese herb Danshen?,and is widely used to treat atherosclerosis.Our previous experience based on luciferase reporter gene p GLuc-Basic system PXR-CYP3A4 pathway demonstrated that Tan ?A is an efficacious PXR agonist that has a potential protective effect on endothelial injuries induced by xenobiotics and endobiotics via PXR activation.In vitro experiment we also illustrate that Tan ?A protects against LCA-induced cholestatic mice model and liver toxicity.But the underlying underline mechanism for its cardiovascular protection activity is not fully understood.Thus,we investigated whether Tan ?A can protect endothelial cells from oxidative stress,apoptosis and inflammation and whether those effects are mediated via the activation of PXR.Methods: We pretreated cells with or without different concentrations of Tan ?A for 24 h,then exposed the cells to 400 ?M H₂O₂ for another 3 h.The percentage of viable cells was evaluated using a cell counting kit-8-?CCK-8?assay kit.The expression of antioxidant stress signaling molecules was measured with q RT-PCR and Western-blot analyses.Cell apoptosis was tested by flow cytometry,Hoechst 33342 staining,and ELISA.High-throughput screening technology was used to detect translocation change of AIF.The inflammatory response was assessed by an ELISA for IL-8.Results:?1?Low concentration of Tan ?A had no obvious effect on endothelial cell viability,and cytotoxicity appears at high concentration.According to the cell viability experiment,we find the optimalconcentration of Tan ?A and H₂O₂ for following experiments.?2?Tan ?A?in 5?10?20 ?M?treated HUVECs for 24 h can increase the expression of PXR of m RNA and protein,and can also up-regulate the expression of CYP3A4,MDR1,GST,GSTM1 and GPx in a concentrationdependent manner.?3?Tan ?A can reduce the productions of ROS,MMP and apoptosis in HUVECs induced by H₂O₂,and this protective effects was significant reduced under PXR knockdown condition.?4?Tan ?A can significantly inhibit the apoptosis related molecules in HUVECs induced by H₂O₂,and this effects was inhibited while PXR was knocked down use si RNA.Conclusion: We confirmed the ability of Tan ?A to protect against endothelial damage through the inhibition of oxidative stress,apoptosis,and inflammation.Importantly,this study is the first to demonstrate that Tan ?A inhibits H₂O₂-triggered HUVEC cells damage and that this was associated with the activation of the important nuclear hormone receptor PXR.Simultaneously,Tan ?A inhibits apoptosis and inflammation induced by H₂O₂ in HUVECs dependent on PXR.This provides further evidence that PXR-mediated endothelial detoxification works as a gatekeeper for vascular defense against oxidative stress and apoptosis.More studies are needed to understand the physiological significance of Tan ?A-activated PXR activation protecting against oxidative stress in vivo.