Tanshinone IIA Protects Endothelial Cells from H₂O₂-Induced Injuries via PXR Activation

Abstract

Tanshinone IIA (Tan IIA) 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. The pregnane X receptor (PXR) is a nuclear receptor that is a key regulator of xenobiotic and endobiotic detoxification. Tan IIA is an efficacious PXR agonist that has a potential protective effect on endothelial injuries induced by xenobiotics and endobiotics via PXR activation. Previously numerous studies have demonstrated the possible effects of Tan IIA on human umbilical vein endothelial cells, but the further mechanism for its exerts the protective effect is not well established. To study the protective effects of Tan IIA against hydrogen peroxide (H₂O₂) in human umbilical vein endothelial cells (HUVECs), we pretreated cells with or without different concentrations of Tan IIA for 24 h, then exposed the cells to 400 μM H₂O₂ for another 3 h. Therefore, our data strongly suggests that Tan IIA may lead to increased regeneration of glutathione (GSH) from the glutathione disulfide (GSSG) produced during the GSH peroxidase-catalyzed decomposition of H₂O₂ in HUVECs, and the PXR plays a significant role in this process. Tan IIA may also exert protective effects against H₂O₂-induced apoptosis through the mitochondrial apoptosis pathway associated with the participation of PXR. Tan IIA protected HUVECs from inflammatory mediators triggered by H₂O₂ via PXR activation. In conclusion, Tan IIA protected HUVECs against H₂O₂-induced cell injury through PXR-dependent mechanisms.

Keywords: Apoptosis; HUVECs; Inflammation; Oxidative stress; PXR; Tanshinone IIA.

Fig. 1.

Tan IIA reduced H₂O₂-induced cell injury in HUVECs. (A) HUVECs were subjected to Tan IIA for different concentrations for 24 h; Cell viability was measured with a CCK-8 assay kit. (B) Cells were transfected with PXR siRNA, with or without pretreatment with Tan IIA for 24 h then treatment with 400 μM H₂O₂ for another 3 h. Data are expressed as percentage of control, and the mean ± SD of 3 replicates. ^#p<0.05 compared with control group. ^***p<0.001 compared with H₂O₂ group.

Fig. 2.

Tan IIA upregulates PXR expression in H₂O₂ treated HUVECs. Cells were pretreated with Tan IIA for 24 h then treated with 400 μM H₂O₂ for another 3 h, RIF treatment was used as a positive control. (A) PXR mRNA was measured by qRT-PCR, with GAPDH as an internal control. (B) Protein expression was measured with Western blot, using histone as an internal control. Data are expressed as percentages of control and are the mean ± SD of 3 replicates. ^**p<0.01, ^***p<0.001 compared with H₂O₂ group.

Fig. 3.

Tan IIA upregulates CYP3A4, MDR1, GST and GSTM1 expression in H₂O₂ treated HUVECs via PXR activation. Cells were transfected with PXR siRNA, with or without pretreatment with Tan IIA for 24 h then treatment with 400 μM H₂O₂ for another 3 h. RIF treatment was used as a positive control. (A-D) CYP3A4, MDR1, GST and GSTM1 mRNA expression was measured by qRT-PCR, using GAPDH as an GAPDH was internal control. Data are expressed as percentages of control and are the mean ± SD of 3 replicates. ^*p<0.05, ^**p<0.01, ^***p<0.001 compared with H₂O₂ group.

Fig. 4.

Tan IIA inhibit H₂O₂-induced oxidative stress via PXR activation. Cells were transfected with PXR siRNA, with or without pretreatment with Tan IIA for 24 h then treated with 400 μM H₂O₂ for another 3 h. ROS production (A), total GSH production (B), and GSSG production (C) were detected by different assays as described in Materials and Methods, (D) GPx mRNA expression was measured by qRT-PCR, GAPDH was used as an internal control. Data are expressed as percentages of control and are the mean ± SD of 3 replicates. ^#p<0.05 compared with control group. ^*p<0.05, ^**p<0.01, ^***p<0.001 compared with H₂O₂ group.

Fig. 5.

Tan IIA inhibits H₂O₂-induced apoptosis via PXR activation. Cells were transfected with PXR siRNA, with or without pretreatment with Tan IIA for 24 h then treatment with 400 μM H₂O₂ for another 3 h. Apoptosis (A) and MMP (B) were detected by different assays as described in Materials and Methods. Cells were stained with Hoechst 33342 and visualized under a fluorescence microscope (C) a: control group; b: H₂O₂ group; c–e: Tan IIA (5, 10, 20 μM)+H₂O₂ group; f: RIF+H₂O₂ group. Condensed or fragmented nuclei were considered as apoptotic cells (magnification: 200×). (D, E, F) Cells were treated with H₂O₂ (400 μM) in the absence or presence of Tan IIA (200 μM) for 24 h, caspase-3/7, 8, and 9 activities were analyzed with a plate reader. (G, H, I) Apoptosis-related proteins bax, cytochrome C and Bcl-2 were measured with Western blotting. Data are expressed as percentages of control and are the mean ± SD of 3 replicates. ^#p<0.05 compared with control group. ^*p<0.05, ^**p<0.01, ^***p<0.001 compared with H₂O₂ group.

Fig. 5.

Tan IIA inhibits H₂O₂-induced apoptosis via PXR activation. Cells were transfected with PXR siRNA, with or without pretreatment with Tan IIA for 24 h then treatment with 400 μM H₂O₂ for another 3 h. Apoptosis (A) and MMP (B) were detected by different assays as described in Materials and Methods. Cells were stained with Hoechst 33342 and visualized under a fluorescence microscope (C) a: control group; b: H₂O₂ group; c–e: Tan IIA (5, 10, 20 μM)+H₂O₂ group; f: RIF+H₂O₂ group. Condensed or fragmented nuclei were considered as apoptotic cells (magnification: 200×). (D, E, F) Cells were treated with H₂O₂ (400 μM) in the absence or presence of Tan IIA (200 μM) for 24 h, caspase-3/7, 8, and 9 activities were analyzed with a plate reader. (G, H, I) Apoptosis-related proteins bax, cytochrome C and Bcl-2 were measured with Western blotting. Data are expressed as percentages of control and are the mean ± SD of 3 replicates. ^#p<0.05 compared with control group. ^*p<0.05, ^**p<0.01, ^***p<0.001 compared with H₂O₂ group.

Fig. 6.

Tan IIA inhibits H₂O₂-induced inflammation via PXR activation. (A, B) Cells with or without pretreatment with Tan IIA for 24 h then treated with 400 μM H₂O₂ for another 3 h. HUVECs were then incubated with fluorescently labeled THP-1 cells (5×10⁵ cells/mL) for 30 min and visualized under a fluorescence microscope, a: control group; b: H₂O₂ group; c–e: Tan IIA (5, 10, 20 μM) + H₂O₂ group; f: RIF+H₂O₂ group. (C) Data are expressed as percentages of control and are the mean ± SD of 3 replicates. ^#p<0.05 compared with control group. ^**p<0.01, ^***p<0.001 compared with H₂O₂ group.