Salvianolic acid B inhibits myocardial I/R-induced ROS generation and cell apoptosis by regulating the TRIM8/GPX1 pathway

Figures

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  Figure 1.. SalB repressed ischemia/reperfusion (I/R)-induced apoptosis and oxidative stress in AC16 cells. I/R-injured AC16 cells were treated with different concentrations of SalB. (A) Apoptotic detection was performed using a flow cytometry. (B) ROS level was detected with DCFH-DA probe. (C) SOD activity was detected using xanthine oxidase method. (D) MDA level was detected using TBA methods. ***p < 0.001 and ****p < 0.0001 vs. Control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. I/R.

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  Figure 2.. SalB inhibited ischemia/reperfusion (I/R)-induced up-regulation of TRIM8 expression. I/R-injured AC16 cells were treated with different concentrations of SalB. (A) The mRNA levels of TRIM8, TRIM11, TRIM32, and TRIM33 were detected by RT-qPCR. (B) The protein levels of TRIM8, TRIM11, TRIM32, and TRIM33 were detected by western blotting. ***p < 0.001 and ****p < 0.0001 vs. Control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. I/R. ns: not significant.

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  Figure 3.. Knock-down of TRIM8 alleviated ischemia/reperfusion (I/R)-induced apoptosis and oxidative stress in AC16 cells. I/R-injured AC16 cells were transduced with lentivirus siTRIM8. (A) Apoptotic detection was performed using a flow cytometry. (B) ROS level was detected with DCFH-DA probe. (C) SOD activity was detected using xanthine oxidase method. (D) MDA level was detected using TBA method. (E) TRIM8 protein level was determined. ****p < 0.0001 vs. Control; ###p < 0.001 vs. I/R + siNC.

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  Figure 4.. SalB alleviated TRIM8 overexpression-induced apoptosis and oxidative stress in AC16 cells. AC16 cells were transduced with lentivirus oeTRIM8, and the treated with 50 μM SalB. (A) Apoptotic detection was performed using a flow cytometry. (B) ROS level was detected with DCFH-DA probe. (C) SOD activity was detected using xanthine oxidase method. (D) MDA level was detected using TBA method. ***p < 0.001 and ****p < 0.0001 vs. vector; ##p < 0.01, and ###p < 0.001 vs. oeTRIM8.

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  Figure 5.. TRIM8 negatively regulated GPX1 via ubiquitination. (A) AC16 cells were treated with lentivirus oeTRIM8. TRIM expression level was assessed. (B) I/R-injured AC16 cells were treated with different concentrations of SalB. The protein levels of GPX1 were detected by western blotting. (C) Co-immunoprecipitation assay was performed to detect the interaction between TRIM8 and GPX1. (D) AC16 cells were treated with lentivirus oeTRIM8 and proteasome inhibitor MG132. TRIM8 and GPX1 protein levels were determined. (E) AC16 cells were treated with lentivirus oeTRIM8. The ubiquitination of GPX1 was detected by ubiquitination assay. ns: not significant.

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  Figure 6.. GPX1 knock-down abolished the protective effects of SalB on ischemia/reperfusion (I/R)-injured cardiomyocytes. I/R-injured AC16 cells were treated with SalB and lentivirus siGPX1. (A) Apoptotic detection was performed using a flow cytometry. (B) ROS level was detected with DCFH-DA probe. (C) TRIM8 protein level was measured. (D) SOD activity was detected using xanthine oxidase method. (E) MDA level was detected using TBA method. (F) GPX1 protein level was measured. ***p < 0.001 and ****p < 0.0001 vs. control; ##p < 0.01 vs. I/R; &&p < 0.001 vs. I/R + SalB.

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  Figure 7.. SalB inhibited cardiomyocyte apoptosis and ROS generation by regulating TRIM8/GPX1 axis in vivo. Ischemia/reperfusion (I/R)-injured rat model was treated with different concentrations of SalB (L: 20 mg/kg SalB; M: 40 mg/kg SalB; H: 60 mg/kg SalB). (A) Apoptotic detection was performed by TUNEL assay. (B) ROS level was detected with dihydroethidium staining. (C) SOD activity was detected using xanthine oxidase method. (D) MDA level was detected using TBA method. (E) TRIM8 and GPX1 protein levels were measured. ***p < 0.001 and ****p < 0.0001 vs. control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. I/R.