Upregulation of serum and glucocorticoid-regulated kinase 1 (SGK1) ameliorates doxorubicin-induced cardiotoxic injury, apoptosis, inflammation and oxidative stress by suppressing glucose regulated protein 78 (GRP78)-mediated endoplasmic reticulum stress

Figure 1.. SGK1 was downregulated in doxorubicin-treated H9c2 cells. H9c2 cells were treated with 1 μM doxorubicin for 3, 6, 12 or 24 h. (a) Western blot assay for determination of SGK1 protein expression in H9c2 cells. (b) RT-qPCR for determination of SGK1 mRNA level in H9c2 cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2.. Upregulation of SGK1 alleviated doxorubicin-induced cardiotoxic injury. (a) H9c2 cells were transfected with Ov-SGK1 or Ov-NC. Western blot assay for determination of SGK1 protein expression in H9c2 cells. (b) H9c2 cells were transfected with Ov-SGK1 or Ov-NC. RT-qPCR for determination of SGK1 mRNA level in H9c2 cells. (c) Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or treated with the specific SGK1 inhibitor EMD638683. CCK-8 assay for determination of the viability of H9c2 cells. (d) Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or treated with the specific SGK1 inhibitor EMD638683. LDH assay kit for determination of the production of intracellular LDH. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3.. Upregulation of SGK1 suppressed the apoptosis of doxorubicin-treated H9c2 cells. Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or treated with the specific SGK1 inhibitor EMD638683. (a, b) TUNEL staining for determination of the apoptosis of H9c2 cells. (c) Western blot assay for determination of Bcl-2, Bax, Cleaved caspase-3, caspase-3, Cleaved caspase-9 and caspase-9 protein expressions in H9c2 cells. ** p < 0.01, *** p < 0.001.

Figure 4.. Upregulation of SGK1 mitigated inflammation and oxidative stress in doxorubicin-treated H9c2 cells. Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or treated with the specific SGK1 inhibitor EMD638683. (a) ELISA kits for determination of TNF-α, IL-1β, IL-6 and IL-10 levels. (b) DCFH-DA for determination of ROS content. (c) MDA assay kit and SOD assay kit for determination of MDA and SOD levels. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5.. Upregulation of SGK1 relieved doxorubicin-induced ERS in H9c2 cells. Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or treated with the specific SGK1 inhibitor EMD638683. Western blot assay for determination of GRP78, p-PERK, PERK, ATF4 and CHOP protein expressions in H9c2 cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6.. SGK1 overexpression alleviated doxorubicin-induced cardiotoxic injury by repressing GRP78-mediated ERS. (a) H9c2 cells were transfected with Ov-GRP78 or Ov-NC. Western blot assay for determination of GRP78 protein expression in H9c2 cells. (b) H9c2 cells were transfected with Ov-GRP78 or Ov-NC. RT-qPCR for determination of GRP78 mRNA level in H9c2 cells. (c) Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or co-transfected with Ov-SGK1 and Ov-GRP78. Western blot assay for determination of GRP78, p-PERK, PERK, ATF4 and CHOP protein expressions in H9c2 cells. (d) Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or co-transfected with Ov-SGK1 and Ov-GRP78. CCK-8 assay for determination of the viability of H9c2 cells. (e) Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or co-transfected with Ov-SGK1 and Ov-GRP78. LDH assay kit for determination of the production of intracellular LDH. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7.. SGK1 overexpression suppressed the apoptosis of doxorubicin-treated H9c2 cells by repressing GRP78-mediated ERS. Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or co-transfected with Ov-SGK1 and Ov-GRP78. (a, b) TUNEL staining for determination of the apoptosis of H9c2 cells. (c) Western blot assay for determination of Bcl-2, Bax, Cleaved caspase-3, caspase-3, Cleaved caspase-9 and caspase-9 protein expressions in H9c2 cells. * p < 0.05, *** p < 0.001.

Figure 8.. SGK1 overexpression mitigated inflammation and oxidative stress in doxorubicin-treated H9c2 cells by repressing GRP78-mediated ERS. Doxorubicin-treated H9c2 cells were transfected with Ov-SGK1 or co-transfected with Ov-SGK1 and Ov-GRP78. (a) ELISA kits for determination of TNF-α, IL-1β, IL-6 and IL-10 levels. (b) DCFH-DA for determination of ROS content. (c) MDA assay kit and SOD assay kit for determination of MDA and SOD levels. * p < 0.05, ** p < 0.01, *** p < 0.001.