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. 2022 Jan;13(1):844-855.
doi: 10.1080/21655979.2021.2013109.

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

Feng Wang  1 , Lili Han  2
Affiliations

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

Feng Wang et al. Bioengineered. 2022 Jan.

Abstract

The clinical application of doxorubicin (Dox) in tumor chemotherapy is limited by time-dependent and dose-dependent cardiotoxicity. Hence, there is an urgent need to elucidate doxorubicin cardiotoxicity and to solve the difficult problem in clinical application. It has been verified that serum and glucocorticoid-regulated kinase 1 (SGK1) possess cardioprotective effects. Here, H9c2 cells were treated with 1 μM doxorubicin for 24 h to establish doxorubicin cardiotoxicity, so as to determine the biological role of SGK1 in doxorubicin cardiomyopathy and to elucidate the underlying molecular mechanism. SGK1 level in doxorubicin-treated H9c2 cells was assessed by performing Western blot assay and RT-qPCR. CCK-8 assay and TUNEL staining were employed to evaluate the cell viability and cell apoptosis. Besides, apoptosis-related proteins were measured by Western blot assay to analyze cell apoptosis. Additionally, the release of TNF-α, IL-1β, IL-6, and IL-10 and the levels of ROS, MDA, and SOD were detected to reflect inflammation and oxidative stress. Moreover, Western blot assay was adopted for determination of ERS-associated proteins. Results revealed that SGK1 was downregulated in doxorubicin-treated H9c2 cells. Upregulation of SGK1 alleviated doxorubicin-induced cardiotoxic injury, cell apoptosis, inflammation and oxidative stress in H9c2 cells. Moreover, SGK1 overexpression mitigated doxorubicin-induced ERS in H9c2 cells. The suppressing effects of SGK1 on doxorubicin-induced cardiotoxic injury, apoptosis, inflammation, oxidative stress and ERS in H9c2 cells were partially abolished upon GRP78 overexpression. To conclude, upregulation of SGK1 may alleviate doxorubicin cardiotoxicity by repressing GRP78-mediated ERS.

Keywords: GRP78; SGK1; cardiotoxicity; doxorubicin; endoplasmic reticulum stress.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
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.
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.
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.
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.
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.
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.
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.
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.
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