Heat Shock Protein 22 Attenuates Doxorubicin-Induced Cardiotoxicity via Regulating Inflammation and Apoptosis

Figures

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  Figure 1. Heat shock protein 22 (Hsp22) improved cardiac function in mice with doxorubicin (DOX)-induced toxicity in vivo. (A) Representative Western blot bands and quantitative results of protein levels of Hsp22 in DOX-induced cardiac injury in C57BL/6 mice treated with DOX. (B) The mRNA level of Hsp22 in the C57BL/6 mice treated with DOX. (C) Representative Western blot bands of protein level of Hsp22 in mice injected with adeno-associated virus (AAV)-Hsp22. (D) Body weight of the four groups (n = 9–12). (E) Heart weight of the four groups (n = 9–12). (F) Statistical results of the heart weight (HW)/tibia length (TL) (n = 9–12). (G) Statistical results of the lung weight (LW)/TL (n = 9–12). (H) Ejection fraction (EF) of mice in the indicated groups (n = 9–12). (I, J) Hemodynamic analysis of mice with or without Hsp22 treatment (n = 7–11). (K) Heart rate of mice with or without Hsp22 treatment. *P < 0.05 versus NS + Ad-GPF, #P < 0.05 versus DOX + Ad-GPF. For panels (A, B), P < 0.05 versus NS.

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  Figure 2. Heat shock protein 22 (Hsp22) suppressed inflammation responses in doxorubicin (DOX)-treated mice. (A–C) Immunohistochemistry analysis of CD45, CD68, and tumor necrosis factor (TNF)-α in DOX-treated hearts. Representative images and quantification (n = 6, 10+ fields per heart) are shown. (D) PCR analysis of inflammation markers [TNF-α, interleukin (IL)-1, IL-6] in DOX-treated hearts (n = 6). (E) Representative images of p-P65 immunofluorescence in mice with or without Ad-Hsp22 transfection (n = 4). (F, G) Western blot and quantitative results (n = 6). *P < 0.05 versus NS + Ad-GPF, #P < 0.05 versus DOX + Ad-GFP.

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  Figure 3. Overexpression of heat shock protein 22 (Hsp22) exhibited anti-apoptosis effect in doxorubicin (DOX)-induced cell death. (A) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the quantitative results in DOX-treated hearts (n = 4). (B) The level of lactate dehydrogenase (LDH) in the indicated groups (n = 6). (C–F) Western blot and quantitative results in the indicated groups (n = 6). (G) Cleaved caspase-3 (c-caspase-3) immunofluorescence (n = 4). *P < 0.05 versus NS + Ad-GFP, #P < 0.05 versus DOX + Ad-GFP.

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  Figure 4. Heat shock protein 22 (Hsp22) regulated the expression of NOD-, LRR-, and Pyrin Domain-Containing Protein 3 (NLRP3) and Toll-Like Receptor (TLR)4 both in vitro and in vivo. (A) Representative Western blot and analysis of NLRP3 in doxorubicin (DOX)-treated hearts (n = 6). (B) Representative Western blot and analysis of TLR4 in DOX-treated hearts (n = 6). (C) Representative Western blot and analysis of NLRP3 in DOX-treated H9c2 cells (n = 6). (D) Representative Western blot and analysis of TLR4 in DOX-treated H9c2 cells (n = 6). For panels (A, B), *P < 0.05 versus NS + Ad-GFP, #P < 0.05 versus DOX + Ad-GFP. For panels (C, D), *P < 0.05 versus PBS + Ad-GFP, #P < 0.05 versus DOX + Ad-GFP.

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  Figure 5. Heat shock protein 22 (Hsp22) inhibited doxorubicin (DOX)-induced inflammation and cell loss via inhibition of the activation of the NOD-, LRR-, and Pyrin Domain-Containing Protein 3 (NLRP3). (A) The mRNA levels of inflammation-related genes in the indicated groups (n = 4). (B, C) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and the quantitative results in the indicated groups (n = 4). (D) Cell viability after the treatment with DOX in the indicated groups (n = 4). (E, F) Representative Western blot and analysis of Bcl-2, Bax, and cytochrome C in the indicated groups (n = 4). *P < 0.05 versus PBS + Ad-NC group, #P < 0.05 versus the DOX + Ad-GFP group.