MSCs enhances the protective effects of valsartan on attenuating the doxorubicin-induced myocardial injury via AngII/NOX/ROS/MAPK signaling pathway

Figures

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  Figure 1. The cell viability determination and effect of Val pre-treatment followed by DOX-exposure on ROS level and apoptosis rate in H9c2 cells. (A) Effect of DOX on H9c2 cells viability for 24 h. (B) Effect of Val on H9c2 cells viability for 24 h. (C) Effect of Val at three concentrations on H9c2 cells over time. (D) Viability of DOX-induced H9c2 cells followed as three of Val concentrations pre-treatment. (E, F) Effect of Val and DOX on tumor cells viability. (G) Effect of Val pre-treatment on the ROS level of DOX-treated H9c2 cells at different time points. (H) Effect of Val on apoptosis of DOX-treated H9c2 cells. (I) Microscopic analysis of Val and DOX-treatment on ROS levels by DCF Fluorescence. (J) The bar graph of the quantitative analysis of apoptosis rate of H9c2 cells. *P<0.05 vs. Control; †P<0.05 vs. DOX. DOX, doxorubicin; Val, valsartan.

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  Figure 2. The effect of Val pre-treatment and DPI followed by DOX-exposure on expression of proteins and ROS level in H9c2 cells. (A) Western blot analysis of Val and DOX-treatment on AT1R, NOX2, NOX4, caspase-3, cleaved caspase-9 protein levels. (B) Densitometry analysis of the protein bands of AT1R, NOX2, NOX4, caspase-3, cleaved caspase-9 proteins. (C) Western blot analysis of DPI and DOX-treatment on caspase-3, cleaved caspase-9 protein levels. (D) Densitometry analysis of the protein bands of caspase-3, cleaved caspase-9 proteins. (E) Effect of DPI and DOX on ROS levels at 12 h time point. (F) Microscopic analysis of DPI and DOX-treatment on ROS levels by DCF Fluorescence. *P<0.05 vs. Control; †P<0.05 vs. Dox. Dox, doxorubicin; Val, valsartan.

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  Figure 3. Knockdown of NOX2 and NOX4 expression could reduce DOX-induced ROS production and expression of apoptotic proteins in H9c2 cells. (A) Fluorescence microscope observation of H9c2 cells after transfecting with Negative siRNA, NOX2 siRNA and NOX4 siRNA (×100). (B) Three distinct NOX2 siRNAs and one negative siRNA were designed, and Nox2-rat-663 was selected as the best interference fragment by PCR. (C) Three distinct NOX4 siRNAs and one negative siRNA were designed, and Nox4-rat-576 was selected as the best interference fragment by PCR. (D) Western blot analysis of NOX2 siRNA and DOX-treatment on NOX2, caspase-3, cleaved caspase-9 protein levels. (E) Densitometry analysis of the protein bands of NOX2, caspase-3, cleaved caspase-9 proteins. (F) Western blot analysis of NOX4 siRNA and DOX-treatment on NOX4, caspase-3, cleaved caspase-9 protein levels. (G) Densitometry analysis of the protein bands of NOX4, caspase-3, cleaved caspase-9 proteins. (H) Effect of NOX2 siRNA, NOX4 siRNA and DOX-treatment on ROS levels at 12 h time point in H9c2 cells. (I) Microscopic analysis of NOX2 siRNA, NOX4 siRNA and DOX-treatment on ROS levels by DCF Fluorescence. *P<0.05 vs. Control; †P<0.05 vs. Dox.…

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  Figure 4. Overexpression of NOX2 and NOX4 could increase DOX-induced ROS production and expression of apoptotic proteins in H9c2 cells. (A) Fluorescence microscope observation of H9c2 cells after transfecting with Empty vector, NOX2 OE and NOX4 OE (×100). (B) The bar graph of NOX2 eukaryotic expression plasmid verified by RT-PCR. (C) The bar graph of NOX4 eukaryotic expression plasmid verified by RT-PCR. (D) Western blot analysis of NOX2 OE and DOX-treatment on NOX2, caspase-3, cleaved caspase-9 protein levels. (E) Densitometry analysis of the protein bands of NOX2, caspase-3, cleaved caspase-9 proteins. (F) Western blot analysis of NOX4 OE and DOX-treatment on NOX4, caspase-3, cleaved caspase-9 protein levels. (G) Densitometry analysis of the protein bands of NOX4, caspase-3, cleaved caspase-9 proteins. (H) Effect of NOX2 OE, NOX4 OE and DOX-treatment on ROS levels at 12 h time point in H9c2 cells. (I) Microscopic analysis of NOX2 OE, NOX4 OE and DOX-treatment on ROS levels by DCF Fluorescence. *P<0.05 vs. Control; †P<0.05 vs. Dox. Dox, doxorubicin; OE, overexpression.

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  Figure 5. MSCs combined with Val effects on ROS generation, AngII, NOX2, NOX4, caspase-3 and cleaved caspase-9 expression in H9c2 cells. (A) Schematic diagram of co-culture of MSCs and H9c2 cells. (B) Western blot analysis of MSCs and Val-treatment on AT1R, NOX2, NOX4, caspase-3, cleaved caspase-9 proteins levels. (C) Effect of MSCs and Val-treatment on ROS levels at 12 h time point in H9c2 cells. (D, E) Densitometry analysis of the protein bands of AT1R, NOX2, NOX4, caspase-3, cleaved caspase-9 proteins. (F) Microscopic analysis of MSCs and Val-treatment on ROS levels by DCF Fluorescence. *P<0.05 vs. Control; †P<0.05 vs. Dox; #P<0.05 vs. Dox+Val, Dox+MSCs. Dox, doxorubicin; Val, valsartan; MSCs, mesenchymal stem cells.

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  Figure 6. Val, NOX (knockdown and overexpression), MSCs effects on MAPK signaling pathway in H9c2 cells. (A) Western blot analysis of DOX and Val-treatment on p-p38, p-JNK, p-ERK proteins levels. (B, C) Western blot analysis of NOX2 siRNA and NOX4 siRNA treatment on p-p38, p-JNK, p-ERK proteins levels. (D, E) Western blot analysis of NOX2 plasmid and NOX4 plasmid treatment on p-p38, p-JNK, p-ERK proteins levels. (F) Western blot analysis of MSCs and Val-treatment on p-p38, p-JNK, p-ERK proteins levels. (G) Densitometry analysis of the protein bands of p-p38, p-JNK, p-ERK after Val and DOX treatment. (H, I) Densitometry analysis of the protein bands of p-p38, p-JNK, p-ERK after NOX2 siRNA and NOX4 siRNA treatment. (J, K) Densitometry analysis of the protein bands of p-p38, p-JNK, p-ERK after NOX2 plasmid and NOX4 plasmid treatment. (L) Densitometry analysis of the protein bands of p-p38, p-JNK, p-ERK after MSCs and Val treatment. *P<0.05 vs. Control, Negative siRNA or Empty vector; †P<0.05 vs. Dox or DOX+Negative siRNA or DOX+Empty vector; #P<0.05 vs. DOX+Val or DOX+MSCs. DOX, doxorubicin; Val, valsartan; MSCs, mesenchymal stem cells.

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  Figure 7. Schematic diagram of the effect of Val combined with MSCs on DOX-induced apoptosis in H9c2 cardiomyocytes.