Metformin Alleviates Epirubicin-Induced Endothelial Impairment by Restoring Mitochondrial Homeostasis

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  Figure 1. Metformin alleviates epirubicininduced HUVEC cell dysfunction. (A) Expression levels of γ−H2A.X. HUVEC cells were respectively incubated with 0.5 μM, 1 μM, 5 μM or 10 μM EPI for 12 h with or without the pretreatment of 1 mM metformin for 12 h. The 5 µM epirubicin treatment group was used as the reference. (B) Levels of ROS. HUVEC cells were treated with 5 μM epirubicin for 6 h with or without the pretreatment of 1 mM metformin for 12 h. ROS levels were then detected by 5 mM DCFH−DA probe. (C) Levels of Ang II in the supernatant. HUVEC cells were treated with 5 μM epirubicin for 24 h with or without the pretreatment of 1 mM metformin for 12 h. Then, the levels of Ang II in the supernatant were detected by ELISA. EPI—epirubicin, MET—metformin, ns—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. Metformin restores mitochondrial biogenesis. (A) Expression levels of TFAM and PGC1α. HUVEC cells were treated with incubated with 0.5 μM, 1 μM, 5 μM or 10 μM EPI, respectively, for 6 h or 24 h with or without the pretreatment of metformin (1 mM) for 12 h. TFAM expression was detected by Western blotting. HUVEC cells were treated with 5 μM epirubicin for 24 h with or without the pretreatment of 1 mM metformin for 12 h, then PGC1α expression was detected by Western blotting. (B) Expression levels of γ−H2A.X. HUVEC cells were incubated with TFAM siRNA or control siRNA for 48 h. HUVEC cells were treated with 5 μM epirubicin for 24 h with or without the pretreatment of 1 mM metformin for 12 h. The levels of γ−H2A.X were detected by Western blotting. The epirubicin treatment group was used as the reference. EPI—epirubicin, MET—metformin, ns—not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 3. Metformin restores mitochondrial dynamics. (A) Mitochondrial morphology analysis by MitoTracker Green. HUVEC cells were treated with 5 μM epirubicin for 24 h with or without the pretreatment of 1 mM metformin for 12 h. Then, the cells were stained with 100 nM MitoTracker Green at 37 °C in the dark for 30 min and imaged. (B) HUVEC cells were treated with 5 μM epirubicin for 24 h with or without the pretreatment of 1 mM metformin for 12 h. The expression levels of mitochondrial dynamics related proteins were detected by Western blotting. (C). HUVEC cells were treated with 5 μM epirubicin for 12 h with the pretreatment of 1 mM metformin for 12 h or 20 μM mdivi−1 for 2 h, respectively. The expression levels of TFAM were detected by Western blotting. (D). HUVEC cells were treated with 5 μM epirubicin for 24 h with the pretreatment of 1 mM metformin for 12 h or 20 μM mdivi−1 for 2 h, respectively. The levels of γ−H2A.X were detected by Western blotting. The epirubicin treatment group was used as the reference. (E). HUVEC cells were treated with 5 μM epirubicin for 24 h with the pretreatment of 1 mM metformin for 12 h or 20 μM mdivi−1 for 2 h, respectively. The levels of Ang II in the…

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  Figure 4. Metformin restores mitochondrial homeostasis via calcineurin/TFEB. (A) Metformin restores the calcineurin/TFEB pathway. HUVEC cells were treated with 5 μM epirubicin for 24 h with pretreatment of 1 mM metformin for 12 h or 5 μM CsA for 2 h, respectively. The expression levels of calcineurin and TFEB were detected by Western blotting. (B) Expression levels of TFAM. The expression of TFEB in HUVEC cells was inhibited by siRNA. Then, HUVEC cells were treated with 5 μM epirubicin for 12 h with or without the pretreatment of 1 mM metformin for 12 h. Western blotting was used to measure the TFAM expression. (C) Expression levels of DRP1. The expression of TFEB in HUVEC cells was inhibited by siRNA. Then, HUVEC cells were treated with 5 μM epirubicin for 24 h with or without the pretreatment of 1 mM metformin for 12 h. DRP1 expression was detected by Western blotting. (D) Expression levels of γ−H2A.X. The expression of TFEB in HUVEC cells was inhibited by siRNA. Then, HUVEC cells were treated with 5 μM epirubicin for 24 h with or without the pretreatment of 1 mM metformin for 12 h. Levels of γ−H2A.X were detected by Western blotting. The epirubicin treatment group was used as the…