Caveolin-1 Alleviates Acetaminophen—Induced Hepatotoxicity in Alcoholic Fatty Liver Disease by Regulating the Ang II/EGFR/ERK Axis

Figures

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  Figure 1. APAP aggravated liver injury in ethanol-induced AFLD mice; the protein expression of CAV1 and ACE2 were decreased while the Ang II was increased in APAP treated AFLD mice, with the oxidative stress levels increasing. Male C57BL/6J mice were fed an alcohol liquid diet or equivalent liquid control diet, while APAP (280 mg/kg) was given by gavage on the last day. (A) Animal experiment process. (B) The bodyweight of mice in each group. (C) The liver index of mice in each group. (D) The serum activity of ALT and AST. (E) The serum activity of TG and TC. (F) Representative images of hematoxylin and eosin staining results (Scale bar = 50 μm). (G) Serum glutathione (GSH). (H) Liver superoxide dismutase (SOD) activity. (I) Malondialdehyde (MDA) levels. (J) Western blot analysis of the relative protein levels of CAV1, ACE2 and Ang II were measured in liver tissue. (K) Representative images of immunohistochemistry results of Ang II for liver tissue sections (Scale bar = 50 μm). Data represent the mean ± SEM (n = 8 mice). * p < 0.05 vs. N group; ** p < 0.01 vs. N group; # p < 0.05 and ## p < 0.01 vs. APAP group. ### p < 0.001.

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  Figure 2. CAV1 reduced lipid deposition and liver injury caused by APAP in AFLD and decreased the level of oxidative stress. (A) The bodyweight of mice in each group. (B) The liver index of mice in each group. (C) The serum activity of ALT. (D) The serum activity of AST. (E) The serum activity of TG. (F) The serum activity of TC. (G) Representative images of hematoxylin and eosin staining results (Scale bar = 50 μm). (H) Western blot analysis of the relative protein levels of CAV1. (I) Serum glutathione (GSH). (J) Liver superoxide dismutase (SOD) activity. (K) Malondialdehyde (MDA) levels. Data represent the mean ± SEM (n = 8 mice). * p < 0.05 vs. N + LV-Control group; ** p < 0.01 vs. N + LV-Control group; # p < 0.05 and ## p < 0.01 vs. EtOH + APAP + LV-Control group.

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  Figure 3. The protein expression of Ang II was inhibited after CAV1 overexpression. (A) Representative images of immunohistochemistry results of Ang II for liver tissue sections (Scale bar = 50 μm). (B) Western blot analysis of the relative protein levels of CAV1, ACE2 and Ang II were measured in liver tissue. (C,D) Representative images of immunofluorescence double staining results of CAV1 and Ang II as well as CAV1 and EGFR. DAPI was used for nuclear staining (blue). Red color represents CAV1 staining. Green color represents Ang II or EGFR staining (Scale bar = 20 μm). Data represent the mean ± SEM (n = 8 mice). * p < 0.05 vs. N + LV-Control group; ** p < 0.01 vs. N + LV-Control group; # p < 0.05 vs. EtOH + APAP + LV-Control group.

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  Figure 4. CAV1 alleviates APAP-induced lipid accumulation in AFLD by affecting the Ang II/EGFR/ERK axis and restoring autophagic flux. (A) Western blot analysis of p-EGFR/EGFR and p-ERK/ERK protein levels. (B) Western blot analysis of P62 and LC3 II / I protein levels. (C) Transmission electron microscopy analysis of mitochondria, autophagosomes and lipid droplets in liver tissues. White arrows show normal mitochondria, yellow arrows show damaged mitochondria, black arrows show lipid droplets and red arrows show autophagosomes. The number of autophagosomes per given area (mean ± S.D.) was determined (Scale bar = 5.0 μm). Data represent the mean ± SEM (n = 8 mice). * p < 0.05 vs. N + LV-Control group; ** p < 0.01 vs. N + LV-Control group; *** p < 0.001 vs. N + LV-Control group; # p < 0.05 and ## p < 0.01 vs. EtOH + APAP + LV-Control group.

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  Figure 5. APAP aggravated lipid deposition and liver injury induced by alcohol and oleic acid in L02 cells, and the protein expression of the CAV1 and ACE2 were decreased while the Ang II was increased. (A) ORO staining in L02 cells (Scale bar = 50 μm). (B) The TG levels treated with EtOH/OA and APAP. (C) Western blot analysis of ACE2, Ang II and CAV1 protein levels. (n = 3 independent experiments). * p < 0.05 vs. N group; ** p < 0.01 vs. N group; **** p < 0.0001 vs. N group; # p < 0.05 vs. APAP group; ## p < 0.01 vs. APAP group; #### p < 0.0001 vs. APAP group.

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  Figure 6. CAV1 alleviated lipid deposition, suppressed the Ang II/EGFR/ERK signaling and restored autophagic flux in EtOH/OA and APAP treated L02 cells. (A) Western blot analysis of CAV1 protein levels. (B) Representative images of ORO staining of lipid droplets in L02 cells (Scale bar = 50 μm). (C) Western blot analysis of ACE2, Ang II and CAV1 protein levels. (D) ROS levels. (E) Western blot analysis of p-EGFR/EGFR, p- ERK/ERK, P62 and LC3 II / I protein levels. Data represent the mean ± SEM (n = 3 independent experiments). * p < 0.05 vs. N + LV-Control group; ** p < 0.01 vs. N + LV-Control group; # p < 0.05 and ## p < 0.01 vs. EtOH/OA + APAP + LV-Control group.

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  Figure 7. CAV1 silencing exacerbated lipid accumulation and activated the Ang II/EGFR/ERK signaling in L02 cells induced by EtOH/OA and APAP. (A) Western blot analysis of CAV1 protein levels. (B) Representative images of ORO staining of lipid droplets in L02 cells (Scale bar = 50 μm). (C) Western blot analysis of ACE2, Ang II and CAV1 protein levels. (D) ROS levels. (E) Western blot analysis of p-EGFR/EGFR, p- ERK/ERK, P62 and LC3 II / I protein levels. Data represent the mean ± SEM (n = 3 independent experiments). * p < 0.05 vs. N + Control-siRNA group; ** p < 0.01 vs. N + Control-siRNA group; *** p < 0.001 vs. N + Control-siRNA group; **** p < 0.0001 vs. N + Control-siRNA group; ns p > 0.05 vs. N + Control-siRNA group; # p < 0.05 and ## p < 0.01 vs. EtOH/OA + APAP + Control-siRNA group.

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  Figure 8. Inhibition of Ang II and ERK1/2 alleviated lipid accumulation and restored autophagic flux in L02 cells. Pretreatment with losartan or PD98059 one hour before cell modeling. (A) Representative images of ORO staining with losartan (2 μM) in L02 cells (Scale bar = 50 μm). (B) Western blot analysis of ACE2, Ang II and p-EGFR/EGFR protein levels. (C) Representative images of ORO staining with PD98059 (40 μM) in L02 cells (Scale bar = 50 μm). (D) Western blot analysis of p-ERK/ERK, P62 and LC3 II/I protein levels. Data represent the mean ± SEM (n = 3 independent experiments). * p < 0.05 vs. N group; ** p < 0.01 vs. N group; *** p < 0.001 vs. N group; # p < 0.05 and ## p < 0.01 vs. EtOH/OA + APAP group.

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  Figure 9. CAV1 alleviates APAP-induced lipid accumulation in AFLD by regulating the Ang II/EGFR/ERK axis, ameliorating oxidative stress, and restoring autophagic flux. EtOH, ethanol; APAP, acetaminophen; ACE2, angiotensin-converting enzyme 2; Ang II, angiotensin II; EGFR, epidermal growth factor receptor; MAPK/ERK, mitogen-activated protein kinase.