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Ablation of apoptosis-stimulating of p53 protein 1 protects mice from acute hepatic injury and dysfunction via NF-кB pathway in CCl4-induced hepatotoxicity
doi: 10.2478/fzm-2021-0007
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Abstract: Acute liver injury (ALI) is characterized by apoptosis, inflammation, and oxidative stress, and pathogenic mechanism of ALI is poorly understood. Apoptosis-stimulating of p53 protein 1 (ASPP1) is involved in environmental responses, tumor growth, and NF-KB activity, which is of critical importance to ALI. However, the role of ASPP1 in ALI remains largely unexplored. The current study aimed to determine the role of ASPP1 in ALI induced by CCl4 and the underlying mechanism. ASPP1 expression was detected in wild type (WT) mice with ALI induced by CCl4. The function of ASPP1 in ALI induced by CCl4 was investigated using conventional knockout ASPP1 mice. ASPP1 expression significantly increased in ALI mice at 24 hours after CCl4 injection. Deletion of ASSP1 ameliorated apoptosis, inflammation, and necrosis in ALI relative to WT mice. In addition, deficiency of ASPP1 improved liver flood flow as well as ALT and AST levels. The levels of phosphorylated p65 and phosphorylated IκBα were lower in ASPP1-/- mice than in WT mice with ALI. These results implicate that deletion of ASPP1 may act via inhibition of the NF-кB pathway and protect mice from ALI, which may be a new potential therapeutic target for the treatment of ALI.
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Key words:
- Apoptosis /
- ASPP1 /
- CCl4 /
- Liver injury /
- Inflammation /
- NF-κB
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Figure 1. Expression profiles of ASPP1 and liver injury markers in wild type mice with CCl4-induced acute hepatic injury.
A, B. Protein expression of ASPP1 in liver tissue of mice 24 hrs after treatment with 5% CCl4 (intraperitoneal injection; dose: 2µL/g body weight) injection. n = 3; *P < 0. 05. C. mRNA expression of ASPP1 in liver tissue of mice 24 hrs after treatment with 5% CCl4 (ip; 2µL/g body weight). n = 3; *P < 0. 05. D, E. Serum ALT/AST activity in liver tissue of mice 24 hrs after treatment with 5% CCl4 (ip; 2µL/g body weight). n = 3; *P < 0. 05. CTL, Control; Hrs, hours; IU, international units; L, litter.
Figure 2. Ablation of ASPP1 alleviates CCl4-induced acute liver injury in wild-type and ASPP knockout mice.
A, B. Protein expression of ASPP1 in WT and ASPP1 knockout (ASPP1-/-) mice. n = 4; *P < 0.05. C. mRNA expression of ASPP1 in WT and ASPP1-/- mice. n =7; *P < 0. 05. D. Comparison of morphological alterations of the livers between WT and ASPP1-/- mice without or with CCl4 treatment 24 hrs post-injection. E, F. Representative images (E) and statistical data on blood flow rate (volume/minute) in the liver. n = 3; *P < 0. 05. Upper panels of E show blood perfusion to liver tissue with red, yellow and blue colors indicating high, moderate and low blood flow rates, respectively. Middle and bottom panels of E display the red-line outlined areas indicating the selected regions for the measurements of blood flow. G. Representative images of Hematoxylin and Eosin (H&E) staining (scale bar = 100µm) showing the histological alterations of liver tissue with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. The outlined area indicates necrosis tissue, and the arrows indicate balloon-like degeneration caused by CCl4. H. Statistical analysis of zone of hepatocellular necrosis grading[39]. n = 9; *P < 0. 05. CTL, control; WT, wild type; KO, knockout; ns, no significance.
Figure 3. Ablation of ASPP1 reduces the levels of liver injury markers and increases the levels of antioxidants.
A, B. Serum levels of ALT and AST with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. ALT, n = 8; AST, n =7, *P < 0. 05. C. Serum level of SOD with and without CCl4 treatment. n = 5; *P < 0. 05. ALT, alanine transaminase; AST, aspartate aminotransferase; SOD, superoxide dismutase.
Figure 4. Ablation of ASPP1 reduces apoptosis in mice with acute liver injury.
A. Representative images of TUNEL staining with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. scale bar = 20µm. Arrows point to the TUNEL-positive cells. B. Statistical data on TUNEL-positive cells. n = 3; *P < 0.05.
C, D. Protein expression of cleaved caspase 3 with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. n = 4; *P < 0.05. E, F. Protein expression of Bax before and after CCl4 treatment. n = 4; *P < 0.05. G. The mRNA of expression of Bax with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. n = 4; *P < 0.05. H, I. Protein expression of BCL-2 with comparisons between WT and ASPP1-/- mice with and without CCl4 treatment. n = 4; *P < 0.05. J. The mRNA expression of BCL-2 with comparisons between WT and ASPP1-/- mice with and without CCl4 treatment. n = 3; *P < 0.05. DAPI, 4', 6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase mediate dUTP nick end labeling; β-actin, beta actin; Bax, BCL-2-associated X protein; BCL-2, B-cell lymphoma 2.
Figure 5. Ablation of ASPP1 lessens inflammation in mice with acute liver injury.
A. mRNA expression of IL-1β with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. n = 3; *P < 0.05. B. mRNA expression of IL-6 with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. n = 3; *P < 0.05. C. mRNA expression of TLR4 with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. n = 5; *P < 0.05. D. mRNA expression of TNF-α with comparisons between WT and ASPP1-/- mice with and withoutCCl4 treatment. n = 4; *P < 0.05. E. mRNA expression of ICAM1 with comparisons between WT and ASPP1-/- mice with and without CCl4 treatment. n = 3; *P < 0.05. IL-1β, interleukin 1 beta; IL-6, interleukin 6; TLR4, Toll like receptors 4; TNF-α, tumor necrosis factor alpha; ICAM1, intercellular adhesion molecule 1.
Figure 6. Effect of IκBα/NF–κB in mice with acute liver injury.
A, B, C. Protein levels of IκBα and p-IκBα and their respective statistical data with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. n = 4; *P < 0.05. D, E, F. Protein levels of NF-κB/P65, p-NF-κB/P65 and their statistical analysis with comparisons between WT and ASPP1-/-mice with and without CCl4 treatment. n = 4; *P < 0.05. IκBα, inhibitor of kappa B alpha (inhibitor protein of NF-κB); p-IκBα, phosphorylated inhibitor of kappa B alpha; NF-κB/P65, nuclear factor kappa B/P65; p-NF-κB/P65, phosphorylated nuclear factor kappa B/P65; β-actin, beta actin.
Table 1. Information on the antibodies used for western blot analysis
Antibody Origin Dilutions Cat. Number Molecular weight Company ASPP1 Rabbit 1:1000 A4355 175kDa Sigma Aldrich Bax Mouse 1:1000 60267-1-1g 23 kDa Protein Tech Bcl-2 Rabbit 1:1000 0712019 26 kDa Cell Signaling Cleaved Caspase 3 Rabbit 1:1000 9664S 19 kDa Cell Signaling IKBα Mouse 1:1000 4814 39 kDa Cell Signaling p- IKBα Rabbit 1:1000 2859 39 kDa Cell Signaling NF-KB P65 Rabbit 1:1000 8242 65 kDa Cell Signaling p- NF-KB P65 Rabbit 1:1000 3033 65 kDa Cell Signaling β-actin Mouse 1:20000 66009-1-1g 42 kDa Protein Tech ASPP1, Apoptosis-stimulating of p53 protein 1; Bax, BCL-2-associated X protein; BCL-2, B-cell lymphoma 2(40); IKBα, Inhibitor of kappa B alpha (inhibitor protein of NF-KB); p-IKBα, phosphorylated inhibitor of kappa B alpha; NF-KB P65, Nuclear factor kappa B P65; p-NF-KB P65, phosphorylated Nuclear factor kappa B P65(18); β-actin, beta actin. 
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Table 2. Sequences of the Primer Pairs used for qRT-PCR
Gene Sense (5'-3') Antisense (5'-3') ASPP1 ATGATATTAACCGTGTTCTTG CACTTCCTCTCCACACTTCAGC Bax TGGAAGAAGATGGGCTGAGG TTCCCACCCCTCCCAATAAT Bcl-2 AACGATTGTGGCAGTCCCTT GAAGTGCTCAGGTGCCATCT β-actin GACAGCAGTTGGTTGGAGCA TTGGGAGGGTGAGGGACTTC TNF-α CTGAACTTCGGGGTGATCGG GGCTTGTCACTCGAATTTTGAGA IL-6 CTGCAAGAGACTTCCATCCAG AGTGGTATAGACAGGTCTGTTGG IL-1β GAAATGCCACCTTTTGACAGTG TGGATGCTCTCATCAGGACAG ICAM-1 TGCCTCTGAAGCTCGGATATAC TCTGTCGAACTCCTCAGTCAC TLR4 ATGGCATGGCTTACACCACC GAGGCCAATTTTGTCTCCACA ASPP1, Apoptosis-stimulating of p53 protein 1; Bax, BCL-2-associated X protein; BCL-2, B-cell lymphoma 2; β-actin, Beta actin; TNF-α, Tumor necrosis factor alpha; IL-6, Interleukin 6; IL-1β, Interleukin 1beta; ICAM1, Intercellular adhesion Molecule; TLR4, Toll like receptors 4.
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