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中国生物工程杂志

China Biotechnology
China Biotechnology  2022, Vol. 42 Issue (4): 9-16    DOI: 10.13523/j.cb.2112036
    
Knockdown of Usp13 Promotes Palmitic Acid-induced Apoptosis in Mouse Hepatocytes
WANG Ting1,LIU Kai2,LI Ke-ying3,CHEN Xu2,REN Guang-ming2,**(),YANG Xiao-ming1,2,3,**()
1 School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
2 Beijing Institute of Lifeomics, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100850, China
3 Qingdao University, Qingdao 266071, China
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Abstract  

Objective: To study the effect of ubiquitin-specific protease 13 (Usp13) on the apoptosis of mouse hepatocytes stimulated by palmitic acid,and to explore the underlying molecular mechanism. Methods: Mouse primary hepatocytes were isolated by two-step collagenase perfusion technique,and then stimulated with palmitic acid after cultured in vitro.Lipid accumulation was detected by triglyceride quantitative method and oil red O staining. Cell viability was measured by MTS and LDH assay. The apoptosis of hepatocytes was tested by Annexin V-FITC/7-AAD assay. Finally the expression levels of anti-apoptotic genes and pro-apoptotic genes were detected by qPCR and the activation of Caspase-3 was detected by Western blotting. Results: Knockdown of Usp13 did not affect the accumulation of triglyceride in palmitic acid-induced hepatocytes,but the cell viability was decreased and apoptosis was increased.Studies show that knockdown of Usp13 resulted in the down-regulated expression of anti-apoptotic genes Bcl-2 and Bcl-xl, and the up-regulated expression of pro-apoptotic genes Bid and p53.The Caspase-3 activity in Usp13 knockdown hepatocytes was enhanced after being exposed to palmitic acid. Conclusion: This study revealed the mechanism of Usp13 regulating the apoptosis of hepatocytes induced by palmitic acid, which laid a foundation for further study of Usp13 regulating the occurrence and development of non-alcoholic steatohepatitis.



Key wordsNon-alcoholic steatohepatitis      Usp13      Apoptosis      Palmitic acid      Mouse primary hepatocytes     
Received: 08 December 2021      Published: 05 May 2022
ZTFLH:  Q819  
Corresponding Authors: Guang-ming REN,Xiao-ming YANG     E-mail: max19920503@163.com;xiaomingyang@sina.com
Cite this article:

WANG Ting,LIU Kai,LI Ke-ying,CHEN Xu,REN Guang-ming,YANG Xiao-ming. Knockdown of Usp13 Promotes Palmitic Acid-induced Apoptosis in Mouse Hepatocytes. China Biotechnology, 2022, 42(4): 9-16.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2112036     OR     https://manu60.magtech.com.cn/biotech/Y2022/V42/I4/9

Gene ID Gene name Upstream (5'-3') Downstream (5'-3')
12043 Bcl-2 ATGCCTTTGTGGAACTATATGGC GGTATGCACCCAGAGTGATGC
12048 Bcl-xl GAAAGCGTAGACAAGGAGATGC ACAAGGGGCGTGGTTCTTAC
22059 p53 ACCGCCGACCTATCCTTACC TCTTCTGTACGGCGGTCTCTC
12122 Bid GCCGAGCACATCACAGACC TGGCAATGTTGTGGATGATTTCT
14433 Gapdh AGGTCGGTGTGAACGGATTTG GGGGTCGTTGATGGCAACA
Table 1 Primer sequences for real-time PCR
Fig.1 Genotype results of Usp13 gene knockout mice (a) Genotype identification of Usp13-/-mice (b) Western blot analysis the expression levels of Usp13 in primary hepatocytes (c) Primary hepatocytes of Usp13+/+ and Usp13-/-mice. Original magnification, ×200. Data are representative of three independent experiments
Fig.2 Knockout of Usp13 did not affect lipid accumulation in hepatocytes (a) Relative intracellular triglyceride levels in hepatocytes (b) Representative oil red O staining images in hepatocytes. Original magnification, ×200. Data are presented as mean ± SD from three independent experiments or representative of three independent experiments. Statistical significance was assessed by two-way ANOVA
Fig.3 Knockdown of Usp13 reduced palmitic acid-induced viability of hepatocytes (a) MTS analysis the viability of hepatocytes induced by palmitic acid (b) Cytosolic LDH release analysis the death of hepatocytes induced by palmitic acid. Data are presented as mean ± SD from three independent experiments or representative of three independent experiments. Statistical significance was assessed by two-way ANOVA and unpaired t-test
Fig.4 Knockdown of Usp13 promotes palmitic acid-induced apoptosis of hepatocytes Flow cytometry analysis hepatocytes apoptosis induced by palmitic acid. Data are presented as mean ± SD from three independent experiments or representative of three independent experiments. Statistical significance was assessed by unpaired t-test
Fig.5 Knockdown of Usp13 enhanced the activation of palmitic acid-induced apoptosis pathway in hepatocytes qPCR analysis the expression of anti-apoptotic genes Bcl-2,Bcl-xl (a) and pro-apoptotic genes Bid,p53 (b). Western blot analysis the Caspase-3 activity in hepatocytes (c). Data are presented as mean ± SD from three independent experiments or representative of three independent experiments. Statistical significance was assessed by two-way ANOVA
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