Effect of hepatitis B x gene-overexpressed hepatocytes on the proliferation and activation of hepatic stellate cells and related mechanism

Tingyu REN; Hezhongrong NIE; Lijia XIAO; Fangnan LIN; Daming WANG; Chunli SONG; Yiwen ZHOU

doi:10.3969/j.issn.1001-5256.2021.07.017

Volume 37 Issue 7

Jul. 2021

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Article Navigation > Journal of Clinical Hepatology > 2021 > 37(7): 1561-1566

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Effect of hepatitis B x gene-overexpressed hepatocytes on the proliferation and activation of hepatic stellate cells and related mechanism

DOI: 10.3969/j.issn.1001-5256.2021.07.017

1.
Department of Clinical Laboratory, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong 518000, China
2.
Genetic Diagnosis Center, The First Hospital of Jilin University, Changchun 130012, China

Research funding:

National Natural Science Foundation of China (81974457);

National Natural Science Foundation of China (81702088);

Natural Science Foundation of Guangdong Province (2018A030313740);

Natural Science Foundation of Guangdong Province (2018A030310446);

Natural Science Foundation of Guangdong Province (2019A1515012079);

Medical Scientific Research Foundation of Guangdong Province (B2018013);

Seeding Program of Shenzhen Hospital of Southern Medical University (2018MM02);

Research Fund of Innovation and Technology Bureau of Baoan District (2019JD443)

Received Date: 2021-02-01
Accepted Date: 2021-03-24
Published Date: 2021-07-20

Abstract

Abstract

Objective To investigate the effect of hepatitis B virus (HBV) infection on the activation of hepatic stellate cells (HSCs) and its mechanism of action. Methods A total of 30 plasma samples of chronic hepatitis B patients, 42 plasma samples of hepatitis B cirrhosis patients, 30 plasma samples of hepatocellular carcinoma patients, and 18 plasma samples of the individuals undergoing physical examination were collected from November 2020 to January 2021, and ELISA was used to measure the content of hepatitis B X protein (HBx), transforming growth factor-β1 (TGFβ1), dopamine beta-hydroxylase (DBH), and hydroxyproline (HYP) in plasma and conditioned medium. LO2 cells were used to establish a cell line with stable overexpression of HBx (LO2-HBx) and negative control cells (LO2-con), and a conditioned medium was prepared for LO2-HBx, LO2-Con, and LO2 cells (Mock), respectively; human HSC cell line LX-2 was incubated and divided into LX-2/LO2-HBx, LX-2/LO2-con, and LX-2/Mock groups, and CCK-8 assay was used to measure the change in cell proliferation. LX-2 cells were stimulated by rhTGFβ1, and the cells in the LX-2/LO2-HBx group were treated with a TGFβ1 receptor inhibitor. Quantitative real-time PCR and Western blot were used to measure the expression of HBx in LO2 cells and the expression of alpha-smooth muscle actin (α-SMA), collagen type Ⅰ alpha 1 (Col1A1), DBH, and TGFβ1 in the above LX-2 cells. An analysis of variance was used for comparison between multiple groups, and the Bonferroni method was used for further comparison; the t-test was used for comparison between two groups; the Pearson method was used for correlation analysis. Results LO2-HBx stably expressed HBx protein and showed an increase in the content of TGFβ1 in supernatant (F=324.701, P < 0.01). The co-cultured LX-2/LO2-HBx group had a significant change in cell morphology, with the presence of cell shrinkage, extended cytoplasmic process, and reduced lipid droplets, and compared with the LX-2/LO2-con group, the LX-2/LO2-HBx group had significant increases in proliferative activity (P < 0.05) and the mRNA and protein expression levels of α-SMA and Col1A1 (mRNA: F=144.712 and 76.680, both P < 0.01; protein: F=234.142 and 528.708, both P < 0.001). The LX-2/LO2-HBx group had significant increases in the content of TGFβ1 (F=29.382, P < 0.01) and DBH (F=42.662, P < 0.01). With the increase in the stimulating concentration of rhTGFβ1, there were significant increases in the expression of α-SMA (F=1 794.031, P < 0.01), Col1A1 (F=91.340, P < 0.01), and DBH (F=2 501.011, P < 0.01), which reached the peak values at the rhTGFβ1 concentration of 10 ng/ml, and after a TGFβ1 receptor inhibitor was added to the conditioned medium, the LO2-HBx group had significant reductions in the expression of DBH and Col1A1 compared with the control group (t=3.603 and 5.798, both P < 0.05). Compared with the healthy control group, the chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma groups had significant increases in the plasma levels of TGFβ1 (F=51.188, P < 0.001), HBx (F=39.227, P < 0.001), DBH (F=34.431, P < 0.001), and HYP (F=16.211, P < 0.001), and a positive correlation was observed between plasma HBx and TGFβ1, between TGFβ1 and DBH, and between HYP and DBH (r= 0.931, 0.863, and 0.765, all P < 0.001). Conclusion HBx protein can promote the secretion of TGFβ1 in LO2 cells, induce the proliferation and activation of LX-2 cells, promote the development of liver fibrosis, and upregulate the expression of TGFβ1 and DBH in LX-2 cells, and rhTGFβ1 stimulation can induce the activation of LX-2 cells and the upregulation of DBH expression.
- Hepatitis B,
- Liver Cirrhosis,
- Hepatic Stellate Cells,
- HBV-X Protein

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Effect of hepatitis B x gene-overexpressed hepatocytes on the proliferation and activation of hepatic stellate cells and related mechanism

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Effect of hepatitis B x gene-overexpressed hepatocytes on the proliferation and activation of hepatic stellate cells and related mechanism

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