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ObjectiveTo investigate the effect of hepatitis B virus (HBV) infection on the activation of hepatic stellate cells (HSCs) and its mechanism of action. MethodsA 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. ResultsLO2-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). ConclusionHBx 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.

Emerging evidence suggests that atherosclerosis, one of the leading phenotypes of cardiovascular diseases, is a chronic inflammatory disease. During the atherosclerotic process, immune cells play critical roles in vascular inflammation and plaque formation. Meanwhile, gastrointestinal disorder is considered a risk factor in mediating the atherosclerotic process. The present study aimed to utilize sivelestat, a selective inhibitor of neutrophil elastase, to investigate its pharmacological benefits on atherosclerosis and disclose the gastrointestinal–vascular interaction. The activation of intestinal neutrophil was increased during atherosclerotic development in Western diet-fed ApoE-/- mice. Administration of sivelestat attenuated atherosclerotic phenotypes, including decreasing toxic lipid accumulation, vascular monocyte infiltration, and inflammatory cytokines. Sivelestat decreased intestinal permeability and endotoxemia in atherosclerotic mice. Mechanistically, sivelestat upregulated the expression of zonula occludens-1 in the atherosclerotic mice and recombinant neutrophil elastase protein-treated intestinal epithelial cells. Meanwhile, treatment of sivelestat suppressed the intestinal expression of inflammatory cytokines and NF-κB activity. In contrast, administration of lipopolysaccharides abolished the anti-atherosclerotic benefits of sivelestat in the Western diet-fed ApoE-/- mice. Further clinical correlation study showed that the circulating endotoxin level and intestinal neutrophil elastase activity were positively correlated with carotid intima-medial thickness in recruited subjects. In conclusion, sivelestat had pharmacological applications in protection against atherosclerosis, and intestinal homeostasis played one of the critical roles in atherosclerotic development.