Human neutrophil peptide-1 promotes alcohol-induced hepatic fibrosis and hepatocyte apoptosis

<div><p>Background and aims</p><p>Neutrophil infiltration of the liver is a typical feature of alcoholic liver injury. Human neutrophil peptide (HNP)-1 is an antimicrobial peptide secreted by neutrophils. The aim of this study was to determine if HNP-1 affects ethanol-induced liver injury and to examine the mechanism of liver injury induced by HNP-1.</p><p>Methods</p><p>Transgenic (TG) mice expressing HNP-1 under the control of a β-actin-based promoter were established. Ethanol was orally administered to HNP-1 TG or wild-type C57BL/6N (WT) mice. SK-Hep1 hepatocellular carcinoma cells were used to investigate the effect of HNP-1 on hepatocytes <i>in vitro</i>.</p><p>Results</p><p>After 24 weeks of ethanol intake, hepatic fibrosis and hepatocyte apoptosis were significantly more severe in TG mice than in WT mice. Levels of CD14, TLR4, and IL-6 in liver tissues were higher in TG mice than in WT mice. Apoptosis was accompanied by higher protein levels of caspase-3, caspase-8, and cleaved PARP in liver tissue. In addition, phosphorylated ASK1, ASK1, phosphorylated JNK, JNK1, JNK2, Bax, Bak and Bim were all more abundant in TG mice than in WT mice. In contrast, the level of anti-apoptotic Bcl2 in the liver was significantly lower in TG mice than in WT mice. Analysis of microRNAs in liver tissue showed that miR-34a-5p expression was significantly higher in TG mice than in WT mice. Furthermore, in the presence of ethanol, HNP-1 increased the apoptosis with the decreased level of Bcl2 in a concentration-dependent manner <i>in vitro</i>.</p><p>Conclusions</p><p>HNP-1 secreted by neutrophils may exacerbate alcohol-induced hepatic fibrosis and hepatocyte apoptosis with a decrease in Bcl2 expression and an increase in miR-34a-5p expression.</p></div

Expression of F4/80, CD68, CD14 and toll-like receptor 4 in liver tissue.

<p>(A) Semi-quantification of positive cells for immunostaining using anti-F4/80 or anti-CD68 antibody. (B) Semi-quantification of CD14 and toll-like receptor 4 protein expression assessed by Western blot analysis. Results are shown as means ± SD (<i>n</i> = 11 in TG mice and <i>n</i> = 6 in WT mice). <i>*P<0</i>.<i>05</i>.</p

Evaluation of apoptosis-related protein using Western blot analysis in liver tissue in the 24-week model.

<p>Results are shown as means ± SD Results are shown as means ± SD (<i>n</i> = 11 in TG mice and <i>n</i> = 6 in WT mice). <i>*P<0</i>.<i>05</i>, <i>**P<0</i>.<i>01</i>, <i>***P<0</i>.<i>001</i>.</p

NFκB and IL-6 expression in liver tissue.

<p>(A) Semi-quantification of positive cells for immunostaining using anti-NFκB-p65 antibody in the 8- and 24-week model. (B) Semi-quantification of the nuclear NFκB-p65 activities in liver tissue. (C) Protein levels of NFκB-p65 and interleukin-6 in liver tissue assessed by Western blot analysis. Results are shown as means ± SD (7 TG mice and 6 WT mice in the 8-week intake group, and 11 TG mice and 6 WT mice in the 24-week intake group). <i>*P<0</i>.<i>05</i>, <i>**P<0</i>.<i>01</i>, <i>***P<0</i>.<i>001</i>.</p

Relative expression of apoptosis-related protein in human hepatic adenocarcinoma cell line SK-Hep-1 assessed by Western blot analysis.

<p>Results are shown as means ± SD (<i>n</i> = 4 for each group). <i>*P<0</i>.<i>001</i>.</p

Apoptosis in human hepatic adenocarcinoma cell line SK-Hep-1 by ethanol and HNP-1 assessed by caspase 3/7 activity and DNA fragmentation.

<p>(A) Activity of caspase 3/7. (B) DNA fragmentation. <i>*P<0</i>.<i>05</i>, ^§A405 nm-A480 nm. Results are shown as means ± SD (<i>n</i> = 4 for each group).</p