[Studying the correlation between ferritin and non-alcoholic fatty liver disease].

Objective: To analyze the correlation between serum ferritin and steatosis in non-alcoholic fatty liver disease. Methods: Data of 167 cases who underwent liver biopsy in the Affiliated Hospital of Hangzhou Normal University were collected. Hydrogen proton magnetic resonance spectroscopy were performed within one week. The pathological results of liver biopsy were used as the gold standard to analyze the case data, serological indicators, magnetic resonance spectroscopy-proton density fat fraction. Results: Pathological monitoring result showed that the serum ferritin in patients without steatosis, and with mild, moderate and severe steatosis were (206.20 ± 189.83), (286.65 ± 200.80), (326.55 ± 214.71), (391.50 ± 184.93) ng/ml, respectively, P < 0.005. Serum ferritin was correlated to body mass index, PDFF, alanine aminotransferase, gamma glutamyltransferase, low-density lipoprotein, high-density lipoprotein. The area under ​​the receiver operating characteristic curve with ferritin for the diagnosis of non-alcoholic fatty liver disease was 0.716, and the optimal diagnostic threshold was 214.56 ng/ml. The sensitivity and specificity were 80.1%, and 68.8%, respectively. There was no statistically significant difference between the intralobular inflammation, fibrosis, and ferritin. Prussian blue iron staining had no apparent deposition of iron particles. Conclusion: Ferritin has significant positive correlation with the results of pathological and magnetic resonance imaging for liver steatosis. Therefore, it can be used as a non-invasive diagnostic method for liver steatosis evaluation.

[Study of liver fat and iron deposition quantification based on magnetic resonance imaging in rats with nonalcoholic fatty liver disease].

Objective: To investigate the accuracy of magnetic resonance imaging (MRI) for quantitative determination of liver fat and iron content through a rat model of non-alcoholic fatty liver disease (NAFLD) induced by methionine-choline deficient (MCD) diet. Methods: Sixty SD rats were randomly divided into experimental (MCD-diet group, n = 30) and normal control group (normal diet, n = 30). Rats were subjected to special MRI examinations at the ends of 2, 4, and 8 weeks. Proton density fat fraction (PDFF) and R2* value were obtained, and then the rats were sacrificed. The liver tissues were stained with HE, Prussian blue, etc. Liver tissue non-heme iron (NHI) homogenate was determined by flame atomic absorption spectrometry. According to different data, one-way analysis of variance, t-test or χ (2) test was used for statistical analysis. Results: PDFF and R2 * values in the MCD diet group at 2, 4 and 8 weeks were 23.37% ± 9.20%, 28.07% ± 6.84%, 25.40% ± 7.04% (P < 0.01) and 90.58 ± 15.92, 104.12 ± 13.47, 106.35 ± 15.76 (P < 0.05), respectively, which were significantly higher than the normal control group PDFF (2.39% ± 0.50%, 2.45% ± 0.45%, 3.26% ± 0.80%) and R2* (48.93 ± 7.90, 54.71 ± 5.91, 64.25 ± 15.76). Additionally, with the disease progression, R2 * had gradually increased, which was consistent with the NHI trend in liver tissue homogenates of each group. Conclusion: MRI, as a non-invasive quantitative method, can accurately assess liver fat and iron content in fatty liver disease, and with the degree of severity of fat changes, iron deposits tend to increase.

[Sivelestat alleviates nonalcoholic steatohepatitis in mice through inhibiting activation of Kupffer cells].

Objective: To investigate the role of neutrophil elastase inhibitor, sivelestat, in preventing and treating nonalcoholic steatohepatitis (NASH) and its underling mechanisms. Methods: A total of forty 4-week-old male C57BL/6J ApoE-/-mice were equally divided into the following four groups: standard chow (SC)+isotonic saline; SC+sivelestat; high-fat, high-cholesterol (HFHC) diet+isotonic saline; and HFHC+sivelestat. These mice were treated with above methods for 12 weeks. Blood and liver tissue samples were collected to measure biochemical parameters, hepatic steatosis and non-alcoholic fatty liver disease (NAFLD) activity score (inflammation) were evaluated by oil red O staining and HE staining, respectively. The mRNA and protein expression levels of hepatic inflammatory cytokines, CD68, and F4/80 were determined by quantitative RT-PCR and immunohistochemistry, respectively. Comparison of means between the four groups was made by one-way analysis of variance, and comparison between any two groups was made by the LSD or SNK method (for data with homogeneity of variance) or the Tamhane or Dunnett method (for data with heterogeneity of variance). Results: Mice fed with an HFHC diet for 12 weeks developed typical pathological features of NASH compared with those fed with SC. Compared with mice fed with HFHC diet without sivelestat, those treated with HFHC and sivelestat exhibited the following features: (1) significantly reduced fast blood glucose, blood cholesterol, and hepatic biochemical parameters, as well as increased insulin sensitivity; (2) significantly reduced NAFLD activity score (5.71±1.11 vs 3.16±1.16, P < 0.05); (3) reduced monocyte chemoattractant protein-1 and tumor necrosis factor -α; (4) significantly reduced mRNA levels of CD68 and F4/80; and (5) reduced expression of CD68 in the liver. Conclusion: Sivelestat alleviates the hepatic steatosis and inflammation of NASH in mice by inhibiting the activation of Kupffer cells.

[Establishment and evaluation of a mouse model of nonalcoholic steatohepatitis-related hepatocellular carcinoma].

OBJECTIVE: To establish an apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) double-knockout (ApoE(-/-)/LDLR(-/-)) mouse model of nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) induced by high-fat and high-cholesterol (HFHC) diet. METHODS: ApoE(-/-) knockout mice were crossed with LDLR(-/-) knockout mice to obtain ApoE(-/-)/LDLR(-/-) mice. The ApoE(-/-)/LDLR(-/-) mice mated with each other, and the offspring were injected with low-dose streptozotocin (STZ) at 2-3 days after birth. Some mice were fed with HFHC diet after weaning as the model group (n = 15), and some mice were fed with normal diet as the control group (n = 15). Mice were sacrificed at the end of weeks 10, 16, and 20 (5 mice at each time point). The body weight was measured. Liver tissue and blood were collected to measure biochemical parameters, evaluate the pathological changes in the liver tissue by HE staining, oil red O staining, and Masson staining, and detect the expression of glypican-3 (a marker of HCC) by immunohistochemical staining. RESULTS: The model group had significantly higher levels of fasting blood glucose and total cholesterol than the control group (P < 0.01). Serum levels of alanine aminotransferase, aspartate aminotransferase, and total triglyceride gradually increased with time in the model group; at week 20, there were significant differences in above three indices between the two groups (P < 0.05). HE staining showed that compared with the control group at the corresponding time point, the model group developed sequential histological changes: NASH at week 10, dysplastic nodules at week 16, and early HCC at week 20. Oil red O staining showed that in the model group, the degree of liver steatosis increased within 10 weeks and gradually decreased later. Masson staining demonstrated that the model group developed pathological changes: mild perisinusoidal fibrosis at week 16 and bridging fibrosis around tumors at week 20. HE staining, oil red O staining, and Masson staining showed that no histological or pathological changes were found in the control group. Glypican-3 was detected in the nodules at week 16 and in the cytoplasm of HCC cells at week 20 in the model group. CONCLUSION: The mouse model of NASH-related HCC can be developed by giving STZ injection to neonatal ApoE(-/-)/LDLR(-/-) mice and feeding them with HFHC diet after weaning for 20 weeks. Early HCC may develop directly from NASH.

Novel antimicrobial peptides with promising activity against multidrug resistant Salmonella enterica serovar Choleraesuis and its stress response mechanism.

AIMS: To evaluate the antibacterial efficacy of novel antimicrobial peptides (AMPs) against multidrug-resistant (MDR) Salmonella enterica serovar Choleraesuis (Salm. Choleraesuis) and to delineate the AMP-responsive mechanisms of wild-type (WT) and MDR strains. METHODS AND RESULTS: Proteomic approaches were performed based on two-dimensional gel electrophoresis and liquid chromatography-electrospray ionization-quadrupole- time-of-flight tandem mass spectrometry to analyse the protein profiles of these two strains upon exposure to AMP GW-Q6. Quantitative real-time PCR was conducted to determine the mRNA expression level of the target genes. Furthermore, lipopolysaccharide (LPS) competition analysis was used to verify whether LPS may serve as the potential binding target when AMP approach and adhere to the bacterial surface. CONCLUSIONS: The minimal inhibitory concentration assay revealed that our AMPs were even more effective against the MDR strains (4-32 µg ml(-1) ), compared with those for the WT (8-64 µg ml(-1) ). LPS dose-dependently suppressed the GW-Q6 antimicrobial activity. Clusters of orthologous groups analysis showed that the majority of the AMP-responsive proteins were involved in cell envelope biogenesis and outer membrane, translation and chaperones. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicated that the novel AMP GW-Q6 serves as a potential candidate for antimicrobial drug development against MDR strains. These findings will also be helpful for expanding our knowledge on the molecular mechanisms of AMP-microbe interaction and the pathogenicity of salmonellosis caused by MDR strains of Salm. Choleraesuis.

[Effect of Chinese herbal medicine for nourishing yin and removing fire on biosynthesis, secretion and regulative mechanism of gonadotropin-releasing hormone in hypothalamus].

OBJECTIVE: To elucidate the effect of Chinese herbal medicine (CHM) for nourishing Yin and removing fire on the biosynthesis, secretion and regulative mechanism of gonadotropin-releasing hormone (GnRH) in hypothalamus. METHODS: The brain slices of medial basal hypothalamus of adolescent rats, which had been fed with CHM, were incubated. The content of GnRH in incubative liquid was determined during the slices were stimulating with high KC1 to observe the change of GnRH biosynthesis from tonic secretory center of GnRH (arcuate nucleus and ventromedial nucleus) in hypothalamus. The integrated optic density of GnRH positive immunoreactive substance in preoptic area of hypothalamus was determined by immunohistochemistry and image processing to observe the change of GnRH content in pulsative secretory center of GnRH (medial preoptic nucleus) in hypothalamus. The push-pull perfusion of medial preoptic area in hypothalamus was performed. The content of GnRH in serial perfusates was determined by radioimmunoassay (RIA) to observe the change of frequency and amplitude of GnRH pulse releasing from medial preoptic area in hypothalamus. The content of aspartic acid, glutamic acid and gamma-amino butyric acid in the perfusate was determined by high performance liquid chromatography-fluorometry, and the content of beta-endorphic in the perfusate was determined by RIA to observe the change of releasing amount of exciting aminoacid neurotransmitter and beta-endorphin from pulsative secretory center of GnRH (medial preoptic area) in hypothalamus. RESULTS: CHM could markedly reduce the content of GnRH in medial basal hypothalamus (arcuate nucleus and veatromeolial nucleus) and preoptic area (meolical preoptic nucleus) of hypothalamus, and could obviously lower the frequency and amplitude of GnRH pulse releasing from medial preoptic nucleus, It also could markedly decrease the releasing amount of aspartic acid and glutamic acid, while obviously increase the releasing amount of gamma-amino butyric acid and beta-endorphin from medial preoptic area of hypothalamus. CONCLUSION: CHM could markedly reduce the activity of GnRH neurons in hypothalamus through inhibiting the releasing of central exciting aminoacid neurotransmitter and promoting the releasing of central inhibiting aminoacid neurotransmitter and beta-endorphin, thereby, obviously decrease the biosynthesis and secretion of GnRH from tonic and pulsative secretory center of GnRH in hypothalamus. It could be one of chief effective mechanism of CHM in efficiently treating the idiopathic precocious puberty.