A gadoxetic acid-enhanced MRI-based model using LI-RADS v2018 features for preoperatively predicting Ki-67 expression in hepatocellular carcinoma.

PURPOSE: To construct a gadoxetic acid-enhanced MRI (EOB-MRI) -based multivariable model to predict Ki-67 expression levels in hepatocellular carcinoma (HCC) using LI-RADS v2018 imaging features. METHODS: A total of 121 patients with HCC who underwent EOB-MRI were enrolled in this study. The patients were divided into three groups according to Ki-67 cut-offs: Ki-67 ≥ 20% (n = 86) vs. Ki-67 < 20% (n = 35); Ki-67 ≥ 30% (n = 73) vs. Ki-67 < 30% (n = 48); Ki-67 ≥ 50% (n = 45) vs. Ki-67 < 50% (n = 76). MRI features were analyzed to be associated with high Ki-67 expression using logistic regression to construct multivariable models. The performance characteristic of the models for the prediction of high Ki-67 expression was assessed using receiver operating characteristic curves. RESULTS: The presence of mosaic architecture (p = 0.045), the presence of infiltrative appearance (p = 0.039), and the absence of targetoid hepatobiliary phase (HBP, p = 0.035) were independent differential factors for the prediction of high Ki-67 status (≥ 50% vs. < 50%) in HCC patients, while no features could predict high Ki-67 status with thresholds of 20% (≥ 20% vs. < 20%) and 30% (≥ 30% vs. < 30%) (p > 0.05). Four models were constructed including model A (mosaic architecture and infiltrated appearance), model B (mosaic architecture and targetoid HBP), model C (infiltrated appearance and targetoid HBP), and model D (mosaic architecture, infiltrated appearance and targetoid HBP). The model D yielded better diagnostic performance than the model C (0.776 vs. 0.669, p = 0.002), but a comparable AUC than model A (0.776 vs. 0.781, p = 0.855) and model B (0.776 vs. 0.746, p = 0.076). CONCLUSIONS: Mosaic architecture, infiltrated appearance and targetoid HBP were sensitive imaging features for predicting Ki-67 index ≥ 50% and EOB-MRI model based on LI-RADS v2018 features may be an effective imaging approach for the risk stratification of patients with HCC before surgery.

Mechanism of thymosin ß4 in ameliorating liver fibrosis via the MAPK/NF-κB pathway.

Liver fibrosis is a grievous global challenge, where hepatic stellate cells (HSCs) activation is a paramount step. This study analyzed the mechanism of Tß4 in ameliorating liver fibrosis via the MAPK/NF-κB pathway. The liver fibrosis mouse models were established via bile duct ligation (BDL) and verified by HE and Masson staining. TGF-ß1-induced activated LX-2 cells were employed in vitro experiments. Tß4 expression was determined using RT-qPCR, HSC activation markers were examined using Western blot analysis, and ROS levels were tested via DCFH-DA kits. Cell proliferation, cycle, and migration were examined by CCK-8, flow cytometry, and Transwell assays, respectively. Effects of Tß4 on liver fibrosis, HSC activation, ROS production, and HSC growth were analyzed after transfection of constructed Tß4-overexpressing lentiviral vectors. MAPK/NF-κB-related protein levels were tested using Western blotting and p65 expression in the nucleus was detected through immunofluorescence. Regulation of MAPK/NF-κB pathway in TGF-ß1-induced LX-2 cells was explored by adding MAPK activator U-46619 or inhibitor SB203580. Furthermore, its regulating in liver fibrosis was verified by treating BDL mice overexpressing Tß4 with MAPK inhibitor or activator. Tß4 was downregulated in BDL mice. Tß4 overexpression inhibited liver fibrosis. In TGF-ß1-induced fibrotic LX-2 cells, Tß4 was reduced and cell migration and proliferation were enhanced with elevated ROS levels, while Tß4 overexpression suppressed cell migration and proliferation. Tß4 overexpression blocked the MAPK/NF-κB pathway activation by reducing ROS production, thus inhibiting liver fibrosis in TGF-ß1 induced LX-2 cells and BDL mice. Tß4 ameliorates liver fibrosis by impeding the MAPK/NF-κB pathway activation.

Molecular biology analysis of ABO blood group variants caused by natural chimaerism.

BACKGROUND AND OBJECTIVES: The chimaerism phenomenon constitutes a significant mechanism underlying ABO phenotype discrepancies; however, its detection has technical challenges. In the current study, we explored different techniques to establish the chimaeric status of ABO blood types. MATERIALS AND METHODS: Fifteen individuals with possible chimaeric ABO blood type, as suggested by standard tube or column agglutination method and RBC adsorption-elution test, were enrolled in the study. The red blood cells from 11 investigated subjects showed mix-field agglutination with anti-A or anti-B in blood typing; weak A or B antigens on the other four individuals' RBCs were detected by adsorption-elution tests. The genetic study was conducted with PCR-SSP genotype, DNA sequencing of the ABO gene, STR analysis and ddPCR. RESULTS: The genetic chimaeric status was confirmed in four (27%) individuals by SSP test alone. The ABO gene sequencing identified an additional ABO allele and enabled chimaerism detection in 10 (67%) subjects. The STR analyses established the chimaerism status in 13 (87%) individuals. In the two cases where neither of the tests mentioned above had positive findings, the ddPCR was adopted, and microchimaerism, with an extremely low degree of chimaerism (0.77% and 0.12%), was revealed. The ddPCR revealed the unequal haplotypes (29.5% B vs. 70.5% O) in one subject and distinguished this B/O-O/O chimaera from certain B subgroups (B/O genotype without any mutation) like B3 . CONCLUSION: The ABO blood type chimaerism can be genetically established by comprehensive molecular methods, including PCR-SSP/DNA sequencing, STR and ddPCR, which is particularly sensitive for the detection of microchimaerism.

MALAT1 promotes liver fibrosis by sponging miR­181a and activating TLR4­NF­κB signaling.

The aim of the present study was to investigate whether long non­coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) could modulate activation and inflammation of hepatic stellate cell (HSCs) via regulation of a microRNA (miR)­181a­toll like receptor (TLR)4/nuclear factor (NF)­κB axis, thereby contributing to the development of liver fibrosis. A total of 151 patients with liver fibrosis were recruited, and the serum levels of alanine transaminase, aspartate aminotransferase and albumin were determined. Transforming growth factor (TGF)­ß1 and LPS were used to activate and induce inflammation in the human HSC cell line LX2. MALAT1 was knocked using small interfering RNA or overexpressed, and an inhibitor and mimic of miR­181a­5p were used to examine the effect of MALAT1 and miR­181a­5p on the activation and inflammation of LX2 cells. Both MALAT1 and miR­181a­5p expression performed well in their ability to differentiate patients with liver fibrosis from healthy volunteers, and MALAT1 expression was associated with the severity of liver fibrosis. The expression levels of TLR4 and NF­κB were increased after stimulation with LPS or TGF­ß1, but MALAT1 knockdown or miR­181a­5p mimic transfection abrogated this increase. Moreover, the TGF­ß1­induced increase in viability, proliferation, migration, adhesion and collagen production, and the LPS­induced inflammation of LX2 cells were all reversed after MALAT1 knockdown or transfection with miR­181a­5p mimic. The MALAT1/miR­181a­5p axis was involved in regulating collagen production and inflammation by activating TLR4/NF­κB signaling, which may be conducive to liver fibrosis treatment in the future.

Combined intake of blueberry juice and probiotics ameliorate mitochondrial dysfunction by activating SIRT1 in alcoholic fatty liver disease.

BACKGROUND: Mitochondrial dysfunction has been implicated as a significant factor in the liver disease process. Blueberry juice and probiotics (BP) synergistically improve liver function in alcoholic fatty liver disease (AFLD), although the mechanism for this effect was unclear. This study aims to investigate the effect and specific mechanisms of BP on AFLD. METHODS: C57/BL6 mice were randomly divided into seven groups: CG (control), MG (AFLD model), BJ (MG mice treated with blueberry), BJB (MG mice treated with BP), SI (AFLD mice treated with SIRT1 siRNA), BJSI (SI mice treated with blueberry), and BJBSI (SI mice treated with BP). The mice were fed an alcohol liquid diet for 10 days to establish the AFLD model, and subjected to BP and SIRT1 siRNA intervention for 10 days. Liver pathology was performed on day 11, and biochemical and molecular analyses of liver mitochondria were employed on day 12. RESULTS: BP significantly ameliorated hepatic mitochondrial injury, mitochondrial swelling, and hepatic necrosis in AFLD. BP alleviated hepatic mitochondrial dysfunction by increasing the expression of succinate dehydrogenase and cytochrome c oxidase, increasing respiratory control rate and the ADP/O ratio, and facilitating the synthesis of energy-related molecules. Besides, BP increased the expression of glutathione and superoxide dismutase, and inhibited malondialdehyde expression and reactive oxygen species activity. BP-induced sirtuin 1 (SIRT1), which activates peroxisome proliferator-activated receptor-gamma coactivator-1α, both of which mediate mitochondrial homeostasis. SIRT1 silencing suppressed the BP-induced changes in liver mitochondria, blunting its efficacy. CONCLUSIONS: The ingredients of BP ameliorate hepatocyte mitochondrial dysfunction in AFLD mice.

Resveratrol contributes to the inhibition of liver fibrosis by inducing autophagy via the microRNA­20a­mediated activation of the PTEN/PI3K/AKT signaling pathway.

Liver fibrosis (LF) is a healing response to wounds resulting in liver injury that can cause liver failure or even cancer without functional prevention. Resveratrol (RSV) has been suggested to exert biological effects against various human diseases. MicroRNA­20a (miRNA/miR­20a) has been shown to promote disease progression. The present study aimed to assess the mechanisms through which RSV induces autophagy and activates the miR­20a­mediated phosphatase and tensin homolog (PTEN)/PI3K/AKT signaling pathway in LF. First, a rat model of carbon tetrachloride (CCL4)­induced LF and a cell model of platelet­derived growth factor (PDGF)­BB­stimulated HSC­T6 cells were established for use in subsequent experiments. Subsequently, RSV at a range of concentrations was injected into the model rats with LF. Indicators related to liver injury, oxidative stress and fibrosis were determined in the rats with LF. The RSV­treated HSC­T6 cells were subjected to transfection with miR­20a mimic and PTEN overexpression plasmid to assess the levels of liver injury and LF. A dual­luciferase reporter gene assay was performed to verify the binding sites between PTEN and miR­20a. RSV was found to alleviate LF in rats, and autophagy was enhanced in the rats with LF following RSV treatment. Furthermore, the activation of the PTEN/PI3K/AKT axis attenuated LF, which was reversed by transfection with miR­20a mimic. RSV reversed the inhibitory effects of miR­20a on PTEN expression, reducing miR­20a expression and promoting PTEN, PI3K and p­AKT protein expression, thus attenuating LF. On the whole, the present study demonstrates that RSV induces autophagy and activates the miR­20a­mediated PTEN/PI3K/AKT signaling pathway to attenuate LF. These findings may lead to the development of potential therapeutic strategies for LF.

EGCG induces ß-defensin 3 against influenza A virus H1N1 by the MAPK signaling pathway.

Epigallocatechin gallate (EGCG) is the main component of green tea, which has been proven to inhibit a variety of viruses, including influenza A virus. However, the mechanism of EGCG against influenza virus remains to be further explored. The mechanism of EGCG against influenza virus was studied. The results showed that EGCG significantly increased the levels of HBD3 mRNA and protein, while the levels of phosphorylation of (p)-p38 MAPK, ERK and JNK after EGCG treatment were significantly up-regulated. p38 MAPK, ERK and JNK inhibitors significantly inhibited the expression of HBD3 induced by EGCG. On the other hand, EGCG significantly inhibited the expression of HA and NP proteins in influenza A virus H1N1, but attenuated the anti-influenza A virus effect of EGCG after silencing HBD3. Thus, the anti-influenza virus effect of EGCG is related to the induction of HBD3 expression. In addition, the expression of EGCG-induced HBD3 is related to the p38 MAPK, ERK and JNK signaling pathways. The research data show that EGCG can induce HBD3 expression through p38 MAPK, ERK and JNK signaling pathway to inhibit the replication of influenza A virus H1N1, providing a new and effective candidate drug for influenza virus.

Protection of hepatocyte mitochondrial function by blueberry juice and probiotics via SIRT1 regulation in non-alcoholic fatty liver disease.

Mitochondrial dysfunction has been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Our previous study has firstly reported that blueberry juice and probiotics (BP) effectively protect liver function in NAFLD. However, the role of BP in hepatic mitochondria is unknown. Here, we aimed to investigate the effects and mechanisms of BP on the mitochondrial function and oxidative stress of rats with NAFLD. The NAFLD rat models were established and treated with BP and SIRT1 siRNA. The mitochondrial ultrastructure was analyzed by electron microscopy, reactive oxygen species (ROS) was detected by immunofluorescence, and biomarkers of mitochondrial function and oxidative stress were examined via quantitative reverse transcription-PCR, western blot, and immunohistochemistry. Results revealed that BP significantly reversed the NAFLD-induced hepatic mitochondrial damage, mitochondrial swelling, and hepatic necrosis. In particular, BP significantly restored the mitochondrial respiratory function of NAFLD rats by decreasing state 4 and 3 respiration rates, by increasing the respiration control ratio (RCR) and the ADP/O ratio, and by enhancing ATP, ADP, AMP, and EC syntheses. Moreover, BP reduced mitochondrial oxidative stress in NAFLD by decreasing the MDA level, elevating the GSH and SOD levels, and suppressing the ROS activity. Importantly, SIRT1 deficiency significantly abolished the effects of BP on the mitochondria and oxidative stress. Furthermore, BP reversed the decline of PGC-1α expression induced by NAFLD, while SIRT1 silencing significantly suppressed the effects of BP on PGC-1α. In conclusion, BP might effectively protect rats against mitochondrial dysfunction during NAFLD as potential ingredients of functional food, by modulating the SIRT1 expression. Potential endogenous modulators of NAFLD pathogenesis may ultimately provide novel tools for therapeutic intervention.

Thymosin-ß4 Mediates Hepatic Stellate Cell Activation by Interfering with CircRNA-0067835/miR-155/FoxO3 Signaling Pathway.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrosis. Our study proved that thymosin beta 4 (Tß4) has anti-fibrogenic effects in HSCs through PI3K/AKT pathway. However, the underlying mechanisms are not fully elucidated. Circular RNAs (circRNAs) play important roles in fine-tuning gene expression and are often deregulated in cancers. However, the expression profile and clinical significance of in liver fibrosis is still unknown. Therefore, we hypothesize that Tß4 influences circRNAs in liver fibrosis. METHODS: Circular RNA microarray was conducted to identify Tß4-related circRNAs. Pathway analysis and miRNA response elements analysis was conducted to predict the potential roles of differentially expressed circRNAs in liver fibrosis. CCK8 assays and flow cytometric assays were conducted to clarify the role of circRNA in liver fibrosis. Bioinformatics analysis and in vitro experiments were conducted to clarify the mechanism of circRNA-mediated gene regulation in liver fibrosis. RESULTS: A total of 644 differentially expressed circRNAs were identified between the Tß4-depleted LX-2 cells and the control LX2 cells. The expression of circRNA-0067835 was significantly increased in the Tß4-depleted LX-2 cells compared with control. Knockdown of circRNA-0067835 observably decreased LX-2 cell proliferation by causing G1 arrest and promoting apoptosis. Bioinformatics online programs predicted that circRNA-0067835 acted as miR-155 sponge to regulate FOXO3a expression, which was validated using luciferase reporter assay. CONCLUSION: Our experiments showed that circRNA-0067835 regulated liver fibrosis progression by acting as a sponge of miR-155 to promote FOXO3a expression, indicating that circRNA-0067835 may serve as a potential therapeutic target for patients with liver fibrosis.

Thymosin-ß4 inhibits proliferation and induces apoptosis of hepatic stellate cells through PI3K/AKT pathway.

Liver fibrosis is a necessary stage for chronic liver diseases, and serious threat to human health. Hepatic fibrosis is a necessary stage for chronic liver diseases. Hepatic stellate cells (HSCs) are the primary cell type responsible for fibrosis. Thymosin beta 4 (Tß4) has a potential role in the pathogenesis of liver fibrosis and that it is especially associated with the activation of HSCs, however, the underlying mechanisms are not fully elucidated. Herein, we investigated the potential role of Tß4 in liver fibrosis by describing the effects of Tß4, and we discuss the possible signaling pathway regulated by Tß4. The expression of Tß4 was significantly decreased in human HSC cell line LX-2 and CCl4-treated mouse liver. The depletion of Tß4 significantly associated with the activation of HSCs via the enhanced expression of α-SMA and vimentin. Tß4 significantly suppressed the viability and migration of HSCs. Understanding the potential effects and regulatory mechanism of Tß4 in liver fibrosis might help to provide a novel treatment for patients with liver fibrosis.

[Recombinant influenza A virus NS2 protein inhibits the production of interferon in lung tissues of infected mice].

OBJECTIVE: To obtain non-structural protein 2 (NS2) of influenza A virus (IAV) via the expression system of Escherichia coli, and observe the effect of NS2 on the production of interferon γ (IFN-γ) in mouse lung tissues. METHODS: A prokaryotic expression plasmid pET22b(+)/NS2 was constructed and transformed into Rosetta-gami(2) of Escherichia coli. Through the inducible expression and purification, NS2 was prepared. Forty BALB/c mice were divided randomly into 8 groups, normal control group, 200 µg/kg NS2 joint virus group, 200 µg/kg NS2 joint RNA group, 100 µg/kg inactivated virus group, 0.2×lethal dose 50% (LD50) virus group, viral RNA group of 50 µg/kg, 200 µg/kg NS2 group, and 100 µg/kg NS2 group. All mice were subjected to nasal inhalation. Reverse transcription-PCR and Western blotting were respectively performed to detect the mRNA and protein expressions of IFN-γ in lung tissues. RESULTS: Recombinant NS2 was obtained by the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) and purification. After the nasal inhalation of NS2 or/and IAV to mice, reverse transcription-PCR and Western blotting showed that the levels of IFN-γ mRNA and protein in the lung tissues of NS2 joint virus group were lower than those of the virus group. CONCLUSION: NS2 could inhibit the expressions of IFN-γ mRNA and protein in lung tissues of mice infected by IAV.