Salivary miRNAs as non-invasive biomarkers of hepatocellular carcinoma: a pilot study

Background Improved detection of hepatocellular carcinoma (HCC) is needed, as current detection methods, such as alpha fetoprotein (AFP) and ultrasound, suffer from poor sensitivity. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate many cellular functions and impact cancer development and progression. Notably, miRNAs are detectable in saliva and have shown potential as non-invasive biomarkers for a number of cancers including breast, oral, and lung cancers. Here, we present, to our knowledge, the first report of salivary miRNAs in HCC and compare these findings to patients with cirrhosis, a high-risk cohort for HCC. Methods We performed small RNA sequencing in 20 patients with HCC and 19 with cirrhosis. Eleven patients with HCC had chronic liver disease, and analyses were performed with these samples combined and stratified by the presence of chronic liver disease. P values were adjusted for multiple comparisons using a false discovery rate (FDR) approach and miRNA with FDR P < 0.05 were considered statistically significant. Differential expression of salivary miRNAs was compared to a previously published report of miRNAs in liver tissue of patients with HCC vs cirrhosis. Support vector machines and leave-one-out cross-validation were performed to determine if salivary miRNAs have predictive potential for detecting HCC. Results A total of 4,565 precursor and mature miRNAs were detected in saliva and 365 were significantly different between those with HCC compared to cirrhosis (FDR P < 0.05). Interestingly, 283 of these miRNAs were significantly downregulated in patients with HCC. Machine-learning identified a combination of 10 miRNAs and covariates that accurately classified patients with HCC (AUC = 0.87). In addition, we identified three miRNAs that were differentially expressed in HCC saliva samples and in a previously published study of miRNAs in HCC tissue compared to cirrhotic liver tissue. Conclusions This study demonstrates, for the first time, that miRNAs relevant to HCC are detectable in saliva, that salivary miRNA signatures show potential to be highly sensitive and specific non-invasive biomarkers of HCC, and that additional studies utilizing larger cohorts are needed.

Development and Validation of a Bioanalytical UHPLC-MS/MS Method Applied to Murine Liver Tissue for the Determination of Indocyanine Green Loaded in H-Ferritin Nanoparticles

Indocyanine green (ICG) is one of the most commonly used fluorophores in near-infrared fluorescence-guided techniques. However, the molecule is prone to form aggregates in saline solution with a limited photostability and a moderate fluorescence yield. ICG was thus formulated using protein-based nanoparticles of H-ferritin (HFn) in order to generate a new nanostructure, HFn-ICG. In this study, an ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system was employed to develop and validate the quantitative analysis of ICG in liver tissue samples from HFn-ICG-treated mice. To precipitate HFn, cold acetone in acidic solution at pH 5.0 was used. The processed liver samples were injected into the UHPLC-MS/MS system for analysis using the positive electrospray ionization mode. Chromatographic separation was achieved on a Waters Acquity UPLC® HSS T3 Column (1.8 μm, 2.1 × 100 mm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. The selected reaction monitoring transitions of m/z 753 →m/z 330 and m/z 827 →m/z 330 were applied for ICG and IR-820 (the internal standard, IS), respectively. The method was selective and linear over a concentration range of 50–1,500 ng/ml. The method was validated for sensitivity, accuracy, precision, extraction recovery, matrix effect, and stability in liver tissue homogenates. ICG extraction recoveries ranged between 85 and 108%. The intra- and inter-day precisions were less than 6.28%. The method was applied to a bio-distribution study to compare the amount of ICG levels from mice treated with HFn-ICG and free ICG. The analyses of the homogenate samples from the two types of treatment showed that the concentration levels of ICG is approximately six-fold higher than those of free ICG (1,411 ± 7.62 ng/ml vs. 235 ± 26.0 ng/ml) at 2 h post injection.

Diagnosis and surgical management of idiopathic bile duct cysts in a bearded dragon (Pogona vitticeps)

The case presented shows the clinical signs, diagnosis and surgical management of idiopathic bile duct cysts in a bearded dragon (Pogona vitticeps). A 12-year-old female bearded dragon presented with constipation resulting from the consumption of sand. A soft mass about 4 cm wide could be palpated in the mid-coelom after the substrate passed. The animal started eating, but soon started showing signs of kyphosis. Ultrasound, radiography and computed tomography examinations revealed that the mass was a cyst 4 cm in diameter. Diagnostic laparotomy was performed and the large cyst and affected liver tissue were removed with a partial hepatectomy. Cytology of the cyst was unremarkable, but histopathological examination showed the lesions to be bile duct cysts. No sign of malignancy or inflammation could be seen.

Distribution Characteristics and Species Diversity of Bacteria in Hepatocellular Carcinoma Tissues

This study was to explore the differences in the distribution and species diversity of bacteria between hepatocellular carcinoma (HCC) tissues and normal liver tissues. 28 HCC patients treated with surgery were selected as the research objects (HCC group), and 19 healthy volunteers with normal physical examinations were included in the control group (Normal group). The tumor specimens were obtained by intraoperative and biopsy puncture, and a 16S ribosomal ribonucleic acid (rRNA) library was constructed. Based on the sequencing data obtained by the IlluminaHi Seq sequencing platform, the differences of bacteria in the liver tissues of the HCC group and the Normal group were analyzed at the level of phyla, family, and genus. The Ace, Chao1, and Shannon of the two groups were compared. The results showed that IlluminaHi Seq sequencing obtained a total of 11,714,659 valid sequences, with an average of 131,625 sequences per sample. The proportions of Bacteroidetes, Firmicutes, and Proteobacteria in HCC group and Normal group were 48.75% versus 34.16%, 37.44% versus 18.02%, and 10.85% versus 39.26%, respectively. The Bacteroidaceae, Prevotellaceae, Lachnospiraceae, and Ruminococcaceae accounted for 22.49%, 20.62%, 16.54%, and 19.93% in Normal group; while those in the HCC tissues accounted for 26.83%, 14.22%, 11.14%, and 13.18%, respectively. The dominant bacteria at the genus level in HCC group and Normal group were Bacteroides and Prevotella-9, with the proportions of 24.19% versus 26.04% and 14.19% versus 8.44%, respectively. The difference in operational taxonomic unit (OTU) numbers of HCC and Normal group were compared and analyzed, which were 1,266 and 1,082, respectively, and the number of common OTU in the two tissues was 875. The Ace in HCC tissue and normal liver tissue were 1063.8±66.79 and 1003.6±52.19, respectively. The Ace in HCC tissue was greater than that in normal liver tissue (P < 0.05). The Chao1 and Shannon in HCC tissue were 1022.9±67.74 and 5.4269±0.3608, respectively; while those in normal liver tissue were 1003.6±66.79 and 5.2842±0.9714, respectively. The Chao1 and Shannon in HCC tissues were much higher than those in Normal group (P < 0.05). It showed that there was no difference in the types of bacterial species in HCC tissues, but the proportions of their flora at the level of phyla, family, and genus changed greatly, which may be related to the occurrence of HCC. This study could provide a reference for the diagnosis and treatment of HCC.

The Study of Yin-Chen-Hao-Tang Preventing and Treating Alcoholic Fatty Liver Disease through PPAR Signaling Pathway Based on Network Pharmacology and RNA-Seq Transcriptomics

Background. Alcoholic fatty liver disease (AFLD) is the first stage of the alcoholic liver disease course. Yin-Chen-Hao-Tang (YCHT) has a good clinical effect on the treatment of AFLD, but its molecular mechanism has not been elucidated. In this study, we tried to explore the molecular mechanism of YCHT in improving hepatocyte steatosis in AFLD mice through network pharmacology and RNA sequencing (RNA-Seq) transcriptomics. Methods. Network pharmacological methods were used to analyze the potential therapeutic signaling pathways and targets of YCHT on AFLD. Then, the AFLD mice model was induced and YCHT was administered concurrently. Liver injury was measured by serum alanine aminotransferase (ALT) activity and liver tissue H&E staining, and liver steatosis was determined by serum triglyceride (TG) level and liver tissue Oil Red staining. The molecular mechanism of YCHT on prevention and treatment of mice AFLD was investigated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differential expression genes data obtained by liver tissue RNA-Seq. Finally, ethanol-induced AFLD AML12 hepatocyte model was established, YCHT with or without PPARα agonist pemafibrate or PPARγ inhibitor GW9662 was administered, Nile Red fluorescent staining was used to evaluate steatosis levels in AML12 hepatocytes, and qRT-PCR was used to detect PPARα and PPARγ gene expression. Results. The results of network pharmacology analysis showed that YCHT may exert its pharmacological effect on AFLD through 312 potential targets which are involved in many signaling pathways including the PPAR signaling pathway. AFLD mice experiments results showed that YCHT markedly decreased mice serum ALT activity and serum TG levels. YCHT also significantly improved alcohol-induced hepatic injury and steatosis in mice livers. Furthermore, KEGG pathway enrichment results of RNA-Seq showed that the PPAR signaling pathway should be the most relevant pathway of YCHT in the prevention and treatment of AFLD. AFLD hepatocyte model experiment results showed that YCHT could remarkably reduce hepatocyte steatosis through reducing PPARγ expression and increasing PPARα expression. Conclusions. Our study discovered that PPARγ and PPARα are the key targets and the PPAR signaling pathway is the main signaling pathway for YCHT to prevent and treat AFLD.

Lipopolysaccharide-Induced Transcriptional Changes in LBP-Deficient Rat and Its Possible Implications for Liver Dysregulation during Sepsis

Sepsis is an organ dysfunction caused by the dysregulated inflammatory response to infection. Lipopolysaccharide-binding protein (LBP) binds to lipopolysaccharide (LPS) and modulates the inflammatory response. A rare systematic study has been reported to detect the effect of LBP gene during LPS-induced sepsis. Herein, we explored the RNA sequencing technology to profile the transcriptomic changes in liver tissue between LBP-deficient rats and WT rats at multiple time points after LPS administration. We proceeded RNA sequencing of liver tissue to search differentially expressed genes (DEGs) and enriched biological processes and pathways between WT and LBP-deficient groups at 0 h, 6 h, and 24 h. In total, 168, 284, and 307 DEGs were identified at 0 h, 6 h, and 24 h, respectively, including Lrp5, Cyp7a1, Nfkbiz, Sigmar1, Fabp7, and Hao1, which are related to the inflammatory or lipid-related process. Functional enrichment analysis revealed that inflammatory response to LPS mediated by Ifng, Cxcl10, Serpine1, and Lbp was enhanced at 6 h, while lipid-related metabolism associated with C5, Cyp4a1, and Eci1 was enriched at 24 h after LPS administration in the WT samples. The inflammatory process was not found when the LBP gene was knocked out; lipid-related metabolic process and peroxisome proliferator-activated receptor (PPAR) signaling pathway mediated by Dhrs7b and Tysnd1 were significantly activated in LBP-deficient samples. Our study suggested that the invading LPS may interplay with LBP to activate the nuclear factor kappa B (NF-κB) signaling pathway and trigger uncontrolled inflammatory response. However, when inhibiting the activity of NF-κB, lipid-related metabolism would make bacteria removal via the effect on the PPAR signaling pathway in the absence of LBP gene. We also compared the serum lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) levels using the biochemistry analyzer and analyzed the expression of high mobility group box 1 (HMGB1) and cleaved-caspase 3 with immunohistochemistry, which further validated our conclusion.

Hepatoprotective Activity of Helicteres isora Ethanol Extract Against Paracetamol-Induced Liver Injury in Mice

Helicteres isora L. is a medicinal plant which is used in several diseases, such as snake-bite, dog-bite, diarrhoea and constipation in a new born baby, gastrointestinal disorders, diabetes, cancer, and infections. This plant has also been used in the management of liver damage through traditional medicine. However, the hepatoprotective activity of H. isora L. ethanolic extract has not been reported so much. The present work was carried to investigate the hepatoprotective effect of H. isora L. against paracetamol-induced liver injury in Swiss mice. Paracetamol (PCM) is widely used as an analgesic and antipyretic drug which at high dose can lead to undesirable side effects, such as hepatotoxicity. Paracetamol induce hepatotoxicity was evaluated by an increase (P<0.05) in AST and ALT serum activity. Paracetamol hepatotoxicity was also manifested by an increase in (P<0.05) lipid peroxidation and depletion of reduced glutathione (GSH) in liver tissue. Ethanol extract of H. isora L. (250, 500 and 1000 mg/kg bw/day) significantly restored the PCM-induced alterations in the biochemical activities of blood and liver tissues. The hepatoprotective effect of H. isora L. was also confirmed by the histopathological examination of liver tissue. Histopathological examination of liver sections in mice administered with 1000 mg/kg bw/day doses of the extract were perfectly protected almost similar to those of untreated mice. The results indicated the hepatoprotective nature of studied plants extract against paracetamol induced toxicity. Our study scientifically validates the folkloric claim as well as traditional uses of H. isora L. as hepatoactive medicine. The results of this study suggests a new direction in the treatment of liver disease in future.

Optimization of Antiosteoporotic Therapy in Patients with Liver Fibrosis

The purpose of the study: to investigate the effect of zolendronic acid on bone mineral density in patients with osteoporosis and fibrotic changes of the liver due to steatohepatitis. Materials and methods. We examined 28 female patients with a mean age of 55.3±4.7 years with decreased bone mineral density, nonalcoholic steatohepatosis and liver fibrosis. All studied patients were in menopause duration of 7.8±3.5 years. For inclusion in the study, all patients were excluded from the presence of comorbidities. The degree of liver fibrosis was determined on the basis of 2D shear wave elastometry by transcutaneous access by the method of shear wave in the SWE mode. The study included patients with liver fibrosis F1 - F3 on METAVIR. Determination of bone mineral density was performed using an X-ray densitometer DEXXUM T by dual energy absorption. Results and discussion. The initial level of liver tissue stiffness in the studied patients was 8.52±1.12 kPa, which corresponded to the stage of fibrosis F2 - F3 according to METAVIR. Isolated decrease in lumbar spine mineral density was diagnosed in 20 patients, 8 patients had a combination of decreased spinal mineral density with decreased femoral mineral density, mean T vertebral T-test was -2.25±0.2, mean femoral neck T-test was -1, 9±0.3. In order to maintain and restore bone mineral density, these patients were advised to minimize the factors that contribute to bone loss, mainly by stopping alcohol and smoking. Patients were advised to exercise as much as possible under the supervision of a rehabilitologist, especially to do exercises aimed at improving the mechanics of the spine. One year after administration of 5 mg zolendronic acid intravenously and daily intake of 1500 mg calcium and 800 IU vitamin D the level of liver tissue stiffness in the studied patients was 7.69±1.14 kPa, which corresponded to the stage of fibrosis F2 - F3 according to METAVIR and not due to a moderate decrease in indicators, there was no statistically significant difference. Isolated decrease in lumbar spine mineral density was diagnosed in 19 patients, in 9 patients there was a combination of decrease in spinal mineral density with decrease in femur mineral density, the average criterion of T vertebrae was 1.1±0.3 (p=0.032), the average criterion of T femoral neck -0.9±0.3 (p=0.029). The study of the level of alaline transaminase, aspartate transaminase, alkaline phosphatase, gamma-glutamyltranspeptidase did not reveal any abnormalities. After administration of zolendronic acid in 7 patients there was an increase in temperature to febrile levels within 2-3 days. None of the patients showed signs of bone fractures of any localization during the observation period. Conclusion. Thus, the administration of zolendronic acid to patients with decreased bone mineral density on the background of fibrous changes in liver tissue due to steatohepatitis is safe and highly effective