USP5: Comprehensive insights into structure, function, biological and disease-related implications, and emerging therapeutic opportunities.

Ubiquitin specific protease 5 (USP5) is a vital deubiquitinating enzyme that regulates various physiological functions by removing ubiquitin chains from target proteins. This review provides an overview of the structural and functional characteristics of USP5. Additionally, we discuss the role of USP5 in regulating diverse cellular processes, including cell proliferation, apoptosis, DNA double-strand damage, methylation, heat stress, and protein quality control, by targeting different substrates. Furthermore, we describe the involvement of USP5 in several pathological conditions such as tumors, pathological pain, developmental abnormalities, inflammatory diseases, and virus infection. Finally, we introduce newly developed inhibitors of USP5. In conclusion, investigating the novel functions and substrates of USP5, elucidating the underlying mechanisms of USP5-substrate interactions, intensifying the development of inhibitors, and exploring the upstream regulatory mechanisms of USP5 in detail can provide a new theoretical basis for the treatment of various diseases, including cancer, which is a promising research direction with considerable potential. Overall, USP5 plays a critical role in regulating various physiological and pathological processes, and investigating its novel functions and regulatory mechanisms may have significant implications for the development of therapeutic strategies for cancer and other diseases.

Role of lncSLCO1C1 in gastric cancer progression and resistance to oxaliplatin therapy.

BACKGROUND: Gastric carcinoma (GC) is one of the most deadly diseases due to tumour metastasis and resistance to therapy. Understanding the molecular mechanism of tumour progression and drug resistance will improve therapeutic efficacy and develop novel intervention strategies. METHODS: Differentially expressed long non-coding RNAs (lncRNAs) in clinical specimens were identified by LncRNA microarrays and validated in different clinical cohorts by quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridisation and bioinformatics analysis. Biological functions of lncRNA were investigated by using cell proliferation assays, migration assays, xenograft tumour models and bioinformatics analysis. Effects of lncSLCO1C1 on GC cell survival were assessed by comet assays and immunofluorescence assays. Underlying molecular mechanisms were further explored by using a number of technologies including RNA pull-down, mass spectrometry analysis, RNA immunoprecipitation, co-immunoprecipitation, miRNA sequencing, luciferase reporter assays and molecular modelling. RESULTS: LncSLCO1C1 was highly upregulated in GC tissue samples and associated with GC patients' poor overall survival. Overexpression of lncSLCO1C1 promoted proliferation and migration, whereas decreased lncSLCO1C1 expression produced the opposite effects. lncSLCO1C1 also mediated tumour resistance to chemotherapy with oxaliplatin by reducing DNA damage and increasing cell proliferation. Despite sequence overlapping between lncSLCO1C1 and PDE3A, alternations of PDE3A expression had no effect on the GC cell progression, indicating that lncSLCO1C1, not PDE3A, related with the progression of GC cells. Mechanistically, lncSLCO1C1 serves as a scaffold for the structure-specific recognition protein 1 (SSRP1)/H2A/H2B complex and regulates the function of SSRP1 in reducing DNA damage. Meanwhile, lncSLCO1C1 functions as a sponge to adsorb miR-204-5p and miR-211-5p that target SSRP1 mRNA, and thus increases SSRP1 expression. Patients with high expressions of both lncSLCO1C1 and SSRP1 have poor overall survival, highlighting the role of lncSLCO1C1 in GC progression. CONCLUSIONS: LncSLCO1C1 promotes GC progression by enhancing cell growth and preventing DNA damage via interacting and scaffolding the SSRP1/H2A/H2b complex and absorbing both miR-211-5p and miR-204-5p to increase SSRP1 expression.

Notch-mediated lactate metabolism regulates MDSC development through the Hes1/MCT2/c-Jun axis.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) play critical roles in tumorigenesis. However, the mechanisms underlying MDSC and TAM development and function remain unclear. In this study, we find that myeloid-specific activation of Notch/RBP-J signaling downregulates lactate transporter MCT2 transcription via its downstream molecule Hes1, leading to reduced intracellular lactate levels, blunted granulocytic MDSC (G-MDSC) differentiation, and enhanced TAM maturation. We identify c-Jun as a novel intracellular sensor of lactate in myeloid cells using liquid-chromatography-mass spectrometry (LC-MS) followed by CRISPR-Cas9-mediated gene disruption. Meanwhile, lactate interacts with c-Jun to protect from FBW7 ubiquitin-ligase-mediated degradation. Activation of Notch signaling and blockade of lactate import repress tumor progression by remodeling myeloid development. Consistently, the relationship between the Notch-MCT2/lactate-c-Jun axis in myeloid cells and tumorigenesis is also confirmed in clinical lung cancer biopsies. Taken together, our current study shows that lactate metabolism regulated by activated Notch signaling might participate in MDSC differentiation and TAM maturation.

Pathological Change of Chronic Hepatitis B Patients with Different Tongue Coatings by Circular Multi-Omics Integrated Analysis.

OBJECTIVE: To compare the circular pathological changes of chronic hepatitis B (CHB) patients according to the tongue diagnosis. METHODS: Totally 41 CHB patients with typical white tongue coating (WTC) or yellow tongue coating (YTC) were enrolled and 14 healthy volunteers with normal tongue manifestation served as controls. The mRNA expression of peripheral leukocytes was detected by GeneChips, and 9 genes were randomly selected for expression validation. Circular metabolites were detected by gas chromatographymass spectrometry. Biological information was analyzed based on ingenuity pathways analysis or metabolomics database and the integrated networks were constructed by ClueGO. RESULTS: A total of 945 and 716 differentially expressed genes were found in patients with WTC and YTC relative to healthy volunteers respectively. The biological information analysis indicated that CHB patients had obviously increased functions in cell death, apoptosis and necrosis (Z-score ⩾2, P<0.05) and decreased activation in T lymphocytes (Z-score ⩽-2, P<0.05), regardless of the tongue manifestation. Compared to patients with WTC, the YTC patients were predicted to be more active in functions related to virus replication (Z-score ⩾2, P<0.05), and the content of circular fatty acids, such as oleic acid (P=0.098) and lauric acid (P=0.035), and citric acid cycle-related metabolites were higher in the YTC patients (P<0.1). The integrated analysis based on differential genes and metabolites indicated that the most difference in the biological function network between the WTC and YTC patients was tumor necrosis factor receptor associated factor 6 mediated-nuclear factor kappa-B activation process. CONCLUSIONS: CHB patients with YTC had more severe inflammation and fatty acids metabolism aberrant than patients with WTC. The results facilitate the modern pathological annotation of Chinese medicine tongue diagnosis theory and provide a reference for the interpretation of pharmacological mechanisms of Chinese medicine treatment.

DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B.

OBJECTIVE: To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines. METHODS: Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. RESULTS: Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δß value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1ß were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1ß concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS. CONCLUSIONS: Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1ß levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB. REGISTRATION NO: ChiCTR-RCS-13004001.

A Combination of Geniposide and Chlorogenic Acid Combination Ameliorates Nonalcoholic Steatohepatitis in Mice by Inhibiting Kupffer Cell Activation.

The incidence of nonalcoholic steatohepatitis (NASH) is increasing worldwide. Activation of Kupffer cells (KCs) is central to the development of diet-induced NASH. We investigated whether a combination of two active chemical components, geniposide and chlorogenic acid (GC), at a specific ratio (67 : 1), ameliorates diet-induced NASH and the underlying mechanisms involved. C57BL/6J mice exposed to a high-fat and high-cholesterol (HFHC) diet containing cholesterol, choline, and high-sugar drinking water, as well as RAW264.7 cells stimulated with lipopolysaccharide (LPS) were studied. The combination exerted a therapeutic effect on HFHC-induced NASH in mice. Simultaneously, GC was found to reduce the expression of cytokines secreted by hepatic macrophages, including tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), IL-1ß, IL-6, monocyte chemotactic protein 1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, GC reduced the number of KCs expressing F4/80. Furthermore, TNF-α, inducible nitric oxide synthase (INOS), IL-1ß, and IL-6 mRNA and TNF-α protein expression levels were suppressed upon GC treatment in RAW264.7 cells. Our findings suggest that GC has a strong anti-inflammatory effect in NASH, and this effect can be attributed to the suppression of KC activity in the liver.

Research on the Mechanism of Qushi Huayu Decoction in the Intervention of Nonalcoholic Fatty Liver Disease Based on Network Pharmacology and Molecular Docking Technology.

OBJECTIVE: To use network pharmacology and molecular docking technology in predicting the main active ingredients and targets of Qushi Huayu Decoction (QHD) treatment in Nonalcoholic Fatty Liver Disease (NAFLD) and explore the potential mechanisms of its multi-component-multi-target-multi-pathway. MATERIALS AND METHODS: The main chemical components of QHD were searched using traditional Chinese medicine system pharmacology technology platform (TCMSP) and PubChem database. The main chemical components of the prescription were ADMET screened by the ACD/Labs software. The main active ingredient was screened by 60% oral bioavailability, and 60% of "bad" ingredients were removed from the drug-like group. Swiss Target Prediction, the SEA, and HitPick systems were sequentially used to search for the target of each active ingredient, and a network map of the QHD's target of the active ingredient was constructed. Genome annotation database platforms (GeneCards, OMIM, and DisGeNET) were used to predict action targets related to fatty liver disease. "Drug-Disease-Target" network diagram could be visualized with the help of Cytoscape (3.7.1) software. UniProt and STRING database platforms were used to build a protein interaction network. The KEGG signal pathway and DAVID platform were analyzed for biological process enrichment. RESULTS: A total of 128 active ingredients and 275 corresponding targets in QHD were discovered through screening. 55 key target targets and 27 important signaling pathways were screened, such as the cancer pathway, P13K-AKT signaling pathway, PPAR signaling pathway, and other related signaling pathways. CONCLUSIONS: The present study revealed the material basis of QHD and discussed the pharmacological mechanism of QHD in fatty liver, thus providing a scientific basis for the clinical application and experimental research of QHD in the future.

Targeted delivery of miR-99b reprograms tumor-associated macrophage phenotype leading to tumor regression.

BACKGROUND: Accumulating evidence has shown that tumor-associated macrophages (TAMs) play a critical role in tumor progression. Targeting TAMs is a potential strategy for tumor immunotherapy. However, the mechanism underlying the TAM phenotype and function needs to be resolved. Our previous studies have demonstrated that miR-125a can reverse the TAM phenotype toward antitumor. Meanwhile, we have found that miR-125a and miR-99b cluster in the first intron of the same host gene, and are transcribed simultaneously in bone marrow-derived macrophages (BMDMs) following LPS+IFNγ stimulation. However, it remains unclear whether miR-99b by itself can exert an antitumor effect by regulating macrophage phenotype. METHODS: miR-99b and/or miR-125a were delivered into TAMs of orthotopic hepatocellular carcinoma (HCC) or subcutaneous Lewis lung cancer (LLC) mice. The effect of treatment was evaluated by live imaging, TUNEL staining and survival tests. The phenotype of the immune cells was determined by qRT-PCR, ELISA, western blot and FACS. The capability of miR-99b-mediated macrophage phagocytosis and antigen presentation was detected by FACS and immunofluorescence staining. The underlying molecular mechanism was examined by qRT-PCR, reporter assay and western blot, and further verified in the tumor model. The expression of miR-99b and its target genes was determined in TAMs sorted from tumor and adjacent tissues in patients with liver cancer. RESULTS: Targeted delivery of miR-99b and/or miR-125a into TAMs significantly impeded the growth of HCC and LLC, especially after miR-99b delivery. More importantly, the delivery of miR-99b re-educated TAM toward antitumor phenotype with enhanced immune surveillance. Further investigation of mechanisms showed that macrophage-specific overexpression of miR-99b promoted M1 while suppressing M2 macrophage polarization by targeting κB-Ras2 and/or mTOR, respectively. miR-99b-overexpressed M1 macrophage was characterized by stronger capability of phagocytosis and antigen presentation. Additionally, delivery of simTOR or siκB-Ras2 into TAMs inhibited miR-99b antagomir-triggered tumor growth. Finally, miR-99b expression was lower in TAMs of patients with liver cancer than that in adjacent tissues, while the expression of κB-Ras2 and mTOR was reversed. CONCLUSIONS: Our results reveal the mechanism of miR-99b-mediated TAM phenotype, indicating that TAM-targeted delivery of miR-99b is a potential strategy for cancer immunotherapy.

Analysis of plasma metabolic profile, characteristics and enzymes in the progression from chronic hepatitis B to hepatocellular carcinoma.

Hepatitis B virus (HBV) infection is an important factor causing hepatocellular carcinoma (HCC). The aim of this study was to investigate the metabolic characteristics and related metabolic enzyme changes during the progression from chronic hepatitis B (CHB) to liver cirrhosis (LC) and, ultimately, to HCC. An untargeted metabolomics assay was performed in plasma from 50 healthy volunteers, 43 CHB patients, 67 LC patients, and 39 HCC patients. A total of 24 differential metabolites (DMs) were identified. Joint pathway analysis suggested striking changes in amino acid metabolism and lipid metabolism from CHB to HCC. The panel of L-serine, creatine and glycine distinguished LC from CHB, and L-serine, cystathionine, creatine and linoleic acid distinguished HCC from LC. Bioinformatic analysis of publicly available data showed that differential metabolite profile-associated enzyme genes, including alanine-glyoxylate aminotransferase-2 (AGXT2), D-amino-acid oxidase (DAO), and cystathionine gamma-lyase (CTH), were downregulated, while bisphosphoglycerate mutase (BPGM), cystathionine-ß-synthase (CBS), phosphoserine phosphatase (PSPH) and acyl-CoA thioesterase 7 (ACOT7) were upregulated, in HCC, all of which correlated with a poor prognosis for HCC patients. Our results indicated that serum metabolites and related enzymes are of considerable significance for the diagnosis and prognosis of HCC and can provide a theoretical basis and therapeutic index for future diagnosis and treatment.

Downregulation of FHL1 protein in glioma inhibits tumor growth through PI3K/AKT signaling.

Human four-and-a-half LIM domains protein 1 (FHL1) is a member of the FHL protein family, which serves an important role in multiple cellular events by interacting with transcription factors using its cysteine-rich zinc finger motifs. A previous study indicated that FHL1 was downregulated in several types of human cancer and served a role as a tumor suppressive gene. The overexpression of FHL1 inhibited tumor cell proliferation. However, to the best of our knowledge, there is no evidence to confirm whether FHL1 affected glioma growth, and the molecular mechanisms through which FHL1 represses tumor development remain unclear. In the present study, the expression level of FHL1 was determined using immunohistochemical staining in 114 tumor specimens from patients with glioma. The results indicated that FHL1 expression was negatively associated with the pathological grade of gliomas. Furthermore, Kaplan-Meier survival curves demonstrated that the patients with an increased FHL1 expression exhibited a significantly longer survival time, suggesting that FHL1 may be a prognostic marker for glioma. The protein level of FHL1 was relatively increased in the U251 glioma cell line compared with that in the U87 cell line. Therefore, FHL1 was knocked down in U251 by siRNA and overexpressed in U87, and it was identified that FHL1 significantly decreased the activation of PI3K/AKT signaling by interacting with AKT. Further experiments verified that FHL1 inhibited the growth of gliomas in vivo by modulating PI3K/AKT signaling. In conclusion, the results of the present study demonstrated that FHL1 suppressed glioma development through PI3K/AKT signaling.

A ketogenic diet attenuates proliferation and stemness of glioma stem­like cells by altering metabolism resulting in increased ROS production.

Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stem­like cells (GSCs), by culturing patient­derived primary GSCs as well as a GSC cell line in glucose­restricted, ß­hydroxybutyrate­containing medium (BHB­Glow) which was used to mimic clinical KD treatment. GSCs cultured in BHB­Glow medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished self­renewal in vitro, and reduced tumorigenic capacity in vivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHB­Glow medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHB­Glow medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxia­inducible factor 1 and B­cell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHB­Glow medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.

Aluminum-Based Metal-Organic Frameworks Derived Al2O3-Loading Mesoporous Carbon as a Host Matrix for Lithium-Metal Anodes.

Li-metal anode attracts great focus owing to its ultra-high specific capacity and the lowest redox potential. However, the uncontrolled growth of Li dendrite leads to severe security issues and limited cycle life. Herein, Al2O3 loading mesoporous carbon (Al2O3@MOF-C) derived from Al-based metal-organic frameworks (Al-MOFs) was investigated as the stable host matrix for Li metal, in which, Al2O3 was served as nano seeds for the Li deposition and decrease the Li nucleation overpotential. Except that, the high specific surface area and wide pore distribution can also buffer the volume changes of Li and fasten electron transfer, hence a dendrite-free morphology was observed even after 50 cycles at 2 mA cm-2. High Li coulombic efficiency of 97.9% after 100 cycles at 1 mA cm-2, 1 mAh cm-2, and 97.6% after 50 cycles at 1 mA cm-2 and 6 mAh cm-2 were performed by Al2O3@MOF-C electrodes. Good performances were also obtained for Li-sulfur and LiFePO4 batteries. The performances of Al2O3@MOF-C@Li were compared with Li foil and Cu@Li in full cell configurations. The electrochemical tests of full cells based on Al2O3@MOF-C@Li indicated that this Al-based functional host matrix can enhance the Li-utilization and lead to significant enhancement of the cycling performance of Li anodes.

Protective effect of genistein on nonalcoholic fatty liver disease (NAFLD).

NAFLD is a vital health problem worldwide; however, no effective treatment is currently available for NAFLD. Intensive studies have indicated the efficacy of genistein (GE), a bioactive isoflavone extracted from soy, in treating NAFLD. In addition to its oestrogen-like effects, GE is known to have multiple molecular effects, for instance, lipid and glucose metabolism-promoting effects and activities against lipid peroxidation, inflammation, fibrosis, and NAFLD-related tumours. Here, this review summarizes the potential role of GE in the treatment and prevention of NAFLD and some of the currently known targets and signalling pathways of GE in NAFLD.

Altered oral microbiota in chronic hepatitis B patients with different tongue coatings.

AIM: To elucidate tongue coating microbiota and metabolic differences in chronic hepatitis B (CHB) patients with yellow or white tongue coatings. METHODS: Tongue coating samples were collected from 53 CHB patients (28 CHB yellow tongue coating patients and 25 CHB white tongue coating patients) and 22 healthy controls. Microbial DNA was extracted from the tongue samples, and the bacterial 16S ribosomal RNA gene V3 region was amplified from all samples and sequenced with the Ion Torrent PGM™ sequencing platform according to the standard protocols. The metabolites in the tongue coatings were evaluated using a liquid chromatography-mass spectrometry (LC-MS) platform. Statistical analyses were then performed. RESULTS: The relative compositions of the tongue coating microbiotas and metabolites in the CHB patients were significantly different from those of the healthy controls, but the tongue coating microbiota abundances and diversity levels were not significantly different. Compared with the CHB white tongue coating patients, the CHB yellow tongue coating patients had higher hepatitis B viral DNA (HBV-DNA) titers (median 21210 vs 500, respectively, P = 0.03) and a significantly lower level of Bacteroidetes (20.14% vs 27.93%, respectively, P = 0.013) and higher level of Proteobacteria (25.99% vs 18.17%, respectively, P = 0.045) in the microbial compositions at the phylum level. The inferred metagenomic pathways enriched in the CHB yellow tongue coating patients were mainly those involved in amino acid metabolism, which was consistent with the metabolic disorder. The abundances of bacteria from Bacteroidales at the order level were higher in the CHB white tongue coating patients (19.2% vs 27.22%, respectively, P = 0.011), whereas Neisseriales were enriched in the yellow tongue coating patients (21.85% vs 13.83%, respectively, P = 0.029). At the family level, the abundance of Neisseriaceae in the yellow tongue patients was positively correlated with the HBV-DNA level but negatively correlated with the S-adenosyl-L-methionine level. CONCLUSION: This research illustrates specific clinical features and bacterial structures in CHB patients with different tongue coatings, which facilitates understanding of the traditional tongue diagnosis.

Potential Biological Effects of (-)-Epigallocatechin-3-gallate on the Treatment of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a major health issue throughout the world. However, no validated treatments for NAFLD are currently available. In-depth studies have demonstrated the efficacy of (-)-epigallocatechin-3-gallate (EGCG), a main bioactive chemical extracted from green tea, in treating NAFLD. EGCG exhibits multi-pronged preventive and therapeutic activities, including promoting lipid and glucose metabolism, anti-lipid peroxidation and anti-inflammation activities, anti-fibrosis, and anti-NAFLD related tumor, thus contributing to the mitigation of NAFLD occurrence and progression. The objectives of this paper are to review and discuss the currently known targets, signaling pathways and roles of EGCG that interfere with NAFLD pathogenesis, then providing additional experimental evidence and the foundation for the further studies and clinical applications of EGCG in the prevention and treatment of NAFLD.

Geniposide and Chlorogenic Acid Combination Ameliorates Non-alcoholic Steatohepatitis Involving the Protection on the Gut Barrier Function in Mouse Induced by High-Fat Diet.

Gut-liver axis is increasingly recognized to be involved in the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). The gut microbiota and intestinal permeability have been demonstrated to be the key players in the gut-liver cross talk in NAFLD. Geniposide and chlorogenic acid (GC) combination is derived from a traditional Chinese medicine, Qushi Huayu Decoction (QHD), which has been used in clinic for NAFLD treatment for decades in China and validated in multiple animal models of NAFLD. GC combination previously has been demonstrated to treat NAFLD via modulation on the gut microbiota composition. In the present study, the effects of GC combination on gut barrier function in NAFLD were evaluated, and QHD and sodium butyrate (NaB), the intestinal mucosa protectant, were used as positive control. The therapeutic effect of GC combination on NAFLD were confirmed by amelioration on non-alcoholic steatohepatitis (NASH) induced by high-fat diet (HFD) in mouse, which was comparable to that of QHD. Simultaneously, GC combination was found to reduce the signaling of gut-derived lipopolysaccharide (LPS) including hepatic LPS binding protein, Toll like receptor 4, interleukin-1ß, tumor necrosis factor -α, and Kupffer cells infiltration. Furthermore, GC combination reduced LPS and D-lactate in plasma, restoring the colonic tight junction (TJ) expression and inhibited colonic TJs disassembly by down-regulation on RhoA/ROCK signaling in NASH induced by HFD. On the other hand, NASH was also alleviated in NaB group. The results of the present study suggested the important role of protection on gut barrier function in NAFLD treatment, which contributed to the therapeutic effects of GC combination on NASH.

Chinese herbal formula Fuzheng Huayu alleviates CCl4-induced liver fibrosis in rats: a transcriptomic and proteomic analysis.

Liver fibrosis is a consequence of chronic liver disease that can progress to liver cirrhosis or even hepatocarcinoma. Fuzheng Huayu (FZHY), a Chinese herbal formula, has been shown to exert anti-fibrotic effects. To better understand the molecular mechanisms underlying the anti-fibrotic effects of FZHY, we analyzed transcriptomic and proteomic combination profiles in CCl4-induced liver fibrosis in rats, which were treated with extracted FZHY powder (0.35 g·kg-1·d-1, ig) for 3 weeks. We showed that FZHY administration significantly improved liver function, alleviated hepatic inflammatory and fibrotic changes, and decreased the hydroxyproline content in the livers of CCl4-treated rats. When their liver tissues were examined using microarray and iTRAQ, we found 255 differentially expressed genes (fold change ≥1.5, P<0.05) and 499 differentially expressed proteins (fold change ≥1.2, P<0.05) in the FZHY and model groups. Functional annotation with DAVID (The Database for Annotation, Visualization and Integrated Discovery) showed that 15 enriched gene ontology terms, including drug metabolic process, response to extracellular stimulus, response to vitamins, arachidonic acid metabolic process, response to wounding, and oxidation reduction might be involved in the anti-fibrotic effects of FZHY; whereas KEGG pathway analysis revealed that eight enriched pathways, including arachidonic acid metabolism, retinol metabolism, metabolism of xenobiotics by cytochrome P450, and drug metabolism might also be involved. Moreover, the protein-protein interaction network demonstrated that 10 core genes/proteins overlapped, with Ugt2a3, Cyp2b1 and Cyp3a18 in retinol metabolism pathway overlapped to a higher degree. Compared to the model rats, the livers of FZHY-treated rats had significantly higher mRNA and protein expression levels of Ugt2a3, Cyp2b1 and Cyp3a18. Furthermore, the concentration of retinoic acid was significantly higher in the FZHY-treated rats compared with the model rats. The results suggest that the anti-fibrotic effects of FZHY emerge through multiple targets, multiple functions, and multiple pathways, including FZHY-regulated retinol metabolism, xenobiotic metabolism by cytochrome P450, and drug metabolism through up-regulated Ugt2a3, Cyp2b1, and Cyp3a18. These genes may play important anti-fibrotic roles in FZHY-treated rats.

Expression and purification of mouse Ttyh1 fragments as antigens to generate Ttyh1-specific monoclonal antibodies.

Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1. With these Ttyh1 protein products, a set of monoclonal antibodies (mAbs) against the mouse Ttyh1 protein was established by using conventional hybridoma techniques. The specificity of the anti-Ttyh1 mAbs was determined based on their activities in Western blotting and immunofluorescent analysis using embryonic brain tissues and cultured mouse neural stem cells (NSCs). We also show that the mouse Ttyh1 protein was expressed in cultured NSCs, most likely in membrane and cytoplasm. In mouse embryonic brains, it appeared that the Ttyh1 protein was specifically expressed in the apical edge of the ventricular zone as puncta-like structures, as determined by using immunofluorescence. Taken together, our study provided a useful tool for further exploration of the biological functions and pathological significance of Ttyh1 in mice.

Mechanisms of CCl4-induced liver fibrosis with combined transcriptomic and proteomic analysis.

The classic toxicity of carbon tetrachloride (CCl4) is to induce liver lesion and liver fibrosis. Liver fibrosis is a consequence of chronic liver lesion, which can progress into liver cirrhosis even hepatocarcinoma. However, the toxicological mechanisms of CCl4-induced liver fibrosis remain not fully understood. We combined transcriptomic and proteomic analysis and biological network technology, predicted toxicological targets and regulatory networks of CCl4 in liver fibrosis. Wistar rats were treated with CCl4 for 9 weeks. Histopathological changes, hydroxyproline (Hyp) contents, serum ALT and AST in the CCl4-treated group were significantly higher than that of CCl4-untreated group. CCl4-treated and -untreated liver tissues were examined by microarray and iTRAQ. The results showed that 3535 genes (fold change ≥ 1.5, P < 0.05) and 1412 proteins (fold change ≥ 1.2, P < 0.05) were differentially expressed. Moreover, the integrative analysis of transcriptomics and proteomics data showed 523 overlapped proteins, enriched in 182 GO terms including oxidation reduction, response to oxidative stress, inflammatory response, extracellular matrix organization, etc. Furthermore, KEGG pathway analysis showed that 36 pathways including retinol metabolism, PPAR signaling pathway, glycolysis/gluconeogenesis, arachidonic acid metabolism, metabolism of xenobiotics by cytochrome P450 and drug metabolism. Network of protein-protein interaction (PPI) and key function with their related targets were performed and the degree of network was calculated with Cytoscape. The expression of key targets such as CYP4A3, ALDH2 and ALDH7A1 decreased after CCl4 treatment. Therefore, the toxicological mechanisms of CCl4-induced liver fibrosis may be related with multi biological process, pathway and targets which may provide potential protection reaction mechanism for CCl4 detoxication in the liver.

Saponin 6 derived from Anemone taipaiensis induces U87 human malignant glioblastoma cell apoptosis via regulation of Fas and Bcl­2 family proteins.

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor, and is associated with a poor prognosis. Saponin 6, derived from Anemone taipaiensis, exerts potent cytotoxic effects against the human hepatocellular carcinoma HepG2 cell line and the human promyelocytic leukemia HL­60 cell line; however, the effects of saponin 6 on glioblastoma remain unknown. The present study aimed to evaluate the effects of saponin 6 on human U87 malignant glioblastoma (U87 MG) cells. The current study revealed that saponin 6 induced U87 MG cell death in a dose­ and time­dependent manner, with a half maximal inhibitory concentration (IC50) value of 2.83 µM after treatment for 48 h. However, saponin 6 was needed to be used at a lesser potency in HT­22 cells, with an IC50 value of 6.24 µM. Cell apoptosis was assessed by flow cytometry using Annexin V­fluorescein isothiocyanate/propidium iodide double staining. DNA fragmentation and alterations in nuclear morphology were examined by terminal deoxynucleotidyl transferase­mediated dUTP nick end labeling and transmission electron microscopy, respectively. The present study demonstrated that treatment with saponin 6 induced cell apoptosis in U87 MG cells, and resulted in DNA fragmentation and nuclear morphological alterations typical of apoptosis. In addition, flow cytometric analysis revealed that saponin 6 was able to induce cell cycle arrest. The present study also demonstrated that saponin 6­induced apoptosis of U87 MG cells was attributed to increases in the protein expression levels of Fas, Fas ligand, and cleaved caspase­3, ­8 and ­9, and decreases in the levels of B­cell lymphoma 2. The current study indicated that saponin 6 may exhibit selective cytotoxicity toward U87 MG cells by activating apoptosis via the extrinsic and intrinsic pathways. Therefore, saponin 6 derived from A. taipaiensis may possess therapeutic potential for the treatment of GBM.