N-glycosylation of SCAP exacerbates hepatocellular inflammation and lipid accumulation via ACSS2-mediated histone H3K27 acetylation.

Sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) is a widely expressed membrane glycoprotein that acts as an important modulator of lipid metabolism and inflammatory stress. N-glycosylation of SCAP has been suggested to modulate cancer development, but its role in NASH is poorly understood. In this study, the N-glycosylation of SCAP was analyzed by using sequential trypsin proteolysis and glycosidase treatment. The liver cell lines expressing wild-type and N-glycosylation sites mutated SCAP were constructed to investigate the N-glycosylation role of SCAP in regulating inflammation and lipid accumulation as well as the underlying mechanisms. The hepatic SCAP protein levels were significantly increased in C57BL/6J mice fed with western diet and sweet water (WD+SW) and diabetic db/db mice, which exhibited typical liver steatosis and inflammation. In vitro, the enhanced N-glycosylation increased the protein stability of SCAP and hence increased its total protein levels, while the ablation of N-glycosylation significantly decreased SCAP protein stability and alleviated lipid accumulation and inflammation in hepatic cell lines. Mechanistically, the presence of SCAP N-glycosylation increased not only the SREBP1-mediated acetyl-CoA synthetase 2 (ACSS2) transcription but also the AMPK-mediated S659 phosphorylation of ACCS2 protein, causing the enhanced ACSS2 levels in nucleus and hence increasing the histone H3K27 acetylation (H3K27ac), which is a key epigenetic modification associated with NASH. Modulating ACSS2 expression or its location in cytoplasm abolished the effects of SCAP N-glycosylation on H3K27ac and lipid accumulation and inflammation. In conclusion, SCAP N-glycosylation aggravates inflammation and lipid accumulation through enhancing ACSS2-mediated H3K27ac in hepatocytes.

Hepatocyte CD36 protects mice from NASH diet-induced liver injury and fibrosis via blocking N1ICD production.

BACKGROUND & AIMS: Fatty acid translocase CD36 (CD36/FAT) is a widely expressed membrane protein with multiple immuno-metabolic functions. Genetic CD36 deficiency is associated with increased risk of metabolic dysfunction-associated fatty liver disease (MAFLD) in patients. Liver fibrosis severity mainly affects the prognosis in patients with MAFLD, but the role of hepatocyte CD36 in liver fibrosis of MAFLD remains unclear. METHODS: A high-fat high-cholesterol diet and a high-fat diet with high-fructose drinking water were used to induce nonalcoholic steatohepatitis (NASH) in hepatocyte-specific CD36 knockout (CD36LKO) and CD36flox/flox (LWT) mice. Human hepG2 cell line was used to investigate the role of CD36 in regulating Notch pathway in vitro. RESULTS: Compared to LWT mice, CD36LKO mice were susceptible to NASH diet-induced liver injury and fibrosis. The analysis of RNA-sequencing data revealed that Notch pathway was activated in CD36LKO mice. LY3039478, an inhibitor of γ-secretase, inhibited Notch1 protein S3 cleavage and Notch1 intracellular domain (N1ICD) production, alleviating liver injury and fibrosis in CD36LKO mice livers. Likewise, both LY3039478 and knockdown of Notch1 inhibited the CD36KO-induced increase of N1ICD production, causing the decrease of fibrogenic markers in CD36KO HepG2 cells. Mechanistically, CD36 formed a complex with Notch1 and γ-secretase in lipid rafts, and hence CD36 anchored Notch1 in lipid rafts domains and blocked Notch1/γ-secretase interaction, inhibiting γ-secretase-mediated cleavage of Notch1 and the production of N1ICD. CONCLUSIONS: Hepatocyte CD36 plays a key role in protecting mice from diet-induced liver injury and fibrosis, which may provide a potential therapeutic strategy for preventing liver fibrogenesis in MAFLD.

Hepatocyte CD36 modulates UBQLN1-mediated proteasomal degradation of autophagic SNARE proteins contributing to septic liver injury.

Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury.Abbreviations: AAV8: adeno-associated virus 8; AOSC: acute obstructive suppurative cholangitis; ATP1A1: ATPase, Na+/K+ transporting, alpha 1 polypeptide; CASP3: caspase 3; CASP8: caspase 8; CCL2: chemokine (C-C motif) ligand 2; cd36-HKO: hepatocyte-specific cd36 knockout; Co-IP: co-immunoprecipitation; CQ: chloroquine; Cys: cysteine; GOT1: glutamic-oxaloacetic transaminase 1, soluble; GPT: glutamic-pyruvic transaminase, soluble; IL1B: interleukin 1 beta; IL6: interleukin 6; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LDH, lactate dehydrogenase; LPS: lipopolysaccharide; LYPLA1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OE: overexpression; qPCR: quantitative polymerase chain reaction; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TNF: tumor necrosis factor; TRIM: tripartite motif-containing; UBA: ubiquitin-associated; UBL: ubiquitin-like; UBQLN: ubiquilin; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

Identification of the prognostic signature based on genomic instability-related alternative splicing in colorectal cancer and its regulatory network.

Background: Colorectal cancer (CRC) is a heterogeneous disease with many somatic mutations defining its genomic instability. Alternative Splicing (AS) events, are essential for maintaining genomic instability. However, the role of genomic instability-related AS events in CRC has not been investigated. Methods: From The Cancer Genome Atlas (TCGA) program, we obtained the splicing profiles, the single nucleotide polymorphism, transcriptomics, and clinical information of CRC. Combining somatic mutation and AS events data, a genomic instability-related AS signature was constructed for CRC. Mutations analyses, clinical stratification analyses, and multivariate Cox regression analyses evaluated this signature in training set. Subsequently, we validated the sensitivity and specificity of this prognostic signature using a test set and the entire TCGA dataset. We constructed a nomogram for the prognosis prediction of CRC patients. Differentially infiltrating immune cells were screened by using CIBERSORT. Inmmunophenoscore (IPS) analysis was used to evaluate the response of immunotherapy. The AS events-related splicing factors (SF) were analyzed by Pearson's correlation. The effects of SF regulating the prognostic AS events in proliferation and migration were validated in Caco2 cells. Results: A prognostic signature consisting of seven AS events (PDHA1-88633-ES, KIAA1522-1632-AP, TATDN1-85088-ES, PRMT1-51042-ES, VEZT-23786-ES, AIG1-77972-AT, and PHF11-25891-AP) was constructed. Patients in the high-risk score group showed a higher somatic mutation. The genomic instability risk score was an independent variable associated with overall survival (OS), with a hazard ratio of a risk score of 1.537. The area under the curve of receiver operator characteristic curve of the genomic instability risk score in predicting the OS of CRC patients was 0.733. Furthermore, a nomogram was established and could be used clinically to stratify patients to predict prognosis. Patients defined as high-risk by this signature showed a lower proportion of eosinophils than the low-risk group. Patients with low risk were more sensitive to anti-CTLA4 immunotherapy. Additionally, HSPA1A and FAM50B were two SF regulating the OS-related AS. Downregulation of HSPA1A and FAM50B inhibited the proliferation and migration of Caco2 cells. Conclusion: We constructed an ideal prognostic signature reflecting the genomic instability and OS of CRC patients. HSPA1A and FAM50B were verified as two important SF regulating the OS-related AS.

Intestinal epithelial ß Klotho is a critical protective factor in alcohol-induced intestinal barrier dysfunction and liver injury.

BACKGROUND: Intestinal barrier dysfunction is crucial in alcohol-associated liver disease (ALD). The decreased beta-Klotho (KLB) expression caused by gene variation is associated with hyperpermeability in patients with irritable bowel syndrome. Here we investigated the roles of intestinal KLB in maintaining the intestinal epithelial barrier in ALD and the underlying mechanisms. METHODS: We constructed the intestine-specific overexpression KLB mice to investigate the role of KLB on alcohol-induced intestinal barrier dysfunction and liver injury in an ALD mouse model. To investigate the molecular mechanism in vitro, Caco2 cells were cultured and infected with the KLB overexpression lentivirus, or transfected with KLB/TRPV6 siRNA, or TRPV6/FXR1 overexpression plasmid, and treated with or without ethanol. FINDINGS: The upregulation of KLB in enterocytes effectively protected mice from alcohol-induced intestinal barrier hyperpermeability, thereby ameliorating hepatic steatosis and inflammation. KLB competitively suppressed FXR1 binding to the TRPV6 mRNA, increasing TRPV6 mRNA stability and protein abundance in intestinal epithelial cells. Furthermore, KLB formed a complex with TRPV6 and tight junction (TJ) proteins, protecting against alcohol-induced TJ proteins endocytosis and degradation as well as intestinal barrier impairment. INTERPRETATION: This work suggested that KLB attenuated alcohol-induced intestinal epithelial barrier dysfunction and liver injury through FXR1/TRPV6/TJ proteins pathway. FUNDING: National Natural Science Foundation of China, Chongqing Natural Science Foundation, Talent Project of Chongqing and the Science and Technology Research Program of Chongqing Municipal Education Commission.

SIRT2 Promotes HBV Transcription and Replication by Targeting Transcription Factor p53 to Increase the Activities of HBV Enhancers and Promoters.

Chronic hepatitis B (CHB) virus infection is one of the leading causes of cirrhosis and liver cancer. Although the major drugs against CHB including nucleos(t)ide analogs and PEG-interferon can effectively control human hepatitis B virus (HBV) infection, complete cure of HBV infection is quite rare. Targeting host factors involved in the viral life cycle contributes to developing innovative therapeutic strategies to improve HBV clearance. In this study, we found that the mRNA and protein levels of SIRT2, a class III histone deacetylase, were significantly upregulated in CHB patients, and that SIRT2 protein level was positively correlated with HBV viral load, HBsAg/HBeAg levels, HBcrAg, and ALT/AST levels. Functional analysis confirmed that ectopic SIRT2 overexpression markedly increased total HBV RNAs, 3.5-kb RNA and HBV core DNA in HBV-infected HepG2-Na+/taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, SIRT2 silencing inhibited HBV transcription and replication. In addition, we found a positive correlation between SIRT2 expression and HBV RNAs synthesis as well as HBV covalently closed circular DNA transcriptional activity. A mechanistic study suggested that SIRT2 enhances the activities of HBV enhancer I/HBx promoter (EnI/Xp) and enhancer II/HBc promoter (EnII/Cp) by targeting the transcription factor p53. The levels of HBV EnI/Xp and EnII/Cp-bound p53 were modulated by SIRT2. Both the mutation of p53 binding sites in EnI/Xp and EnII/Cp as well as overexpression of p53 abolished the effect of SIRT2 on HBV transcription and replication. In conclusion, our study reveals that, in terms of host factors, a SIRT2-targeted program might be a more effective therapeutic strategy for HBV infection.

CD36 Senses Dietary Lipids and Regulates Lipids Homeostasis in the Intestine.

Dietary lipids absorbed in the intestine are closely related to the development of metabolic syndrome. CD36 is a multi-functional scavenger receptor with multiple ligands, which plays important roles in developing hyperlipidemia, insulin resistance, and metabolic syndrome. In the intestine, CD36 is abundant on the brush border membrane of the enterocytes mainly localized in proximal intestine. This review recapitulates the update and current advances on the importance of intestinal CD36 in sensing dietary lipids and regulating intestinal lipids uptake, synthesis and transport, and regulating intestinal hormones secretion. However, further studies are still needed to demonstrate the complex interactions between intestinal CD36 and dietary lipids, as well as its importance in diet associated metabolic syndrome.

Src-mediated Tyr353 phosphorylation of IP3R1 promotes its stability and causes apoptosis in palmitic acid-treated hepatocytes.

Palmitic acid (PA)-induced hepatocyte apoptosis is critical for the progression of nonalcoholic fatty liver disease (NAFLD). Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is an intracellular Ca2+-release channel and is involved in PA-induced hepatocyte apoptosis. While the expression of IP3R1 is elevated in patients with NAFLD and in hepatocytes treated with PA, it remains unclear how PA promotes the expression of IP3R1. In present study, our results showed that PA induced mitochondrial dysfunction and apoptosis, which is accompanied with the increase of the IP3R1 expression in hepatic cells. The inhibition of IP3R1 expression using siRNA ameliorated the PA-induced mitochondrial dysfunction. Furthermore, PA enhanced the stability of the IP3R1 protein instead of an increase in its mRNA levels. PA also promoted the phosphorylation of IP3R1 at the Tyr353 site and increased the phosphorylation of src in hepatic cells. Moreover, an inhibitor of src kinase (SU6656) significantly reduced the Tyr353 phosphorylation of IP3R1 and decreased its stability. In addition, SU6656 improved mitochondrial function and reduced apoptosis in hepatocytes. Conclusion: PA promotes the Tyr353 phosphorylation of IP3R1 by activating the src pathway and increasing the protein stability of IP3R1, which consequently results in mitochondrial Ca2+ overload and mitochondrial dysfunction in hepatic cells. Our results also suggested that inhibition of the src/IP3R1 pathway, such as by SU6656, may be a novel potential therapeutic approach for the treatment of NAFLD.

Prognostic Dynamic Nomogram Integrated with Inflammation-Based Factors for Non-Small Cell Lung Cancer Patients with Chronic Hepatitis B Viral Infection.

Chronic inflammation plays an important role in tumor progression. The aim of this study was to develop an effective predictive dynamic nomogram integrated with inflammation-based factors to predict overall survival (OS) of non-small cell lung cancer (NSCLC) patients with chronic hepatitis B viral (HBV) infection. We retrospectively analyzed NSCLC patients with HBV infection from Sun Yat-sen University Cancer Center between 2008 and 2010. Univariate and multivariate Cox survival analyses were performed to identify prognostic factors associated with OS of patients. All of the independent prognostic factors were utilized to build the dynamic nomogram. The predictive accuracy of the dynamic nomogram was evaluated concordance index (C-index), decision curve analysis and were compared with previous reported model and traditional TNM staging system. According to the total points (TPS) by dynamic nomogram, we further stratified patients into different risk groups. A total of 203 patients were included. Multivariate Cox analysis showed TNM stage (P = 0.019), treatment (P < 0.001), C-reactive protein (P = 0.020) and platelet (P = 0.012) were independent prognostic factors of OS. The dynamic nomogram was established by involving all the factors above. The C-index of dynamic nomogram for predicting OS was 0.76 (95%CI: 0.72-0.80), which was statistically higher than that of traditional TNM staging system (0.70, 95%CI: 0.66-0.74, P<0.001). Decision curve analysis demonstrated that the dynamic nomogram was better than the TNM staging system. The predictive accuracy of the current model keeping almost the same accuracy as previous one. Based on the total points (TPS) of dynamic nomogram, we divided the patients into 3 subgroups: low risk (TPS ≤ 107), intermediate risk (107< TPS ≤ 149), and high risk (TPS > 149). The differences of OS rates were significant in the subgroups. We propose a novel dynamic nomogram model based on inflammatory prognostic factors that is highly predictive of OS in NSCLC patients with HBV infection and outperforms the traditional TNM staging system.

Establishment and validation of a predictive nomogram model for non-small cell lung cancer patients with chronic hepatitis B viral infection.

BACKGROUND: This study aimed to establish an effective predictive nomogram for non-small cell lung cancer (NSCLC) patients with chronic hepatitis B viral (HBV) infection. METHODS: The nomogram was based on a retrospective study of 230 NSCLC patients with chronic HBV infection. The predictive accuracy and discriminative ability of the nomogram were determined by a concordance index (C-index), calibration plot and decision curve analysis and were compared with the current tumor, node, and metastasis (TNM) staging system. RESULTS: Independent factors derived from Kaplan-Meier analysis of the primary cohort to predict overall survival (OS) were all assembled into a Cox proportional hazards regression model to build the nomogram model. The final model included age, tumor size, TNM stage, treatment, apolipoprotein A-I, apolipoprotein B, glutamyl transpeptidase and lactate dehydrogenase. The calibration curve for the probability of OS showed that the nomogram-based predictions were in good agreement with the actual observations. The C-index of the model for predicting OS had a superior discrimination power compared with the TNM staging system [0.780 (95% CI 0.733-0.827) vs. 0.693 (95% CI 0.640-0.746), P < 0.01], and the decision curve analyses showed that the nomogram model had a higher overall net benefit than did the TNM stage. Based on the total prognostic scores (TPS) of the nomogram, we further subdivided the study cohort into three groups: low risk (TPS ≤ 13.5), intermediate risk (13.5 < TPS ≤ 20.0) and high risk (TPS > 20.0). CONCLUSION: The proposed nomogram model resulted in more accurate prognostic prediction for NSCLC patients with chronic HBV infection.

Antioxidant and anti-aging activities of the polysaccharide TLH-3 from Tricholoma lobayense.

Polysaccharides from edible fungi usually exhibit many bioactivities. Our previous studies found that polysaccharide TLH-3 extracted from Tricholoma lobayense possessed noticeable antioxidant activity. To further explore its biological activities, the antioxidant and anti-aging activities of TLH-3 were evaluated in vitro and in vivo. The results of antioxidant activity in vitro showed that TLH-3 could enhance the cell viability, reduce the production of reactive oxygen species (ROS) and inhibit oxidative damage induced by tert-butylhydroperoxide (t-BHP) in human embryonic lung fibroblasts (HELF) cells. The anti-aging capability was measured in d-galactose (d-gal)-induced aged mice model, and the experimental data showed that TLH-3 significantly inhibited the formation of malondialdehyde (MDA) and raised the activities of superoxide dismutase (SOD) and catalase (CAT) in mice liver and serum (p<0.05). This study suggested that TLH-3 possessed apparent antioxidant and anti-aging activities and could be exploited as a potent dietary supplement to attenuate aging and prevent age-related diseases in humans.

Antioxidant capacity and cytotoxicity of sulfated polysaccharide TLH-3 from Tricholoma lobayense.

Polysaccharide TLH-3 from the fruit body of Tricholoma lobayense Heim has shown outstanding antioxidant activity. In order to further explore it, TLH-3 was successfully modified to obtain a sulfated derivative (STLH-3). The chemical characteristics of STLH-3 and TLH-3 were determined by high performance liquid chromatography and infrared spectroscopy. Antioxidant activity and cytotoxicity of the samples were investigated in vitro. The antioxidant activities of STLH-3 were significantly improved. In the four indicators of antioxidant activity, the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion scavenging activities of STLH-3 have exceeded that of Vitamin C (Vc). The cytotoxicity against tumor cells including human cervical carcinoma cell line (HeLa) and human breast cancer cell line (MCF-7) of STLH-3 was obviously stronger than that of TLH-3. STLH-3 had no toxic on human embryonic lung fibroblasts (HELF) cells. These results suggested that STLH-3 would be a promising bioactive macromolecule for potential applications in the field of health care and pharmaceutical.

Prediction of cancer cell sensitivity to natural products based on genomic and chemical properties.

Natural products play a significant role in cancer chemotherapy. They are likely to provide many lead structures, which can be used as templates for the construction of novel drugs with enhanced antitumor activity. Traditional research approaches studied structure-activity relationship of natural products and obtained key structural properties, such as chemical bond or group, with the purpose of ascertaining their effect on a single cell line or a single tissue type. Here, for the first time, we develop a machine learning method to comprehensively predict natural products responses against a panel of cancer cell lines based on both the gene expression and the chemical properties of natural products. The results on two datasets, training set and independent test set, show that this proposed method yields significantly better prediction accuracy. In addition, we also demonstrate the predictive power of our proposed method by modeling the cancer cell sensitivity to two natural products, Curcumin and Resveratrol, which indicate that our method can effectively predict the response of cancer cell lines to these two natural products. Taken together, the method will facilitate the identification of natural products as cancer therapies and the development of precision medicine by linking the features of patient genomes to natural product sensitivity.