High Fat Cholesterol Research Articles

Evaluation of the role of hepatic Gstm4 in diet-induced obesity and dyslipidemia

Obesity and its related diseases continue to rise worldwide, necessitating further investigation to develop new therapeutic strategies. The dysregulation of redox homeostasis is tightly associated with metabolic diseases. Glutathione, an antioxidant, acts as a cofactor for antioxidant and detoxification enzymes such as glutathione S-transferases (GSTs)—a superfamily including Gstm4. So far, the physiological role of Gstm4 remains largely unknown. Human genetics is a powerful tool to discover novel therapeutic targets for metabolic diseases. The single nucleotide polymorphism rs650985, located within the sixth intron of the human gene Gstm4, was associated with plasma lipids, indicating that targeting Gstm4 might intervene in the progression of dyslipidemia. Furthermore, we found that Gstm4 is highly expressed in the liver and enriched in hepatocytes—the parenchymal cells of the liver. We established the mouse model with the hepatic deletion of Gstm4 and found that this mouse model did not present altered body weight, serum lipid profile, or liver fat content in the context of chow or high-fat high cholesterol diet feeding, indicating that hepatic Gstm4 is dispensable for diet-induced obesity and dyslipidemia. Further analysis revealed that hepatic deletion of Gstm4 upregulates the level of protein but not mRNA of Npc1l1—a critical protein mediating cholesterol uptake, suggesting that there might be a link between Gstm4 and lipid metabolic diseases in certain contexts.

Systematic pharmacology and experimental validation to elucidate the inflammation-associated mechanism of Huanglian Wendan (HLWD) decoction in the treatment of MAFLD associated with atherosclerosis

Ethnopharmacological relevanceMetabolic-associated fatty liver disease (MAFLD) and atherosclerosis are very common disorders that frequently coexist. The therapeutic efficacy of Huanglian Wendan (HLWD) decoction, a traditional Chinese medicine (TCM) prescription, is satisfactory in treating MAFLD associated with atherosclerosis. However, the underlying mechanisms through which HLWD exerts its effects need to be elucidated. Given the complex composition of HLWD and its multiple therapeutic targets, pharmacological investigation is challenging. Aim of this studyThis study aimed to identify the effective compounds in HLWD and elucidate the mechanisms involved in its therapeutic effect on MAFLD associated with atherosclerosis. Materials and methodsWe used a systematic pharmacology method to identify effective compounds present in HLWD and determine the mechanism by which it affects MAFLD associated with atherosclerosis. The effective components of HLWD were identified through ultrahigh-performance liquid chromatography-q exactive-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). Next, a comprehensive in silico method was used to predict potential related targets and disease targets for these compounds to establish corresponding pathways. The accuracy of our assumed systemic pharmacology results was determined by conducting follow-up experiments. ResultsBy conducting UHPLC-Q-Orbitrap HRMS combined with network analysis, we identified 18 potentially active components of HLWD and assessed the inflammatory regulatory mechanism by which it affects MAFLD associated with atherosclerosis on the basis of 52 key targets. We used a high-fat, high-cholesterol (HFHC)-induced mice model of MAFLD associated with atherosclerosis to confirm our results. We found that administering HLWD significantly improved the appearance of their liver and reduced their body weight, liver weight, blood lipids, hepatic damage, and hepatic pathology. HLWD also decreased atherosclerotic lesion areas, foam cells, and inflammatory cells in the aorta. HLWD showed anti-inflammatory effects, suppressed M1 polarization, and promoted M2 polarization in the liver and aorta. HLWD might also regulate peroxisome proliferator-activated receptor-γ (PPARγ)/nuclear factor kappa-B (NF-κB) signaling to influence macrophage polarization and inflammation. ConclusionsOur results showed that HLWD protected against HFHC diet-induced MAFLD associated with atherosclerosis by regulating PPARγ/NF-κB signaling, thus adjusting macrophage polarization and inflammation. Additionally, pharmacochemistry research, network pharmacology analysis, and experimental verification can be combined to form a comprehensive model used in studies on TCM.

Protective role of S-adenosylmethionine on high fat/high cholesterol diet-induced hepatic and aortic lesions and oxidative stress in guinea pigs.

S-adenosylmethionine (SAM) is the main methyl group donor and has antioxidant potential. In this study, preventive and regressive potential of SAM were investigated in high fat/high cholesterol (HFHC) diet-induced non-alcoholic fatty liver disease (NAFLD) in guinea pigs. They were injected with SAM (50 mg/kg, i.p.) for 6 weeks along with HFHC diet or 4 weeks after HFHC diet. Serum transaminase activities, total cholesterol (TC), triglyceride (TG), cytochrome p450-2E1 (CYP2E1) and hydroxyproline (Hyp) levels, prooxidative and antioxidative parameters, protein expressions of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) together with histopathological changes were examined in the liver. SAM treatment diminished HFHC diet-induced increases in serum transaminase activities and hepatic TC, TG, CYP2E1, Hyp, α-SMA and TGF-β1 expressions and ameliorated prooxidant-antioxidant balance. Histopathological scores for hepatic steatosis, inflammation, and fibrosis were decreased by SAM treatment. Increases in TC, diene conjugate levels, and lipid vacuoles within the tunica media of the aorta were reduced in HFHC-fed animals treated with SAM. These protective effects were also detected in the regression period of HFHC-guinea pigs due to SAM. In conclusion, SAM treatment was found to be effective in prevention and regression of HFHC-induced hepatic and aortic lesions together with decreases in oxidative stress in guinea pigs with NAFLD.

Integrating Lipidomics, Metabolomics, and Network Pharmacology to Reveal the Mechanism of Cannabidiol against Inflammation in High-Fat, High-Cholesterol Diet-Induced Mice.

Inflammation plays a critical role in the development of numerous diseases. Cannabidiol (CBD), found in hemp, exhibits significant pharmacological activities. Accumulating evidence suggests that CBD has anti-inflammatory and cardiovascular protection effects, but the potential mechanisms require further exploration. In this study, we aimed to reveal the mechanisms of CBD against high-fat, high-cholesterol (HFC) diet-induced inflammation combining metabolomics with network pharmacology. First, plasma lipidomics results indicated that oxidized lipids could serve as potential biomarkers for HFC diet-induced inflammation, and CBD reversed the elevated levels of oxidized lipids. The HFC diet was also found to enhance intestinal permeability, facilitating the entry of lipopolysaccharides (LPSs) into the circulatory system and subsequently increasing systemic inflammation. Additionally, cell metabolomic results indicated that CBD could reverse 10 important differential metabolites in LPS-induced RAW 264.7 cells. Using network pharmacology, we identified 49 core targets, and enrichment analysis revealed that arachidonic acid was the most significantly affected by CBD, which was closely associated with inflammation. Further integrated analysis focused on three key targets, including PTGS2, ALOX5, and ALOX15. Molecular docking showed high affinities between key targets and CBD, and qPCR further demonstrated that CBD could reverse the mRNA expression of these key targets in RAW 264.7 cells. Collectively, this finding integrates lipidomics and metabolomics with network pharmacology to elucidate the anti-inflammatory effects of CBD and validates key therapeutic targets.

Hepatic miR-363 promotes nonalcoholic fatty liver disease by suppressing INSIG1

Nonalcoholic fatty liver disease (NAFLD) constitutes one of major worldwide health problem which typically progressively results in nonalcoholic steatohepatitis (NASH) and eventually cirrhosis and liver cancer. Liver-specific deletion of INSIG1 promotes SREBP1 nuclear translocation to activate downstream lipogenic genes expression, leading to lipid accumulation. However, the underlying pathogenesis of NAFLD, and particularly involved in miRNA participation are still to be thoroughly explored. Here, we found that miR-363-3p was significantly overexpressed in high-fat, high-cholesterol (HFHC) diet mice liver tissue and fatty acid-induced steatosis cells. miR-363-3p directly targets INSIG1 to inhibit its expression, thereby facilitating the cleavage of SREBP and nuclear translocation to activate subsequent transcription of lipogenic genes in vitro and in vivo. In addition, we identified apigenin, a natural flavonoid compound, inhibited miR-363-3p expression to up-regulate INSIG1 and suppress nuclear translocation of SREBP1, thereby down-regulated lipogenic genes expression in steatosis cells and HFHC diet mice liver tissues. Taken together, our results demonstrated that miR-363-3p as a key regulator of hepatic lipid homeostasis targeted INSIG1, and apigenin alleviated NAFLD through the miR-363-3p/INSIG1/SREBP1 pathway. This indicates that reduction of miR-363-3p levels as a possible treatment of hepatic steatosis and provides a potential new therapeutic strategy for targeting miRNA to ameliorate NAFLD.

Native and Gd-EOB-DTPA-Enhanced T1 mapping for Assessment of Liver Fibrosis in NAFLD: Comparative Analysis of Modified Look-Locker Inversion Recovery and Water-specific T1 mapping

Rationale and ObjectivesTo investigate the diagnostic performance of water-specific T1 mapping for staging liver fibrosis in a non-alcoholic fatty liver disease (NAFLD) rabbit model, in comparison to Modified Look-Locker Inversion recovery (MOLLI) T1 mapping. Materials and methodsSixty rabbits were randomly divided into the control group (12 rabbits) and NAFLD model groups (8 rabbits per subgroup) corresponding to different durations of high-fat high cholesterol diet feeding. All rabbits underwent MRI examination including MOLLI T1 mapping and 3D multi-echo variable flip angle (VFAME- GRE) sequences were acquired before and 20min after the administration of Gd- EOB-DTPA. Histological assessments were performed to evaluate steatosis, inflammation, ballooning, and fibrosis. Statistical analysis included the intraclass correlation coefficient, analysis of variance, spearman correlation, multiple linear regression, and receiver operating characteristic curve. ResultsA moderate correlation was observed between conventional native T1 and MRI-PDFF (r=-0.513, P<0.001), as well as between conventional native T1 and liver steatosis grades (r=-0.319, P=0.016). However, no significant correlation was found between the native wT1 and PDFF (r=0.137, P=0.314), or between the native wT1 and steatosis grades (r=0.106, P=0.435). In the multiple regression analysis, liver fibrosis, and hepatocellular ballooning were identified as independent factors influencing native wT1 in this study (R2=0.545, P<0.05), while steatosis was independently associated with conventional native T1 (R2=0.321, P<0.005). The AUC values for native T1, native wT1, HBP T1, and HBP wT1 were 0.549(0.410-0.682), 0.811(0.684-0.903), 0.775(0.644-0.876), and 0.752(0.619-0.858) for F1 or higher, 0.581(0.441-0.711), 0.828(0.704-0.916), 0.832(0.708-0.919), and 0.854(0.734-0.934) for F2 or higher, respectively. ConclusionThe native wT1 may provide a more reliable assessment of early liver fibrosis in the context of NAFLD compared to conventional native T1.

Intestinal NSD2 Aggravates Nonalcoholic Steatohepatitis Through Histone Modifications.

Mounting clinical evidence suggests that a comprised intestinal barrier contributes to the progression of nonalcoholic steatohepatitis (NASH); nevertheless, the precise mechanism remains elusive. This study unveils a significant upregulation of nuclear receptor-binding SET domain protein 2 (NSD2) in the intestines of obese humans and mice subjected to a high-fat cholesterol diet (HFCD). Intestine-specific NSD2 knockout attenuated the progression of intestinal barrier impairment and NASH, whereas NSD2 overexpression exacerbated this progression. Mechanistically, NSD2 directly regulates the transcriptional activation of Ern1 by demethylating histone H3 at lysine 36 (H3K36me2), thus activating the ERN1-JNK axis to intensify intestinal barrier impairment and subsequently foster NASH progression. These findings elucidate the crucial role of NSD2-mediated H3K36me2 in intestinal barrier impairment, suggesting that targeting intestinal NSD2 can represent a novel therapeutic approach for NASH.

Enterocyte-specific FATP4 deficiency elevates blood lipids via a shift from polar to neutral lipids in distal intestine.

Fatty acid transport protein (FATP)4 was thought to mediate intestinal lipid absorption, which was disputed by a study using keratinocyte-Fatp4-rescued Fatp4-/- mice. These knockouts when fed with a Western diet showed elevated intestinal triglyceride (TG) and fatty acid levels. To investigate a possible role of FATP4 on intestinal lipid processing, ent-Fatp4 (KO) mice were generated by Villin-Cre-specific inactivation of the Fatp4 gene. We aimed to measure circulating and intestinal lipids in control and KO mice after acute or chronic fat intake or during aging. Remarkably, ent-Fatp4 mice displayed an approximately 30% decrease in ileal behenic, lignoceric, and nervonic acids, ceramides containing these FA, as well as, ileal sphingomyelin, phosphatidylcholine, and phosphatidylinositol levels. Such decreases were concomitant with an increase in jejunal cholesterol ester. After a 2-wk recovery from high lipid overload by tyloxapol and oral-lipid treatment, ent-Fatp4 mice showed an increase in plasma TG and chylomicrons. Upon overnight fasting followed by an oral fat meal, ent-Fatp4 mice showed an increase in plasma TG-rich lipoproteins and the particle number of chylomicrons and very low-density lipoproteins. During aging or after feeding with a high-fat high-cholesterol (HFHC) diet, ent-Fatp4 mice showed an increase in plasma TG, fatty acids, glycerol, and lipoproteins as well as intestinal lipids. HFHC-fed KO mice displayed an increase in body weight, the number of lipid droplets with larger sizes in the ileum, concomitant with a decrease in ileal ceramides and phosphatidylcholine. Thus, enterocyte FATP4 deficiency led to a metabolic shift from polar to neutral lipids in distal intestine rendering an increase in plasma lipids and lipoproteins.NEW & NOTEWORTHY Enterocyte-specific Fatp4 deficiency in mice increased intestinal lipid absorption with elevation of blood lipids during fasting and aging, as well as after an acute oral fat-loading or chronic HFHC feeding. Lipidomics revealed that knockout mice displayed a shift from very long-chain to long-chain fatty acids, and from polar to neutral lipids, predominantly in the ileum. Thus, FATP4 may have a physiological function in the control of blood lipids via metabolic shifts in distal intestine.

Gentiopicroside improves NASH and liver fibrosis by suppressing TLR4 and NLRP3 signaling pathways

BackgroundNon-alcoholic steatohepatitis (NASH) and liver fibrosis are progressive conditions associated with non-alcoholic fatty liver disease (NAFLD), characterized by hepatocyte pyroptosis and hepatic stellate cell (HSC) activation. Gentiopicroside (GPS) has emerged as a potential treatment for NASH, yet its underlying mechanism remains unclear. AimTo confirm that GPS can improve NASH and liver fibrosis by blocking the NLRP3 signaling pathway Study designInitially, different animal models were used to study the effects and mechanisms of GPS on NASH and fibrosis. Subsequent in vitro experiments utilized co-cultures and other techniques to delve deeper into its mechanism, followed by validation of the findings in mouse liver tissues. MethodsC57BL/6 mice were fed high-fat, high-cholesterol (HFHC), or methionine-choline-deficient (MCD) diets to induce NASH and fibrosis. RAW264.7 cells and born marrow bone marrow-derived macrophages (BMDMs) were stimulated with LPS and ATP to induce inflammation, then co-cultured with primary hepatocytes and HSCs, treated with GPS, and its efficacy and mechanism were analyzed. ResultsIn vivo, GPS alleviated NASH and liver fibrosis by inhibiting the NLRP3 pathway. In vitro, GPS attenuated inflammation induced by BMDMs by inhibiting TLR4 and NLRP3 signaling pathways, and Co-culture studies suggested that GPS reduced hepatocyte pyroptosis and HSC activation, which was also confirmed in liver tissues ConclusionGPS improves NASH and liver fibrosis by inhibiting the TLR4 and NLRP3 signaling pathways. The specific mechanism may be related to the suppression of macrophage-mediated inflammatory responses, thereby reducing hepatocyte pyroptosis and HSC activation.

Tomato juice regulates bile acid-TGR5 signaling and reverses cognitive deficit and neuroinflammation in high-fat high-cholesterol diet-induced mice

Cognitive deficits and neuroinflammation are significant complications associated with metabolic diseases induced by a high-fat, high-cholesterol (HFHC) diet. Although tomato juice (TJ) has shown potential benefits for brain health, its specific interventional role remains unclear. This research examined the capability of TJ to mitigate HFHC-induced cognitive deficits and delineated TJ's specific mechanisms against neuroinflammation. The results demonstrated that TJ effectively ameliorated cognitive impairments triggered by HFHC diets, improving spatial working memory, novel object recognition, and reducing anxiety-like behaviors in open space and elevated maze tests. Moreover, TJ mitigated neuronal damage, decreased the activity of immune cells, and diminished the level of COX-2 and iNOS in the hippocampus. Additionally, TJ decreased serum TNF-α and lipopolysaccharide levels, reduced liver inflammation, and improved intestinal barrier markers Claudin-1 and Mucin-2 expression. Fecal metabolomics demonstrated that TJ significantly upregulated bile acid metabolism-related pathways. TJ also altered the profile of serum bile acids, elevating the levels of Takeda G protein-coupled receptor 5 (TGR5)-activating bile acids, including deoxycholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid. TJ upregulated TGR5 protein expression and inhibited the phosphorylation of protein kinase B and nuclear factor kappa B proteins in the hippocampus. Notably, the positive effects of TJ on cognition and TGR5 activation were nullified in an antibiotic-treated mouse model, underscoring the essential role of the gut-brain axis in mediating TJ's neuroprotective effects. These findings propose that bile acid-TGR5 signaling may be associated with the anti-neuroinflammatory effects of TJ, which may represent a novel approach for neurogenic disease prevention.

Therapeutic Targeting of PKM2 Ameliorates NASH Fibrosis Progression in a Macrophage-Specific and Liver-Specific Manner

Nonalcoholic steatohepatitis (NASH) may soon become the leading cause of end-stage liver disease worldwide with limited treatment options. Liver fibrosis, which is driven by chronic inflammation and hepatic stellate cell (HSC) activation, critically determines morbidity and mortality in patients with NASH. Pyruvate kinase M2 (PKM2) is involved in immune activation and inflammatory liver diseases; however, its role and therapeutic potential in NASH-related fibrosis remain largely unexplored. Bioinformatics screening and analysis of human and murine NASH livers indicated that PKM2 was upregulated in nonparenchymal cells (NPCs), especially macrophages, in the livers of patients with fibrotic NASH. Macrophage-specific PKM2 knockout (PKM2FL/FLLysM-Cre) significantly ameliorated hepatic inflammation and fibrosis severity in three distinct NASH models induced by a methionine- and choline-deficient (MCD) diet, a high-fat high-cholesterol (HFHC) diet, and a western diet plus weekly carbon tetrachloride injection (WD/CCl4). Single-cell transcriptomic analysis indicated that deletion of PKM2 in macrophages reduced profibrotic Ly6Chigh macrophage infiltration. Mechanistically, PKM2-dependent glycolysis promoted NLR family pyrin domain containing 3 (NLRP3) activation in proinflammatory macrophages, which induced HSC activation and fibrogenesis. A pharmacological PKM2 agonist efficiently attenuated the profibrotic crosstalk between macrophages and HSCs in vitro and in vivo. Translationally, ablation of PKM2 in NPCs by cholesterol-conjugated heteroduplex oligonucleotides, a novel oligonucleotide drug that preferentially accumulates in the liver, dose-dependently reversed NASH-related fibrosis without causing observable hepatotoxicity. The present study highlights the pivotal role of macrophage PKM2 in advancing NASH fibrogenesis. Thus, therapeutic modulation of PKM2 in a macrophage-specific or liver-specific manner may serve as a novel strategy to combat NASH-related fibrosis.

Doenjang Ameliorates Diet-Induced Hyperlipidemia and Hepatic Oxidative Damage by Improving Lipid Metabolism, Oxidative Stress, and Inflammation in ICR Mice.

Hyperlipidemia, characterized by elevated cholesterol, lipids, and triglycerides in the bloodstream, is linked to hepatic oxidative damage. Doenjang, a traditional Korean condiment made from fermented soybeans, is known for its health benefits, yet its anti-hyperlipidemic effects remain understudied. Our study aimed to assess the hypolipidemic and hepatic protective effects of Doenjang on male ICR mice fed a high-fat cholesterol diet for 8 weeks. Mice were divided into three groups: the normal diet (ND), the high-fat cholesterol diet (HD), and the Doenjang-supplemented HD diet (DS) group. Doenjang supplementation significantly regulated total cholesterol, triglycerides, LDL cholesterol, and HDL cholesterol levels compared to the HD group. It also downregulated lipogenic genes, including PPARγ, FAS, and ACC, and positively influenced the cholesterol metabolism-related genes HMGCR and LXR. Moreover, Doenjang intake increased serum glutathione levels, activated oxidative stress defense genes (NRF2, SOD, GPx1, and CAT), positively modulated inflammation genes (NF-kB and IL6) in hepatic tissue, and reduced malondialdehyde levels. Our findings highlight the effectiveness of traditional Doenjang in preventing diet-induced hyperlipidemia and protecting against hepatic oxidative damage.

A high-dose of ursodeoxycholic acid treatment alleviates liver inflammation by remodeling gut microbiota and bile acid profile in a mouse model of non-alcoholic steatohepatitis

Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid commonly used for treating cholestatic liver disease. However, its efficacy on non-alcoholic steatohepatitis (NASH) was controversial. This study aimed to investigate the impact of a high dosage of UDCA on a mouse model of NASH. Forty 6-week-old mice were fed a high-fat high-cholesterol (HFHC) diet for 12 weeks to establish a mouse model of NASH, and then divided into four groups: two groups transitioned to a normal diet, and the other two groups maintained the HFHC diet. Each group was administered a daily dosage of 300 mg/kg of UDCA or saline for a period of 8 weeks. The 16 s ribosomal RNA genes extracted from mice fecal pellets were sequenced using next-generation sequencing techniques. Serum bile acid profiles were quantified using liquid chromatography electrospray ionization tandem mass spectrometry method. The results showed that UDCA treatment ameliorated liver inflammation, without affecting liver fibrosis. UDCA treatment reduced the relative abundance of the genera Bacteroides, Parabacteroides, and Intestinimonas, whereas increased the relative abundance of the genera norank_f_Muribaculaceae and Parasutterella in the HFHC-maintaining groups. The serum levels of total bile acids and total primary bile acids increased, whereas those of endogenous primary bile acids decreased after UDCA treatment. Correlation analysis showed that primary bile acids were negatively correlated with the genera norank_f_Christensenellaceae and unclassified_f_Ruminococcaceae. In conclusion, a high dosage of UDCA can alleviate liver inflammation, probably by modifying the composition of gut microbiota and serum bile acid profiles in NASH mice.

Gentiopicroside improves non-alcoholic steatohepatitis by activating PPARα and suppressing HIF1.

Gentiopicroside (GPS) is a highly water-soluble small-molecule drug and the main bioactive secoiridoid glycoside of Gentiana scabra that has been shown to have hepatoprotective effects against non-alcoholic steatohepatitis (NASH), a form of non-alcoholic fatty liver disease (NAFLD) that can progress to cirrhosis and hepatocellular carcinoma. However, the effects of GPS on NASH and the underlying mechanisms remain obscure. Firstly, a high-fat, high-cholesterol (HFHC) diet and a high-sugar solution containing d-fructose and d-glucose were used to establish a non-alcoholic steatohepatitis (NASH) mice model. Secondly, we confirmed GPS supplementation improve metabolic abnormalities and reduce inflammation in NASH mice induced by HFHC and high-sugar solution. Then we used metabolomics to investigate the mechanisms of GPS in NASH mice. Metabolomics analysis showed GPS may work through the Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway and glycine, serine, and threonine metabolism. Functional metabolites restored by GPS included serine, glycine, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Western blot and qRT-PCR analysis confirmed GPS improve NASH by regulating PPARα and Hypoxia-Inducible Factor-1α (HIF-1α) signaling pathways. In vitro, studies further demonstrated EPA and DHA enhance fatty acid oxidation through the PPARα pathway, while serine and glycine inhibit oxidative stress through the HIF-1α pathway in palmitic acid-stimulated HepG2 cells. Our results suggest GPS's anti-inflammatory and anti-steatosis effects in NASH progression are related to the suppression of HIF-1α through the restoration of L-serine and glycine and the activation of PPARα through increased EPA and DHA.

Intestinal epithelial Krüppel-like factor 4 alleviates endotoxemia and atherosclerosis through improving NF-κB/miR-34a-mediated intestinal permeability.

Maintenance of intestinal barrier function contributes to gastrointestinal homeostasis and therefore cardiovascular diseases. A number of studies show that intestinal permeability is affected by excessive inflammatory responses. Krüppel-like factor (KLF) 4 is one of the critical transcriptional factors, which controls multiple immune responses. In this study we investigated the role of KLF4 in regulating intestinal inflammation and permeability during the atherosclerotic process. Atherosclerotic model was established in ApoE-/- mice by feeding a high fat high cholesterol (HFHC) diet. We showed that colon expression levels of KLF4 and tight junction proteins were significantly decreased whereas inflammatory responses increased in atherosclerotic mice. Overexpression of colon epithelial Klf4 decreased atherosclerotic plaque formation and vascular inflammation in atherosclerotic mice, accompanied by remarkable suppression of intestinal NF-κB activation. We found that overexpression of epithelial Klf4 in atherosclerotic mice significantly increased intestinal tight junction expression and ameliorated endotoxemia, whereas replenishment of LPS abolished these benefits. Overexpression of Klf4 reversed LPS-induced permeability and downregulation of ZO-1 and Occludin in Caco-2 cells in vitro. HFHC diet stimulated the expression of epithelial microRNA-34a, whereas silence of epithelial Klf4 abolished the benefits of microRNA-34a sponge, a specific miR-34a inhibitor, on intestinal permeability and atherosclerotic development. A clinical cohort of 24 atherosclerotic patients supported colon KLF4/NF-κB/tight junction protein axis mediated intestine/cardiovascular interaction in patients with atherosclerosis. Taken together, intestinal epithelial KLF4 protects against intestinal inflammation and barrier dysfunction, ameliorating atherosclerotic plaque formation.

Lactobacillus acidophilus suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinoma through producing valeric acid

Gut probiotic depletion is associated with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we investigated the prophylactic potential of Lactobacillus acidophilus against NAFLD-HCC. NAFLD-HCC conventional and germ-free mice were established by diethylnitrosamine (DEN) injection with feeding of high-fat high-cholesterol (HFHC) or choline-deficient high-fat (CDHF) diet. Orthotopic NAFLD-HCC allografts were established by intrahepatic injection of murine HCC cells with HFHC feeding. Metabolomic profiling was performed using liquid chromatography-mass spectrometry. Biological functions of L.acidophilus conditional medium (L.a CM) and metabolites were determined in NAFLD-HCC human cells and mouse organoids. L.acidophilus supplementation suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice. This was confirmed in orthotopic allografts and germ-free tumourigenesis mice. L.a CM inhibited the growth of NAFLD-HCC human cells and mouse organoids. The protective function of L.acidophilus was attributed to its non-protein small molecules. By metabolomic profiling, valeric acid was the top enriched metabolite in L.a CM and its upregulation was verified in liver and portal vein of L.acidophilus-treated mice. The protective function of valeric acid was demonstrated in NAFLD-HCC human cells and mouse organoids. Valeric acid significantly suppressed NAFLD-HCC formation in HFHC-fed DEN-treated mice, accompanied by improved intestinal barrier integrity. This was confirmed in another NAFLD-HCC mouse model induced by CDHF diet and DEN. Mechanistically, valeric acid bound to hepatocytic surface receptor GPR41/43 to inhibit Rho-GTPase pathway, thereby ablating NAFLD-HCC. L.acidophilus exhibits anti-tumourigenic effect in mice by secreting valeric acid. Probiotic supplementation is a potential prophylactic of NAFLD-HCC. Shown in Acknowledgments.

Hepatocyte Deubiquitinating Enzyme OTUD5 Deficiency is a Key Aggravator for Metabolic Dysfunction-Associated Steatohepatitis by Disturbing Mitochondrial Homeostasis

BACKGROUND & AIMSMetabolic dysfunction-associated steatohepatitis (MASH) is a common chronic liver disease worldwide. No effective pharmacological therapies for MASH have been developed; to develop such promising drugs, the underlying mechanisms regulating MASH need to be elucidated. Here, we aimed to determine the role of OTUD5 in MASH progression and identify a specific mechanism. METHODSThe expression levels of OTUD subfamily under palmitic acid/oleic acid (PAOA) stimulation were screened. OTUD5 expression was assessed in human liver tissues without steatosis, those with simple steatosis, and those with MASH. MASH models were developed in hepatocyte-specific Otud5-knockout mice that were fed high-fat high-cholesterol (HFHC) and high-fat high-cholesterol plus high-fructose/sucrose (HFF) diet for 16 weeks. RESULTSThe expression of OTUD5 was downregulated in fatty liver and was negatively related to the progression of MASH. Lipid accumulation and inflammation were exacerbated by Otud5 knockdown but attenuated by Otud5 overexpression under PAOA treatment. Hepatocyte-specific Otud5 deletion markedly exacerbated steatosis, inflammation, and fibrosis in the livers of two MASH mouse models. We identified voltage-dependent anion channel 2 (VDAC2) as an OTUD5-interacting partner; OTUD5 cleaved the K48-linked polyubiquitin chains from VDAC2, and it inhibited subsequent proteasomal degradation. The anabolic effects of OTUD5 knockdown on PAOA-induced lipid accumulation were effectively reversed by VDAC2 overexpression in primary hepatocytes. Metabolomic results revealed that VDAC2 is required for OTUD5-mediated protection against hepatic steatosis by maintaining mitochondrial function. CONCLUSIONSOTUD5 may ameliorate MASH progression via VDAC2-maintained mitochondrial homeostasis. Targeting OTUD5 may be a viable MASH-treatment strategy.

Edible Bird’s Nest Effectively Attenuates Atherosclerosis through Modulation of Cholesterol Metabolism via Activation of PPARγ/LXRα Signaling Pathway In Vivo

Atherosclerosis is the underlying cause of the majority of cardiovascular diseases (CVDs) due to high-cholesterol deposition in the aorta, resulting in hardening and narrowing of blood vessels and subsequently leading to an obstructed blood flow. There are few previous publications showing the preventive effects of edible bird’s nest (EBN) on CVD, but the study on cholesterol homeostasis related to atherosclerosis is limited. In this study, we have determined the mechanisms by which EBN modulates cholesterol metabolism by identifying genes and biomarkers. Male New Zealand white rabbits were fed either a high-fat high-cholesterol (HFC) diet or a HFC diet supplemented with EBN (500 mg/kg b.w/day) for 12 weeks. For comparison purposes, a group of rabbits receiving a HFC diet was supplemented with simvastatin (10 mg/kg b.w/day) as a standard drug treatment for hypercholesterolemia. EBN supplementation significantly improved lipid and coagulation profiles, reduced hepatosteatosis, and stabilized atherosclerotic plaque formation in hypercholesterolemic rabbits. Cholesterol accumulation in liver and aorta tissues, as well as hepatic HMGCR levels, was reduced in the hypercholesterolemic rabbits supplemented with EBN. Gene expression analysis showed that EBN induced many genes related to cholesterol uptake (LDLR, LOX-1, and CD36), cholesterol efflux (ABCA1, LCAT, and CYP7A1), and cholesterol-sensing signaling (LXRα and PPARγ). In conclusion, our studies suggest that EBN supplementation could be an effective food product for the treatment or prevention of atherosclerosis by regulating cholesterol metabolism.

CARD9 deficiency aggravated nonalcoholic steatohepatitis in mice through increasing inflammatory response

Nonalcoholic steatohepatitis (NASH), a subtype of nonalcoholic fatty liver disease (NAFLD), is the leading cause of liver-related morbidity worldwide. Caspase recruitment domain family member 9 (CARD9), a myeloid cell-specific signaling protein, belongs to the CARD protein family. However, its role in NASH is unknown. Therefore, this study aimed to investigate the role of CARD9 in the development of NASH. NASH models were established using CARD9-knockout and wild-type mice. They were either fed a methionine/choline deficient (MCD) diet for 6 weeks or a high-fat high-cholesterol (HFHC) diet for 16 weeks. Liver fibrosis model was also developed using CCl4. CARD9 deficiency accelerated steatohepatitis development in MCD or HFHC diet-fed mice, accompanied by an upregulation of fibrosis, adipogenesis, and proinflammatory genes. CARD9 deficiency was found to exacerbate CCl4-induced liver fibrosis. In vitro studies demonstrated that CARD9 deficiency induced the expression of S100a8/a9 through Toll-like receptor in Kupffer cells treated with palmitate. This led to an increased expression of proinflammatory, fibrosis, and lipid metabolism-related genes in NASH progression. These results highlight the role of CARD9 in the development of NASH and provide new insights into the therapeutic strategies for NASH.

The S100 calcium-binding protein A6 plays a crucial role in hepatic steatosis by mediating lipophagy.

S100 calcium-binding protein A6 (S100A6) is a calcium-binding protein that is involved in a variety of cellular processes, such as proliferation, apoptosis, and the cellular response to various stress stimuli. However, its role in NAFLD and associated metabolic diseases remains uncertain. In this study, we revealed a new function and mechanism of S100A6 in NAFLD. S100A6 expression was upregulated in human and mouse livers with hepatic steatosis, and the depletion of hepatic S100A6 remarkably inhibited lipid accumulation, insulin resistance, inflammation, and obesity in a high-fat, high-cholesterol (HFHC) diet-induced murine hepatic steatosis model. In vitro mechanistic investigations showed that the depletion of S100A6 in hepatocytes restored lipophagy, suggesting S100A6 inhibition could alleviate HFHC-induced NAFLD. Moreover, S100A6 liver-specific ablation mediated by AAV9 alleviated NAFLD in obese mice. Our study demonstrates that S100A6 functions as a positive regulator of NAFLD, targeting the S100A6-lipophagy axis may be a promising treatment option for NAFLD and associated metabolic diseases.