The micro-743a-3p-GSTM1 pathway is an endogenous protective mechanism against alcohol-related liver disease in mice.

BACKGROUND AND AIMS: Epidemiological evidence suggests that the phenotype of glutathione S-transferase mu 1 (GSTM1), a hepatic high-expressed phase II detoxification enzyme, is closely associated with the incidence of alcohol-related liver disease (ALD). However, whether and how hepatic GSTM1 determines the development of ALD is largely unclear. This study was designed to elucidate the role and potential mechanism(s) of hepatic GSTM1 in the pathological process of ALD. METHODS: GSTM1 was detected in the liver of various ALD mice models and cultured hepatocytes. Liver-specific GSTM1 or/and micro (miR)-743a-3p deficiency mice were generated by adenoassociated virus-8 delivered shRNA, respectively. The potential signal pathways involving in alcohol-regulated GSTM1 and GSTM1-associated ALD were explored via both genetic manipulation and pharmacological approaches. RESULTS: GSTM1 was significantly upregulated in both chronic alcohol-induced mice liver and ethanol-exposed murine primary hepatocytes. Alcohol-reduced miR-743a-3p directly contributed to the upregulation of GSTM1, since liver specific silencing miR-743a-3p enhanced GSTM1 and miR-743a-3p loss protected alcohol-induced liver dysfunctions, which was significantly blocked by GSTM1 knockdown. GSTM1 loss robustly aggravated alcohol-induced hepatic steatosis, oxidative stress, inflammation, and early fibrotic-like changes, which was associated with the activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38. GSTM1 antagonized ASK1 phosphorylation and its downstream JNK/p38 signaling pathway upon chronic alcohol consumption via binding with ASK1. ASK1 blockage significantly rescued hepatic GSTM1 loss-enhanced disorders in alcohol-fed mice liver. CONCLUSIONS: Chronic alcohol consumption-induced upregulation of GSTM1 in the liver provides a feedback protection against hepatic steatosis and liver injury by counteracting ASK1 activation. Down-regulation of miR-743a-3p improves alcohol intake-induced hepatic steatosis and liver injury via direct targeting on GSTM1. The miR-743a-3p-GSTM1 axis functions as an innate protective pathway to defend the early stage of ALD.

Increased soluble endoglin levels in newly-diagnosed type 2 diabetic patients are associated with endothelial dysfunction.

Endothelial dysfunction (ED) contributes to the pathologic process underlying macrovascular complications, a common complication of type 2 diabetes mellitus (T2DM). Soluble endoglin (sEng) shed from the extracellular domain of the entire endoglin molecule blocks endothelial protection mediated by transforming growth factor-beta 1 (TGF-ß1). The reactive hyperemia index (RHI), which is determined by reactive hyperemia peripheral arterial tonometry (RH-PAT), is a new index with which to evaluate ED. This study determined the changes in serum sEng levels in newly-diagnosed (untreated) T2DM patients and the correlation with the RHI. The T2DM group included 34 newly-diagnosed T2DM patients, while the control group included 53 healthy adults. The clinical data from the two groups were evaluated retrospectively. The intima-media thickness (IMT) of the common carotid artery (CCA) and the ankle-brachial index (ABI) of both legs were used to assess structural vascular changes. The serum sEng level was determined using an ELISA kit. Endothelial function was assessed using RH-PAT and the RHI was computed. The serum sEng level in the T2DM group was significantly greater than the control group, although the RHI was significantly lower in the T2DM group (p < 0.05). The serum sEng level was negatively correlated with the RHI in T2DM patents (r = 0.354, p = 0.041). The serum sEng level, CCA-IMT, and ABI were not significantly correlated with T2DM (p > 0.05). In summary, among newly-diagnosed T2DM patients, the serum sEng levels were inversely correlated with the RHI, and an elevated sEng level may be associated with ED.

Design and Synthesis of Novel Indole Ethylamine Derivatives as a Lipid Metabolism Regulator Targeting PPARα/CPT1 in AML12 Cells.

Peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) are important targets of lipid metabolism regulation for nonalcoholic fatty liver disease (NAFLD) therapy. In the present study, a set of novel indole ethylamine derivatives (4, 5, 8, 9) were designed and synthesized. The target product (compound 9) can effectively activate PPARα and CPT1a. Consistently, in vitro assays demonstrated its impact on the lipid accumulation of oleic acid (OA)-induced AML12 cells. Compared with AML12 cells treated only with OA, supplementation with 5, 10, and 20 µM of compound 9 reduced the levels of intracellular triglyceride (by 28.07%, 37.55%, and 51.33%) with greater inhibitory activity relative to the commercial PPARα agonist fenofibrate. Moreover, the compound 9 supplementations upregulated the expression of hormone-sensitive triglyceride lipase (HSL) and adipose triglyceride lipase (ATGL) and upregulated the phosphorylation of acetyl-CoA carboxylase (ACC) related to fatty acid oxidation and lipogenesis. This dual-target compound with lipid metabolism regulatory efficacy may represent a promising type of drug lead for NAFLD therapy.

Association between gestational blood lipids and TSH levels and pregnancy outcome of patients with subclinical hypothyroidism.

Objective: To investigate the association between gestational blood lipids and thyroid stimulating hormone (TSH) levels and pregnancy outcomes of patients with subclinical hypothyroidism (SCH). Methods: In this retrospective observational study, we analyzed the clinical data of 82 patients (case group) with gestational SCH treated in our hospital from January 2021 to January 2022 at gestational weeks 25-33 and grouped them according to whether SCH was well controlled by treatment (case Group-A: well controlled, n=55; case Group-B: poorly controlled, n=27), and the clinical data of 41 pregnant women (control group) undergoing physical examination during the same period. After comparing the blood lipids and TSH levels of the three groups, we compared their adverse pregnancy outcomes to assess the possible correlations between blood lipids and TSH levels and pregnancy outcomes. Results: The levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and TSH in the case Group-B were significantly higher than those in the case Group-A and the control group (P<0.05). Compared with case Group-B and the control group, the incidence of premature delivery, abortion and neonatal growth restriction was higher in case Group-A (P<0.05). Among 82 patients in the case group 42 presented adverse pregnancy outcomes. The levels of TC, TG, LDL-C and TSH in mothers and infants in the adverse outcome group were significantly higher than those in the favorable outcome group (P<0.05). Our Pearson analysis results showed that the levels of TC, TG and LDL-C were positively correlated with the TSH levels and the pregnancy outcomes, and that TSH was positively correlated with pregnancy outcomes (P<0.05). Conclusion: The levels of TC, TG, LDL-C and TSH in patients with poorly controlled SCH were increased during pregnancy, and were associated with the pregnancy outcomes and positively correlated with each other.

Ectopic accumulation of ceramide in cardiomyocytes modulates alcoholic cardiomyopathy via the TLR4-dependent pathway.

BACKGROUND AND AIMS: Excessive alcohol consumption predisposes drinkers to develop alcoholic cardiomyopathy. Although cardiomyocyte loss is the hallmark of cardiomyopathy, the underlying mechanism remains elusive. This study examined the potential mechanism of alcohol-induced cardiomyocyte death in a mouse model of alcoholic cardiomyopathy. METHODS: We established the alcoholic cardiomyopathy mouse model using C57BL/6J mice and confirmed it via echocardiography and histological examination. The cardiac ceramide content and profile were analyzed with a triple-quadrupole mass spectrometer. The molecular mechanism underlying the accumulation of ceramide due to chronic alcohol consumption and ceramide-induced cardiomyocyte death were investigated by in vivo and in vitro models. Finally, we established a TLR4 mutation model to explore the function of TLR4 in CH3/HeJ mice. RESULTS: Cardiac lipotoxicity that followed alcohol exposure resulted mainly in C16:0-, C18:0-, and C24:1-ceramide aggregation. Genes encoding the sphingosine hydrolysis enzymes (SMPD1 and SMPD2) rather than de novo synthetic biomarkers were markedly upregulated. Exogenous ceramide mimics (C6-ceramide) werenderlying the accumulation of ceramide observed to cause H9C2 cardiomyocyte-like cell death, which was consistent with results under palmate acid (PA) treatment. As a ceramide precursor, PA induces intracellular ceramide generation through TLR4 signaling, which can be abolished by an inhibitor of ceramide synthesis. Furthermore, mechanistic investigations demonstrated that pharmacological or genetic inhibition of TLR4 attenuated PA-induced cell death and corresponding ceramide production. Moreover, global mutation of TLR4 in CH3/HeJ mice significantly reduced the accumulation of C24:0, C24:1, OH_C24:1, and total ceramide following alcohol challenge. CONCLUSIONS: Our findings demonstrate that ceramide accumulation plays a crucial role in alcoholic cardiomyopathy, effects that are partially mediated through the TLR4-dependent pathway.

Long non-coding RNA-EN_181 potentially contributes to the protective effects of N-acetylcysteine against non-alcoholic fatty liver disease in mice.

N-acetylcysteine (NAC) possesses a strong capability to ameliorate high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in mice, but the underlying mechanism is still unknown. Our study aimed to clarify the involvement of long non-coding RNA (lncRNA) in the beneficial effects of NAC on HFD-induced NAFLD. C57BL/6J mice were fed a normal-fat diet (10 % fat), a HFD (45 % fat) or a HFD plus NAC (2 g/l). After 14-week of intervention, NAC rescued the deleterious alterations induced by HFD, including the changes in body and liver weights, hepatic TAG, plasma alanine aminotransferase, plasma aspartate transaminase and liver histomorphology (haematoxylin and eosin and Oil red O staining). Through whole-transcriptome sequencing, 52 167 (50 758 known and 1409 novel) hepatic lncRNA were detected. Our cross-comparison data revealed the expression of 175 lncRNA was changed by HFD but reversed by NAC. Five of those lncRNA, lncRNA-NONMMUT148902·1 (NO_902·1), lncRNA-XR_001781798·1 (XR_798·1), lncRNA-NONMMUT141720·1 (NO_720·1), lncRNA-XR_869907·1 (XR_907·1), and lncRNA-ENSMUST00000132181 (EN_181), were selected based on an absolute log2 fold change value of greater than 4, P-value < 0·01 and P-adjusted value < 0·01. Further qRT-PCR analysis showed the levels of lncRNA-NO_902·1, lncRNA-XR_798·1, and lncRNA-EN_181 were decreased by HFD but restored by NAC, consistent with the RNA sequencing. Finally, we constructed a ceRNA network containing lncRNA-EN_181, 3 miRNA, and 13 mRNA, which was associated with the NAC-ameliorated NAFLD. Overall, lncRNA-EN_181 might be a potential target in NAC-ameliorated NAFLD. This finding enhanced our understanding of the biological mechanisms underlying the beneficial role of NAC.

Combined signatures of serum proteome and transcriptome in patients with recurrent aphthous ulcer.

OBJECTIVES: Recurrent aphthous ulcer (RAU) is a common oral disease with unclear mechanism. This study aimed to explore the serum signatures of RAU patients via proteomic and transcriptomic analysis. METHODS: This study was based on clinical observation. Part of serum was used for clinical tests, while the rest was processed for isobaric tags for relative and absolute quantitation (ITRAQ) labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) combined with microRNA (miRNA) microarrays. Bioinformatic analysis was then used to obtain significant signatures, which was verified by ELISA, qRT-PCR, and dual-luciferase reporter gene assays. RESULTS: Clinical data showed that triglyceride level, white blood cell count, and neutrophils percentage were increased in RAU group, while lymphocytes percentage was decreased. ITRAQ-2D LC-MS/MS identified 22 upregulated and 33 downregulated proteins in RAU group. Simultaneously, miRNA microarrays identified 64 upregulated and 31 downregulated miRNAs. After integrative bioinformatic analysis and verification, three miRNA-protein pairs, mainly involved in oxidative stress and inflammation responses, were obtained. Additionally, the interaction network indicated the crucial role of complement and coagulation cascade pathway in RAU. CONCLUSIONS: Our study revealed that complement and coagulation cascade pathway, oxidative stress, and inflammation responses may act as vital factors in pathogenesis of RAU.

[Atractylenolide â improves acetaminophen-induced acute liver injury in mice by inhibiting MAPK/NF-κB signaling pathway].

This study explored the protective effect of atractylenolide Ⅰ(AO-Ⅰ) against acetaminophen(APAP)-induced acute liver injury(ALI) in mice and its underlying mechanism. C57 BL/6 J mice were randomly divided into a control group, an APAP group(500 mg·kg~(-1)), a low-dose combination group(500 mg·kg~(-1) APAP + 60 mg·kg~(-1) AO-Ⅰ), and a high-dose combination group(500 mg·kg~(-1) APAP + 120 mg·kg~(-1) AO-Ⅰ). ALI was induced by intraperitoneal injection of APAP(500 mg·kg~(-1)). AO-Ⅰ by intragastric administration was performed 2 hours before APAP treatment, and the control group received the same dose of solvent by intragastric administration or intraperitoneal injection. The protective effect of AO-Ⅰ against APAP-induced ALI was evaluated by detecting alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels in the plasma and H&E staining in liver tissues of mice. The malondialdehyde(MDA) and glutathione(GSH) content and catalase(CAT) activity in mouse liver tissues were detected to evaluate the effect of AO-Ⅰ on APAP-induced oxidative stress in the liver. The proteins in the liver p38 mitogen-activated protein kinase(p38 MAPK), c-jun N-terminal kinase(JNK), and nuclear factor kappa-B p65(NF-κB p65) signaling pathways were measured by Western blot, and the liver inflammatory cytokines interleukin-1ß(IL-1ß) and interleukin-6(IL-6) were detected by real-time PCR. Compared with the APAP group, the combination groups showed reduced APAP-induced ALT level and liver MDA content, potentiated liver CAT activity, and elevated GSH content. Mechanistically, AO-Ⅰ treatment significantly inhibited APAP-up-regulated MAPK phosphorylation and NF-κB p65, and significantly reduced the transcriptional activities of IL-1ß and IL-6, downstream targets of NF-κB p65. AO-Ⅰ can improve APAP-induced ALI and the underlying mechanism is related to the inhibition of the MAPK/NF-κB p65 signaling pathway in APAP-challenged mice.

Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy.

Nonalcoholic fatty liver disease (NAFLD) is related to elevated cytoplasmic calcium signaling in hepatocytes, which may be mediated by store-operated calcium channel (SOCC) and inositol triphosphate receptor (IP3R). However, the regulatory effect of calcium signaling on lipid accumulation and degeneration in hepatocytes and the underlying molecular mechanism remain unknown. Autophagy inhibition promotes lipid accumulation and steatosis in hepatocytes. However, the association between elevated calcium signaling and autophagy inhibition in hepatocytes and its effect on hepatocyte fatty lesions remain unclear. Here, we established a mouse hepatocyte fatty gradient model using oleic acid. SOCC and IP3R channel opening and cytoplasmic calcium levels gradually increased with the hepatocyte pimelosis degree, whereas autophagy gradually decreased. We also established an optimal oleic acid (OOA) hepatocyte model, observing significantly increased SOCC and IP3R channel opening and calcium influx alongside significantly decreased autophagy and aggravated cellular fatty lesion. Calcium channel blockers (CCBs) and calcium channel gene silencing reagents (CCGSRs), respectively, reversed these effects, indicating that elevated cytoplasmic calcium signaling promotes NAFLD occurrence and the development by inhibiting hepatocyte autophagy. In the OOA model, upregulated extracellular regulated protein kinases 1/2 (ERK1/2), which can be regulated by SOCC and IP3R proteins transient receptor potential canonical 1 (TRPC1)/IP3R with elevated cytoplasmic calcium signaling, over-inhibited forkhead/winged helix O (FOXO) signaling and over-activated mammalian target of rapamycin complex 1 (mTORC1) signaling. Over-inhibited FOXO signaling significantly downregulated autophagy-related gene 12, which inhibits autophagosome maturation, while over-activated mTORC1 signaling over-inactivated Unc-51 like autophagy activating kinase 1, which inhibits preautophagosome formation. CCBs and CCGSRs recovered autophagy by significantly downregulating ERK1/2 to block abnormal changes in FOXO and mTORC1 signaling. Our findings indicate that upregulated SOCC and IP3R channels and subsequent elevated cytoplasmic calcium signaling in hepatocyte fatty lesions inhibits hepatocyte autophagy through (TRPC1/IP3R)/ERK/(FOXO/mTORC1) signaling pathways, causes lipid accumulation and degeneration in hepatocytes, and promotes NAFLD occurrence and development.

RNA Sequencing Reveals a Comprehensive Circular RNA Expression Profile in a Mouse Model of Alcoholic Liver Disease.

BACKGROUND AND AIMS: Alcoholic liver disease (ALD) is the most common liver disease and a severe mortality burden in the world. However, ALD is rarely detected at its early stages. Thus, exploration of an early event for ALD may help the prognosis and further therapy of ALD. Several circRNAs were proven as novel molecular biomarkers for the progression of chronic nonalcoholic fatty liver disease. Whether circRNAs are involved explicitly in ALD remains unknown. METHODS: The expression profile of circRNAs in an ALD mouse model was depicted by circRNA sequencing. The dysregulated circRNAs were verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bioinformatics and circRNA/microRNA (miRNA) crosstalk analyses were applied to predict the potential functions of circRNAs. Finally, the miRNA expression was confirmed by miRNA sequencing. RESULTS: Compared with the control group, 6 members of circRNAs were up-regulated, and 4 were down-regulated in the ALD model. GO enrichment analyses revealed these circRNAs were predominantly enriched in the endoplasmic reticulum, arachidonic acid metabolism, and cytochrome P450 metabolism pathway. Among these circRNAs, the differential expression of 5 circRNAs was validated and consistent with qRT-PCR, and only the up-regulated mou_circ_1657 is included in the circBase. Further, the crosstalk analysis of circRNA-miRNA revealed 7 miRNAs were targeted by mou_circ_1657, of which miR-19-5b was the only miRNA that was down-regulated in the ALD mice according to the miRNA sequencing data, suggesting it needs further attention in ALD. CONCLUSIONS: This study demonstrates that a cluster of circRNAs is aberrantly expressed in the livers of ALD mice. mou_circ_1657/miR-19-5b may play a critical role in the development of ALD. Our study provides new insight into the future investigation and therapy on ALD.

Characterization of the anti-Staphylococcus aureus fraction from Penthorum chinense Pursh stems.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) causes serious infections in hospitals. Penthorum chinense Pursh (PCP), employed by the Miao ethnic minority in China, presents antibacterial activities. In this study, the anti-Staphylococcus aureus activities in the pinocembrin-7-O residue-rich fraction from PCP (PGF) were evaluated and characterized. METHODS: The PGF was prepared with 70% ethanol reflux extraction followed by fractional extraction and column chromatography. Pinocembrin-7-O residue components were identified with electrospray ionization mass spectrometry (ESI-MS). Anti-S. aureus activities of the fraction and the main components were evaluated in vitro with serially diluted microbroth assays. Cytotoxicity was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) chromogenic assays using the NCTC 1469 cell line. RESULTS: This study indicated that the PGF and three components (S1, S2, and S3) presented anti-S. aureus activities, including against clinically isolated MRSA strains. The molecular masses of S1, S2, and S3 were identical to those of pinocembrin-7-O-[4″,6″-hexahydroxydiphenoyl (HHDP)]-ß-D-glucose, pinocembrin-7-O-[3″-O-galloyl-4″,6″-(s)-HHDP]-ß-D-glucose, and Thonningianin A, respectively. The PGF, S1, S2, and S3 all presented an identical minimum inhibitory concentration (MIC) against S. aureus ATCC 25923 and ATCC 43300, which was 62.5 µg/mL. The minimum bactericidal concentrations (MBCs) of the PGF and S3 against ATCC 25923 were 125 and 250 µg/mL, and the MBCs of the PGF, S2, and S3 against ATCC 43300 were 250, 500, and 250 µg/mL, respectively. A time-kill assay consistently indicated that none of the bacterial clones of ATCC 25923 and ATCC 43300 could survive under 2× and 4× MIC PGF treatment for 24 h, respectively. In contrast, 104 CFU (colony-forming units) of ATCC 25923 and ATCC 43300 were killed by 8× and 4× MIC S3 within 24 h, respectively. Additionally, 1×, 2×, and 4× MIC the PGF presented similar postantibiotic effects (PAEs) on the strain ATCC 25923. However, the PAE of the PGF on the strain ATCC 43300 was concentration dependent (1× < 2× < 4× MIC). Finally, the PGF (200 µg/mL) and S3 (60 µg/mL) showed no cytotoxicity against human hepatoma cells. CONCLUSIONS: The PGF and S3 from PCP present potential for the treatment of S. aureus and MRSA infections. The components S1 and S2 present inhibition activities against S. aureus.

[Mechanisms of tanshinone â ¡_A in reducing 4-HNE-induced hepatocyte damage by activating PPARα].

Tanshinone Ⅱ_A( Tan Ⅱ_A),the liposoluble constituents of Salvia miltiorrhiza,can not only ameliorate the lipidic metabolism and decrease the concentration of lipid peroxidation,but also resist oxidation damage,scavenge free radicals and control inflammation,with a protective effect on prognosis after liver function impairment. Therefore,the studies on the exact mechanism of Tan Ⅱ_A in protecting the liver can provide important theoretical and experimental basis for the prevention and treatment effect of Tan Ⅱ_A for liver injury. In the present study,the protective effects and mechanism of Tan Ⅱ_A on 4-hydroxynonenal( 4-HNE)-induced liver injury were investigated in vitro. Normal liver tissues NCTC 1469 cells were used to induce hepatocytes oxidative damages by 4-HNE treatment. The protective effect of Tan Ⅱ_A on hepatocytes oxidative damages was detected by release amount of lactate dehydrogenase( LDH) analysis and hoechst staining. The protein expression changes of peroxisome proliferator-activated receptor α( PPARα) and peroxisome proliferator response element( PPRE) were analyzed by Western blot analysis in NCTC 1469 cells before and after Tan Ⅱ_A treatment. The gene expression changes of fatty aldehyde dehydrogenase( FALDH) were analyzed by Real-time polymerase chain reaction( PCR) analysis. The results showed that 4-HNE increased the release amount of LDH,lowered the cell viability of NCTC 1469 cells,and Tan Ⅱ_A reversed 4-HNE-induced hepatocyte damage. Western blot analysis and RT-PCR analysis results showed that 4-HNE decreased the expression of PPARα and FALDH and increased the expression of 4-HNE. However,the expression of PPARα and FALDH were increased significantly and the expression of 4-HNE was decreased obviously after Tan Ⅱ_A treatment. This study confirmed that the curative effect of Tan Ⅱ_A was obvious on hepatocytes damage,and the mechanism may be associated with activating PPARα and FALDH expression as well as scavenging 4-HNE.

The TLR4-IRE1α pathway activation contributes to palmitate-elicited lipotoxicity in hepatocytes.

Lipotoxicity induced by saturated fatty acids (SFAs) plays a pathological role in the development of non-alcoholic fatty liver disease (NAFLD); however, the exact mechanism(s) remain to be clearly elucidated. Toll-like receptor (TLR) 4 plays a fundamental role in activating the innate immune system. Intriguingly, hepatocytes express TLR4 and machinery for TLR4 signalling pathway. That liver-specific TLR4 knockout mice are protective against diet-induced NAFLD suggests that hepatocyte TLR4 signalling pathway plays an important role in NAFLD pathogenesis. Herein, using cultured hepatocytes, we sought to directly examine the role of TLR4 signalling pathway in palmitate-elicited hepatotoxicity and to elucidate underlying mechanism(s). Our data reveal that palmitate exposure up-regulates TLR4 expression at both mRNA and protein levels in hepatocytes, which are associated with NF-κB activation. The inhibition of TLR4 signalling pathway through both pharmacological and genetic approaches abolished palmitate-induced cell death, suggesting that TLR4 signalling pathway activation contributes to palmitate-induced hepatotoxicity. Mechanistic investigations demonstrate that inositol-requiring enzyme 1α (IRE1α), one of three major signal transduction pathways activated during endoplasmic reticulum (ER) stress, is the downstream target of palmitate-elicited TLR4 activation and mechanistically implicated in TLR4 activation-triggered cell death in response to palmitate exposure. Collectively, our data identify that the TLR4-IRE1α pathway activation contributes to palmitate-elicited lipotoxicity in hepatocytes. Our findings suggest that targeting TLR4-IRE1α pathway can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism.

Sirtuin 3 acts as a negative regulator of autophagy dictating hepatocyte susceptibility to lipotoxicity.

Lipotoxicity induced by saturated fatty acids (SFAs) plays a central role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, the exact mechanisms remain to be fully elucidated. Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide-dependent deacetylase located primarily inside mitochondria. In this study, we demonstrated that an SFA-rich high-fat diet (HFD) was more detrimental to the liver than an isocaloric unsaturated HFD rich in fatty acids. Unexpectedly, SIRT3 expression and activity were significantly elevated in the livers of mice exposed to the SFA-rich HFD. Using cultured HepG2 and AML-12 hepatocytes, we demonstrated that unlike monounsaturated fatty acids, SFAs up-regulate SIRT3 expression and activity. SIRT3 overexpression renders both the liver and hepatocytes susceptible to palmitate-induced cell death, which can be alleviated by SIRT3 small interfering RNA (siRNA) transfection. In contrast, SIRT3 suppression protects hepatocytes from palmitate cytotoxicity. Further studies revealed that SIRT3 acts as a negative regulator of autophagy, thereby enhancing the susceptibility of hepatocytes to SFA-induced cytotoxicity. Mechanistic investigations revealed that SIRT3 overexpression causes manganese superoxide dismutase deacetylation and activation, which depleted intracellular superoxide contents, leading to adenosine monophosphate-activated protein kinase (AMPK) inhibition and mammalian target of rapamycin C1 activation, resulting in autophagy suppression. In contrast, SIRT3 siRNA gene silencing enhanced autophagy flux. A similar result was observed in the liver tissue of SIRT3 knockout mice. CONCLUSION: Our data indicate that SIRT3 is a negative regulator of autophagy whose activation by SFAs contributes to lipotoxicity in hepatocytes and suggest that restraining SIRT3 overactivation can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism. (Hepatology 2017;66:936-952).

Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation.

Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation.

Nuclear factor (erythroid-derived 2)-like 2 activation-induced hepatic very-low-density lipoprotein receptor overexpression in response to oxidative stress contributes to alcoholic liver disease in mice.

UNLABELLED: Chronic alcohol consumption leads to hypertriglyceridemia, which is positively associated with alcoholic liver disease (ALD). However, whether and how it contributes to the development of fatty liver and liver injury are largely unknown. In this study we demonstrate that chronic alcohol exposure differently regulates the expression of very-low-density lipoprotein receptor (VLDLR) in adipose tissue and the liver. Whereas adipose tissue VLDLR is significantly down-regulated, its hepatic expression is dramatically increased after chronic alcohol feeding. While HepG2 cells stably overexpressing VLDLR manifests increased intracellular triglyceride accumulation, VLDLR-deficient mice are protective against fatty liver and liver injury after chronic alcohol exposure. Mechanistic investigations using both in vitro and in vivo systems reveal that oxidative stress-induced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation plays a critical role in alcohol-induced VLDLR up-regulation in hepatocytes, but not in adipocytes. Oxidative stress enhances VLDLR gene expression and protein abundance in primary hepatocytes, concomitant with the Nrf2 activation. Conversely, Nrf2 gene silencing abrogates oxidative stress-induced VLDLR up-regulation in the liver, but not in adipose tissue. In mice, alcohol exposure induces hepatic oxidative stress and Nrf2 activation. Supplementation of N-acetylcysteine alleviates fatty liver and liver injury induced by chronic alcohol exposure, which is associated with suppressed Nrf2 activation and attenuated VLDLR increase in the liver. Furthermore, in comparison to wild-type counterparts, Nrf2-deficient mice demonstrate attenuated hepatic VLDLR expression increase in response to chronic alcohol exposure. CONCLUSION: Chronic alcohol consumption differently alters VLDLR expression in adipose tissue and the liver. Oxidative stress-induced Nrf2 activation is mechanistically involved in VLDLR overexpression in hepatocytes in response to chronic alcohol consumption. Hepatic VLDLR overexpression plays an important role in the pathogenesis of ALD.

The Combination of Bioactive Herbal Compounds with Biomaterials for Regenerative Medicine.

Regenerative medicine aims to restore the function of diseased or damaged tissues and organs by cell therapy, gene therapy, and tissue engineering, along with the adjunctive application of bioactive molecules. Traditional bioactive molecules, such as growth factors and cytokines, have shown great potential in the regulation of cellular and tissue behavior, but have the disadvantages of limited source, high cost, short half-life, and side effects. In recent years, herbal compounds extracted from natural plants/herbs have gained increasing attention. This is not only because herbal compounds are easily obtained, inexpensive, mostly safe, and reliable, but also owing to their excellent effects, including anti-inflammatory, antibacterial, antioxidative, proangiogenic behavior and ability to promote stem cell differentiation. Such effects also play important roles in the processes related to tissue regeneration. Furthermore, the moieties of the herbal compounds can form physical or chemical bonds with the scaffolds, which contributes to improved mechanical strength and stability of the scaffolds. Thus, the incorporation of herbal compounds as bioactive molecules in biomaterials is a promising direction for future regenerative medicine applications. Herein, an overview on the use of bioactive herbal compounds combined with different biomaterial scaffolds for regenerative medicine application is presented. We first introduce the classification, structures, and properties of different herbal bioactive components and then provide a comprehensive survey on the use of bioactive herbal compounds to engineer scaffolds for tissue repair/regeneration of skin, cartilage, bone, neural, and heart tissues. Finally, we highlight the challenges and prospects for the future development of herbal scaffolds toward clinical translation. Overall, it is believed that the combination of bioactive herbal compounds with biomaterials could be a promising perspective for the next generation of regenerative medicine.

The application of deep learning in abdominal trauma diagnosis by CT imaging.

BACKGROUND: Abdominal computed tomography (CT) scan is a crucial imaging modality for creating cross-sectional images of the abdominal area, particularly in cases of abdominal trauma, which is commonly encountered in traumatic injuries. However, interpreting CT images is a challenge, especially in emergency. Therefore, we developed a novel deep learning algorithm-based detection method for the initial screening of abdominal internal organ injuries. METHODS: We utilized a dataset provided by the Kaggle competition, comprising 3,147 patients, of which 855 were diagnosed with abdominal trauma, accounting for 27.16% of the total patient population. Following image data pre-processing, we employed a 2D semantic segmentation model to segment the images and constructed a 2.5D classification model to assess the probability of injury for each organ. Subsequently, we evaluated the algorithm's performance using 5k-fold cross-validation. RESULTS: With particularly noteworthy performance in detecting renal injury on abdominal CT scans, we achieved an acceptable accuracy of 0.932 (with a positive predictive value (PPV) of 0.888, negative predictive value (NPV) of 0.943, sensitivity of 0.887, and specificity of 0.944). Furthermore, the accuracy for liver injury detection was 0.873 (with PPV of 0.789, NPV of 0.895, sensitivity of 0.789, and specificity of 0.895), while for spleen injury, it was 0.771 (with PPV of 0.630, NPV of 0.814, sensitivity of 0.626, and specificity of 0.816). CONCLUSIONS: The deep learning model demonstrated the capability to identify multiple organ injuries simultaneously on CT scans and holds potential for application in preliminary screening and adjunctive diagnosis of trauma cases beyond abdominal injuries.

Xiezhuo Tiaozhi formula inhibits macrophage pyroptosis in the non-alcoholic fatty liver disease by targeting the SIRT1 pathway.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a challenging disease to interfere with and represents a potential long-term risk factor for hepatic fibrosis and liver cancer. The Xiezhuo Tiaozhi (XZTZ) formula, a water extract from crude herbs, has been widely used as an anti-NAFLD agent through clinical observation. However, the underlying pharmacological mechanisms of the XZTZ formula and its impact on the potential pathways against NAFLD have not been elucidated. PURPOSE: Our study aims to investigate the pharmacological effects and underlying regulatory mechanisms of the XZTZ formula to treat NAFLD. METHODS: The possible active components and pharmacological mechanisms of the XZTZ formula against NAFLD were identified using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and molecular docking. To further explore the potential mechanisms, forty-eight 6-week-old male C57BL/6 J mice were given individual attention with high-fat and high-sugar diet (HFHSD) or relevant control (Ctrl) diets for 16 weeks to successfully construct a NAFLD mouse model. Subsequently, the levels of serum biochemicals, pathological changes in the liver, and pyroptosis levels were assessed in mice to investigate the therapeutic effects of the XZTZ formula. Further, LPS-induced RAW264.7 cells and Immortalized Mouse Kupffer cells (ImKC) were used to verify the potential mechanisms of the XZTZ formula against NAFLD in vitro. RESULTS: We identified 7 chemical compounds and 2 potential therapeutic targets as plausible therapeutic points for the treatment of NAFLD using the XZTZ formula. Subsequent histopathological analysis revealed marked hepatic steatosis and lipid accumulation in the HFHSD mice liver, while conditions were effectively ameliorated by administration of the XZTZ formula. Additionally, our work demonstrated that the XZTZ formula could attenuate M1 polarization, promote M2 polarization, and suppress pyroptosis via the SIRT1 pathway in tissue samples. Moreover, validation performed through LPS-induced RAW264.7 and ImKC cells by showing that silencing SIRT1 weaken the effects of the XZTZ formula on relative pyroptosis affirmed that its role was associated with the SIRT1 pathway in macrophage. CONCLUSION: These findings suggest that the XZTZ formula alleviated hepatic steatosis and lipid accumulation in NAFLD mice. These ameliorations are associated with mechanisms involving the attenuation of M1 polarization, promotion of M2 polarization, and anti-pyroptosis effects through the SIRT1 pathway.