Diisoprenyl-cyclohexene-type meroterpenoids from a mangrove endophytic fungus Aspergillus sp. GXNU-Y65 and their anti-nonalcoholic steatohepatitis activity in AML12 cells.

Nine previously undescribed diisoprenyl-cyclohexene-type meroterpenoids, aspergienynes A-I, together with five known analogues, were obtained from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y65. The diisoprenyl-cyclohexene-type meroterpenoids were elucidated based on multispectroscopic analysis, and the previously undescribed compounds' absolute configurations were established via electronic circular dichroism calculations. Biological activity results indicated that aspergienyne C (compound 3) had strong anti-nonalcoholic steatohepatitis activity against AML12 cells treated with PA (Palmitic acid) + OA (Oleic acid). At the same concentration of 20 µM, 3 significantly reduced triglyceride (TG) content compared with fenofibrate (positive control) in PA + OA treated AML12 cells, and obviously increased phosphorylation of acetyl-CoA carboxylase.

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.

Inhibition of the Akt/NF-κB pathway is involved in the anti-gastritis effects of an ethanolic extract of the rhizome of Atractylodes macrocephala.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastritis can lead to ulcers and the development of gastric cancer. The rhizome of Atractylodes macrocephala Koidz. (Asteraceae), a traditional Chinese medicinal herb, is prescribed for the treatment of gastric disorders, hepatitis and rheumatism. Its bio-active compounds are considered to be particularly effective in this regard. However, the molecular processes of the herb's anti-inflammatory activity remain obscure. This study elucidates a mechanism upon which an ethanolic extract of this herb (Am-EE) exerts anti-inflammation effects in RAW264.7 macrophage cells (RAW cells) stimulated by lipopolysaccharide (LPS) treatment and HCl Ethanol-stimulated gastritis rats. AIM OF THE STUDY: To investigate the anti-gastritis activities of Am-EE and explore the mode of action. MATERIALS AND METHODS: Ethanol (95%) was used to prepare Am-EE. The quality of the extract was monitored by HPLC analysis. The in vivo effects of this extract were examined in an HCl Ethanol-stimulated gastritis rat model, while LPS-stimulated RAW cells were used for in vitro assays. Cell viability and nitric oxide (NO) production were observed by MTT and Griess assays. Real-time PCR was used to examine mRNA expression. The PGE2 ELISA kit was employed to detect prostaglandin E2 (PGE2). Enzyme activities and protein contents were examined by immunoblotting. Luciferase reporter gene assays (LRA) were employed to observe nuclear transcription factor (NF)-κB activity. The SPSS (SPSS Inc., Chicago, Illinois, United States) application was used for statistical examination. RESULTS: HPLC analysis indicates that Am-EE contains atractylenolide-1 (AT-1, 1.33%, w/w) and atractylenolide-2 (AT-2, 1.25%, w/w) (Additional Figure. A1). Gastric tissue damage (induced by HCl Ethanol) was significantly decreased in SD rats following intra-gastric application of 35 mg/kg Am-EE. Indistinguishable to the anti-inflammation effects of 35 mg/kg ranitidine (gastric medication). Am-EE treatment also reduced LPS-mediated nitric oxide (NO) and prostaglandin E2 (PGE2) production. The mRNA and protein synthesis of inducible cyclooxygenase (COX)-2 and NO synthase (iNOS) was down-regulated following treatment in RAW cells. Am-EE decreased NF-κB (p50) nuclear protein levels and inhibited NF-κB-stimulated LRA activity in RAW cells. Lastly, Am-EE decreased the up-regulated levels of phosphorylated IκBα and Akt proteins in rat stomach lysates and in LPS challenged RAW cell samples. CONCLUSION: Our study illustrates that Am-EE suppresses the Akt/IκBα/NF-κB pathway and exerts an anti-inflammatory effect. These novel conclusions provide a pharmacological basis for the clinical use of the A. macrocephala rhizome in the treatment and prevention of gastritis and gastric cancer.

[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.

NLRP3 inflammasome activation in gestational diabetes mellitus placentas is associated with hydrogen sulfide synthetase deficiency.

The placenta may play a key role in the activation of inflammation and initiation of insulin resistance (IR) during gestational diabetes mellitus (GDM) pathogenesis. Interleukin (IL)-1ß and IL-18, regulated by NLR family pyrin domain containing-3 (NLRP3) inflammasome, are important inflammatory cytokines in the initiation of maternal IR during GDM. However, the mechanism responsible for the regulatory of NLRP3 inflammasome in placenta remains unknown. Hydrogen sulfide (H2S) exerts anti-inflammatory function partially via suppressing the activation of the NLPR3 inflammasome. The present study aimed to investigate the role of NLRP3 inflammasome, H2S synthetase cystathionine-γ-lyase (CSE) and cystathionine-ß-synthetase (CBS) in placenta in the pathogenesis of GDM. Clinical placenta samples were collected from pregnant women with GDM (n=16) and healthy pregnant women at term (n=16). Western blot analysis was performed to detect the protein expression levels of NLRP3, cleaved caspase-1, CBS and CSE in the placenta samples. Pearson's correlation analysis was performed to assess the correlation between NLRP3 inflammasome and H2S synthetase. Human placental cells were cultured and treated with different concentrations of NaHS (0, 10, 25 and 50 nmol/l) or L-cysteine (0, 0.25, 0.50 and 1.00 mmol/l). In addition, western blot analysis was performed to detect the protein expression levels of NLRP3 and cleaved caspase-1, while ELISA was performed to measure the production of IL-1ß and IL-18 in the culture media. The results demonstrated that the expression levels of NLRP3 and cleaved caspase-1 increased, while the expression levels of CBS and CSE decreased in the placenta samples. In addition, the expression levels of NLRP3 and cleaved caspase-1 were inversely correlated with the expression levels of CBS and CSE. Notably, NaHS and L-cysteine significantly suppressed the expression levels of NLRP3 and cleaved caspase-1, and the production of IL-1 and IL-18 in human placental cells. Taken together, the results of the present study suggest that H2S synthetase deficiency in placenta may contribute to excessive activation of NLRP3 inflammasome in GDM.

Dendrobium officinale Kimura et Migo and American ginseng mixture: A Chinese herbal formulation for gut microbiota modulation.

Dendrobium officinale Kimura et Migo (D. officinale) is a famous traditional Chinese medicine (TCM). A mixture of D. officinale and American ginseng has been shown to enhance cell-mediated immunity, humoral immunity, and monocyte/macrophage functions in mice. Here, the effects of a D. officinale and American ginseng mixture on the structure of gut microbial community in dogs were examined using high-throughput 16S rRNA gene amplicon sequencing. The data revealed that while the mixture did not change the diversity of gut microbial community significantly, differences among individuals were significantly reduced. Furthermore, the mixture-responsive operational taxonomic units (OTUs) exhibited a phase-dependent expression pattern. Fifty-five OTUs were found to exhibit a mixture-induced expression pattern, among which one third were short-chain fatty acid (SCFA)-producing genera and the others were probiotic genera included Lactobacillus spp., Sutterella, Alistipes, Anaerovorax, Bilophila, Coprococcus, Gordonibacter, Oscillibacter, among others. By contrast, 36% of the OTUs exhibiting a mixture-repressed expression pattern were disease-associated microorganisms, and six genera, namely Actinomyces, Escherichia/Shigella, Fusobacterium, Slackia, Streptococcus and Solobacterium, were associated with cancer. In addition, five genera were closely associated with diabetes, namely Collinsella, Rothia, Howardella, Slackia and Intestinibacter. Our results indicate that this D. officinale and American ginseng mixture may be used as a prebiotic agent to enhance SCFA-producing genera and prevent gut dysbiosis.

High expression of FOXO3 is associated with poor prognosis in patients with hepatocellular carcinoma.

The role of forkhead box O3 (FOXO3) as a tumor suppressor gene and its association with the human lifespan is well documented. However, several studies have indicated that high expression of FOXO3 is also significantly associated with tumorigenesis. The aim of the present study was to determine the clinical significance of FOXO3 in the development and prognosis of hepatocellular carcinoma (HCC). mRNA expression data of FOXO3 from The Cancer Genome Atlas database was analyzed through the UALCAN online tool to compare the expression of FOXO3 between HCC and normal liver tissues. Subsequently, the expression of FOXO3 at the protein level was investigated via immunohistochemical staining of 314 HCC and 150 non-cancerous liver tissue samples. The association between protein expression and clinicopathological parameters was analyzed using the χ2 test, and the effect of FOXO3 expression on survival was assessed via Kaplan-Meier analysis. The expression of FOXO3 mRNA was significantly higher in HCC in comparison with healthy tissues. High FOXO3 protein expression was revealed in 43/150 non-cancerous liver tissues, and in 238/314 HCC samples. A significant association was demonstrated between FOXO3 expression and metastasis, Tumor-Node-Metastasis stage, Edmondson grade, α-fetoprotein level and overall survival. In conclusion, the high expression of FOXO3 predicts a poor prognosis in patients with HCC, indicating this protein as a potential therapeutic target in HCC.

[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.

Adipose tissue-liver axis in alcoholic liver disease.

Alcoholic liver disease (ALD) remains an important health problem worldwide. The disease spectrum is featured by early steatosis, steatohepatitis (steatosis with inflammatory cells infiltration and necrosis), with some individuals ultimately progressing to fibrosis/cirrhosis. Although the disease progression is well characterized, no effective therapies are currently available for the treatment in humans. The mechanisms underlying the initiation and progression of ALD are multifactorial and complex. Emerging evidence supports that adipose tissue dysfunction contributes to the pathogenesis of ALD. In the first part of this review, we discuss the mechanisms whereby chronic alcohol exposure contributed to adipose tissue dysfunction, including cell death, inflammation and insulin resistance. It has been long known that aberrant hepatic methionine metabolism is a major metabolic abnormality induced by chronic alcohol exposure and plays an etiological role in the pathogenesis of ALD. The recent studies in our group documented the similar metabolic effect of chronic alcohol drinking on methionine in adipose tissue. In the second part of this review, we also briefly discuss the recent research progress in the field with a focus on how abnormal methionine metabolism in adipose tissue contributes to adipose tissue dysfunction and liver damage.

[Synergistic action of 4-hydroxynonenal and tumor necrosis factor involving the NF-kB/IkBa signaling pathway in alcohol-induced liver injury].

OBJECTIVE: To investigate the effects and mechanism of intracellular 4-hydroxynonenal (4-HNE) accumulation on tumor necrosis factor (TNF)-induced hepatotoxicity in alcoholic liver disease (ALD). METHODS: An ALD model was established in male C57BL/6 mice (6-8 weeks old) by feeding an ethanol-containing diet for 5 weeks; mice given regular (non-ethanol) diet served as controls. ALD-related changes in 4-HNE and TNF levels were detected by western blotting. The underlying mechanisms of this molecular effect were examined by pre-treating HepG2 cells with 4-HNE followed by exposure to various concentrations of TNF. Effects on cell death were evaluated by MTT assay. Effects on TNF-mediated upstream factors' expression were detected by ELISA, western blotting, and real-time PCR. Effects on the TNF-induced inhibitor of NF-kB (IkBa) activity (phosphorylation status) and its formation of adducts were detected by western blotting and immunoprecipitation. RESULTS: ALD mice showed increased hepatic 4-HNE and TNF levels, and the increases were associated with extent of liver injury. Cell culture studies revealed that 4-HNE, at non-toxic concentrations, sensitized hepatocytes to TNF killing, which was associated with suppressed NF-kB trans activity. Furthermore, 4-HNE prevented phosphorylation of IkBa without affecting upstream IkB kinase activity. The ALD-enhanced 4-HNE content was found to associated with increased formation of 4-HNE-IkBa adduction for both the 4-HNE - treated hepatocytes in culture and in the livers of ALD mice. CONCLUSION: Alcohol-induced increase in 4-HNE accumulation represents a potent and clinically relevant mechanism of sensitizing hepatocytes to TNF-induced toxicity. These data support the notion that decreasing or eliminating accumulated intracellular 4-HNE can serve as a potential therapeutic option for ALD.

Progress of research in treatment of hyperlipidemia by monomer or compound recipe of Chinese herbal medicine.

Hyperlipidemia (HLP) is the No.1 risk factor for patients with atherosclerosis (AS) and is directly related to the occurrence of coronary artery disease (CAD) and cerebrovascular disease. Therefore, prevention and treatment of AS is of great importance and of practical significance in controlling the incidence and mortality of CAD. With its peculiar syndrome-dependent therapy, traditional Chinese medicine (TCM) has accumulated abundant practical experiences in this field and good clinical effects have been achieved. Chinese herbal medicine, with its particularly unique advantages and high potentials yet to be tapped, displays its huge strength in HLP prevention and treatment. The progress of studies concerning prevention and treatment of HLP by Chinese herbal medicines, in the form of monomers or compound recipes, is reviewed in this paper.