Isoquercitrin Attenuates Steatohepatitis by Inhibition of the Activated NLRP3 Inflammasome through HSP90.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease with a global prevalence of 25%. However, the medicines approved by the FDA or EMA are still not commercially available for the treatment of NAFLD. The NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome plays a crucial role in inflammatory responses, and the mechanisms related to steatohepatitis have been sufficiently clarified. NLRP3 has been widely evaluated as a potential target for multiple active agents in treating NAFLD. As a quercetin glycoside, isoquercitrin (IQ) has a broad inhibitory effect on oxidative stress, cancers, cardiovascular diseases, diabetes, and allergic reactions in vitro and in vivo. This study aimed to investigate the undercover mechanism of IQ in the treatment of NAFLD, particularly in anti-steatohepatitis, by suppressing the NLRP3 inflammasome. In this study, a methionine-choline-deficient induced steatohepatitis mice model was used to explore the effect of IQ on NAFLD treatment. Further mechanism exploration based on transcriptomics and molecular biology revealed that IQ inhibited the activated NLRP3 inflammasome by down-regulating the expression of heat shock protein 90 (HSP90) and suppressor of G-two allele of Skp1 (SGT1). In conclusion, IQ could alleviate NAFLD by inhibiting the activated NLRP3 inflammasome by suppressing the expression of HSP90.

A novel alcohol steamed preparation from Gastrodia elata Blume: Pharmacological assessment of a functional food.

Rhizoma Gastrodia (Orchidaceae; Gastrodia elata Blume), the rhizome of Gastrodia elata Blume (GE), is traditionally used as both a medicinal and functional food, with proven efficacy in treating mental disorders. In traditional processing, GE is washed, steamed with water, dried, and sliced. In this study, a novel processing technology-alcohol steamed GE (AGE) was proposed as an alternative. Totally, 17 compounds were identified in fresh GE and AGE. Compared with fresh GE, the relative content of parishin A and parishin E decreased after alcohol steaming, whereas gastrodin (GAS), p-hydroxylbenzyl alcohol (HBA), Parishin B, and Parishin C were increased. Additionally, the pentobarbital-induced sleep mice model and Chronic Restraint Stress (CRS) model were applied to evaluate the pharmacological effects of fresh GE and steamed GE, and both fresh and steamed GE showed an intensive hypnotic and anti-anxiety effect. Furthermore, the anti-anxiety mechanism based on serum metabolic was investigated and the tryptophan metabolic pathway was considered the response to the anti-anxiety effect of GE. Although the optimization of the processing technology of AGE still needs to be further explored, the current results have provided new thoughts for the processing technology and clinical application of GE.

Chemometric Discrimination of Cichorium glandulosum Boiss. et Huet and Cichorium intybus L. via Their Metabolic Profiling, Antioxidative, and Hypoglycemic Activities.

Cichorium glandulosum Boiss. et Huet (CG) and Cichorium intybus L. (CI) are widely used as the main raw material of functional food with hepatoprotective and hypoglycemic effects. Due to the lack of comparison on the chemical ingredients and efficacy, they were often used imprecisely and interchangeably. It is necessary to distinguish between them. With the plant metabolomics based on high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) and multivariate chemometric techniques, the chemical ingredients were characterized and 59 compounds between CG and CI were classified. As for antioxidative and hypoglycemic activities in vitro, CI extraction exhibited better antioxidant activity than CG, while CG extraction showed stronger hypoglycemic activity. Furthermore, a bivariate correlation between the chemical composition and efficacy of the extract was also analyzed, and three differentially strong correlation components between CI and CG were prepared, and the antioxidative and hypoglycemic efficacies were compared in vivo and different active phenotypes were obtained. Finally, we revealed chemical and biological differences between CG and CI, providing a basis for achieving better quality control and developing more effective functional foods.

Esculetin Alleviates Nonalcoholic Fatty Liver Disease on High-Cholesterol-Diet-Induced Larval Zebrafish and FFA-Induced BRL-3A Hepatocyte.

Non-alcoholic fatty liver disease (NAFLD), defined in recent years as metabolic-associated fatty liver disease (MAFLD), is one of the most common liver diseases in the world, with no drugs on market. Esculetin (ESC) is an active compound discovered in a variety of natural products that modulates a wide range of metabolic diseases and is a potential drug for the treatment of NAFLD. In this study, we used an HCD-induced NAFLD larval zebrafish model in vivo and an FFA-induced BRL-3A hepatocyte model in vitro to evaluate the anti-NAFLD effect of ESC. Lipid lowering, anti-oxidation and anti-inflammation effects were revealed on ESC and related gene changes were observed. This study provides a reference for further study and development of ESC as a potential anti-NAFLD/MAFLD drug.

Dendrobium catenatum Lindl. Water Extracts Attenuate Atherosclerosis.

OBJECTIVES: Dendrobium catenatum Lindl. (DH) is a Chinese herbal medicine, which is often used to make tea to improve immunity in China. Rumor has it that DH has a protective effect against cardiovascular disease. However, it is not clear how DH can prevent cardiovascular disease, such as atherosclerosis (AS). Therefore, the purpose of this study is to study whether DH can prevent AS and the underlying mechanisms. METHODS: Zebrafish larvae were fed with high-cholesterol diet (HCD) to establish a zebrafish AS model. Then, we used DH water extracts (DHWE) to pretreat AS zebrafish. The plaque formation was detected by HE, EVG, and oil red O staining. Neutrophil and macrophage counts were calculated to evaluate the inflammation level. Reactive oxygen species (ROS) activity, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity in zebrafish were measured to reflect oxidative stress. The cholesterol accumulation and the levels of lipid, triglyceride (TG), and total cholesterol (TC) were measured to reflect lipid metabolism disorder. Then, parallel flow chamber was utilized to establish a low shear stress- (LSS-) induced endothelial cell (EC) dysfunction model. EA.hy926 cells were exposed to LSS (3 dyn/cm2) for 30 min and treated with DHWE. The levels of ROS, SOD, MDA, glutathione (GSH), and glutathiol (GSSG) in EA.hy926 cells were analysed to determine oxidative stress. The release of nitric oxide (NO), endothelin-1 (ET-1), and epoprostenol (PGI2) in EA.hy926 cells was measured to reflect EC dysfunction. The mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in EA.hy926 cells was detected to reflect EC dysfunction inflammation. RESULTS: The results showed that DHWE significantly reduced cholesterol accumulation and macrophage infiltration in early AS. Finally, DHWE significantly alleviate the lipid metabolism disorder, oxidative stress, and inflammation to reduce the plaque formation of AS zebrafish larval model. Meanwhile, we also found that DHWE significantly improved LSS-induced EC dysfunction and oxidative stress in vitro. CONCLUSION: Our results indicate that DHWE could be used as a prevention method to prevent AS.

Kaempferol-3-O-Glucuronide Ameliorates Non-Alcoholic Steatohepatitis in High-Cholesterol-Diet-Induced Larval Zebrafish and HepG2 Cell Models via Regulating Oxidation Stress.

NAFLD (non-alcoholic fatty liver disease) is one of the most prominent liver diseases in the world. As a metabolic-related disease, the development of NAFLD is closely associated with various degrees of lipid accumulation, oxidation, inflammation, and fibrosis. Ilex chinensis Sims is a form of traditional Chinese medicine which is used to treat bronchitis, burns, pneumonia, ulceration, and chilblains. Kaempferol-3-O-glucuronide (K3O) is a natural chemical present in Ilex chinensis Sims. This study was designed to investigate the antioxidative, fat metabolism-regulating, and anti-inflammatory potential of K3O. A high-cholesterol diet (HCD) was used to establish steatosis in larval zebrafish, whereby 1mM free fatty acid (FFA) was used to induce lipid accumulation in HepG2 cells, while H2O2 was used to induce oxidative stress in HepG2. The results of this experiment showed that K3O reduced lipid accumulation and the level of reactive oxygen species (ROS) both in vivo (K3O, 40 µM) and in vitro (K3O, 20 µM). Additionally, K3O (40 µM) reduced neutrophil aggregation in vivo. K3O (20 µM) also decreased the level of malondialdehyde (MDA) and significantly increased the level of glutathione peroxidase (GSH-px) in both the HCD-induced larval zebrafish model and H2O2-exposed HepG2 cells. In the mechanism study, keap1, nrf2, tnf-α, and il-6 mRNA were all significantly reversed by K3O (20 µM) in zebrafish. Changes in Keap1 and Nrf2 mRNA expression were also detected in H2O2-exposed HepG2 cells after they were treated with K3O (20 µM). In conclusion, K3O exhibited a reduction in oxidative stress and lipid peroxidation, and this may be related to the Nrf2/Keap1 pathway in the NAFLD larval zebrafish model.

A simple, efficient and rapid HPLC-UV method for the detection of 5-HT in RIN-14B cell extract and cell culture medium.

5-Hydroxytryptamine (also known as 5-HT, serotonin) is one of the monoamine neurotransmitters which is distributed widely in plasma and brain of mammals and plays important roles in physiological manipulations. In the present method, we describe the development of a simple, efficient and rapid high performance liquid chromatographic method coupled with ultraviolet (HPLC-UV) detector for the qualitative and quantitative analysis of 5-HT in both cell extract and cell culture medium (RIN-14B). The experiments use repeated freeze-thaw cycles followed by centrifugation and direct injection of the supernatant into the chromatography. An analytical C18 column (Agilent Zorbax Extend, 4.6 × 250 mm, 5 µm.) was taken for chromatographic separation; the mobile phase was 0.05 mol/L potassium dihydrogen phosphate (KH2PO4)/acetonitrile (90:10 v/v). Isocratic elution is established at the flow rate of 1.0 mL/min. The time required for this chromatographic run is 8 min. Over the concentration range of 0.1-10 µg/mL, the calibration curve is linear in this method. Other unique characteristics and advantages include high accuracy (92.02-103.28%) and high precision (intra- and inter-day coefficients of variation ≤ 4.69%). This method is applicable for the investigation of drug/condition-response relationships in the function of synthesis and secretion of 5-HT in cultured RIN-14B cells in various in vitro studies.

A Comprehensive Study of High Cholesterol Diet-Induced Larval Zebrafish Model: A Short-Time In Vivo Screening Method for Non-Alcoholic Fatty Liver Disease Drugs.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease around the world. However, there is still no drug for NAFLD in the market, the study of potential therapeutic drugs on NAFLD is extraordinarily pressing and urgent. The rodent models for NAFLD drugs' study are always with a long time cost. Therefore, we aim to establish a short-time NAFLD drug screening model. A laboratory-made high cholesterol diet was used on larval zebrafish for 3 weeks to establish the NAFLD screen model. Lipid metabolism, oxidant stress, and pathology were studied to comprehensively demonstrate the whole spectrum of NAFLD on this model. Bezafibrate and pioglitazone were used to evaluate the model. Moreover, mechanism research was performed on this model.The NAFLD larval zebrafish model was established with the comprehensive process of NAFLD. Moreover, multiple index on lipid metabolism, oxidant stress, hepatic steatosis, and hepatic inflammation can be easily tested for drug screening. Furthermore, this model can be used to perform the mechanism research by testing mRNA expression. The NAFLD larval zebrafish model is a comprehensive short-time screening method for NAFLD drugs.

Lipid Modulating Anti-oxidant Stress Activity of Gastrodin on Nonalcoholic Fatty Liver Disease Larval Zebrafish Model.

Non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the world. However, there are still no drugs for NAFLD/NASH in the market. Gastrodin (GAS) is a bioactive compound that is extracted from Gastrodia elata, which is used as an active compound on nervous system diseases. Recent reports showed that GAS and Gastrodia elata possess anti-oxidant activity and lipid regulating effects, which makes us curious to reveal the anti-NAFLD effect of GAS. A high cholesterol diet (HCD) was used to induce a NAFLD larval zebrafish model, and the lipid regulation and anti-oxidant effects were tested on the model. Furthermore, qRT-PCR studied the underlying mechanism of GAS. To conclude, this study revealed a lipid regulation and anti-oxidant insights of GAS on NAFLD larval zebrafish model and provided a potential therapeutic compound for NAFLD treatment.

Pharmacokinetic comparison of quercetin, isoquercitrin, and quercetin-3-O-ß-D-glucuronide in rats by HPLC-MS.

BACKGROUND: Quercetin (Qr), isoquercitrin (IQ), and quercetin-3-O-ß-D-glucuronide (QG) are powerful phytochemicals that have been shown to exhibit disease prevention and health promotion properties. However, there may exist transformations between Qr, IQ, and QG in vivo. And the pharmacokinetic profiles of Qr, IQ, and QG have not been systematically compared. The pharmacokinetics study would be helpful to better understand the pharmacological actions of them. METHODS: Herein, we developed a reliable HPLC-MS method to compare the pharmacokinetics of Qr, IQ, and QG after separate (50 mg/kg) oral administration of them in rats, using puerarin as internal standard. The detection was performed using negative selected ion monitoring. This method was validated in terms of selectivity, linearity, precision, accuracy, extraction recovery, matrix effect, and stability; and shows reliabilities in monitoring the pharmacokinetic behaviors of these three compounds. RESULTS: Our results showed that after separate oral administration of Qr, IQ, and QG, all of the compounds could be detected in plasma. In addition, QG could be detected in the Qr group; Qr and QG could be measured in the IQ group; and Qr could be found in rat plasma after 1.5 h of QG administration. Moreover, the AUC0-t of Qr in the; Qr group (2,590.5 ± 987.9 mg/L*min), IQ group (2,212.7 ± 914.1 mg/L*min), and QG group (3,505.7 ± 1,565.0 mg/L*min) was larger than the AUC0-t of QG in the; Qr group (1,550.0 ± 454.2 mg/L*min), IQ group (669.3 ± 188.3 mg/L*min), and QG group (962.7 ± 602.3 mg/L*min). The AUC0-t of IQ was the lowest among all groups. DISCUSSION: Quercetin, IQ, and QG can all be absorbed into plasma. A mutual transformation exists between Qr and QG, and IQ can be metabolized into Qr and QG in SD rats. These results would provide a meaningful basis for understanding the pharmacological actions of these three compounds.

Isoquercetin Improves Hepatic Lipid Accumulation by Activating AMPK Pathway and Suppressing TGF-ß Signaling on an HFD-Induced Nonalcoholic Fatty Liver Disease Rat Model.

Isoquercetin (IQ), a glucoside derivative of quercetin, has been reported to have beneficial effects in nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the potential improvement of IQ in liver lipid accumulation, inflammation, oxidative condition, and activation in Kupffer cells (KCs) on a high-fat diet (HFD) induced NAFLD models. Male Sprague-Dawley (SD) rats were induced by HFD, lipopolysaccharides/free fatty acids (LPS/FFA) induced co-culture cells model between primary hepatocytes and Kupffer cells was used to test the effects and the underlying mechanism of IQ. Molecular docking was performed to predict the potential target of IQ. Significant effects of IQ were found on reduced lipid accumulation, inflammation, and oxidative stress. In addition, AMP-activated protein kinase (AMPK) pathway was activated by IQ, and is plays an important role in lipid regulation. Meanwhile, IQ reversed the increase of activated KCs which caused by lipid overload, and also suppression of Transforming growth factor beta (TGF-ß) signaling by TGF-ß Recptor-1 and SMAD2/3 signaling. Finally, TGF-ßR1 and TGF-ßR2 were both found may involve in the mechanism of IQ. IQ can improve hepatic lipid accumulation and decrease inflammation and oxidative stress by its activating AMPK pathway and suppressing TGF-ß signaling to alleviate NAFLD.

Lipid-modulate activity of Cichorium glandulosum Boiss. et Huet polysaccharide in nonalcoholic fatty liver disease larval zebrafish model.

Nonalcoholic fatty liver disease (NAFLD)1 is the most common chronic liver disease worldwide. Cichorium glandulosum Boiss. et Huet (CG) is a common traditional Uighur medicine, and it has been widely used as active therapy on various hepatic diseases. Recently, lipid-lowering effect has been revealed on CG. Polysaccharides are principal component of CG which could be the possible lipid-lowering compound in CG. In this study, extraction and purification of CG polysaccharides (CGP) was performed, and the lipid regulation effect of CGP was investigated on NAFLD zebrafish model. The results showed that CGP significantly decreased the levels of TC, TG, and decreased the mRNA expression of srebf-1, and fas, increased the expression of pparab. The findings suggest that the lipid-lowering effects of CGP mainly depend on facilitation of lipolysis (mainly beta-oxidation) or inhibition of lipogenesis. Furthermore, CGP could prevent and causes the regression of steatosis in NAFLD via its lipid metabolism regulation effect.

Protective effects of cichoric acid on H2O2-induced oxidative injury in hepatocytes and larval zebrafish models.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease with a broad spectrum of liver injury. Oxidant stress is believed to be the pathogenesis of NAFLD as the "second hit". Hydrogen peroxide is widely used as an oxidant reagent to induce the oxidant injury of cells and larval zebrafish. Recently, cichoric acid is being studied extensively for its obesity attenuating, hepatic steatosis reduction and anti-oxidant effects. In this study, to identify whether CRA could protect the H2O2 induced oxidant injury via anti-oxidant impact by using L02 and HepG2 hepatocytes as in vitro and larval zebrafish as in vivo injury models, and evaluated the protective and anti-oxidant effects of CRA by pretreated it on both in vitro and in vivo models. CRA was found to reduce the production of ROS and MDA, activate the anti-oxidant enzymes SOD and GSH-px, and pathways Keap1-Nrf2 and HO-1. These results demonstrated that CRA might protect the liver injury by its anti-oxidant effect, which could be a potential therapeutic agent of NAFLD.

Comprehensive Study of Multiple Stages Progressing to Nonalcoholic Steatohepatitis with Subsequent Fibrosis in SD Rats.

Because of the absence of the time course of histological nonalcoholic fatty hepatitis with subsequent fibrotic progression, the effective approaches available for controlling the onset and progression of non-alcoholic steatohepatitis (NASH) remain limited. Therefore, we detected the serum and liver tissue related lipid metabolism disorder, liver pathology and relative molecular makers alteration dynamically in a high fat-sucrose diet during different time points. High fat-sucrose diet significantly increased the serum lipid level on day 10. The excess lipid accumulation in liver was referred to as simple steatosis after the feeding of a high fat-sucrose diet for 20 days. The high fat-sucrose diet induced a hepatic inflammation response on day 30. Similarly, hepatic fibrosis was also initiated on day 30 and gradually formed from the 30th to the 50th day. Oxidative stress may be related with the process from NASH to liver fibrosis. Insulin resistance was involved in the progression from hepatic steatosis to NASH with hepatic fibrosis from the 20th to the 50th day. In conclusion, we established a high fat-sucrose diet induced nonalcoholic fatty hepatitis with liver fibrosis rat model, which presented the time course of histological nonalcoholic steatohepatitis and the initiation and progression change of characteristic molecular makers in the process from steatosis to hepatic fibrosis.