Combined Hydroxyethyl Starch Luteolin Nanocrystals for Effective Anti-Hyperuricemia Effect in Mice Model.

Introduction: Although flavonoid compounds exhibit various pharmacological activities, their clinical applications are restricted by low oral bioavailability owing to their poor solubility. Nanocrystals (NCs) represent an excellent strategy for enhancing the oral bioavailability of flavonoids. Hydroxyethyl starch (HES), a biomaterial compound used as a plasma expander, could be an ideal stabilizer material for preparing flavonoid NCs. Methods: HES was used to stabilize flavonoid nanocrystals (NCs), using luteolin (LUT) as a model drug. After full characterization, the freeze-drying and storage stability, solubility, intestinal absorption, pharmacokinetics, and in vivo anti-hyperuricemic effect of the optimized HES-stabilized LUT NCs (LUT-HES NCs) were investigated. Results: Uniformed LUT-HES NCs were prepared with mean particle size of 191.1±16.8 nm, zeta potential of about -23 mV, drug encapsulation efficiency of 98.52 ± 1.01%, and drug loading of 49.26 ± 0.50%. The freeze-dried LUT-HES NCs powder showed good re-dispersibility and storage stability for 9 months. Notably, compared with the coarse drug, LUT-HES NCs exhibited improved saturation solubility (7.49 times), increased drug dissolution rate, enhanced Caco-2 cellular uptake (2.78 times) and oral bioavailability (Fr=355.7%). Pharmacodynamic studies showed that LUT-HES NCs remarkably lowered serum uric acid levels by 69.93% and ameliorated renal damage in hyperuricemic mice. Conclusion: HES is a potential stabilizer for poorly soluble flavonoid NCs and provides a promising strategy for the clinical application of these compounds. LUT-HES NCs may be an alternative or complementary strategy for hyperuricemia treatment.

Sweet triterpenoid glycoside from Cyclocarya paliurus ameliorates obesity-induced insulin resistance through inhibiting the TLR4/NF-κB/NLRP3 inflammatory pathway.

Our prophase studies have manifested that the sweet triterpenoid glycoside from the leaves of Cyclocarya paliurus (CPST) effectively improved the disorders of glucolipid metabolism in vitro and in patients. The current purpose was to further detect its mechanisms involved. The results demonstrated that CPST could ameliorate high-fat diet (HFD)-induced insulin resistance (IR), which was linked to reducing HFD-induced mice's body weight, serum glucose (GLUO), triglyceride (TG), total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL-C), lowering the area under the oral glucose tolerance curve and insulin tolerance, elevating the percentage of brown adipose, high-density lipoprotein cholesterol (HDL-C), reducing fat droplets of adipocytes in interscapular brown adipose tissue (iBAT) and cross-sectional area of adipocytes. Further studies manifested that CPST obviously downregulated TLR4, MyD88, NLRP3, ASC, caspase-1, cleased-caspase-1, IL-18, IL-1ß, TXNIP, and GSDMD protein expressions and p-NF-кB/NF-кB ratio in iBAT. These aforementioned findings demonstrated that CPST ameliorated HFD induced IR by regulating TLR4/NF-κB/NLRP3 signaling pathway, which in turn enhancing insulin sensitivity and glucose metabolism.

Naringenin prevents non-alcoholic steatohepatitis by modulating the host metabolome and intestinal microbiome in MCD diet-fed mice.

Non-alcoholic steatohepatitis (NASH) is a severe inflammatory phase of the non-alcoholic fatty liver disease (NAFLD) spectrum and can progress to advanced stages of NAFLD if left untreated. This study uses multi-omics data to elucidate the underlying mechanism of naringenin's reported benefit in alleviating (NASH). Male mice were fed a NASH-inducing (methionine-choline-deficient) MCD diet with or without naringenin supplementation for 6 weeks. Naringenin prevented NASH-induced histopathological liver damage and reversed the abnormal levels of hepatic triglyceride (TG)/total cholesterol (TC), serum TG/TC, serum alanine aminotransferase/aspartate transaminase, and hepatic malondialdehyde and glutathione. Importantly, naringenin intervention significantly modulated the relative abundance of gut microbiota and the host metabolomic profile. We detected more than 700 metabolites in the serum and found that the gut genus levels of Anaeroplasma and the [Eubacterium] nodatum group were closely associated with xanthine, 2-picoline, and securinine, respectively. Tuzzerella alterations showed the highest number of associations with host endogenous metabolites such as FAHFA (8:0/10:0), FFA (20:2), carnitine C8:1, tridecanedioic acid, securinine, acetylvaline, DL-O-tyrosine, and Phe-Asn. This study indicates that the interplay between host serum metabolites and gut microbiota may contribute to the therapeutic effect of naringenin against NASH.

Ergothioneine suppresses hepatic stellate cell activation via promoting Foxa3-dependent potentiation of the Hint1/Smad7 cascade and improves CCl4-induced liver fibrosis in mice.

Ergothioneine (EGT) is a bioactive compound derived from certain edible mushrooms. The activation of hepatic stellate cells (HSCs) is critically involved in the etiology of liver fibrosis (LF). Here, we report that in LX-2 HSCs, EGT upregulates the expression of Hint1 and Smad7 and suppresses their activation provoked by TGFß1. The EGT-triggered inhibition of HSC activation is abolished by knocking down the expression of Hint1. Overexpression of Hint1 increases Smad7 and represses TGFß1-provoked activation of LX-2 HSCs. In silico predictions unveiled that in the promoter region of the human Hint1 gene, there are two conserved cis-acting elements that have the potential to interact with the transcription factor Foxa3 termed hFBS1 and hFBS2, respectively. The knockdown of Foxa3 obviously declined Hint1 expression at both mRNA and protein levels. Transfection of Foxa3 or EGT treatment increased the activity of the luciferase reporter driven by the Hint1 promoter in an hFBS2-dependent manner. The knockdown of Foxa3 eliminated EGT-mediated upregulation of Hint1 promoter activity. Additionally, EGT triggered the nuclear translocation of Foxa3 without obviously affecting its expression level. Molecular docking analysis showed that EGT has the potential to directly interact with the Foxa3 protein. Moreover, Foxa3 played a critical role in EGT-mediated hepatoprotection. EGT modulated the Foxa3/Hint1/Smad7 signaling in mouse primary HSCs and inhibited their activation. The gavage of EGT considerably relieved CCl4-induced LF in mice. Our data provide new insights into the anti-LF activity of EGT. Mechanistically, EGT triggers the nuclear translocation of Foxa3 in HSCs, which promotes Hint1 transcription and subsequently elevates Smad7.

Biomarkers for Diagnosing and Treating Fetal Growth Restriction.

Fetal growth restriction (FGR), a common obstetric complication, significantly increases the risks of fetal intrauterine death and neonatal death, and fetuses with growth restriction are prone to cognitive retardation and various diseases in adulthood. The early determination of FGR risk is contentious in clinical research, and few indicators are available for the early prediction and diagnosis of FGR. This review focuses on the prediction and diagnosis of FGR, as well as the significance of biomarkers for FGR, such as those related to gene regulation, apoptosis, mitochondrial function, and inflammation. Although many of these biomarkers are still in the early stages of research, they are good predictors of the threats to fetal health and safety, and they provide new insights for the treatment of FGR.

Tormentic acid, a triterpenoid isolated from the fruits of Chaenomeles speciose, protected indomethacin-induced gastric mucosal lesion via modulating miR-139 and the CXCR4/CXCL12/PLC/PKC/Rho a/MLC pathway.

CONTEXT: Tormentic acid (TA), an effective triterpenoid isolated from Chaenomeles speciosa (Sweet) Nakai (Rosaceae) fruits, exerts an effective treatment for gastric damage. OBJECTIVE: To investigate the gastroprotective effect of TA on indomethacin (IND) damaged GES-1 cells and rats, and explore potential mechanisms. MATERIALS AND METHODS: TA concentrations of 1.563-25 µM were used. Cell proliferation, apoptosis and migration were performed using MTT, colony formation, wound healing, migration, Hoechst staining assays. SD rats were divided into control, IND, TA (1, 2 and 4 mg/kg) + IND groups, once a day for 21 continuous days. Twenty-four hours after the last administration, all groups except the control group were given IND (100 mg/kg) by gavage. Gastric juice parameters, gastric ulcer, gastric blood flow (GBF), blood biochemical parameters and cytokine analysis and gastric mucosal histopathology were detected for 2 h and 6 h after IND oral administration. The mRNA and protein expression of miR-139 and the CXCR4/CXCL12/PLC/PKC/Rho A/MLC pathway were analyzed in the IND-damaged GES-1 cells and gastric tissue of rats. RESULTS: TA might ameliorate the gastric mucosal injury by accelerating the IND-damaged GES-1 cell proliferation and migration, ameliorating GBF, ulcer area and pathologic changes, the redox system and cytokine levels, the gastric juice parameters, elevating the gastric pH in IND damaged rats; suppressed miR-139 mRNA expression, elevated CXCR4 and CXCL12 mRNA and protein expression, p-PLC, p-PKC, Rho A, MLCK and p-MLC protein expression. DISCUSSION AND CONCLUSIONS: TA may have potential use as a clinical drug candidate for gastric mucosal lesion treatment.

Quercetin ameliorates nonalcoholic fatty liver disease (NAFLD) via the promotion of AMPK-mediated hepatic mitophagy.

The global incidence of nonalcoholic fatty liver disease (NAFLD) has been surging in recent years, however, no drug is currently approved to treat this disease. Quercetin, a natural flavonoid abundant in plants and fruits, has been reported to alleviate NAFLD, however, the exact molecular mechanism remains unclear. This study aims to further elucidate its potential mechanism of action. The beneficial effects and the underlying mechanism of quercetin in alleviating NAFLD were explored both in vitro and in vivo, by employing chemical inhibitors of autophagosomes (3-methyladenine, 3-MA), autolysosomes (chloroquine, CQ), AMPK (Compound C, CC) and SIRT1 (selisistat, EX-527). The levels of intracellular lipids, reactive oxygen species, mitochondria function, autophagy, and mitophagy were assessed by fluorescent labeling and examined using flow cytometry or confocal microscopy. Key protein expressions of autophagy, mitophagy, and inflammation were also determined. In vivo, quercetin was shown to dose-dependently effectively alleviate NAFLD, but intraperitoneal injection of 3-MA could block the beneficial effects of quercetin on body weight, liver weight, serum ALT/AST, hepatic ROS and inflammation. In vitro, quercetin could reduce intracellular lipids (Nile Red staining) and ROS/DHE accumulation, which could be also blocked by 3-MA or CQ. Furthermore, we found that CC could abrogate the protective effects of quercetin on lipid and ROS accumulation in vitro. Also, CC abolished the proautophagic and anti-inflammatory effects of quercetin, as shown by western blot determination and Lyso-Tracker labeling. Importantly, mitophagy, a specific form of mitochondria-targeted autophagy, was enhanced by quercetin, as demonstrated by PINK1/Parkin protein variation and immunofluorescence colocalization of autophagosomes and mitochondria, which could also be blocked by the intervention of CC. This study demonstrates that quercetin prevents NAFLD through AMPK-mediated mitophagy and suggests that promoting mitophagy via an upregulation of AMPK may be a promising therapeutic strategy against NAFLD.

Molecular mechanisms of hepatoprotective effect of tectorigenin against ANIT-induced cholestatic liver injury: Role of FXR and Nrf2 pathways.

Cholestatic liver injury is caused by toxic action or allergic reaction, resulting in abnormality of bile formation and excretion. Few effective therapies have become available for the treatment of cholestasis. Herein, we found that tectorigenin (TG), a natural isoflavone, showed definite protective effects on alpha-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury, significantly reversing the abnormality of plasma alanine/aspartate aminotransferase, total/direct bilirubin and alkaline phosphatase, as well as hepatic reactive oxygen species, catalase and superoxide dismutase. Importantly, the targeted metabolomic determination found that BA homeostasis could be well maintained in TG-treated cholestatic mice, especially the levels of glycocholic acid, tauromuricholic acid, taurocholic acid, taurolithocholic acid, tauroursodeoxycholic acid and taurodeoxycholic acid. Overall, primary/secondary and amidated/unamidated bile acid (BA) levels were significantly altered upon ANIT stimulation but could be restored by TG intervention to certain extents. In addition, TG boosted the expression of farnesoid x receptor (FXR), which in turn upregulated multidrug resistance protein 2 (MRP2) and bile salt export pump (BSEP) to accelerate the excretion of BA. Meanwhile, TG enhanced the expression of Nrf2 and its upstream genes PI3K/Akt and downstream target genes HO-1, NQO1, GCLC and GCLM to strengthen the antioxidant capacity. Taken together, TG plays a vital role in maintaining BA homeostasis and ameliorating cholestatic liver injury through regulating FXR-mediated BA efflux and Nrf2-mediated antioxidative pathways.

Ganoderma lucidum polysaccharide peptide alleviates hyperuricemia by regulating adenosine deaminase and urate transporters.

Hyperuricemia (HUA) affects human health and is involved in the pathogenesis of common chronic diseases. Previous studies showed that Ganoderma lucidum extract lowered HUA in animals. However, the active ingredient and pharmacological mechanism of Ganoderma lucidum extract in the improvement of HUA are unknown. The purpose of this study was to determine the anti-HUA efficacy and related mechanism of Ganoderma lucidum polysaccharide peptide (GLPP) using a potassium oxonate (PO)-induced mouse model and an adenosine-induced cell model. The experimental results showed that blood uric acid (UA) was decreased up to 40.6% by GLPP in HUA mice in a dose-dependent manner. Additionally, GLPP significantly reduced UA production by inhibiting the hepatic and blood adenosine deaminase (ADA) activity and increased UA excretion by decreasing the expression of glucose transporter 9 (GLUT9) and increasing the expression of organic anion transporter 1 (OAT1) in kidney. The adenosine-induced cell model showed that the inhibitory effect of GLPP on ADA activity may be the main reason for the alleviation of HUA by GLPP. Furthermore, PO-induced renal histopathological damage was also alleviated by GLPP in a dose-dependent manner. The experimental results in this study indicated that GLPP exerted anti-HUA effects via regulating the UA production and excretion, suggesting that GLPP could be developed into a therapeutic agent for HUA.

Xanthomicrol suppresses human hepatocellular carcinoma cells migration and invasion ability via Μu-opioid receptor.

BACKGROUND: Xanthomicrol is one of the methoxylated flavones and a promising cancer chemopreventive agent, but its anti-migration and anti-invasion ability on human hepatocellular carcinoma (HCC) remains unknown. OBJECTIVES: This study aims to explore Xanthomicrol's effects on migration and invasion ability of the human HCC Huh7 cell line. METHODS: Viability of Huh7 cells was measured by cell counting kit-8 (CCK8) assay. Cell apoptosis was assayed with flow cytometry analysis. The ability of migration and invasion of Huh7 cells was then detected through Transwell assays. Epithelial-mesenchymal transition (EMT)-related proteins were also detected through Western blot. KEY FINDINGS: Xanthomicrol inhibits the migration and invasion of Huh7 cells. The overexpression of Μu-opioid receptor (MOR) increases Huh7 cells' proliferation and enhances migration and invasion ability, while xanthomicrol treatment decreases the expression of MOR. Moreover, xanthomicrol can reverse migration, invasion and EMT-related protein expression by overexpressed MOR. CONCLUSIONS: These results suggest that xanthomicrol is a potential MOR antagonist, and it possesses potent anti-migration and anti-invasion ability on Huh7 cells.

Preparation, Characterization, and In Vitro Pharmacodynamics and Pharmacokinetics Evaluation of PEGylated Urolithin A Liposomes.

Urolithin A (Uro-A), a metabolite of ellagitannins in mammals' intestinal tract, displays broad biological properties in preclinical models, including anti-oxidant, anti-inflammatory, and anti-tumor effects. However, the clinical application of Uro-A is restricted because of its low aqueous solubility and short elimination half-life. Our purpose was to develop a delivery system to improve the bioavailability and anti-tumor efficacy of Uro-A. To achieve this goal, urolithin A-loaded PEGylated liposomes (Uro-A-PEG-LPs) were prepared for the first time and its physicochemical properties and anti-tumor efficacy in vitro were evaluated. The morphology of Uro-A-PEG-LPs displayed a uniform sphere under transmission electron microscope. The particle size, polydispersity index, zeta potential, and encapsulation efficiency of Uro-A-PEG-LPs were 122.8 ± 7.4 nm, 0.25 ± 0.16, - 25.5 ± 2.3 mV, and 94.6 ± 1.6%, respectively. Moreover, Uro-A-PEG-LPs possessed higher stability and could be stably stored at 4°C for a long time. In vitro release characteristics indicated that Uro-A-PEG-LPs possessed superior sustained release properties. The results of confocal laser scanning microscopy experiment showed that the coumarin 6-loaded PEGylated liposomes (C6-PEG-LPs) have superior cellular uptake than that of conventional liposomes. In addition, in vitro tests demonstrated that Uro-A-PEG-LPs elevated cytotoxicity and pro-apoptotic effect in human hepatoma cells comparing with free Uro-A. Furthermore, the results of pharmacokinetic experiments showed that the t1/2, AUC0-t, and MRT0-t of Uro-A-PEG-LPs increased to 4.58-fold, 2.33-fold, and 2.43-fold than those of free Uro-A solution, respectively. Collectively, these manifested that PEGylated liposomes might be a potential delivery system for Uro-A to prolonging in vivo circulation time, promoting cellular uptake, and enhancing its anti-tumor efficacy.

Orlistat delays hepatocarcinogenesis in mice with hepatic co-activation of AKT and c-Met.

Orlistat (Xenical™), a US Food and Drug Administration (FDA)-approved anti-obesity drug, shows efficacy against multiple tumor types, including hepatocellular carcinoma (HCC), due to its ability to inhibit fatty acid synthase (FASN) activity. However, whether orlistat affects hepatocellular malignant transformation during hepatocarcinogenesis in vivo is unknown. This study assessed the antisteatotic and antitumorigenic efficacy of orlistat in a rapid HCC FVB/N mouse model established via hydrodynamic transfection of activated forms of AKT and c-Met proto-oncogenes. Human hepatoma cell lines were used for mechanical validation in vitro. Hematoxylin and eosin staining, immunohistochemistry, and immunoblotting were applied for the mechanistic investigation. The results revealed that when orlistat was administered in the early stage of AKT/c-Met-triggered hepatocarcinogenesis, it resulted in the elimination of hepatic tumor burden. Mechanistically, orlistat efficiently elevated PTEN expression and suppressed AKT/SREBP1/FASN signaling both in vivo and in vitro, impairing AKT/c-Met-driven de novo lipogenesis and aberrant proliferation. Altogether, this study demonstrates the antilipogenic and antiproliferative efficacy of orlistat in hepatocarcinogenesis, suggesting that orlistat may be beneficial for the treatment of HCC, especially in NAFLD-related HCCs featuring activated AKT/mTOR cascade and increased lipogenesis in livers.

CircRNA_000864 Upregulates B-cell Translocation Gene 2 Expression and Represses Migration and Invasion in Pancreatic Cancer Cells by Binding to miR-361-3p.

BACKGROUND: Pancreatic cancer is a fatal disease with a very poor prognosis due to its characteristic insidious symptoms, early metastasis, and chemoresistance. Circular RNAs (circRNAs) are involved in the development of pancreatic cancer. AIM: Hence, the aim of this study is to elucidate the mechanism of circRNA_000864 in regulating BTG2 expression in pancreatic cancer by binding to miR-361-3p. METHODS: CircRNA_000864, miR-361-3p, and BTG2 expression patterns in the pancreatic cancer tissues were detected by RT-qPCR. Correlation among circRNA_000864, miR-361-3p, and BTG2 was evaluated by RNA-pull down assay, RNA Immunoprecipitation assay, and dual-luciferase reporter gene assay. After ectopic expression and depletion experiments, 5-ethynyl-2'-deoxyuridine assay, Transwell assay, and flow cytometry were employed to assess the cell proliferation, migration and invasion, cell cycle, and apoptosis. Xenotransplantation of nude mice was conducted to detect the effects of circRNA_000864, miR-361-3p, and BTG2 on tumor growth. RESULTS: CircRNA_000864 and BTG2 were poorly expressed, and miR-361-3p was highly expressed in the pancreatic cancer tissues. CircRNA_000864 bound to miR-361-3p could target BTG2. Cell proliferation, migration, and invasion were inhibited, and the cell cycle arrest and apoptosis were stimulated after overexpression of circRNA_000864 or BTG2 or downregulation of miR-361-3p. Overexpression of circRNA_000864 or downregulation of miR-361-3p also decreased the tumor growth in vivo. CONCLUSIONS: Conjointly, our findings elicited that the overexpression of circRNA_000864 could promote BTG2 expression to inhibit pancreatic cancer development by binding to miR-361-3p, which represents an appealing therapeutic target for the treatment of pancreatic cancer.

Antitumor immunostimulatory activity of polysaccharides from Panax japonicus C. A. Mey: Roles of their effects on CD4+ T cells and tumor associated macrophages.

In this study, chemical properties of polysaccharides from rhizomes of Panax japonicus C. A. Mey (PSPJ) were investigated and the antitumor immunostimulatory activity of PSPJ was assessed in mice bearing H22 hepatoma cells. Chemical properties of PSPJ were determined by GC, FT-IR, 1H NMR and 13C NMR analysis. Furthermore, we showed that PSPJ repressed H22 tumor growth in vivo with undetectable toxic effects on tumor-bearing mice. PSPJ upregulated host thymus/spleen indexes and ConA/LPS-induced splenocyte proliferation. Cytotoxic activities of natural killer and CD8+ T cells against H22 hepatoma cells were also elevated. Tumor transplantation led to substantial apoptosis of CD4+ T cells and dysregulation of the cytokine profile secreted by CD4+ T cells. These abnormalities were alleviated by PSPJ in a dose-dependent manner. In tumor-associated macrophages (TAMs), PSPJ reduced the production of immunosuppressive factors such as TGF-ß, IL-10 and PEG2. In addition, M2-like polarization of TAMs was also considerably declined in response to PSPJ. Our findings clearly demonstrated the antitumor immunostimulatory activity of PSPJ and supported considering PSPJ as an adjuvant reagent in clinical treatment of malignant diseases.