JiaGaSongTang improves chronic cholestasis via enhancing FXR-mediated bile acid metabolism.

BACKGROUND: Bile acid (BA) enterohepatic circulation disorders are a main feature of chronic cholestatic diseases. Promoting BA metabolism is thus a potential method of improving enterohepatic circulation disorders, and treat enterohepatic inflammation, oxidative stress and fibrosis due to cholestasis. PURPOSE: To investigate the effect of JiaGaSongTang (JGST) and its blood-absorbed ingredient 6-gingerol on α-naphthylisothiocyanate (ANIT)-induced chronic cholestasis, as well as elucidate the underlying regulatory mechanism. METHODS: Chronic cholestasis was induced in mice via subcutaneous injection of ANIT (50 mg/kg) every other day for 14 d. Treatment groups were administered JGST orally daily. Damage to the liver and intestine was observed using histopathological techniques. Biochemical techniques were employed to assess total BA (TBA) levels in the serum, liver, and ileum samples. Liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze fecal BA components. Bioinformatic methods were adopted to screen the core targets and pathways. The blood-absorbed ingredients of JGST were scrutinized via LC-MS/MS. The effects of the major JGST ingredients on farnesoid X receptor (FXR) transactivation were validated using dual luciferase reporter genes. Lastly, the effects of the FXR inhibitor, DY268, on JGST and 6-gingerol pharmacodynamics were observed at the cellular and animal levels. RESULTS: JGST ameliorated pathological impairments in the liver and intestine, diminishing TBA levels in the serum, liver and gut. Fecal BA profiling revealed that JGST enhanced the excretion of toxic BA constituents, including deoxycholic acid. Bioinformatic analyses indicated that JGST engaged in anti-inflammatory mechanisms, attenuating collagen accumulation, and orchestrating BA metabolism via interactions with FXR and other pertinent targets. LC-MS/MS analysis identified six ingredients absorbed to the bloodstream, including 6-gingerol. Surface plasmon resonance (SPR) and dual luciferase reporter gene assays confirmed the abilities of 6-gingerol to bind to FXR and activate its transactivation. Ultimately, in both cellular and animal models, the therapeutic efficacy of JGST and 6-gingerol in chronic cholestasis was attenuated in the presence of FXR inhibitors. CONCLUSION: The findings, for the first time, demonstrated that 6-gingerol, a blood-absorbed ingredient of JGST, can activate FXR to affect BA metabolism, and thereby attenuate ANIT-induced liver and intestinal injury in chronic cholestasis mice model via inhibition of inflammation, oxidative stress, and liver fibrosis, in part in a FXR-dependent mechanism.

Aqueous extract of Paeoniae Radix Rubra prevents deep vein thrombosis by ameliorating inflammation through inhibiting GSK3ß activity.

BACKGROUND: Deep vein thrombosis (DVT) is a kind of blood stasis syndrome. Paeoniae Radix Rubra (PRR) has long been widely used for eliminating blood stasis in China, but its effect on DVT has not yet been reported. PURPOSE: The present study aimed to assess the potential inhibitory effect of the aqueous extract of PRR (i.e.,PRR dispensing granule, PRRDG) on DVT and explore the underlying mechanism. STUDY DESIGN/METHODS: The chemical profile of PRRDG was analyzed by high-performance liquid chromatography. Sprague-Dawley rats were intragastrically treated with PRRDG (0.625, 1.25 and 1.875 g crude drug/kg/d) once daily for 7 consecutive days. On the sixth day, a model of inferior vena cava (IVC) stenosis-induced DVT was established. All rats were sacrificed on the seventh day. Serum was collected for enzyme-linked immunosorbent assay. Thrombus-containing IVC was weighed and further processed for histopathologic examination, immunohistochemical analysis and western blotting. LiCl and LY294002 were adopted to block and increase the activity of glycogen synthase kinase 3ß (GSK3ß), respectively. RESULTS: The chemical profile analysis showed that paeoniflorin, benzoylpaeoniflorin, albiflorin, gallic acid and catechin were the main constituents of PRRDG. LiCl decreased thrombus weight, reduced the number of inflammatory cells in thrombus and vein wall, down-regulated phosphorylated NF-κB p65 (p-p65) protein expression. Similarly, PRRDG decreased thrombus weight and tissue factor (TF) protein expression. PRRDG reduced the protein expression levels of P-selectin, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in venous endothelium, serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and the number of inflammatory cells in thrombus and vein wall. Moreover, PRRDG down-regulated p-p65 protein expression and up-regulated phosphorylated GSK3ß (p-GSK3ß) protein expression. LY294002 abrogated the inhibitory effects of PRRDG on thrombus weight, TF protein expression, TNF-α and IL-1ß serum levels, inflammatory cells influxes, and p-p65 protein expression. CONCLUSION: PRRDG prevents DVT by ameliorating inflammation through inhibiting GSK3ß activity.

A portable and quantitative detection of microRNA-21 based on cascade enzymatic reactions with dual signal outputs.

MicroRNAs (miRNAs) are physiological status-related molecules which can be used as biomarkers for diseases, such as cancers. The point-of-care testing (POCT) of miRNAs has great application potential in early diagnosis and process monitoring of diseases. In this paper, a fast and dual signal outputs detection for microRNA-21 (miRNA-21) was established by using both personal glucose meter (PGM) and fluorescence spectrometer. In such an assay protocol, a dual-functional hairpin structure was rationally designed to recognize miRNA-21 and serve as the carrier of the reporter adenosine monophosphate (AMP). The hairpin structure can be specifically degraded by exonuclease T (Exo T) after hybridization with the target miRNA-21, releasing a large amount of AMP as the reporter. Then a smart signal conversion machinery composed of four enzymes and the corresponding substrates was employed to produce dual output signals through enzymatic cascade reactions. The machinery includes two parts: an adenosine triphosphate (ATP) generation system and a glucose consumption/NADPH production system. The produced AMP in the former step triggers the production of ATP, and subsequently the consumption of glucose and the production of NADPH. The changes of both glucose and NADPH are proportional to the concentration of miRNA-21, and can be determined by PGM and fluorescence spectrometer, respectively. Besides, the build-in substrate-recycling mechanism achieves signal amplification of the cascade enzymatic reactions. Under the optimal experimental conditions, the PGM signal is linearly correlated with the concentration of miRNA-21 in the range from 5 to 150 nM, with the limit of detection (LOD) of 3.65 nM. The LOD of fluorescence detection mode is even lowered to 0.03 nM. The miRNA-21-spiked serum samples, as well as the actual serum samples from cancer patients, have been successfully detected by this detection strategy. Thus the established assay provides a POCT solution for cancer diagnosis and prognosis.

Aqueous extract of Whitmania pigra Whitman ameliorates ferric chloride-induced venous thrombosis in rats via antioxidation.

Whitmania pigra Whitman (W. pigra) has been widely employed in decoction for the treatment of blood stasis syndrome for many years in China. The aim of the present study was to explore the anti-venous thrombosis (VT) mechanism of the aqueous extract of W. pigra (AEW) in rats. Rats were orally administered with AEW. A inferior vena cava (IVC) thrombosis model was established. Thrombosed IVC was weighed and histopathological analyses were performed. Blood coagulation, blood fibrinolysis, blood cell count, blood viscosity and platelet activity were evaluated. Reactive oxygen species (ROS) accumulation was analyzed. Malondialdehyde (MDA) content in thrombosed IVC and antioxidants in serum were detected. Protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in thrombosed IVC was determined. AEW significantly reduced thrombus weight. It did not affect blood coagulation, blood fibrinolysis, blood cell count, platelet activity, or whole blood viscosity. However, AEW remarkably alleviated vascular injury, reduced ROS accumulation and MDA content, enhanced the total antioxidant capacity and the activities of superoxide dismutase, glutathione peroxidase and glutathione reductase. It increased the glutathione/oxidized glutathione ratio and the protein expression levels of Nrf2 and HO-1. In summary, W. pigra may prevent VT via Nrf2-mediated antioxidation.

Naringenin protects RPE cells from NaIO3-induced oxidative damage in vivo and in vitro through up-regulation of SIRT1.

BACKGROUND: Dry age-related macular degeneration (dAMD) leads to serious burden of visual impairment and there is no definitive treatment. Previous studies have showed that naringenin (NAR) significantly increased electroretinography (ERG) c-wave in sodium iodate (NaIO3)-treated rats and viability of NaIO3-treated ARPE-19 cells. But the underlying mechanism is still unknown. PURPOSE: We tested the hypothesis that anti-oxidation mediated by Sirtuin 1 (SIRT1) was important to the protective effect of NAR on dAMD. STUDY DESIGN/METHODS: NaIO3-induced mice retinopathy and ARPE-19 cells injury models were established. In vivo, the protective effect of NAR eye drops on retina was evaluated by flash ERG (FERG) recording and histopathological examination. In vitro, viability of ARPE-19 cells, and the levels of lactic dehydrogenase (LDH), reactive oxygen species (ROS) and carbonyl protein were detected. Protein expression of SIRT1 was analyzed by immunochemical staining, immunofluorescence and western blotting. RESULTS: NAR eye drops improved retinal function and morphology and normalized the protein expression of SIRT1 in mice exposed to NaIO3. NAR promoted the survival of ARPE-19 cells in a concentration-dependent manner. NAR up-regulated SIRT1 protein expression, and decreased levels of ROS and carbonyl protein. Moreover, EX527, a selective inhibitor of SIRT1, abolished the effects of NAR on the cell viability and ROS. In addition, SRT1720, a selective agonist of SIRT1, improved the viability of cells and suppressed the production of ROS. CONCLUSION: Our findings indicate that SIRT1-mediated anti-oxidation contributes to the protective effect of NAR eye drops on dAMD.

Spatholobi Caulis dispensing granule reduces deep vein thrombus burden through antiinflammation via SIRT1 and Nrf2.

BACKGROUND: Deep vein thrombosis (DVT) is a kind of blood stasis syndrome. Spatholobi Caulis (SC) has been widely used for the treatment of blood stasis syndrome in China, but the underlying mechanism remains poorly understood. PURPOSE: The aim of present study was to investigate the anti-DVT mechanism of Spatholobi Caulis dispensing granule (SCDG). STUDY DESIGN/METHODS: A rat model of inferior vena cava (IVC) stenosis-induced DVT and a cell model of oxygen-glucose deprivation (OGD) were performed. Rats were orally administered with SCDG solution once daily for seven consecutive days. IVC stenosis-induced DVT was operated on the sixth day. Thrombi were harvested and weighed on the seventh day. Pathological changes were observed by hematoxylin-eosin (HE) staining. Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß of serum were analyzed by enzyme-linked immunosorbent assay. C-reactive protein (CRP) was measured with turbidimetric immunoassay. Protein expressions in thrombosed IVCs and/or OGD-stimulated EA. hy926 cells were evaluated by western blot and/or immunofluorescence analyses. RESULTS: SCDG dramatically decreased thrombus weight. SCDG decreased tissue factor (TF) protein expression, inflammatory cells influxes in thrombosed vein wall and serum levels of inflammatory cytokines and CRP. Further, SCDG up-regulated Sirtuin 1 (SIRT1) protein expression and down-regulated acetylated-NF-κB p65 (Ace-p65) protein expression. Moreover, SCDG up-regulated nuclear factor-erythroid 2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expressions, and down-regulated phosphorylated-NF-κB p65 (p-p65) protein expression. In the OGD cell model, SCDG medicated serum decreased the protein expression of TF. SCDG medicated serum enhanced SIRT1 protein expression and reduced Ace-p65 nuclear protein expression. SCDG medicated serum promoted protein expressions of nuclear Nrf2 and total HO-1, and inhibited translocation of p65. Furthermore, inhibiting SIRT1 and Nrf2 reversed the protective effect of SCDG medicated serum on OGD-induced EA. hy926 cells. CONCLUSION: SCDG may prevent DVT through antiinflammation via SIRT1 and Nrf2.

Inferior vena cava stenosis-induced deep vein thrombosis is influenced by multiple factors in rats.

BACKGROUND: The pathogenesis of deep vein thrombosis (DVT) is incompletely understood, requiring reliable animal models. Inferior vena cava (IVC) stenosis model mimics human DVT. OBJECTIVE: To provide optimal conditions for establishing a rat model of IVC stenosis-induced DVT. METHODS: Effects of suture, and body weight, sex and side branches of rats on the IVC stenosis model were evaluated. 1 d after modeling, the weight and length of thrombosed IVCs and side branch distance were measured. Histopathological change and leukocytes influxes were observed by hematoxylin and eosin staining. Ly-6G-positive neutrophils were located by immunofluorescence. A multiple regression linear model was then built. RESULTS: IVCs stenosed with silk or monofilament sutures presented no difference in leukocyte influxes. Thrombus of 220-340 g rats was significantly heavier than that of 180-220 g rats. Although no statistic difference was found in thrombus weight between male and female rats weighing 180-260 g, males weighing 260-300 g formed larger thrombi than weight-matched females. Thrombus weight and length of rats except 180-220 g females was not impacted by side branch ligation and side branch distance. The regression model showed that sex and body weight were key factors affecting thrombus weight. CONCLUSIONS: Male and female rats weighing 220-260 g are more suitable for establishing a model of DVT induced by stenosing IVC with silk and without side branch ligation.

miR-130a alleviates neuronal apoptosis and changes in expression of Bcl-2/Bax and caspase-3 in cerebral infarction rats through PTEN/PI3K/Akt signaling pathway.

Effect of micro ribonucleic acid (miR)-130a on neuronal apoptosis in rats with cerebral infarction (CI) was studied to explore whether phosphatase and tensin homolog deleted on chromosome ten (PTEN)/phosphatidylinositol 3-hydroxy kinase (PI3K)/protein kinase B (Akt) is involved in the regulation of neuronal apoptosis. Thirty-six Sprague-Dawley (SD) rats were randomly divided into blank control group, model group and miR-130a low-expression group. miR-130a was determined by quantitative polymerase chain reaction (qPCR), the content of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10 was detected using the enzyme-linked immunosorbent assay (ELISA) kits, and the neuronal apoptosis level in each group was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The neurobehavioral score was significantly lower in model group than that in blank control group (P<0.01), while it was significantly higher in miR-130a low-expression group than that in model group (P<0.01). Compared with blank control group, the model group had obviously increased content of TNF-α and IL-6 (P<0.01), decreased content of IL-10 (P<0.01), more apoptotic neurons (P<0.01), higher expression of caspase-3 (P<0.01), and obviously lower Bcl-2/Bax (P<0.01). Moreover, expression of phosphorylated (p)-PTEN, PI3K and p-Akt in brain tissues was remarkably lower in the model group than those in the blank control group (P<0.01). The expression level of miR-130a in brain tissues of CI rats is significantly increased. miR-130a promotes the release of inflammatory factors and facilitates neuronal apoptosis through suppressing the PTEN/PI3K/Akt signaling pathway.

Downregulated miRNA-324-5p aggravates neuronal injury induced by oxygen-glucose deprivation via modulating RAN.

Differentially expressed miRNAs in the GEO profile of ischemic stroke were analyzed to clarify the specific role of microRNA-324-5p (miRNA-324-5p) in ischemic stroke and the potential mechanism. After screening out miRNA-324-5p, its level in peripheral blood of stroke patients and in vitro oxygen-glucose deprivation (OGD)-induced primary rat neurons was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Regulatory effects of miRNA-324-5p on viability, and apoptosis of OGD-induced neurons were evaluated by CCK-8 and Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, respectively. Glucose uptake and caspase-3 activity in OGD-induced neurons transfected with miRNA-324-5p mimics or inhibitor were also examined. The binding of miRNA-324-5p to its target gene RAN was analyzed by dual-luciferase reporter gene assay and western blot analysis. By analyzing the data of GSE46266 profile, miRNA-324-5p expression was shown markedly lower in MCAO rats relative to controls. Identically, we also observed the downregulated miRNA-324-5p in peripheral blood of stroke patients and in vitro OGD-induced primary neurons. Overexpression of miRNA-324-5p accelerated viability, induced apoptosis and strengthened glucose uptake ability of OGD-induced neurons. Knockdown of miRNA-324-5p, conversely, obtained the opposite results. Furthermore, we confirmed the binding of miRNA-324-5p to RAN, the target gene that was negatively regulated by miRNA-324-5p. Importantly, RAN overexpression partially reversed the regulatory effect of miRNA-324-5p on viability and glucose uptake of OGD-induced neurons. miRNA-324-5p is downregulated after ischemic stroke, which aggravates the disease condition by inhibiting neuronal proliferation and glucose uptake via upregulating RAN.

Protective Effects of Li-Fei-Xiao-Yan Prescription on Lipopolysaccharide-Induced Acute Lung Injury via Inhibition of Oxidative Stress and the TLR4/NF-κB Pathway.

Li-Fei-Xiao-Yan prescription (LFXY) has been clinically used in China to treat inflammatory and infectious diseases including inflammatory lung diseases. The present study was aimed at evaluating the potential therapeutic effects and potential mechanisms of LFXY in a murine model of lipopolysaccharide- (LPS-) induced acute lung injury (ALI). In this study, the mice were orally pretreated with LFXY or dexamethasone (positive drug) before the intratracheal instillation of LPS. Our data indicated that pretreatment with LFXY enhanced the survival rate of ALI mice, reversed pulmonary edema and permeability, improved LPS-induced lung histopathology impairment, suppressed the excessive inflammatory responses via decreasing the expression of proinflammatory cytokines (TNF-α, IL-1ß, and IL-6) and chemokine (MIP-2) and inhibiting inflammatory cells migration, and repressed oxidative stress through the inhibition of MPO and MDA contents and the upregulation of antioxidants (SOD and GSH) activities. Mechanistically, treatment with LFXY significantly prevented LPS-induced TLR4 expression and NF-κB (p65) phosphorylation. Overall, the present study suggests that LFXY protected mice from acute lung injury induced by LPS via inhibition of TLR4/NF-κB p65 activation and upregulation of antioxidative enzymes and it may be a potential preventive and therapeutic agent for ALI in the clinical setting.

Polydatin attenuates d-galactose-induced liver and brain damage through its anti-oxidative, anti-inflammatory and anti-apoptotic effects in mice.

Accumulating evidence has shown that chronic injection of d-galactose (d-gal) can mimic natural aging, with accompanying liver and brain injury. Oxidative stress and apoptosis play a vital role in the aging process. In this study, the antioxidant ability of polydatin (PD) was investigated using four established in vitro systems. An in vivo study was also conducted to investigate the possible protective effect of PD on d-gal-induced liver and brain damage. The results showed that PD had remarkable in vitro free radical scavenging activity on 2,2-diphenyl-1-picryl-hydrazyl (DPPH˙), 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS+˙) radical ions, and hydroxyl and superoxide anions. Results in vivo indicated that, in a group treated with d-gal plus PD, PD remarkably decreased the depression of body weight and organ indexes, reduced the levels of the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and alleviated alterations in liver and brain histopathology. PD also significantly decreased the level of MDA and elevated SOD, GSH-Px, CAT activity and T-AOC levels in the liver and brain. In addition, the levels of inflammatory mediators, such as TNF-α, IL-1ß and IL-6 in serum were markedly reduced after PD treatment. Western blotting results revealed that PD treatment noticeably attenuated the d-gal-induced elevation of Bcl-2/Bax ratio and caspase-3 protein expression in liver and brain. Overall, our findings indicate that PD treatment could effectively attenuate d-gal-induced liver and brain damage, and the mechanism might be associated with decreasing the oxidative stress, inflammation and apoptosis caused by d-gal. PD holds good potential for further development into a promising pharmaceutical candidate for the treatment of age-associated diseases.

[Purification and physi-chemical properties of polysaccharides SJZPS-Vb-1-2 from immunocompetence parts of sijunzi decoction].

OBJECTIVE: To supply basic research materials for clarifying the immunocompetence materials of Sijunzi Decoction. METHODS: Two polysaccharides(SJZPS-Vb-1-2) were purified with Sephadex DEAE A 25 and Sephadex G 200 from SJZPS-Vb part (with the highest immunocompetence). Their molecular weights were determined. The sugar compositions were analyzed with GC. The linkage positions of the component sugars were determined by methylation and GC/MS. RESULTS: SJZPS-Vb-1-2 was uonic-containing heteropolysaccharides with the component sugars of glucose, galactose and mannose in different molar ratios. The molecular weight of SJZPS-VB-1 was 38,300, SJZPS-VB-2, 26,000. The molar ratio of SJZPS-Vb-1 was as glu:gal:man 1:046:2.15 and SJZPS-Vb-2, glu:gal:man 1:1.41:4.18.

[Studies on purgative activity of rhubarb extracts by different technologies].

OBJECTIVE: To study on purgative activity of rhubarb extracts by solvent extraction, SFE-CO2 and SFE-CO2 & residue resin purification. METHODS: The effects of the extracts by the three technologies on creepage of mouse small intestine and rat large intestine were studied by injecting charcoal ink into the intestines. And the volume of the mouse small intestine was observed. The effects of the extracts were also studied on water absorption of mouse small intestine and large intestine by weighing the intestines. RESULTS: The purgative activity of the extracts by the three technologies was SFE-CO2 & residue resin purification > solvent extraction > SFE-CO2. CONCLUSION: Extracting different polar components separately might get a good result.