Zhi-Liang Sun

Berberine inhibits dyslipidemia in C57BL/6 mice with lipopolysaccharide induced inflammation

BACKGROUND Inhibiting the action of proprotein convertase subtilisin/kexin type 9 (PCSK9) on the low-density lipoprotein receptor (LDLR) has emerged as a novel therapeutic target for hypercholesterolemia. Here we investigated the effect of berberine, natural plant extracts, on PCSK9-LDLR pathway in C57BL/6 mice with lipopolysaccharide (LPS) induced inflammation. METHODS Forty female mice were divided into four groups (n =10): control, LPS (5 mg/kg), LPS + berberine 10 (5 mg/kg LPS plus 10 mg/kg berberine), and LPS + berberine 30 (5 mg/kg LPS plus 30 mg/kg berberine). Changes in the levels of blood lipids [total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C)]; pro-inflammatory cytokines [interferon-γ (IFNγ), tumor necrosis factor α (TNFα), and interleukin-1α (IL-1α)], 8-isoprostane, hepatic expressions of PCSK9 and LDLR were determined. RESULTS Berberine pretreatment reduced the expression of hepatic PCSK9, decreased the plasma TC, TG, LDL-C, IFNγ, TNFα, IL-1α, and 8-isoprostane concentrations; increased HDL-C level and LDLR expression in mice. CONCLUSION The present results suggest that berberine inhibits dyslipidemia in C57BL/6 mice with LPS induced inflammation through regulating PCSK9-LDLR pathway.

Protective effects of kaempferol against endothelial damage by an improvement in nitric oxide production and a decrease in asymmetric dimethylarginine level

Previous investigations have shown that asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) synthases (NOS) and that ADMA is a risk factor for endothelial dysfunction. The objective of this study was to investigate the protective effect of kaempferol, a naturally occurring flavonoid antioxidant agent, against endothelial damage and the mechanisms involved. The experiments were performed in aorta and plasma from C57BL/6J control and apolipoprotein E-deficient (ApoE(-/-)) mice treated or not with kaempferol (50 or 100mg/kg, intragastrically) for 4 weeks, and in human umbilical vein endothelial cells (HUVECs) pretreated or not with kaempferol (1, 3 or 10 microM) for 1h and exposed to lysophosphatidylcholine (LPC) (10 microg/mL) for 24h. Kaempferol treatment improved endothelium-dependent vasorelaxation, increased the maximal relaxation value, and decreased the half-maximum effective concentration concomitantly with an increase in nitric oxide plasma concentration, a decrease in ADMA and malondialdehyde (MDA) plasma concentrations, and increase in the expression of aortic endothelial NOS (eNOS) as well as dimethylarginine dimethylaminohydrolase II (DDAH II) in ApoE(-/-) mice. In addition, LPC caused a reduction in NO production, an increase in ADMA concentration concomitantly with a decreased expression of eNOS and DDAH II in HUVECs, and the effect of LPC was abolished by kaempferol. Treatment with kaempferol also significantly decreased reactive oxygen species production in mice aorta and in HUVECs. The present results suggest that the protective effect of kaempferol against endothelial damage may be associated with an improvement in NO production and a decrease in ADMA level.

Kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of apolipoprotein E deficient mice

Recent studies show that osteopontin (OPN) and its receptor cluster of differentiation 44 (CD44) are two pro-inflammatory cytokines contributing to the development of atherosclerosis. The objective of this study was to explore the inhibitory effect of kaempferol, a naturally occurring flavonoid compound, on atherogenesis and the mechanisms involved. The experiments were performed in aorta and plasma from C57BL/6J control and apolipoprotein E-deficient (ApoE(-/-)) mice treated or not with kaempferol (50 or 100mg/kg, intragastrically) for 4 weeks. Kaempferol treatment decreased atherosclerotic lesion area, improved endothelium-dependent vasorelaxation, and increased the maximal relaxation value concomitantly with decrease in the half-maximum effective concentration, plasma OPN level, aortic OPN expression, and aortic CD44 expression in ApoE(-/-) mice. In addition, treatment with kaempferol also significantly decreased reactive oxygen species production in mice aorta. The present results suggest that kaempferol regulates OPN-CD44 pathway to inhibit the atherogenesis of ApoE(-/-) mice.

Identification of sanguinarine metabolites in pig liver preparations by accurate mass measurements using electrospray ionization hybrid ion trap/time-of-flight mass spectrometry

RATIONALE Sanguinarine (SA) is currently used in veterinary medicine for animal husbandry as a natural component of feed additive Sangrovit. To date, SA metabolism in food-producing animals has not yet been reported. Therefore, the purpose of the present study was to investigate the metabolism of SA in pig liver microsomes and cytosol. METHODS The SA incubations mixtures of microsomes and cytosol were processed by trichloroacetic acid (TCA) and acetonitrile. Then, the samples were analyzed using a sensitive and reliable method based on liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC-IT/TOFMS). The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and product ions generated from precursor ions with those of the parent drug. RESULTS Seven metabolites were identified in pig liver preparations. Dihydrosanguinarine (DHSA, m/z 334) was the main metabolite formed in liver microsomes and the only one in cytosol. One oxidative metabolite and two O-demethylenated metabolites of SA (m/z 320) were found in the TCA-treated microsomal samples. However, SA pseudobase and two additional O-demethylenated metabolites of DHSA (m/z 322) were found only in the acetonitrile-treated microsomal samples. CONCLUSIONS It was demonstrated that different metabolites of SA were identified depending on the acidic or neural extraction conditions. A metabolic pathway of SA in pig was tentatively proposed based on these characterized metabolites and early reports.

Reductive metabolism of the sanguinarine iminium bond by rat liver preparations

BACKGROUND Sanguinarine (SA) is a quaternary benzo[c]phenanthridine alkaloid that is mainly present in the Papaveraceae family. SA has been extensively studied because of its antimicrobial, anti-inflammatory, antitumor, antihypertensive, antiproliferative and antiplatelet activities. Metabolic studies demonstrated that SA bioavailability is apparently low, and the main pathway of SA metabolism is iminium bond reduction resulting in dihydrosanguinarine (DHSA) formation. Nevertheless, the metabolic enzymes involved in SA reduction are still not known in detail. Thus, the aim of this study was to investigate the rat liver microsomes and cytosol-induced SA iminium bond reduction, and to examine the effects of cytosol reductase inhibitors on the reductive activity. METHODS DHSA formation was quantified by HPLC. The possible enzymes responsible for DHSA formation were examined using selective individual metabolic enzyme inhibitors. RESULTS When SA was incubated with liver microsomes and cytosol in the absence of NAD(P)H, DHSA, the iminium bond reductive metabolite was formed. The reductase activity of the liver microsomes and cytosol was also enhanced significantly in the presence of NADH. The amount of DHSA formed in the liver cytosol was 4.6-fold higher than in the liver microsomes in the presence of NADH. The reductase activity in the liver cytosol was inhibited by the addition of flavin mononucleotide and/or riboflavin. Inhibition studies indicated that menadione, dicoumarol, quercetin and 7-hydroxycoumarin inhibited rat liver cytosol-mediated DHSA formation in the absence of NADH. However, only menadione and quercetin inhibited rat liver cytosol-mediated DHSA formation in the presence of NADH. CONCLUSIONS These results suggest that the SA iminium bond reduction proceeds via two routes in the liver cytosol. One route is direct non-enzymatic reduction by NAD(P)H, and the other is enzymatic reduction by possible carbonyl and/or quinone reductases in the liver cytosol.

Identification of allocryptopine and protopine metabolites in rat liver S9 by high‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

RATIONALE Allocryptopine (AL) and protopine (PR) have been extensively studied because of their anti-parasitic, anti-arrhythmic, anti-thrombotic, anti-inflammatory and anti-bacterial activity. However, limited information on the pharmacokinetics and metabolism of AL and PR has been reported. Therefore, the purpose of the present study was to investigate the in vitro metabolism of AL and PR in rat liver S9 using a rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC/QqTOFMS) method. METHODS The incubation mixture was processed with 15% trichloroacetic acid (TCA). Multiple scans of AL and PR metabolites and accurate mass measurements were automatically performed simultaneously through data-dependent acquisition in only a 30-min analysis. The structural elucidations of these metabolites were performed by comparing their changes in accurate molecular masses and product ions with those of the precursor ion or metabolite. RESULTS Eight and five metabolites of AL and PR were identified in rat liver S9, respectively. Among these metabolites, seven and two metabolites of AL and PR were identified in the first time, respectively. The demethylenation of the 2,3-methylenedioxy, the demethylation of the 9,10-vicinal methoxyl group and the 2,3-methylenedioxy group were the main metabolic pathways of AL and PR in liver S9, respectively. In addition, the cleavage of the methylenedioxy group of the drugs and subsequent methylation or O-demethylation were also the common metabolic pathways of drugs in liver S9. In addition, the hydroxylation reaction was also the metabolic pathway of AL. CONCLUSIONS This was the first investigation of in vitro metabolism of AL and PR in rat liver S9. The detailed structural elucidations of AL and PR metabolites were performed using a rapid and accurate HPLC/QqTOFMS method. The metabolic pathways of AL and PR in rat were tentatively proposed based on these characterized metabolites and early reports. Copyright

Effect of Danofloxacin on Reactive Oxygen Species Production, Lipid Peroxidation and Antioxidant Enzyme Activities in Kidney Tubular Epithelial Cell Line, LLC-PK1

The purpose of this study was to investigate the possibility that oxidative stress was involved in danofloxacin‐induced toxicity in renal tubular cells epithelial cell line (LLC‐PK1). Confluent LLC‐PK1 cells were incubated with various concentrations of danofloxacin. The extent of oxidative damage was assessed by measuring the reactive oxygen species (ROS) level, lipid peroxidation, cell apoptosis and antioxidative enzyme activities. Danofloxacin induced a concentration‐dependent increase in the ROS production, not even cytotoxic conditions. Similarly, danofloxacin caused an about 4 times increase in the level of thiobarbituric acid reactive substances at the concentration of 400 μM for 24 hr, but it did not induce cytotoxicity and apoptosis. Antioxidant enzymes activities, such as superoxide dismutase (SOD) and catalase (CAT), were increased after treatment with 100, 200 and 400 μM of danofloxacin for 24 hr. The activity of glutathione peroxidase (GPX) was significantly decreased in a concentration‐dependent manner. In addition, ROS production, lipid peroxidation and GPX decline were inhibited by additional glutathione and N‐acetyl cysteine. These data suggested that danofloxacin could not induce oxidative stress in LLC‐PK1 cells at the concentration (≤400 μM) for 24 hr. The increase levels of ROS and lipid peroxidation could be partly abated by the increase activities of SOD and CAT.

Angiopoietin-like protein 2 may mediate the inflammation in murine mastitis through the activation of interleukin-6 and tumour necrosis factor-α.

Mastitis is the inflammation of the mammary gland. Recent research has shown that Angiopoietin-like protein 2 (ANGPTL2) is a key inflammatory mediator. In the present study, we tested whether there is a correlation between increased ANGPTL2 expression and inflammation in response to Staphylococcus aureus in murine mastitis and the mechanisms involved. Thirty mice were divided into two groups: blank control group, challenged group. The entire infused mammary glands were removed to observe the changes of histopathology, myeloperoxidase (MPO) activity, production of tumour necrosis factor-α (TNF-α) and interleukin (IL)-6, and genes expression of ANGPTL2, TNF-α and IL-6. In challenged group, the structure of mammary glands was damaged and the large areas of cell fragments were observed. The MPO activity, IL-6 and TNF-α concentrations, ANGPTL2, IL-6, and TNF-α mRNA levels were significantly elevated in challenged group compared with blank control group. The present findings indicate ANGPTL2 may mediate the inflammation in murine mastitis through the activation of IL-6 and TNF-α.

NQO1 involves in the imine bond reduction of sanguinarine and recombinant adeno-associated virus mediated NQO1 overexpression decreases sanguinarine-induced cytotoxicity in rat BRL cells

UNLABELLED Although sanguinarine (SANG) can be transformed to dihydrosanguinarine (DHSA) in human and animals, the enzyme involved in the imine bond reduction of SANG is still unknown. In this study, we found that rat NAD(P)H quinone oxidoreductase 1 expressed by prokaryotic system can transform SANG to DHSA in an NADPH dependent manner. We also found out that there was more DHSA in rAAV-NQO1 infected than rAAV-CYP1A1 and rAAV-control infected BRL cells. SANG decreased rat BRL cell proliferation and augmented cell apoptosis in a time and dose dependent manner. However, the influence of DHSA to BRL cells is not significant difference than SANG. SANG-induced apoptosis was correlated with the up-regulation of Bax/Bcl2 ratio and the down-regulation of Bcl2. SANG can also dose dependently down regulate NQO1 expression, but CYP1A1 expression was a little up regulated. Since CYP1A1 involving in SANG oxidative reactions and NQO1 involving in the transform of SANG to DHSA, we hypothesized that up regulation of NQO1 could reduce SANG cytotoxicity and up regulation of CYP1A1 could increase SANG cytotoxitity. Our further study showed that recombinant adeno-associated virus (rAAV) mediated overexpression of NQO1 significantly increased cell proliferation and decreased Bax/Bcl2 ratio, apoptosis, and cytotoxicity, whereas rAAV mediated CYP1A1 overexpression had opposite effects. These data illustrated that NQO1 involved in the imine bond reduction of sanguinarine and this was a less toxic metabolizing pathway than CYP1A1-metabolizing pathway.

Protective effect of soluble fiber from Undaria pinnatifida on vascular endothelium in hypercholesterolemic rabbits.

Dietary fiber could improve endothelial function and abnormal production of nitric oxide (NO) and elevated endothelin-1 (ET-1) concentration induce endothelial dysfunction. In the present study, we tested the effect of soluble fiber extracted from Undaria pinnatifida (UP) on endothelial function and NO and ET-1 production in hypercholesterolemic rabbits. After treatment with UP soluble fiber (5 or 10%) for 8 wk, endothelium-dependent vasorelaxation in isolated aortic rings, the concentrations of NO and ET-1 and malondialdehyde (MDA), and the expression of endothelial NO synthase (eNOS) gene were measured. The UP soluble fiber (5 or 10%) treatment significantly attenuated the impairment of endothelium-dependent vasorelaxation concomitantly with increase of plasma NO concentration and expression of aortic endothelial nitric oxide synthase (eNOS), reduction of plasma MDA level and ET-1 concentration and aortic ET-1 concentration. The present study indicates that the protective effects of UP soluble fiber on ...