Effect of Astragali-Cordyceps Mixtura on TGF-β/Smad signal pathway in the lung of asthma airway remodeling

Aim of the study

We try to find out the influence of traditional Chinese Medicine Astragali-Cordyceps Mixtura (ACM) on TGF-β/Smad signal pathway in the lung of asthma airway remodeling.

Materials and methods

Mice were sensitized and challenged by OVA to establish a model of asthma. To assess the effects of ACM on the mice, animals of the ACM groups were treated with ACM. Data were achieved by using techniques as follow: counting cell number of BALF, assaying the amount of collagen deposition by Masson's staining, performing RT-PCR and immunohistochemistry for mRNA and protein expression of TGF-β1, Smad3 and Smad7.

Results

The depositions of collagen in airway wall greatly increased at the model group compared with that of the normal group. In contrast, these decreased at the ACM groups. As compared with the control group, TGF-β1 expression also decreased at both mRNA and protein level at the ACM-M group versus increased both at the model group. Whereas, Smad7 significantly decreased only at the model group and partly restored at the ACM-M group.

Conclusions

ACM greatly improves the symptoms of asthma airway remodeling by inhibiting the expression of TGF-β1 and upregulating the amount of Smad7.     

Metabolite Characterization of Penta-O-galloyl-??￠-D-glucose in Rat Biofluids by HPLC/QT of MS

Objective: To understand the in vivo metabolic fate of 1,2,3,4,6-Penta-O-galloyl-β-d-glucose(PGG)naturally existed in many medicinal herbs and food plants such as Rhus chinensis,Paeonia suffruticosa,Paeonia lactiflora and Mango.Methods: The metabolites of PGG in rat biofluids were characterized using high performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry(HPLC-QTOF-MS).Results: Ten metabolites in urine,five metabolites in feces and two metabolites in plasma,were observed when the rats were administrated with a single intravenous injection of PGG(20 mg/kg).Conclusion: PGG is firstly metabolized to gallic acid,then gallic acid undergoes sulfation,glucuronidation and methylation by rat liver.The determination of metabolites and the proposed metabolic pathway of PGG in vivo will be benefit to gain deeper insights into its pharmacological activities.     

Low-dose of multi-glycoside of Tripterygium wilfordii Hook. f., a natural regulator of TGF-β1/Smad signaling activity improves adriamycin-induced glomerulosclerosis in vivo

Ethnopharmacological relevance

Transforming growth factor (TGF)-β1/Smad signaling pathway plays a critical role in the prolonged glomerulosclerosis (GS), which is an important determinant during the progression in chronic kidney disease (CKD). For recent 30 years, multi-glycoside of Tripterygium wilfordii Hook. f. (GTW), an extract from Chinese herbal medicine has been proved clinically effective in improving GS in CKD in China. However, therapeutic mechanisms involved in vivo are still unclear. In this study, we aimed to explain the dose-effects and molecular mechanisms of GTW on GS by regulating TGF-β1/Smad signaling activity in adriamycin (ADR)-induced nephropathy (ADRN).

Materials and methods

Rats with ADRN, created by unilateral nephrectomy and twice adriamycin injections (ADR, 4 mg/kg and 2 mg/kg) within 4 weeks, were divided into four groups, the Sham group, the Vehicle group, the low-dose GTW-treated group, and the high-dose GTW-treated group, and that, sacrificed at the end of the 6th week after administration. Proteinuria, blood biochemical parameters, glomerulosclerotic morphological makers, podocyte shape, and nephrin expression were examined, respectively. Protein expressions of key signaling molecules in TGF-β1/Smad pathway, such as TGF-β1, Smad3, phosphorylated-Smad2/3 (p-Smad2/3), and Smad7, were also evaluated individually.

Results

The results indicated that the characterizations of ADRN involved the typical prolonged GS, a small amount of abnormal proteinuria, and the failing renal function; TGF-β1/Smad signaling molecules, especially Smad3, p-Smad2/3, and Smad7 were activated in vivo, accompanied by the exasperation of glomerulosclerotic lesion; GTW at high-dose (100 mg/kg) and low-dose (50 mg/kg) could slightly ameliorate the prolonged GS and nephrin expression, furthermore, the anti-proliferative action of GTW at high-dose was superior to that at low-dose, but caused the significant liver injury; in ADRN model rats, protein expressions of TGF-β1, p-Smad2/3, and Smad7 in the kidneys could be regulated with the treatment of GTW at low-dose.

Conclusion

This study farther demonstrated that the low-dose of GTW, as a natural regulator in vivo, could effectively and safely ameliorate the prolonged GS in FSGS model, via the potential molecular mechanisms involving the reduction of ECM components and the suppression of TGF-β1 over-expression, as well as the bidirectional regulation of TGF-β1/Smad signaling activity.     

Effect of bioactive peptide of Carapax Trionycis on TGF-β1-induced intracellular events in hepatic stellate cells

Ethnopharmacological relevance

In traditional Chinese medicines for hepatic fibrosis therapy, Carapax Trionycis is used usually as an indispensable component and has a long history of medical use in China. Previous studies have demonstrated that extracts of Carapax Trionycis were able to protect liver against fibrosis in CCl₄ animal models.

Aim of the study

The purpose of this study is to verify the inhibitory effect and the underlying mechanisms of Carapax Trionycis extract peptide (CTEP) on activated hepatic stellate cells which play a central role in liver fibrogenesis.

Materials and methods

Hepatic stellate cells induced by TGF-β₁ were applied to evaluate the anti-fibrotic effect of CTEP in vitro. MTS assay, enzyme-linked immunosorbent assay and western blotting were then used to further investigate the molecular mechanisms.

Results

The results show that the contents of collagen I, collagen III and TIMP-1 were significantly inhibited and the level of collagen I, collagen III, p-Smad 3, TIMP-1 and α-SMA proteins decreased significantly in a concentration-dependence manner after treatment with CTEP. Interestingly, the level of Smad 3 protein was not different significantly.

Conclusions

Our data indicate that CTEP efficiently inhibits cultured HSC-T₆ cell activation and proliferation via the TGF-β₁/Smad pathway as well as by the elimination of the extracellular matrix.     

In vivo and in vitro antidiabetic effect of Cistus laurifolius L. and detection of major phenolic compounds by UPLC–TOF-MS analysis

Ethnopharmacological relevance

In Turkish folk medicine, various parts of Cistus laurifolius L. are used to treat gastric ulcer and various types of pains. Additionally the tea prepared from the leaves is used to decrease symptoms of diabetes.

Materials and methods

In the present study, the hypoglycemic effects of aqueous and ethanol extracts of Cistus laurifolius were investigated in normal, glucose loaded hyperglycemic and streptozocin (STZ)-induced diabetic rats. α-Glucosidase and α-amylase enzyme inhibitory effects were determined to evaluate the mechanism of action. Total phenolic content of the extracts were determined by using Folin–Ciocalteu reagent and Ultra Performance Liquid Chromatography–Time of Flight Mass Spectrometer (UPLC–TOF-MS) was used to detect the major phenolic compounds in the extract.

Results

Results indicated that blood glucose levels of the STZ-induced diabetic rats were decreased by ethanol extract at of 250 and 500 mg/kg doses as compared to control group (16%–34%). In glucose loaded animals, extracts have shown a weak hypoglycemic effect (11%–20%). Additionally, the ethanol extract of Cistus laurifolius is found to be a potent inhibitor of α-glucosidase and α-amylase, possibly due to several polyphenolic compounds present within the extract. Twelve major flavonoids (apigenin, quercetin, kaempferol, naringenin, quercitrin and their derivatives), gallic, ellagic and chlorogenic acid in chromatographic fingerprint were analyzed by the on-line UPLC–TOF-MS system.

Conclusions

Due to having inhibitory effect on blood glucose level and carbohydrate digesting enzymes (α-glucosidase and α-amylase), Cistus laurifolius leaves might be beneficial for diabetic patients.     

Pharmacokinetic study of four flavones of Glycyrrhiza in rat plasma using HPLC–MS

Aim of the study

This study aimed to develop a specific HPLC–MS method for simultaneous quantification of four flavones of Glycyrrhiza in rat plasma after oral administration and to describe the pharmacokinetics of four flavones in rat plasma.

Materials and methods

A simple, sensitive and selective method for simultaneous determination of four flavones of Glycyrrhiza in rat plasma, i.e., liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin, by high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) with negative electrospray ionization mode, was developed and validated. The method was applied to investigate the pharmacokinetics of four flavones in rat plasma after oral administration of Glycyrrhiza flavones. Chromatographic separation was accomplished on an Agilent TC-C18 column (4.6 mm×250 mm, and 5 μm), with gradient elution by using a mixture of methanoic acid (A) and acetonitrile (B) as the mobile phase at a flow rate of 0.8 mL/min.

Results

The calibration curves for four flavones had good linearity higher than 0.997 in the measured range. Relative standard deviations (RSDs) of the intra- and inter-day precision at different levels were all less than 4.8%. The pharmacokinetic profile of four flavones in rat plasma was fitted with a two-compartment model detected by a simple, rapid and accurate HPLC–MS method. Time (h) to reach peak concentration (μg/mL) of liquiritin (2.69±0.04), isoliquiritin (10.16±0.02), liquiritigenin (2.83±0.02), and isoliquiritigenin (0.28±0.01) was 2.02±0.23, 1.97±0.20, 0.48±0.02, and 1.93±0.36, respectively. The distribution and elimination half-life (h) and area under the concentration–time curve (μg/mL–h) from t=0 to last time of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were 1.02±0.48/2.27±0.53/16.97±0.43, 2.04±1.01/2.38±0.80/69.20±5.24, 0.35±0.10/4.26±0.16/14.83±0.11, and 1.18±0.32/3.04±0.22/2.10±0.09, respectively. Isoliquiritin presented the phenomenon of double peaks and the others appeared together in a single and plateau absorption phase. Isoliquiritigenin had the lowest oral bioavailability because of C_max and AUC_0−∞. Liquiritigenin had the fastest absorption and distribution rate and the lowest elimination rate according to T_max, t_1/2α, and t_1/2β.

Conclusions

This paper first reported on identification and determination of four flavones of Glycyrrhiza in rat plasma and their respective pharmacokinetic characteristics. The results provided a meaningful basis for better understanding the absorption mechanism of Glycyrrhiza and evaluating the clinical application of this medicine.     

Pharmacological evaluation of sedative–hypnotic activity and gastro-intestinal toxicity of Rhizoma Paridis saponins

Ethnopharmacological relevance

Rhizoma Paridis saponins (RPS) have been well studied for antimicrobial, anti-hemorrhagic, and anticancer effects. However, scientific information on RPS regarding the toxic and neuropharmacological effects is limited. In this study, the acute oral toxicity, sedative–hypnotic activity and gastro-intestinal toxicity of RPS were investigated.

Materials and methods

The acute toxicity was carried out by administering single doses (800–5000 mg/kg) of RPS to adult mice. Rotarod test and sodium pentobarbital-induced hypnosis activity were used to evaluate the neuropharmacological effects on mice. Gastric emptying and intestinal transit were used to investigate the gastric–intestinal system effects.

Results

A single oral administration of RPS dose-dependently caused adverse effects on the general behavior and mortality rate of mice. LD₅₀ value of oral acute toxicity was 2182.4 mg/kg, with 95% confidence limit of 1718.4–2807.8 mg/kg. In the test of sleeping mice, RPS acted in synergy with sodium pentobarbital at doses 250 and 500 mg/kg while motor coordination was not influenced within 120 min after treatment with RPS. Regarding the gastric–intestinal toxicity, RPS (100, 250, and 500 mg/kg) significantly inhibited gastric emptying but did not affect the intestinal transit.

Conclusions

RPS, which is a hypotoxic anticancer drug, possesses the sedative–hypnotic activity and gastric stimulus side effect.     

Influence of processing on pharmacokinetic of typical constituents in radix polygoni multiflori after oral administration by LC–ESI–MS/MS

Ethnopharmacological relevance

The processed radix polygoni multiflori (P-RPM) are produced from the raw radix polygoni multiflori (R-RPM) steamed with black bean juice, but the two traditional Chinese medicines are used to treat the different diseases in clinic. In order to clarify the influence of processing on pharmacological properties of radix polygoni multiflori, an investigation was carried out to compare the pharmacokinetics of typical constituents after oral administration of P-RPM and R-RPM extracts

Materials and methods

A simple, rapid and sensitive high performance liquid chromatography–tandem mass spectroscopy (LC–MS/MS) was developed and validated for the simultaneous determination of gallic acid, polydatin, 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (PM-SG), resveratrol, and emodin in rat plasma. Male Sprague-Dawley rats were orally administered the two extracts with approximately the same dosage.

Results

It was found that gallic acid was distributed as opened one-compartment model while polydatin, PM-SG and emodin were fitted to an open two-compartment model after oral administration of raw and processed radix polygoni multiflori extract. Cmax and AUC of gallic acid were increased (P<0.01), but C_max and AUC of PM-SG were descreased (P<0.05). AUC of polydatin and emodin were similar with that of PM-SG. However, resveratrol was not detected in plasma collected at certain intervals following oral administration of the two extracts.

Conclusions

These results indicate that influence of the processing could improve the bioavailability of gallic acid and reduce the absorption of PM-SG, polydatin and emodin in rats. The LC–MS/MS method could be used to evaluate the effect of processing on pharmacokinetic of typical constituents in radix polygoni multiflori after oral administration.     

Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-γ down-regulation in NC/Nga mice

Ethnopharmacological relevance

Uncariae rhynchophylla (UR) is an herb which has blood pressure lowering and anti-inflammatory effects and has been prescribed traditionally to treat stroke and vascular dementia.

Aim of study

In the present study, we examined whether UR suppress Atopic dermatitis (AD)-like skin lesions in NC/Nga mice treated with 2, 4-dinitrofluorobenzene (DNFB) under SPF conditions.

Material and methods

The effect of UR in DNFB- treated NC/Nga mice was determined by measuring the skin symptom severity, levels of serum IgE, and of the amounts of IL-4 and IFN-γ secreted by activated T cells in draining lymph nodes.

Results

Oral administration of UR to DNFB-treated NC/Nga mice was found to inhibit ear thickness increases and the skin lesions induced by DNFB. IFN-γ production by CD4+ T cells from the lymph nodes of DNFB-treated NC/Nga mice was significantly inhibited by UR treatment, although levels of IL-4 and total IgE in serum were not.

Conclusion

UR may suppress the development of AD-like dermatitis in DNFB-treated NC/Nga mice by reducing IFN-γ production.     

Bioguided isolation of myricetin-3-O-β-galactopyranoside with antinociceptive activity from the aerial part of Davilla elliptica St.-Hil

Ethnopharmacological relevance

Davilla elliptica St.-Hil. (Dilleniaceae) is a medicinal plant traditionally used in Brazil to treat inflammatory processes, to relieve pain, as diuretic, gastro- and hepatoprotective agents.

Aim of the study

To undertake the fractionation of the ethanolic extract from Davilla elliptica leaves guided by an antinociceptive assay.

Materials and methods

The antinociceptive activity was evaluated through the formalin test in mice. Extract fractionation was performed by percolation through silica gel and partition between immiscible solvents, followed by successive column chromatography over Sephadex LH-20 and preparative RP-HPLC. Structure elucidation of the isolated compound was accomplished by spectroscopic data.

Results

The EtOAc and MeOH fractions derived from the crude extract reduced significantly the licking time in the late phase of the formalin test. The bioguided fractionation of the MeOH fraction resulted in the isolation of myricetin-3-O-β-galactopyranoside, which produced significant inhibition on nociception induced by formalin (ID₅₀=0.26 mg/kg; p.o.).

Conclusions

These results point out that myricetin-3-O-β-galactopyranoside contributes for the antinociceptive effect of Davilla elliptica extract, a constituent considerably more potent than diclofenac, employed as reference drug.     

2,3,4',5-tetrahydroxystilbene-2-O-β-D-glucoside inhibits proliferation of vascular smooth muscle cells: involvement of NO/cGMP/PKG pathway

The proliferation of vascular smooth muscle cells (VSMCs) induced by injury to the intima of arteries is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis. 2,3,4',5-Tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component extracted from Polygonum multiflorum, has been found to have an antiatherosclerotic effect. The aim of this study was to investigate the effects of TSG on platelet derived growth factor (PDGF)-BB induced VSMCs proliferation and to explore the possible mechanisms of such effects. Pretreatment of VSMCs with TSG significantly inhibited PDGF-BB-induced cell proliferation in a concentration-dependent but not time-dependent manner. In addition, flow cytometry analysis of the DNA content revealed blocking of the PDGF-BB-inducible cell cycle progression by TSG. On the contrary, an inhibitory effect of TSG on VSMCs proliferation and expression of cell cycle regulators were markedly attenuated by addition of an nitric oxide (NO) synthase inhibitor, a soluble guanylate cyclase inhibitor and a cyclic GMP (cGMP)-dependent protein kinase (PKG) inhibitor: N(G)-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4] oxadiazolo [4,3-α] quinoxalin-1-one (ODQ) and KT5823, respectively. It was also demonstrated that TSG enhanced NO and cGMP formation through up-regulating endothelial NO synthase expression in VSMCs. The findings indicate that TSG inhibited VSMCs proliferation induced by PDGF-BB may involve the NO/cGMP/PKG signal pathway.     

Antidiabetic effect of propolis: reduction of expression of glucose-6-phosphatase through inhibition of Y279 and Y216 autophosphorylation of GSK-3α/β in HepG2 cells

Propolis is a sticky, resinous material that honey bees collect from various plants, and mix with wax and other secretions. The aim of this study was to evaluate the antidiabetic effect of propolis through an analysis of the expression and enzyme activity of glucose-6-phosphatase (G6Pase) and to elucidate the mechanism by which propolis inhibits G6Pase gene expression. When HepG2 cells were incubated in high glucose media (25 mm), G6Pase expression was induced. Propolis significantly reduced the expression and enzyme activity of G6Pase; however, the hypoglycemic effect was not abolished by the phosphoinositide 3-kinase inhibitor, LY294002, and by the mitogen-activated protein kinase (MAPK) inhibitor, U0126. Propolis inhibited the activity of GSK3α and β via the inhibition of serine and tyrosine phosphorylation, specifically, Y279 for GSK3α and Y216 for GSK3β. The phosphorylations of Y279 and Y216 occur through autophosphorylation by GSK3α/β and are involved in their own activity. Although propolis showed antioxidant activity, antidiabetic effect of propolis was not influenced by hydrogen peroxide and N-acetylcysteine. These results suggest that propolis inhibits the expression of G6Pase by inhibiting the autophosphorylation of Y279 and Y216 of GSK3α and β, respectively, which are involved in the activation of GSK3. These findings suggest that propolis may be a potential antidiabetic agent for the treatment of insulin-insensitive diabetes.     

Hypoglycemic activities of flowers of Xanthoceras sorbifolia and identification of anti-oxidant components by off-line UPLC-QTOF-MS/MS-free radical scavenging detectionOA

Objective: To identify phytochemical constituents present in the extract of flowers of Xanthoceras sorbifolia and evaluate their anti-oxidant and anti-hyperglycemic capacities.Methods: The AlCl₃ colorimetric method and Prussian Blue assay were used to determine the contents of total flavonoids and total phenolic acids in extraction layers, and the bioactive layers was screened through anti-oxidative activity in vitro. The Waters ACQUITY UPLC system and a Waters ACQUITY UPLC BEH C_...     

Mechanisms of anti-inflammatory property of Anacardium occidentale stem bark: Inhibition of NF-κB and MAPK signalling in the microglia

Ethnopharmacological relevance

Anacardium occidentale is used in traditional African medicine for the treatment of arthritis, fever, aches, pains, and inflammation of the extremities.

Aim of the study

In this study, we investigated the molecular mechanisms responsible for anti-inflammatory effects of a stem bark extract of A. occidentale (ANE) in LPS-stimulated microglia.

Materials and methods

Nitric oxide (NO), prostaglandin E₂ and cytokine (TNFα and IL-6) production were evaluated in supernatants from LPS-stimulated BV-2 cells. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and microsomal prostaglandin E2 synthase (mPGES-1) protein expressions in rat primary microglia were measured using western blot. The effects of ANE on NF-κB activation and nuclear translocation were evaluated in the luciferase reporter gene assay and ELISA, while ability of ANE to influence IκB phosphorylation was determined using ELISA specific for phospho-IκB. The involvement of MAPK phosphorylation in the anti-inflammatory actions of ANE was evaluated using specific ELISA for phospho-p38, phospho-p42/44 and phospho-JNK. The MTT assay was used to determine the effect of ANE on BV-2 microglia viability.

Results

ANE (25–100 μg/ml) produced significant (p<0.05) reduction in the production of NO, PGE₂, TNFα and IL-6 in BV-2 microglia stimulated with LPS for 24 h. Pre-treatment with ANE caused a significant (p<0.05) inhibition of COX-2, iNOS and mPGES-1 protein expressions in the rat primary microglia. Further experiments showed that ANE inhibited COX-2 and iNOS protein expression via IκB-mediated nuclear translocation and transactivation of NF-κB. Our studies also revealed that ANE produced significant (p<0.05) and dose-dependent inhibition of p38, p42/44 and JNK MAPK phosphorylation in LPS-activated BV-2 microglia.

Conclusions

We conclude that ANE has an anti-inflammatory property related to inhibition of inflammation-associated cytokine production as well as iNOS and COX-2 gene expression by blocking NF-κB and MAPK pathways in the microglia. It is also suggested that mPGES-1 inhibition contributes to the effect of ANE on PGE₂ production in the microglia.     

Mechanism of action of （-）-epigallocatechin-3-gallate：auto-oxidation-dependent activation of extracellular signal-regulated kinase 1/2 in Jurkat cells

AIM：（-）-Epigallocatechin-3-gallate（EGCG）, a major compound of tea polyphenols, exhibited antitumor activity in previous studies. In these studies, EGCG usually inhibits EGFR, and impairs the ERK1/2 phosphorylation in tumor cells. The aim was to clarify the mechanism of ERK1/2 activation induced by EGCG. METHODS： Jurkat and 293 T cells were treated with EGCG in different culture conditions. Western Blotting（WB） was employed to analyze ERK1/2 and MEK phosphorylation. Cetuximab and FR180204 were used to inhibit cell signaling. The stability of EGCG was assessed by HPLC. The concentration of hydrogen peroxide generated by the auto-oxidation of EGCG was determined by photocolorimetric analysis. RESULTS： Activation of ERK1/2 was observed to be both time- and dose-dependent. Stimulation of cell signaling was dependent on MEK activity, but independent of EGFR activity. Unexpectedly, EGCG was depleted within one hour of incubation under traditional culture conditions. Auto-oxidation of EGCG generated a high level of hydrogen peroxide in the medium. Addition of catalase and SOD to the acidic medium inhibited the oxidation of EGCG. However, this particular condition also prevented the phosphorylation of ERK1/2. The generation of ROS by hydrogen peroxide may also induce ERK1/2 activation in Jurkat cells. CONCLUSION： ERK1/2 phosphorylation was caused by auto-oxidation of EGCG. Traditional culture conditions were determined to be inappropriate for EGCG research.     

Implication of phosphatidylinositol-3 kinase/Akt/glycogen synthase kinase-3β pathway in ginsenoside Rb1's attenuation of beta-amyloid-induced neurotoxicity and tau phosphorylation

Ginseng has long been used to alleviate many ailments, particularly those associated with aging and memory deterioration. In the present study we aimed to investigate the neuroprotective effects of ginsenoside Rb1, against Aβ(1-42) toxicity in cultured cortical neurons and also the potential involvement of PI3K/Akt/GSK-3β signal pathway. Cortical neurons were pre-treated with ginsenoside Rb1 (20, 40, 100 μM) or LiCl (1, 5, 10 mM) for 24 h, and then were co-treated with 20 μM Aβ(1-42) for 12 h. In some experiments to evaluate the mechanism of Rb1 action, a PI3K inhibitor (LY294002 10 μM) was co-administered with Rb1 for the 24-h pretreatment. We revealed that Rb1 significantly attenuated Aβ(1-42)-induced neurotoxicity and tau hyperphosphorylation at multiple AD-related sites in a dose-dependent manner. Simultaneously, it increased the levels of phospho-Ser(473)-Akt and down-regulated GSK-3β activity by PI3K activation. The neuroprotective effects of Rb1 against Aβ(1-42)-induced neurotoxicity and tau hyperphosphorylation were blocked by LY294002 (10 μM), a PI3K inhibitor. In addition, Rb1 reversed the Aβ(1-42)-induced decrease in phosphorylation cyclic AMP response element binding (CREB) protein, which could also be blocked by the PI3K inhibitor. All these findings suggest that Rb1 may represent a potential treatment strategy for Alzheimer's disease.     

Characterization of thirty-nine polymethoxylated flavonoids （PMFs） in the branches of Murraya paniculata by HPLC-DAD-ESI-MS/MS

目的:定性分析千里香枝中的多甲氧基黄酮类成分。方法:建立一种灵敏的HPLC-DAD-ESI-MS/MS方法,筛选千里香枝中的多甲氧基黄酮。结果:分别研究总结了多甲氧黄酮、二氢黄酮、查耳酮以及多甲氧基黄酮苷的诊断性断裂途径。结合这些特征和EIC-MS/MS实验,筛选了39个多甲氧基黄酮类成分,包括24个多甲氧基黄酮、10个多甲氧基二氢黄酮或查耳酮、5个多甲氧基黄酮苷。结论:本研究为从复杂物质体系中筛选多甲氧基黄酮提供了一种快速有效的方法。     

Apoptotic and antihepatofibrotic effect of honokiol via activation of GSK3β and suppression of Wnt/β-catenin pathway in hepatic stellate cells

Though honokiol, derived from the Magnolia tree, was known to suppress renal fibrosis, pulmonary fibrosis, non-alcoholic steatoheptitis, inflammation and cancers, the underlying antifibrotic mechanisms of honokiol are not fully understood in hepatic stellate cells until now. Thus, in the present study, inhibitory mechanism of honokiol on liver fibrosis was elucidated mainly in hepatic stellate cells (HSCs) by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle analysis and western-blotting. Honokiol exerted cytotoxicity in LX-2, HSC-T6 and Hep-G2 cells. Honokiol increased sub G1 population and activated caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) in HSCs. Moreover, honokiol attenuated the expression of alpha smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β1), phospho-Smad3, phospho-AKT, cyclin D1, c-Myc, Wnt3a, β-catenin, and activated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) in HSCs. Conversely, GSK3β inhibitor SB216763 reversed the effect of honokiol on PARP, α-SMA, phospho-GSK3β, β-catenin and sub G1 population in LX-2 cells. Overall, honokiol exerts apoptotic and antifibrotic effects via activation of GSK3β and inhibition of Wnt3a/β-catenin signalling pathway.     

The impact of Zataria multiflora Boiss extract on in vitro and in vivo Th1/Th2 cytokine (IFN-γ/IL4) balance

Ethnopharmacological relevance

Anti-inflammatory, anti-oxidant and effect of Zataria multiflora on Th₁/Th₂ balance were previously described. Different therapeutic effects of this plant have been described in Iranian traditional medicine. To evaluate the immune modulatory effects of Zataria multiflora on Th1/Th2 balance, which may be implicated in inflammatory disorders, in vitro and in vivo studies were carried out.

Materials and methods

The effects of three concentrations of the extract, dexamethasone, and saline on interleukin 4 (IL-4) and interferon γ (IFN-γ) gene expression were evaluated in phytohemagglutinin (PHA) stimulated and non-stimulated human peripheral blood mononuclear cells (hPBMCs). RNA was extracted from the hPBMCs to make cDNA for real time PCR relative quantification. Furthermore, the effect of the extract on serum level of IL-4 and IFN-γ was assessed in ovalbumin (OA) sensitized guinea pigs (n=6 for each group).

Results

Dexamethasone showed significant inhibitory effect on both IFN-γ and IL-4 gene expression and serum level of the cytokines and significantly enhanced IFN-γ/IL-4 ratio (p<0.05–p<0.001). The extract inhibited IL-4 and enhance IFN-γ gene expression and IFN-γ/IL-4 ratio too (p<0.05–p<0.001). In sensitized animals also serum level of IL-4 were significantly decreased after treatment with both dexamethasone and extract, but serum level of IFN-γ and IFN-γ/IL-4 ratio were significantly increased due to extract treatment (p<0.01 for medium and p<0.001 for high concentration).

Conclusions

These results indicated consistent in vitro and in vivo data for selective immune modulatory effect of the extract of Zataria multiflora which increased IFN-γ, decreased IL-4, and enhanced the ratio of IFN-γ to IL-4 (Th₁/Th₂ balance). Therefore, the extract of Zataria multiflora may have therapeutic value in inflammatory responses such as allergy, autoimmunity and infectious diseases associated with Th₁/Th₂ imbalance.