iTRAQ Quantitative Proteomic Analysis of Different Expressed Proteins and Signal Pathways in Bakuchiol-Induced Hepatotoxicity.

Bakuchiol (BAK) is an abundant natural compound. BAK has been reported to have several biological activities such as anticancer, antiaging, anti-inflammatory, and prevention of bone loss. However, it causes hepatotoxicity, the mechanism of which is not known. In this study, we explored the mechanism of BAK hepatotoxicity by treating rats with 52.5 mg/kg and 262.5 mg/kg of BAK, administered continuously for 6 weeks. We examined the liver pathology and biochemical composition of bile to determine toxicity. Mechanisms of BAK hepatotoxicity were analyzed based on relative and absolute quantification (iTRAQ) protein equivalent signatures and validated in vitro using LO2 cells. iTRAQ analysis revealed 281 differentially expressed proteins (DEPs) in liver tissue of the BAK-treated group, of which 215 were upregulated, and 66 were downregulated. GO and KEGG enrichment analysis revealed that bile secretion, lipid metabolism, and cytochrome P450 signaling pathways were enriched in DEPs. Among them, peroxisome proliferator-activated receptor α (PPARα), farnesoid X receptor (FXR), and cholesterol 7α-hydroxylase (CYP7a1) were closely associated with the development and progression of BAK-induced hepatic metabolic dysfunction and abnormal bile metabolism. This study shows that BAK can induce hepatotoxicity through multiple signaling pathways.

Integrating Network Pharmacology and In Vivo Model to Investigate the Mechanism of Biheimaer in the Treatment of Functional Dyspepsia.

Objective: Biheimaer (BHM) is a hospital formulation for clinical treatment of dyspepsia and acid reflux, based on Compatibility Theory of Traditional Chinese Medicine. This study anticipated to elucidate the molecular mechanism of BHM against Functional dyspepsia via combined network pharmacology prediction with experimental verification. Methods: Based on network pharmacology, the potential active components and targets of BHM in the treatment of functional dyspepsia were explored by prediction and molecular docking technology. The results of protein-protein interaction analysis, functional annotation, and pathway enrichment analysis further refined the main targets and pathways. The molecular mechanism of BHM improving functional dyspepsia mice induced by L-arginine + atropine was verified on the basis of network pharmacology. Results: In this study, 183 effective compounds were screened from BHM; moreover, 1007 compound-related predicted targets and 156 functional dyspepsia-related targets were found. The results of enrichment analysis and in vivo experiments showed that BHM could regulate intestinal smooth muscle contraction to play a therapeutic role in functional dyspepsia by reducing the expression of NOS3, SERT, TRPV1, and inhibiting the inflammatory cytokine (IL-1ß, TNF-α) to intervene the inflammatory response in mice. Conclusions: This study revealed the molecular biological mechanisms of the Traditional Chinese Medicine formulation of BHM in functional dyspepsia by network pharmacology and experimental verification, meanwhile provided scientific support for subsequent clinical medication.

Effect of Artemisia rupestris L. Extract on Gastrointestinal Hormones and Brain-Gut Peptides in Functional Dyspepsia Rats.

Artemisia rupestris L. is the perennial herb of rupestris belonging to Artemisia (Compositae), which is wildly distributed in Xinjiang (China), middle Asia, and Europe. It is known to have anti-inflammatory, hepatoprotective, immune function regulation, and gastrointestinal function regulation effects. AR is used to treat digestive diseases, but the effects of AR on antifunctional dyspepsia (FD) activity have not yet been reported. In this study, we aimed to investigate the therapeutic effects of Artemisia rupestris L. extract (ARE) on gastrointestinal hormones and brain-gut peptide in functional dyspepsia (FD) rats. Sixty Sprague-Dawley rats were randomly divided into 6 groups. An FD rat model was established by irregular tail clamp stimulation for 14 days except the blank group. After FD rat models, the blank group and model group were given menstruum, and the medicated rats were given corresponding medicine for 14 days. The general observations, bodyweight, and food intake were observed, and the content of serum gastrin (GAS), plasma motilin (MTL), plasma vasoactive intestinal peptide (VIP), and plasma somatostatin (SS) by the enzyme-linked immunosorbent assay was observed. The content of plasma VIP and plasma SS in the ARE group was significantly lower than in the model group, and the content of serum GAS and plasma MTL was increased in the ARE group; the GAS expression of antrum and hypothalamus was increased in the ARE group, and SS expression of antrum and hypothalamus was decreased in the ARE group by immunohistochemical detection; the results of semiquantitative reverse transcription polymerase chain reaction (RT-PCR) indicate that ARE inhibits the mRNA expression of VIP. Our results suggest that ARE can recover gastrointestinal hormone levels and regulation of the peripheral and central nervous system and alter gut peptide levels, which confirm the therapeutic effect of ARE on functional dyspepsia.

Ellagic Acid Inhibits Trichophyton rubrum Growth via Affecting Ergosterol Biosynthesis and Apoptotic Induction.

BACKGROUND: Trichophyton rubrum, among other dermatophytes, is a major causative agent for superficial dermatomycoses like onychomycosis and tinea pedis, especially among pediatric and geriatric populations. Ellagic acid (EA) and shikonin (SK) have been reported to have many bioactivities, including antifungal activity. However, the mechanism of EA and SK on Trichophyton rubrum has not yet been reported. OBJECTIVES: The purposes of this study were to evaluate the antifungal activities of EA and SK against Trichophyton rubrum and to illuminate the underlying action mechanisms. METHODS: The effect of EA (64, 128, and 256 µg/mL) and SK (8, 4, and 2 µg/mL) on Trichophyton rubrum was investigated with different doses via detecting cell viability, ultrastructure with using a scanning electron microscope (SEM), cell apoptosis and necrosis by using the flow cytometry instrument technique (FCIT), and the ergosterol biosynthesis pathway-related fungal cell membrane key gene expressions in vitro. RESULTS: SEM detection revealed that the T. rubrum cell surface was shrivelled, folded, and showed deformation and expansion, visible surface peeling, and broken hyphae, and cell contents overflowed after being treated with EA and SK; the cell apoptosis rate was significantly increased in dose-dependent manner after T. rubrum was treated with EA and SK; the qPCR results showed that mRNA expression of MEP4 and SUB1 was downregulated in EA- and SK-treated groups. CONCLUSIONS: Overall, our results revealed the underlying antifungal mechanism of EA and SK, which may be related to the destruction of the fungal cell membrane and inhibition of C14 demethylase and the catalytic rate of squalene cyclooxidase in the ergosterol biosynthesis pathway via downregulation of MEP4 and SUB1, suggesting that EA and SK have the potential to be developed further as a natural antifungal agent for clinical use.

Antifatigue effects of anshenyizhi compound in acute excise-treated mouse via modulation of AMPK/PGC-1α-related energy metabolism and Nrf2/ARE-mediated oxidative stress.

The anshenyizhi compound (AC), a mixture from Chinese medicine herbs, has numerous biological effects. In the present study, the acute exercise-treated mice model was established to explore the antifatigue properties of AC and its underlying mechanisms. AC increased exercise endurance in the weight-loaded forced swimming test and rota-rod test. The antifatigue properties of AC were closely correlated with enhancing the body's exercise endurance by increasing the levels of cyanmethemoglobin, testosterone/corticosterone, and creatine kinase, while decreasing the levels of lactic acid, lactate dehydrogenase, and blood urea nitrogen in serum. Moreover, our results confirmed the antioxidant ability of AC by improving the activities of superoxide dismutase while reducing reactive oxygen species and malondialdehyde levels in serum. The AC also improved the storage of glycogen by increasing the levels of succinate dehydrogenase, and malate dehydrogenase in liver and muscle. Additionally, AC displayed the antifatigue and antiapoptosis effects via regulating Nrf2-mediated oxidative stress, AMPK-related glucose metabolism, and p53 pathways. Our experimental results first provided a support that AC had effects on antifatigue through regulating AMPK/PGC-1α-related energy metabolism and Nrf2/ARE-mediated oxidative stress. Consequently, AC could be developed into a new functional food supplement for the prevention and treatment of diseases related to fatigue in the future.

Bakuchiol Contributes to the Hepatotoxicity of Psoralea corylifolia in Rats.

Psoralea corylifolia L. (Fructus Psoraleae) is widely used in Asia, but there are concerns about hepatotoxicity caused by constituents such as psoralens and bakukiol. Bakuchiol (BAK) has antiinflammatory, antipyretic, antibacterial antiviral, anticancer, and estrogenic activity but appears to be hepatotoxic in in vitro tests. This study investigated the hepatotoxicity in vivo in rats. Using intragastrically administered bakuchiol at doses of 52.5 and 262.5 mg/kg for 6 weeks. Bodyweight, relative liver weight, biochemical indicators, histopathology, mRNA expression of CYP7A1, HMG-CoA reductase, BSEP, PPARα, SREBP-2, and MRP3 were measured. Many abnormalities were observed in the bakuchiol-treated groups including suppression of weight gain and food intake, change of some parameters in serum biochemistry, and increased weight of liver. The mRNA expression of CYP7A1, HMG-CoA reductase, PPARα, and SREBP-2 decreased in bakuchiol-treated group, the expression of BSEP increased in bakuchiol-treated low dosage, and the expression of BSEP decreased in bakuchiol-treated high dosage. In conclusion, we provide evidence for the first time that bakuchiol can induce cholestatic hepatotoxicity, suggesting potential hepatotoxicity. The mechanism may be related to effects on liver lipid metabolism, but further investigation is necessary. Copyright © 2017 John Wiley & Sons, Ltd.

The Protective Effect of Lavender Essential Oil and Its Main Component Linalool against the Cognitive Deficits Induced by D-Galactose and Aluminum Trichloride in Mice.

Lavender essential oil (LO) is a traditional medicine used for the treatment of Alzheimer's disease (AD). It was extracted from Lavandula angustifolia Mill. This study was designed to investigate the effects of lavender essential oil (LO) and its active component, linalool (LI), against cognitive impairment induced by D-galactose (D-gal) and AlCl3 in mice and to explore the related mechanisms. Our results revealed that LO (100 mg/kg) or LI (100 mg/kg) significantly protected the cognitive impairments as assessed by the Morris water maze test and step-though test. The mechanisms study demonstrated that LO and LI significantly protected the decreased activity of superoxide dismutase (SOD), glutathione peroxidase (GPX), and protected the increased activity of acetylcholinesterase (AChE) and content of malondialdehyde (MDA). Besides, they protected the suppressed nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression significantly. Moreover, the decreased expression of synapse plasticity-related proteins, calcium-calmodulin-dependent protein kinase II (CaMKII), p-CaMKII, brain-derived neurotrophic factor (BDNF), and TrkB in the hippocampus were increased with drug treatment. In conclusion, LO and its active component LI have protected the oxidative stress, activity of cholinergic function and expression of proteins of Nrf2/HO-1 pathway, and synaptic plasticity. It suggest that LO, especially LI, could be a potential agent for improving cognitive impairment in AD.

Antifungal Activity of Gallic Acid In Vitro and In Vivo.

Gallic acid (GA) is a polyphenol natural compound found in many medicinal plant species, including pomegranate rind (Punica granatum L.), and has been shown to have antiinflammatory and antibacterial properties. Pomegranate rind is used to treat bacterial and fungal pathogens in Uyghur and other systems of traditional medicine, but, surprisingly, the effects of GA on antifungal activity have not yet been reported. In this study, we aimed to investigate the inhibitory effects of GA on fungal strains both in vitro and in vivo. The minimal inhibitory concentration (MIC) was determined by the NCCLS (M38-A and M27-A2) standard method in vitro, and GA was found to have a broad spectrum of antifungal activity, with MICs for all the tested dermatophyte strains between 43.75 and 83.33 µg/mL. Gallic acid was also active against three Candida strains, with MICs between 12.5 and 100.0 µg/mL. The most sensitive Candida species was Candida albicans (MIC = 12.5 µg/mL), and the most sensitive filamentous species was Trichophyton rubrum (MIC = 43.75 µg/mL), which was comparable in potency to the control, fluconazole. The mechanism of action was investigated for inhibition of ergosterol biosynthesis using an HPLC-based assay and an enzyme linked immunosorbent assay. Gallic acid reduced the activity of sterol 14α-demethylase P450 (CYP51) and squalene epoxidase in the T. rubrum membrane, respectively. In vivo model demonstrated that intraperitoneal injection administration of GA (80 mg/kg d) significantly enhanced the cure rate in a mice infection model of systemic fungal infection. Overall, our results confirm the antifungal effects of GA and suggest a mechanism of action, suggesting that GA has the potential to be developed further as a natural antifungal agent for clinical use. Copyright © 2017 John Wiley & Sons, Ltd.

Antidepressant-like effects and cognitive enhancement of the total phenols extract of Hemerocallis citrina Baroni in chronic unpredictable mild stress rats and its related mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Depression induce distressed emotional state and cognitive deficits simultaneously, which both should be improved in the treatment. Hemerocallis citrina Baroni (HC) is a traditional herbal medicine in Eastern-Asia areas and the total phenols extract of HC (HCPE) contains the main active ingredients. It has been reported that HC has the emotional improvement effect. But the cognitive effect of HC was seldom researched. AIM OF THE STUDY: We designed to evaluate the antidepressant and cognitive improvement effect of HCPE using a chronic unpredictable mild stress (CUMS) model, and the potential mechanisms were explored by investigating the corticosterone (CORT), monoamine neurotansmitters, brain-derived neurotropic factor (BDNF) and oxidative stress. MATERIALS AND METHODS: The depression rats were induced by CUMS procedures and treated with HCPE (10, 20, 40mg/kg/day, by gastric gavage). The antidepressant effect was evaluated by sucrose preference test, open field test and body weight, while the cognitive improvement was investigated using morris water maze test. Besides, the levels of monoamine neurotransmitters in the hippocampus and frontal cortex were measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The serum CORT and BDNF in hippocampus were test using enzyme-linked immunosorbent assay (ELISA) kits. The oxidative stress indicators in frontal cortex were also analyzed. RESULTS: HCPE (40mg/kg) improved the emotion and cognition related behaviors in depression effectively. Moreover, HCPE increased the neurotransmitters concentration (5-HT, DA and NE) in the hippocampus and frontal cortex compared with CUMS rats. Meanwhile, the CUMS induced changes of serum corticosterone level and the hippocampus BDNF level were reversed. Besides, HCPE reduced malondialdehyde (MDA) in the frontal cortex of model rats. CONCLUSION: It suggested that HCPE could improve the depression-like emotional status and associated cognitive deficits in CUMS rats, which might be mediated by regulation of neurotransmitters and BDNF levels in brain, alleviation of corticosterone level as well as the alleviation of oxidative stress.

Protective effect of lavender oil on scopolamine induced cognitive deficits in mice and H2O2 induced cytotoxicity in PC12 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lavender essential oil (LO), an aromatic liquid extracted from Lavandula angustifolia Mill., has been traditionally used in the treatments of many nervous system diseases, and recently LO also reported to be effective for the Alzheimer's disease (AD). AIM OF THE STUDY: The improvement effect of lavender oil (LO) on the scopolamine-induced cognitive deficits in mice and H2O2 induced cytotoxicity in PC12 cells have been evaluated. The relevant mechanism was also researched from the perspective of antioxidant effect and cholinergic system modulation. MATERIALS AND METHODS: Cognitive deficits were induced in C57BL/6J mice treated with scopolamine (1mg/kg, i.p.) and were assessed by Morris water maze (MWM) and step-through passive avoidance tests. Then their hippocampus were removed for biochemical assays (acetylcholinesterase (AChE), superoxide dismutase (SOD), glutathione peroxidase (GPX) and malondialdehyde (MDA)). In vitro, the cytotoxicity were induced by 4h exposure to H2O2 in PC12 and evaluated by cell viability (MTT), lactate dehydrogenase (LDH) level, nitric oxide (NO) release, reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP). RESULTS: The results demonstrated that LO (100mg/kg) could improve the cognitive performance of scopolamine induced mice in behavioral tests. Meanwhile, it significantly decreased the AChE activity, MDA level, and increase SOD and GPX activities of the model. Moreover, LO (12µg/mL) protected PC12 cells from H2O2 induced cytotoxicity by reducing LDH, NO release, intracellular ROS accumulation and MMP loss. CONCLUSIONS: It was suggested that LO could show neuroprotective effect in AD model in vivo (scopolamine-treated mice) and in vitro (H2O2 induced PC12 cells) via modulating oxidative stress and AChE activity.

Antifungal Activity of Ellagic Acid In Vitro and In Vivo.

Ellagic acid (EA) has been shown to have antioxidant, antibacterial, and anti-inflammatory activities. In Uighur traditional medicine, Euphorbia humifusa Willd is used to treat fungal diseases, and recent studies suggest that it is the EA content which is responsible for its therapeutic effect. However, the effects of EA on antifungal activity have not yet been reported. This study aimed to investigate the inhibitory effect of EA on fungal strains both in vitro and in vivo. The minimal inhibitory concentration (MIC) was determined by the National Committee for Clinical Laboratory Standards (M38-A and M27-A2) standard method in vitro. EA had a broad spectrum of antifungal activity, with MICs for all the tested dermatophyte strains between 18.75 and 58.33 µg/ml. EA was also active against two Candida strains, with MICs between 25.0 and 75.0 µg/ml. It was inactive against Candida glabrata. The susceptibility of six species of dermatophytes to EA was comparable with that of the commercial antifungal, fluconazole. The most sensitive filamentous species was Trichophyton rubrum (MIC = 18.75 µg/ml). Studies on the mechanism of action using an HPLC-based assay and an enzyme linked immunosorbent assay showed that EA inhibited ergosterol biosynthesis and reduced the activity of sterol 14α-demethylase P450 (CYP51) in the Trichophyton rubrum membrane, respectively. An in vivo test demonstrated that topical administration of EA (4.0 and 8.0 mg/cm(2) ) significantly enhanced the cure rate in a guinea-pig infection model of Trichophyton rubrum. The results suggest that EA has the potential to be developed as a natural antifungal agent.

[Action of Euphorbia humifusa effective fraction on membrane biosynthesis and the gene expression of proteases MEP and SUB of Trichophyton rubrum].

This study is to investigate the effect of Euphorbia humifusa effective fraction (EHEF) on the CYP51 enzyme activity, the lanosterol content and the MEP, SUB gene expression of Trichophyton rubrum. Trichophyton rubrum was treated by EHEF for 7 days at 26 degrees C. The activity of CYP51 enzyme of Trichophyton rubrum in the cell membrane was determined by using ELISA kit, and the lanosterol content was investigated by using high performance liquid chromatography (HPLC), and the MEP, SUB gene expression of Trichophyton rubrum was detected with the reverse transcription polymerase chain reaction (RT-PCR) method. Results showed that EHEF can decrease the membrane CYP51 enzyme activity, and it also can accumulate the fungal lanosterol in a dose-dependent manner, and it also can decrease the gene expression of MEP and SUB. The antifungal mechanism of EHEF may be related to the inhibition on CYP51 enzyme activity, and to the effects on fungal cell membrane ergosterol biosynthesis. It may also play an antifungal effect by inhibiting the MEP, SUB gene expression of fungal proteases.

[Grey relational analysis on fingerprint characteristics of different eluted parts of Euphorbia humifusa and antifungal effect].

OBJECTIVE: To determine the contribution of chemical components represented by characteristic peaks of fingerprint of different eluted parts of Euphorbia humifusa to the antifungal effect to provide theoretical basis and data support for the traditional Chinese medicine quality control mode. METHOD: HPLC was introduced to analyze fingerprint of different eluted parts of E. humifusa. The antifungal effect was observed using NCCLS M38-A method. The correlation between fingerprint and antifungal effect of E. humifusa was identified using grey relational analysis. RESULT: The antifungal effect of different eluted parts of E. humifusa is resulted from the combined action of multiple chemical components. Chemical components representing a contribution to pharmacy dynamics were ordered by representative spectrum 2 > 5 > 3 > 4 > 1 for anti-Trichophyton rubrum and anti-T. mentagrophytes 5 > 2 > 3 > 4 > 1. CONCLUSION: Fingerprint of different eluted parts of E. humifusa is related with their antifungal effect to some extent.