GLP-1/GLP-1RAs: New Options for the Drug Treatment of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) has recently emerged as a global public health concern. Currently, the cornerstone of NAFLD treatment is lifestyle modification and, if necessary, weight loss. However, compliance is a challenge, and this approach alone may not be sufficient to halt and treat the more serious disease development, so medication is urgently needed. Nevertheless, no medicines are approved to treat NAFLD. Glucagon-like peptide-1 (GLP-1) is an enteropeptide hormone that inhibits glucagon synthesis, promotes insulin secretion, and delays gastric emptying. GLP-1 has been found in recent studies to be beneficial for the management of NAFLD, and the marketed GLP-1 agonist drugs have different degrees of effectiveness for NAFLD while lowering blood glucose. In this article, we review GLP-1 and its physiological roles, the pathogenesis of NAFLD, the correlation between NAFLD and GLP-1 signaling, and potential strategies for GLP-1 treatment of NAFLD.

Sinisan ameliorates colonic injury induced by water immersion restraint stress by enhancing intestinal barrier function and the gut microbiota structure.

CONTEXT: Sinisan (SNS) has been used to treat psychosomatic diseases of the digestive system. But little is known about how SNS affects water immersion restraint stress (WIRS). OBJECTIVE: To study the effects of SNS on colonic tissue injury in the WIRS model. MATERIALS AND METHODS: Forty-eight Kunming (KM) mice were randomized into 6 groups (n = 8): The control and WIRS groups receiving deionized water; the SNS low-dose (SL, 3.12 g/kg/d), SNS middle-dose (SM, 6.24 g/kg/d), SNS high-dose (SH, 12.48 g/kg/d), and diazepam (DZ, 5 mg/kg/d) groups; each with two daily administrations for 5 consecutive days. The 5 treatment groups were subjected to WIRS for 24 h on day 6. The effects of SNS on colon tissue injury caused by WIRS were assessed by changes in colon histology, inflammatory cytokines, brain-gut peptides, and tight junction (TJ) proteins levels. 16S rRNA gene sequencing was used to detect the regulation of the gut microbiota. RESULTS: SNS pretreatment significantly reduced TNF-α (0.75- to 0.81-fold), IL-6 (0.77-fold), and IFN-γ (0.69-fold) levels; and increased TJ proteins levels, such as ZO-1 (4.06- to 5.27-fold), claudin-1 (3.33- to 5.14-fold), and occludin (6.46- to 11.82-fold). However, there was no significant difference between the levels of substance P (SP) and vasoactive intestinal peptide (VIP) in the control and WIRS groups. SNS regulated the composition of gut microbiota in WIRS mice. CONCLUSION: The positive effects of SNS on WIRS could provide a theoretical basis to treat stress-related gastrointestinal disorders.

Protective Effects and Mechanisms of Melatonin on Stress Myocardial Injury in Rats.

ABSTRACT: Prolonged and intense stress can exceed the body's normal self-regulation and limited compensatory and repair capacity, resulting in pathological damage to the body. In this study, we established a rat stress myocardial injury (SMI) model to explore the protective effect of melatonin (MLT) on SMI and its possible mechanisms of action. Adult female Sprague Dawley (SD) rats were randomly divided into 5 groups: blank control group (NC), SMI group, MLT low-dose group, MLT medium-dose group, and MLT high-dose group, and 10 rats in each group were used to establish a SMI model by the water immersion restraint method. We observed the changes in body weight and tail vein glucose of each group. Serum levels of corticosterone (Cort), creatine kinase isoenzyme (CK-MB), and Troponin Ⅰ (Tn-Ⅰ) and activity of lactic acid dehydrogenase were measured by ELISA. Transcriptome sequencing was used to find differentially expressed genes in the control and model groups, and the results were verified by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). HE staining was used to visualize the pathological changes in the heart tissue of each group, and Western blot was used to study the differences in protein expression in the cardiomyocytes of each group to further corroborate the results. The body weight growth rate of rats in the SMI group was significantly lower than that of the NC group ( P < 0.01), and the body weight growth rate of rats in the MLT high-dose group was significantly higher than that of the SMI group ( P < 0.05) with no significant difference compared with the NC group rats. The mean blood glucose of rats in the SMI group was significantly higher compared with the NC group ( P < 0.001), while the mean blood glucose of rats in the MLT administration groups was dose-dependently reduced compared with the SMI group. By RNA-seq and bioinformatics tools such as KEGG and Gene ontology, we found that the circadian clock-related genes Ciart , Arnt1 , Per1 , and Dbp were significantly downregulated in the SMI group during water immersion stress, and differentially expressed genes were enriched in the p38MAPK signaling pathway and p53 signaling pathway. Moreover, genes related to inflammation and apoptosis were differentially expressed. ELISA results showed that Cort, CK-MB, and Tn-Ⅰ levels were significantly higher in the SMI group compared with the NC group ( P < 0.01) and melatonin reduced the levels of Cort, CK-MB, and Tn-Ⅰ and decreased lactic acid dehydrogenase activity in rat serum. HE staining results showed that melatonin could attenuate stress-generated myocardial injury. Western blot showed that melatonin reduced the expression of p38MAPK, p53, Bax, and caspase-3 and increased the expression of Bcl-2 protein in rat heart. Melatonin can inhibit myocardial injury caused by water immersion, and its mechanism of action may be related to the regulation of the expression of circadian clock genes such as Ciart , Arnt1 , Per1 , and Dbp ; the inhibition of the expression of proapoptotic proteins such as p38MAPK, p53, Bax, and caspase-3; and the increase of the expression of Bcl-2 antiapoptotic protein.

Micro-interaction of mucin tear film interface with particles: The inconsistency of pharmacodynamics and precorneal retention of ion-exchange, functionalized, Mt-embedded nano- and microparticles.

Physiological reflexes and anatomical barriers render traditional eye drop delivery inefficient. We previously reported that drug-loaded nanoparticles and microspheres prepared from montmorillonite and Eudragit polymers exhibited good sustained-release and lowered intraocular pressure. Here, we compared the performance of optimized formulations to select the most suitable formulation for glaucoma therapy. We found that the microspheres had much higher encapsulation efficiency and drug loading than nanoparticles. Moreover, cytocompatibility experiments demonstrated that nanoparticles showed more severe cytotoxicity than microspheres, probably due to their smaller particles, enhanced cell uptake, and intracellular solubility. Interestingly, the pre-corneal retention time of nanoparticles reflected a clear advantage over microspheres, while the duration of the pharmacological effect of nanoparticles was not as good as that of microspheres: compared with the nanoparticle depressurization duration of only 8 h, the microspheres continuously depressurized for 12 h. The slower release of the microspheres and its micro-interaction mechanism with the discontinuous mucin layer of the tear film led to the inconsistency between duration of pharmacodynamics and fluorescence ocular retention time. In summary, the lower cytotoxicity and longer pharmacological effect of microspheres indicate their potential advantages for glaucoma applications.

The Role of Potassium Channels in Chronic Stress-Induced Brain Injury.

Chronic stress-induced brain injury (CSBI) is the organic damage of brain tissue caused by long-term psychological and environmental stress. However, there is no effective drug for the treatment of CSBI. The present study aimed to investigate possible mechanisms of CSBI and to explore related therapeutic targets. A rat model of CSBI was established by combining chronic restraint and cold water immersion. Our CSBI model was validated via Nissl staining, Western blotting, and behavioral tests. RNA sequencing (RNA-seq) was used to identify differentially expressed genes (DEGs) within brain tissue during CSBI. Both Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to determine signaling pathways associated with CSBI-induced DEGs. Agonists/antagonists were used to validate the pharmacodynamics of potential therapeutic targets. A combination of chronic restraint and cold water immersion successfully induced a rat model of CSBI, as indicated by various markers of brain injury and cell apoptosis that were verified via Nissl staining, Western blotting, and behavioral tests. RNA-seq analysis identified 1131 DEGs in CSBI rats. Of these DEGs, 553 genes were up-regulated and 778 genes were down-regulated. GO and KEGG pathway analyses revealed that significant DEGs were predominantly related to membrane-bound ion channels, among which the potassium channel function was found to be significantly affected. Pharmacological experiments revealed that retigabine, a voltage-gated potassium channel opener, demonstrated a protective effect in CSBI rats. Taken together, our findings suggest that potassium channel function is disrupted in CSBI, and that potassium channel regulators may function as anti-CSBI drugs.

Flavin adenine dinucleotide ameliorates hypertensive vascular remodeling via activating short chain acyl-CoA dehydrogenase.

AIMS: Flavin adenine dinucleotide (FAD), participates in fatty acid ß oxidation as a cofactor, which has been confirmed to enhance SCAD activity and expression. However, the role of FAD on hypertensive vascular remodeling is unclear. In this study, we investigated the underlying mechanisms of FAD on vascular remodeling and endothelial homeostasis. MAIN METHODS: Morphological examination of vascular remodeling were analyzed with hematoxylin and eosin (HE) staining, Verhoeff's Van Gieson (EVG) staing, Dihydroethidium (DHE) staining and Sirius red staining. HUVECs apoptotic rate was detected by flow cytometry and HUVECs reactive oxygen species (ROS) was detected by DHE-probe. Enzymatic reactions were used to detect SCAD enzyme activity. The protein level was detected by Western Blots, the mRNA level was detected by quantitative real-time PCR. KEY FINDINGS: In vivo experiments, FAD significantly decreased blood pressure and ameliorated vascular remodeling by increasing SCAD expression, Nitric Oxide (NO) production and reducing ROS production. In vitro experiments, FAD protected against the tBHP induced injury in HUVEC, by increasing the activity of SCAD, increasing the elimination of free fatty acid (FFA), scavenging ROS, reducing apoptotic rate, thereby improving endothelial cell function. SIGNIFICANCE: FAD has a new possibility for preventing and treating hypertensive vascular remodeling.

[Effects of short-chain acyl-CoA dehydrogenase on human umbilical vein endothelial cell apoptosis].

OBJECTIVE: To observe the changes of short-chain acyl-CoA dehydrogenase (SCAD) expression on human umbilical vein endothelial cell (HUVEC) apoptosis and investigate its relationship with apoptosis. METHODS: The HUVEC was cultured normally for 2-3 days. The apoptotic model of HUVEC was established by tert-butyl hydrogen peroxide (tBHP). The HUVEC was treated by different concentrations of tBHP (0, 10, 20, 30, 40, 50 µmol/L) for 12 hours and different time (0, 3, 6, 9, 12 hours) with 50 µmol/L tBHP to establish the apoptotic model of HUVEC. The cell viability was detected by methyl thiazolyl tetrazolium (MTT), the mRNA expression of SCAD was determined by real-time polymerase chain reaction (PCR), the protein expression of SCAD was achieved by Western Blot. The best concentrate and time were determined to interfere the HUVEC to achieve the apoptotic model of HUVEC. The SCAD gene of HUVEC was knocked down by RNA interference sequence (siRNA274, siRNA414, siRNA679). The mRNA expression of SCAD, the protein expression of SCAD and the activity of SCAD enzyme were detected to achieve the best RNA interference sequence. The HUVEC was intervened by the best RNA interference sequence and tBHP. The cell activity and apoptosis rate, the enzyme activity of SCAD, the mRNA and protein expression of SCAD, the contents of reactive oxygen species (ROS), aderosine triphosphate (ATP) and free fatty acid (FFA) were detected to observe the effect of SCAD on apoptosis of HUVEC. RESULTS: (1) The cell viability, the mRNA expression and the protein expression of SCAD were decreased gradually in a concentration and time dependent manner with the increase of tBHP concentration and the prolongation of intervention time. The decline was most significant in the group of the 50 µmol/L tBHP to interfere HUVEC for 12 hours. (2) The siRNA679 transfection was the most significant in reducing SCAD mRNA and protein expressions among the three interference sequences (siRNA274, siRNA414, siRNA679). (3) Compare with blank control group, the cell viability was significantly decreased in the siRNA679 group (A value: 0.48±0.09 vs. 1.00±0.09, P < 0.01), the apoptotic rate of HUVEC was significantly increased [(29.96±2.09)% vs. (2.90±1.90)%, P < 0.01], the expression of SCAD mRNA and SCAD protein, the activity of SCAD enzyme and the content of ATP were significantly decreased [SCAD mRNA (2-ΔΔCt): 0.50±0.16 vs. 1.34±0.12, SCAD/α-Tubulin: 0.67±0.11 vs. 1.00±0.06, the activity of SCAD enzyme (kU/g): 0.38±0.04 vs. 0.53±0.04, the content of ATP (µmol/g): 0.14±0.02 vs. 0.19±0.01, all P < 0.05], the contents of FFA and ROS were significantly increased [FFA (nmol/g): 0.84±0.07 vs. 0.47±0.04, ROS (average fluorescence intensity): 647.5±23.7 vs. 434.2±46.5, both P < 0.01]. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as HUVEC treated with tBHP. CONCLUSIONS: Down-regulation of SCAD may play an important role in HUVEC apoptosis. Increase in the expression of SCAD may become an important part in intervening HUVEC apoptosis.

[Change of short-chain acyl-CoA dehydrogenase in heart failure after myocardial infarction in rats and the intervention of aerobic exercise].

OBJECTIVE: To Study the changes of short-chain acyl-CoA dehydrogenase (SCAD) in heart failure (HF) after myocardial infarction (MI), and the effect of aerobic exercise on SCAD. METHODS: Healthy male Sprague-Dawley (SD) rats were divided into sham operation group (Sham group), sham operation swimming group (Sham+swim group), HF model group (LAD group) and HF swimming group (LAD+swim group) by random number table method, with 9 rats in each group. The left anterior descending branch of coronary artery (LAD) was ligated to establish a rat model of HF after MI. In Sham group, only one loose knot was threaded under the left coronary artery, and the rest operations were the same as those in LAD group. Rats in Sham+swim group and LAD+swim group were given swimming test for 1 week after operation (from 15 minutes on the 1st day to 60 minutes on the 5th day). Then they were given swimming endurance training (from the 2nd week onwards, 60 minutes daily, 6 times weekly, 10 weeks in a row). Tail artery systolic pressure (SBP) was measured before swimming endurance training and every 2 weeks until the end of the 10th week. Ten weeks after swimming training, echocardiography was performed to measure cardiac output (CO), stroke volume (SV), left ventricular ejection fraction (LVEF), shortening fraction (FS), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV). Morphological changes of heart were observed by Masson staining. Apoptosis of myocardial cells was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling stain (TUNEL) and apoptosis index (AI) was calculated. Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the mRNA and protein expression of myocardial SCAD respectively. In addition, the enzyme activity of SCAD, the content of adenosine triphosphate (ATP) and free fatty acid (FFA) in serum and myocardium were detected according to the kit instruction steps. RESULTS: Compared with Sham group, Sham+swim group showed SBP did not change significantly, with obvious eccentric hypertrophy and increased myocardial contractility, and LAD group showed persistent hypotension, obvious MI, thinning of left ventricle, and decreased myocardial systolic/diastolic function. Compared with LAD group, SBP, systolic/diastolic function and MI in LAD+swim group were significantly improved [SBP (mmHg, 1 mmHg = 0.133 kPa): 119.5±4.4 vs. 113.2±4.5 at 4 weeks, 120.3±4.0 vs. 106.5±3.7 at 6 weeks, 117.4±1.3 vs. 111.0±2.3 at 8 weeks, 126.1±1.6 vs. 119.4±1.9 at 10 weeks; CO (mL/min): 59.10±6.31 vs. 33.19±4.76, SV (µL): 139.42±17.32 vs. 84.02±14.26, LVEF: 0.523±0.039 vs. 0.309±0.011, FS: (28.17±2.57)% vs. (15.93±3.64)%, LVEDD (mm): 8.80±0.19 vs. 9.35±0.30, LVESD (mm): 5.90±0.77 vs. 7.97±0.60, LVEDV (µL): 426.57±20.84 vs. 476.24±25.18, LVESV (µL): 209.50±25.18 vs. 318.60±16.10; AI: (20.4±1.4)% vs. (31.2±4.6)%; all P < 0.05]. Compared with Sham group, the mRNA and protein expression of myocardium SCAD, the activity of SCAD in Sham+swim group were significantly increased, the content of ATP was slightly increased, the content of serum FFA was significantly decreased, and the content of myocardial FFA was slightly decreased; conversely, the mRNA and protein expression of myocardium SCAD, the activity of SCAD and the content of ATP in LAD group were significantly decreased, the content of serum and myocardial FFA were significantly increased. Compared with LAD group, the mRNA and protein expression of myocardium SCAD, the content of ATP were significantly increased in LAD+swim group [SCAD mRNA (2-ΔΔCt): 0.52±0.16 vs. 0.15±0.01, SCAD/GAPDH (fold increase from Sham group): 0.94±0.08 vs. 0.60±0.11, ATP content (µmol/g): 52.8±10.1 vs. 14.7±6.1, all P < 0.05], the content of serum and myocardial FFA were significantly decreased [serum FFA (nmol/L): 0.11±0.03 vs. 0.29±0.04, myocardial FFA (nmol/g): 32.7±8.2 vs. 59.7±10.7, both P < 0.05], and the activity of SCAD was slightly increased (kU/g: 12.3±4.3 vs. 8.9±5.8, P > 0.05). CONCLUSIONS: The expression of SCAD in HF was significantly down-regulated, and the expression was significantly up-regulated after aerobic exercise intervention, indicating that swimming may improve the severity of HF by up-regulating the expression of SCAD.

Effects of berberine on tumor growth and intestinal permeability in HCT116 tumor-bearing mice using polyamines as targets.

The prognosis of colorectal cancer (CRC) is seriously affected by high intestinal mucosal permeability accompanied by increasing tumor load. Berberine, a natural plant-derived product, can protect the intestinal mucosal barrier and suppress tumor growth, but its effects on the intestinal mucosal barrier dysfunction of CRC have not yet been evaluated. Herein, we assessed the effects of berberine on the intestinal mucosal permeability of HCT116 tumor-bearing mice and the underlying mechanism. Berberine (6.25, 12.5, 25 mg/kg) was administered to tumor-bearing mice for 3 weeks by intraperitoneal injection, and saline was given to controls and models. Compared with the control group, tumor-bearing mice had increased intestinal mucosal permeability in the third week. Meanwhile, the body weight decreased by 4%-7%, the concentration of D-lactic acid in plasma increased, and the expressions of ZO1 and Occludin were down-regulated. The intestinal mucosa was impaired. Compared with the model group, berberine inhibited tumor growth in a dose-dependent manner (6.25, 12.5, 25 mg/kg), reduced the permeability of intestinal mucosa, and alleviated intestinal mucosal damage. HPLC showed that berberine decreased the content of polyamines in tumor tissue, whereas increased that in intestinal mucosa tissue. Western blot showed that berberine inhibited the expressions of ODC, C-MYC and HIF-1α, but up-regulated those of OAZ1 and SSAT. In short, berberine may exert antitumor effects by suppressing tumor growth and elevating the intestinal mucosal permeability.

Downregulation of Glutathione S-transferase A1 suppressed tumor growth and induced cell apoptosis in A549 cell line.

Glutathione S-transferase A1 (GSTA1) is a phase II detoxification enzyme and serves a crucial role in anti-cancer drug resistance. In our previous study, GSTA1 was identified to be highly expressed in various subtypes of non-small-cell lung cancer cell lines compared with human embryonic lung fibroblast cell line MRC-5. The aim of the present study was to investigate the effect of GSTA1 expression on the proliferation and apoptosis of A549 cells. GSTA1 expression was knocked down or with overexpressed using lentivirus particles. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the protein, and mRNA levels of GSTA1 in A549 cells, respectively. The effect of GSTA1 manipulation on cell proliferation and apoptosis were investigated in vitro using MTT assays, Hoechst 33258 staining and flow cytometry, and in vivo using A549 cell line xenografts in nude mice. The results of the western blot analysis and RT-qPCR revealed that stable cell models of GSTA1 knockdown, and overexpression were established. The data of the MTT assay indicated that the downregulation of GSTA1 significantly inhibited cell proliferation compared with si-control-transfected cells. These si-GSTA1 A549 cells exhibited typical morphological changes of apoptosis, including chromatin condensation and shrunken nuclei compared with the si-control counterparts. An AnnexinV-fluorescein isothiocyanate assay verified that the downregulation of GSTA1 significantly induced cell apoptosis in vitro. In addition, overexpression of GSTA1 significantly promoted tumor growth in vivo. Accordingly, downregulation of GSTA1 suppressed tumor growth. In conclusion, GSTA1 plays an important role in regulation of cell proliferation and cell apoptosis in A549 cell line.

Curcumol inhibits the proliferation of gastric adenocarcinoma MGC-803 cells via downregulation of IDH1.

Curcumol, a polyphenol compound derived from the rhizome of Curcuma, has been established as an antitumor compound against multiple types of cancer, including gastric (GC), lung, liver and breast cancer. However, the molecular mechanisms undelying its anticancer activity in GC are still unclear. In this study, the antitumor efficacy of curcumol was ascertained in human gastric adenocarcinoma MGC-803 cells. An MTT assay was used to assess the viability of the MGC-803 cells treated by curcumol. The results of the Annexin V/propidium iodide (PI) staining followed by fluorescence activated cell sorting (FACS) analysis demonstrated that the cell cycle was arrested in the G2/M phase by curcumol. Annexin V-FITC/PI double staining followed by FACS analysis revealed that curcumol induced apoptosis of MGC-803 cells. FACS analysis after the cells were loaded with a DFCH-DA probe revealed that the level of reactive oxygen species (ROS) increased after the cells were treated with curcumol. In adittion, FACS analysis after the cells were loaded with JC-1 revealed that the level of mitochondrial membrane potential (MMP) decreased after the cells were treated with curcumol. Furthermore, the downregulation of isocitrate dehydrogenase 1 (IDH1) was observed in the MGC-803 cells after being treated with curcumol as determined by western blotting and RT-qPCR. In conclusion, we elucidated the antitumor effect of curcumol on MGC-803 cells and the involved mechanisms related to the induction of apoptosis, the increase of ROS, the decrease of MMP and the downregulation of IDH1.

Apigenin Inhibits Human SW620 Cell Growth by Targeting Polyamine Catabolism.

Apigenin is a nonmutagenic flavonoid that has antitumor properties. Polyamines are ubiquitous cellular polycations, which play an important role in the proliferation and differentiation of cancer cells. Highly regulated pathways control the biosynthesis and degradation of polyamines. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the metabolism, and spermidine/spermine-N1-Acetyl transferase (SSAT) is the rate-limiting enzyme in the catabolism of polyamines. In the current study, the effect of increasing concentrations of apigenin on polyamine levels, ODC and SSAT protein expression, mRNA expression, cell proliferation and apoptosis, and the production of reactive oxygen species (ROS) was investigated in SW620 colon cancer cells. The results showed that apigenin significantly reduced cell proliferation, decreased the levels of spermidine and spermine, and increased previously downregulated putrescine contents. Apigenin also enhanced SSAT protein and mRNA levels and the production of reactive oxygen species in SW620 cells, though it had no significant effect on the levels of ODC protein or mRNA. Apigenin appears to decrease the proliferation rate of human SW620 cells by facilitating SSAT expression to induce polyamine catabolism and increasing ROS levels to induce cell apoptosis.

Cytotoxic and Antitumor Effects of Curzerene from Curcuma longa.

Curzerene is a sesquiterpene and component used in oriental medicine. It was originally isolated from the traditional Chinese herbal medicine Curcuma rhizomes. In this study, anticancer activity of curzerene was examined in both in vitro and in vivo models. The result of the MTT assay showed that curzerene exhibited antiproliferative effects in SPC-A1 human lung adenocarcinoma cells in a time-dependent and dose-dependent manner. The anticancer IC50s were 403.8, 154.8, and 47.0 µM for 24, 48, and 72 hours, respectively. The flow cytometry analysis indicated curzerene arrested the cells in the G2/M cell cycle and promoted or induced apoptosis of SPC-A1 cells. The percentage of cells arrested in the G2/M phase increased from 9.26 % in the control group cells to 17.57 % in the cells treated with the highest dose (100 µM) of curzerene. Western blot and RT-PCR analysis demonstrated that curzerene induced the downregulation of GSTA1 protein and mRNA expressions in SPC-A1 cells. Tumor growth was significantly inhibited in SPC-A1 cell-bearing nude mice by using curzerene (135 mg/kg daily), meanwhile, curzerene did not significantly affect body mass and the organs of the mice, which may indicate that curzerene has limited toxicity and side effects in vivo. In conclusion, curzerene could inhibit the proliferation of SPC-A1 human lung adenocarcinoma cells line in both in vitro and in vivo models. Focusing on its relationship with GSTA1, curzerene could induce the downregulation of GSTA1 protein and mRNA expressions in SPC-A1 cells. Curzerene might be used as an anti-lung adenocarcinoma drug candidate compound for further development.

Effects of short-chain acyl-CoA dehydrogenase on cardiomyocyte apoptosis.

Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid ß-oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert-butyl hydroperoxide (tBHP)-induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down-regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA-1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl-2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP-activated protein kinase α (p-AMPKα) and Peroxisome proliferator-activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways.

Absorption mechanism of oxymatrine in cultured Madin-Darby canine kidney cell monolayers.

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin-Darby canine kidney (MDCK) cell monolayers. Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0 × 10(-5)-1.0 × 10(-3) M in 2 h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10(-4) M was studied. Moreover, Papp of OMT was determined in the absence/presence of cyclosporine and surfactants at 100 µM to further confirm the relative transport mechanism. Results The Papp AP→BL ranged from (3.040 ± 0.23) × 10(-6) to (3.697 ± 0.19) × 10(-6 )cm/s as the concentration varied from 10(-5) to 10(-3) M. OMT showed similar Papp at 4 and 37 °C (p > 0.05). Increasing the apical pH 7.4 and 8.0 resulted in Papp versus pH 5.0 (p < 0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, Papp was (0.318 ± 0.033) × 10(-5), (0.464 ± 0.048) × 10(-5), (0.897 ± 0.115) × 10(-5) and (1.341 ± 0.122) × 10(-5 )cm/s, respectively. In the presence of surfactants, Papp significantly increased up to 1.5-4.3-fold (p < 0.05). Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.

Simultaneous determination of thirteen flavonoids from Xiaobuxin-Tang extract using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry.

A simple and reliable high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis method was established to simultaneously determine thirteen flavonoids of Xiaobuxing-Tang in intestine perfusate, namely onpordin, 3'-O-methylorobol, glycitein, patuletin, genistein, luteolin, quercetin, nepitrin, quercimeritrin, daidzin, patulitrin, quercetagitrin and 3-glucosylisorhamnetin. Detection was performed on a quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ionization mode. Negative ion ESI was used to form deprotonated molecules at m/z 315 for onpordin, m/z 299 for 3'-O-methylorobol, m/z 283 for glycitein, m/z 331 for patuletin, m/z 269 for genistein, m/z 285 for luteolin, m/z 301 for quercetin, m/z 477 for nepitrin, m/z 463 for quercimeritrin, m/z 461 for daidzin, m/z 493 for patulitrin, m/z 479 for quercetagitrin, m/z 477 for 3-glucosylisorhamnetin and m/z 609.2 for rutin. The linearity, sensitivity, selectivity, repeatability, accuracy, precision, recovery and matrix effect of the assay were evaluated. The proposed method was successfully applied to simultaneous determination of these thirteen flavonoids, and using this method, the intestinal absorption profiles of thirteen flavonoids were preliminarily predicted.

Curcumol induces apoptosis in SPC-A-1 human lung adenocarcinoma cells and displays anti-neoplastic effects in tumor bearing mice.

Curcumol is a sesquiterpene originally isolated from curcuma rhizomes, a component of herbal remedies commonly used in oriental medicine. Its beneficial pharmacological activities have attract significant interest recently. In this study, anti-cancer activity of curcumol was examined with both in vitro and in vivo models. It was found that curcumol exhibited time- and concentration-dependent anti-proliferative effects in SPC-A-1 human lung adenocarcinoma cells with cell cycle arrest in the G0/G1 phase while apoptosis-induction was also confirmed with flow cytometry and morphological analyses. Interestingly, curcumol did not display growth inhibition in MRC-5 human embryonic lung fibroblasts, suggesting the anti-proliferative effects of curcumol were specific to cancer cells. Anti-neoplastic effects of curcumol were also confirmed in tumor bearing mice. Curcumol (60 mg/kg daily) significantly reduced tumor size without causing notable toxicity. In conclusion, curcumol appears a favorable anti-cancer candidate for further development.

Effects of ERK1/2/PPARα/SCAD signal pathways on cardiomyocyte hypertrophy induced by insulin-like growth factor 1 and phenylephrine.

AIMS: Short-chain acyl-CoA dehydrogenase (SCAD) is a key enzyme in fatty acid oxidation. In the present study we aim to investigate the changes in SCAD between pathological and physiological cardiomyocyte hypertrophy. We also explore the different signaling pathways of pathological and physiological cardiomyocyte hypertrophy. MAIN METHODS: After neonatal rat cardiomyocytes were treated as setups, cell surface area, expression of SCAD, PPARα, phospho-ERK1/2, activity of SCAD, free fatty acid content and ATP content in the cardiomyocytes were measured. KEY FINDINGS: Neonatal rat cardiomyocytes treated by PE showed an increased cell surface area and free fatty acid content, increased ERK1/2 phosphorylation, decreased expression of PPARα, decreased expression and activity of SCAD and decreased levels of ATP. Neonatal rat cardiomyocytes treated by IGF-1 showed the reverse effects except for the cell surface area. PPARα inhibitor GW6471 and PPARα activator Fenofibrate treatments abrogated the effects induced by IGF-1 and PE in cardiomyocytes respectively, as well as ERK1/2 activator EGF and ERK1/2 inhibitor PD98059. SIGNIFICANCE: SCAD has different changes between pathological and physiological cardiomyocyte hypertrophy. The ERK1/2/PPARα/SCAD signaling pathways play different roles in pathological and physiological cardiomyocyte hypertrophy. SCAD may be used as a new target to prevent the development of pathological cardiac hypertrophy.

The effect of acori graminei rhizoma and extract fractions on spatial memory and hippocampal neurogenesis in amyloid beta 1-42 injected mice.

Acori graminei Rhizoma (AGR), the dry rhizoma of Acorus gramineus Soland (Araceae), has been used as an Asian traditional herbal medicine against senile dementia, stroke, and cardiovascular disease. Previous studies have revealed neuroprotective effects of AGR on neuronal damage and learning impairment, while mostly focused on the effect of volatile oil fraction of AGR. This study aimed to investigate the neuroprotective effects of different extract fractions from AGR against Alzheimer disease-like symptoms induced by Amyloid Beta (Aß) 1-42 intra-hippocampal injection. On day 7 after intra-hippocampal injection of saline or Aß1-42, spatial memory was assessed by the first Morris water maze, followed by 3-week intra-gastric administration of saline or water extract, volatile oil fraction, or defatted decoction fraction of AGR respectively. Mice were subsequently subjected to the second Morris water maze task. Levels of Aß1-42 and expressions of doublecortin and nestin in the hippocampus were examined using immunohistochemistry. Our results suggested that treatment with these different extract fractions from AGR could ameliorate cognitive impairment and down-regulate expressions of doublecortin and nestin in the hippocampus of Aß1-42 injected mice, in which water extract and volatile oil fractions were more effective in spatial memory than defatted decoction fraction.

Expression and function of GSTA1 in lung cancer cells.

Glutathione S-transferase A1 (GSTA1) appears to be primarily involved in detoxification processes, but possible roles in lung cancer remain unclear. The objective of this study was to investigate the expression and function of GSTA1 in lung cancer cells. Real-time PCR and Western blotting were performed to assess expression in cancer cell lines and the normal lung cells, then verify the A549 cells line with stable overexpression. Localization of GSTA1 proteins was assessed by cytoimmunofluorescence. Three double-strand DNA oligoRNAs (SiRNAs) were synthesized prior to being transfected into A549 cells with Lipofectamine 2000, and then the most efficient SiRNA was selected. Expression of the GSTA1 gene in the transfected cells was determined by real-time PCR and Western blotting. The viability of the transfected cells were assessed by MTT. Results showed that the mRNA and protein expression of A549 cancer cells was higher than in MRC-5 normal cells. Cytoimmunofluorescence demonstrated GSTA1 localization in the cell cytoplasm and/or membranes. Transfection into A549 cells demonstrated that down-regulated expression could inhibit cell viability. Our data indicated that GSTA1 expression may be a target molecule in early diagnosis and treatment of lung cancer.