Structural damage in the C. elegans epidermis causes release of STA-2 and induction of an innate immune response.

The epidermis constantly encounters invasions that disrupt its architecture, yet whether the epidermal immune system utilizes damaged structures as danger signals to activate self-defense is unclear. Here, we used a C. elegans epidermis model in which skin-penetrating infection or injury activates immune defense and antimicrobial peptide (AMP) production. By systemically disrupting each architectural component, we found that only disturbance of the apical hemidesmosomes triggered an immune response and robust AMP expression. The epidermis recognized structural damage through hemidesmosomes associated with a STAT-like protein, whose disruption led to detachment of STA-2 molecules from hemidesmosomes and transcription of AMPs. This machinery enabled the epidermis to bypass certain signaling amplification and directly trigger AMP production when subjected to extensive architectural damage. Together, our findings uncover an evolutionarily conserved mechanism for the epithelial barriers to detect danger and activate immune defense.

Engineering Escherichia coli native metabolism for efficient biosynthesis of orotate.

Orotate (OA) is a precursor of pyrimidine nucleotides and is widely used in food, pharmaceutical, and cosmetic industries. Although various microorganisms have been used for OA production, the production efficiency needs to be further improved for industrial application. In this study, we engineered Escherichia coli native metabolism for efficient OA production. The entire pathway was divided into the downstream OA synthesis, the midstream aspartate/glutamine supply, and the upstream glycolysis modules. First, the downstream module was optimized by disrupting pyrE to block OA consumption and release the feedback inhibition, and tuning expression of the biosynthetic genes. Second, the midstream pathway was enhanced by increasing the supply of the precursors and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). More importantly, we observed that pyrE disruption may lead to metabolic disorder as indicated by the accumulation of large amount of acetate. This problem was solved by reducing the flux of glycolysis. With these efforts, the final strain produced 80.3 g/L OA with a yield of 0.56 g/g glucose in fed-batch fermentation, which are the highest titer and yield reported so far. This work paves the way for industrial production of OA and represents as a good example of modulating cell metabolism for efficient chemical production.

DMC (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone) enhances exercise tolerance via the AMPK-SIRT1-PGC-1α pathway in mice fed a high-fat diet.

Obesity is caused by an imbalance between energy intake and energy expenditure. This study aimed to determine the effects and mechanisms of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) on exercise tolerance in high-fat diet (HFD)-fed mice. Male C57BL/6J mice were randomly divided into two categories (7 groups [n = 8]): sedentary (control [CON], HFD, 200 mg/kg DMC, and 500 mg/kg DMC) and swimming (HFD, 200 mg/kg DMC, and 500 mg/kg DMC). Except the CON group, all other groups were fed HFD with or without DMC intervention for 33 days. The swimming groups were subjected to exhaustive swimming (three sessions/week). Changes in swimming time, glucolipid metabolism, body composition, biochemical indicators, histopathology, inflammation, metabolic mediators, and protein expression were assessed. DMC combined with regular exercise improved endurance performance, body composition, glucose and insulin tolerance, lipid profile, and the inflammatory state in a dose-dependent manner. Further, DMC alone or combined with exercise could restore normal tissue morphology, reduce fatigue-associated markers, and boost whole-body metabolism and the protein expression of phospho-AMP-activated protein kinase alpha/total-AMP-activated protein kinase alpha (AMPK), sirtuin-1 (SIRT1), peroxisome-proliferator-activated receptor gamma coactivator 1alpha (PGC-1α), and peroxisome proliferator-activated receptor alpha in the muscle and adipose tissues of HFD-fed mice. DMC exhibits antifatigue effects by regulating glucolipid catabolism, inflammation, and energy homeostasis. Furthermore, DMC exerts a synergistic exercise-related metabolic effect via the AMPK-SIRT1-PGC-1α signaling pathway, suggesting that DMC is a potential natural sports supplement with mimicked or augmented exercise effects for obesity prevention.

Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid.

Ferulic acid (FA) is a natural methylated phenolic acid which represents various bioactivities. Bioproduction of FA suffers from insufficient methyl donor supplement and inefficient hydroxylation. To overcome these hurdles, we first activate the S-adenosylmethionine (SAM) cycle in E. coli by using endogenous genes to supply sufficient methyl donor. Then, a small protein Fre is introduced into the pathway to efficiently regenerate FADH2 for the hydroxylation. Remarkably, regeneration of these two cofactors dramatically promotes FA synthesis. Together with decreasing the byproducts formation and boosting precursor supply, the titer of FA reaches 5.09 g/L under fed-batch conditions, indicating a 20-fold improvement compared with the original producing E. coli strain. This work not only establishes a promising microbial platform for industrial level production of FA and its derivatives, but also highlights a convenient and effective strategy to enhance the biosynthesis of chemicals requiring methylation and FADH2-dependent hydroxylation.

Shikonin ameliorates oxidative stress and neuroinflammation via the Akt/ERK/JNK/NF-κB signalling pathways in a model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Shikonin plays protective roles in age-associated diseases. Therefore, we investigate the biological functions of shikonin and its mechanisms involved in PD pathogenesis. The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to mimic PD-like conditions in animal models. The learning and memory capacities were assessed by Morris water-maze test, pole test, locomotor activity test and rotarod test. Neuroinflammation was determined by measuring the levels of tumour necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The quantification of superoxide dismutase, malondialdehyde and glutathione in substantia nigra was performed to estimate oxidative damage. Histopathologic changes were examined by haematoxylin and eosin staining. Immunofluorescence staining was conducted to determine the activation of astrocytes, tyrosine hydroxylase (TH)-positive neurons, and nuclear translocation of p65. Immunohistochemistry was performed to evaluate dopamine transporter (DAT)-positive neurons. Protein levels were measured by western blotting. Shikonin alleviates the cognitive and behavioural impairments. The death of dopaminergic neurons in nigra was attenuated by shikonin. The MPTP-induced neuroinflammation and oxidative stress in substantia nigra were alleviated by shikonin administration. Shikonin ameliorated the neuronal damage in nigra and inhibited the activation of astrocyte. Shikonin modulated the protein kinase B (Akt)/extracellular regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/nuclear factor κB (NF-κB) pathways. Shikonin ameliorates dopaminergic neuronal apoptosis by inhibiting oxidative stress and neuroinflammation via the Akt/ERK/JNK/NF-κB pathways in PD. The study has several limitations. First, in a previous study, levels of phosphorylated ERK were increased by MPTP. In our current study, we observed decreased p-ERK in nigra following MPTP treatment. Therefore, further investigation in the mechanisms of shikonin against PD progression is required. Second, the biological functions of shikonin need more exploration, including mitochondrial function and autophagy. Moreover, specific molecular targets for shikonin remain uncertain.

Mulberry Leaf Extract Alleviates Staphylococcus aureus-Induced Conjunctivitis in Rabbits via Downregulation of NLRP3 Inflammasome and Upregulation of the Nrf2 System and Suppression of Pro-Inflammatory Cytokines.

INTRODUCTION: Mulberry (Morus alba L.) leaves are widely used in traditional Chinese medicine for their antioxidant, anti-inflammatory, antibacterial, anti-obesity, antidiabetic, antiatherosclerotic, and anticancer properties. The current study aimed to investigate the effect of mulberry leaf extract (MLE) on Staphylococcus aureus (S. aureus)-induced conjunctivitis (5 × 109 colony-forming units, 0.5 mL/eye) in a rabbit model. METHODS: Rabbits were treated with MLE (5 mL/kg·d-1 and 10 mL/kg·d-1), 0.9% saline, pearl bright eye (PBE) drops, or erythromycin eye ointment (EEO) group for 5 days. The ocular infection symptoms, bacterial negative conversion rate, and conjunctival histopathological changes of rabbits in each group were observed. The expression of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, NOD-like receptor leucine-rich pyrin domain-containing protein 3 (NLRP3), interleukin (IL)-18, IL-6, IL-1ß, TNFα, Keap1, and nuclear factor erythroid 2-related factor 2 (Nrf2) in conjunctival tissue of rabbits were detected by quantitative real-time reverse transcription PCR and/or Western blot analysis. RESULTS: The results showed that MLE treatment significantly reduced the clinical sign scores of conjunctivitis, alleviated clinical signs, and decreased bacterial load, and histological damage in a time- and dose-dependent manner was compared to that in the control group. The antibacterial and anti-inflammatory activities of MLE (10 mL/kg·d-1) were similar to those of the positive control drug PBE and EEO. In addition, MLE significantly decreased the levels of pro-inflammatory cytokines, downregulated the NLRP3 inflammasome, and upregulated the Nrf2 system. CONCLUSIONS: MLE is effective in alleviating S. aureus-induced conjunctivitis in rabbits, and this mechanism is associated with the inhibition of the NLRP3 inflammasome and activation of the Nrf2 system to regulate pro-inflammatory signaling.

Screening of Bletilla striata, Bletilla ochracea and Oreorchis foliosa differential metabolites based on metabolomics.

As a representative medicinal plant in the Orchidaceae, Bletilla striata plays a variety of pharmacological roles in the clinic. However, the emergence of counterfeit species is affecting the basic medicinal materials source identification process, for which Bletilla ochracea and Oreorchis foliosa of the Orchidaceae are two representative species. For this study, 13 representative B. striata samples, three B. ochracea samples and three O. foliosa samples were selected for the systematic determination of polysaccharide yields and monosaccharide composition, and further detection of secondary metabolites by HPLC-MS. The results revealed that there was a significant difference in the yields of polysaccharides between B. striata and B. ochracea (p = 0.006). Although the polysaccharides of both species were composed of glucose and mannose, the molar ratio of the two monosaccharides was different, suggesting that the structures of the polysaccharides were different. The metabolomics results showed that there were no differences in the types of metabolites between B. striata and B. ochracea; however, there were differences in the contents of these metabolites. Although there was no significant difference in the polysaccharide yields of B. striata and O. foliosa (p = 0.074) and the monosaccharide composition was the same (glucose and mannose), many different metabolites were screened out between them: six compounds such as C36 H34 O11 existed only in B. striata, while substance C39 H54 O22 was unique to O. foliosa. Therefore, based on the analysis of the polysaccharide content and monosaccharide composition, combined with phase metabolomics research, a preliminary distinction between B. striata, B. ochracea and O. foliosa was achieved.

Identification and Characterization of Jasmonic Acid Biosynthetic Genes in Salvia miltiorrhiza Bunge.

Jasmonic acid (JA) is a vital plant hormone that performs a variety of critical functions for plants. Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as Danshen, is a renowned traditional Chinese medicinal herb. However, no thorough and systematic analysis of JA biosynthesis genes in S. miltiorrhiza exists. Through genome-wide prediction and molecular cloning, 23 candidate genes related to JA biosynthesis were identified in S. miltiorrhiza. These genes belong to four families that encode lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-OPDA reductase3 (OPR3). It was discovered that the candidate genes for JA synthesis of S. miltiorrhiza were distinct and conserved, in contrast to related genes in other plants, by evaluating their genetic structures, protein characteristics, and phylogenetic trees. These genes displayed tissue-specific expression patterns concerning to methyl jasmonate (MeJA) and wound tests. Overall, the results of this study provide valuable information for elucidating the JA biosynthesis pathway in S. miltiorrhiza by comprehensive and methodical examination.

Integrative Effects and Vagal Mechanisms of Transcutaneous Electrical Acustimulation on Gastroesophageal Motility in Patients With Gastroesophageal Reflux Disease.

INTRODUCTION: Impaired esophageal and gastric motilities are known to contribute to symptoms of gastroesophageal reflux disease (GERD). However, there is a lack of GERD therapy, targeting both gastric and esophageal functions. This study was designed to investigate the effects of transcutaneous electrical acustimulation (TEA) on symptoms of GERD and gastroesophageal functions and possible mechanisms in patients with GERD. METHODS: Thirty patients with GERD with ineffective esophageal motility were equally divided and randomized into a 4-week sham-TEA or 4-week TEA treatment. The GERD questionnaire (GerdQ), GERD health-related quality-of-life questionnaire, high-resolution esophageal manometry, a nutrient drink test, the electrogastrogram, and ECG were performed to assess the severity of reflux symptoms, low esophageal sphincter (LES) pressure, distal contractile integral (DCI), gastric accommodation, gastric slow waves (GSW), and autonomic functions, respectively. RESULTS: Compared with sham-TEA, the 4-week TEA treatment significantly decreased the GerdQ score (P = 0.011) and GERD health-related quality of life (P = 0.028) and improved nutrient drink-induced fullness (P < 0.001) and belching (P < 0.001) in patients with GERD. Although only acute TEA significantly enhanced LES pressure (P < 0.05), both acute and chronic TEA remarkedly increased DCI (P < 0.05) and reduced the incidence of ineffective esophageal contractions during wet swallows (P = 0.02). In addition, chronic TEA significantly increased gastric accommodation and the percentage of postprandial normal GSW compared with sham-TEA and baseline. Concurrently, TEA-enhanced vagal activity (P = 0.02) and the vagal activity positively correlated with LES pressure (r = 0.528; P = 0.003) and DCI (r = 0.522; P = 0.003). DISCUSSION: The TEA treatment performed in this study improves reflux-related symptoms, increases DCI, reduces the incidence of ineffective esophageal contractions during wet swallows, and improves gastric accommodation and slow waves. The improvement in GERD symptoms might be attributed to the integrative effects of TEA on these gastroesophageal functions mediated via the vagal mechanism.

HDL-C to hsCRP ratio is associated with left ventricular diastolic function in absence of significant coronary atherosclerosis.

BACKGROUND: High-density lipoprotein cholesterol (HDL-C) is considered as a protective marker of coronary atherosclerotic disease (CAD). It is still not clear if HDL-C is associated with left ventricular (LV) diastolic function in an inflammation-related manner in absence of significant coronary atherosclerosis. METHODS: 392 patients who complained of chest pain and were suspected of CAD without heart failure were enrolled in this study. Coronary angiography or coronary artery CT scan was performed to detect coronary atherosclerosis. Transthoracic echocardiography was performed to evaluate cardiac function. Plasma level of HDL-C and high-sensitive C-reactive protein (hsCRP) were determined in each subject. Relationship between HDL-C/hsCRP ratio and LV diastolic function in subjects without significant coronary atherosclerosis was investigated. RESULTS: 204 subjects without significant coronary plaques were analyzed finally, including 84 males and 120 females whose ages ranged from 30 to 84 years old. When divided into HDL-C/hsCRP quartiles, those in the fourth quartile demonstrated the best diastolic function (E/e' 10.14 ± 2.87, P = 0.02 ). HDL-C/hsCRP was the most significant factor correlated with E/e' in univariate regression analysis (r = - 0.232, P < 0.001) and multiple regression analysis adjusted by other factors (standardized ß = - 0.258 , P < 0.0005 ). In logistic regression, HDL-C/hsCRP was proved to be a protective factor of LV diastolic dysfunction E/e' > 14 (OR = 0.649, 95%CI 0.444-0.948,P = 0.025 ). The sensitivity and specificity of using HDL-C/hsCRP < 0.98 to predict LV diastolic dysfunction were 64.3% and 56.2%, respectively. HDL-C/hsCRP ratio presented a reduced trend as increasing rate of CV risk factors. CONCLUSIONS: HDL-C/hsCRP ratio strongly correlates with LV diastolic function in absence of significant coronary atherosclerosis. Low HDL-C/hsCRP ratio tends to relate with LV diastolic dysfunction.

Synergistic effect of phytochemicals on cholesterol metabolism and lipid accumulation in HepG2 cells.

BACKGROUND: Crocin (CRO), chlorogenic acid (CGA), geniposide (GEN), and quercetin (QUE) are all natural compounds with anti-obesity properties, in particular, hypolipidemic effects, which have been widely used for the treatment of obesity-related metabolic diseases. However, it is not yet known whether these compounds interact synergistically. Here, we investigated the effects and molecular mechanisms of CRO, CGA, GEN, QUE, and a combination of all four compounds (CCGQ), on lipid accumulation in human hepatoma (HepG2 cells). METHODS: The optimal concentration of CRO, CGA, GEN, QUE to stimulate HepG2 cells proliferation was determined using MTT assay. HepG2 cells were pretreated with 10 µmol/L simvastatin, 1 µmol/L CRO, 30 µmol/L CGA, 10 µmol/L GEN, 10 µmol/L QUE, and CCGQ (a combination of 1 µmol/L CRO, 30 µmol/L CGA, 10 µmol/L GEN, and 10 µmol/L QUE) for 24 or 48 h. Oil red O staining and extracellular TC and TG levels were detected. The RT-PCR was used to observe on cholesterol metabolism-related gene expression. Immunocytochemistry and western-blot assayed the 3-hydroxy-3-methylglutaryl-coenzyme (HMGCR) protein expression in HepG2 cells. RESULTS: Compared to those of control, we demonstrated that treating HepG2 cells for 48 h with CCGQ resulted in a strong synergistic effect, causing a marked decrease in lipid deposition in comparison to individual treatments, in both triglyceride and total cholesterol (CRO, 5.74- and 1.49-folds; CGA, 3.38- and 1.12-folds; GEN, 4.04- and 1.44-folds; QUE, 3.36- and 1.24-folds; simvastatin, 5.49- and 1.83-folds; and CCGQ, 7.75- and 2.20-folds), and Oil red O staining assays. In addition, CCGQ treatment increased ATP-binding cassette transporter (ABCA1), cholesterol 7α-hydroxylase (CYP7A1), and AMP-activated protein kinase 2α (AMPKα2) mRNA expression, while decreasing sterol regulatory element binding protein 2 (SREBP2), and liver X receptor alpha (LXRα) mRNA expression. Notably, CCGQ was more effective in decreasing HMGCR expression than the individual treatments. CONCLUSION: The CCGQ combination has potential, both as a complementary therapy for hyperlipemia, and in preventing further obesity-related complications.

Improvement of Trehalose Production by Immobilized Trehalose Synthase from Thermus thermophilus HB27.

Trehalose is a non-reducing disaccharide with a wide range of applications in the fields of food, cosmetics, and pharmaceuticals. In this study, trehalose synthase derived from Thermus thermophilus HB27 (TtTreS) was immobilized on silicalite-1-based material for trehalose production. The activity and the stability of TtTreS against pH and temperature were significantly improved by immobilization. Enzyme immobilization also led to a lower concentration of byproduct glucose, which reduces byproduct inhibition of TtTreS. The immobilized TtTreS still retained 81% of its initial trehalose yield after 22 cycles of enzymatic reactions. The immobilized TtTreS exhibited high operational stability and remarkable reusability, indicating that it is promising for industrial applications.

The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen-glucose deprivation-induced injury.

CONTEXT: The effects of icariin, a chief constituent of flavonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown. OBJECTIVE: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability. MATERIALS AND METHODS: PC12 cells were treated with icariin (10-7, 10-6 or 10-5 mol/L) for 3 h (1 h before oxygen-glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3 ± 1.9 ng/L, HSP-60: 199 ± 16 ng/L, HSP-70: 195 ± 17 ng/L, NSE: 1487 ± 125 ng/L), and cell viability was significantly decreased (0.26 ± 0.03), while icariin (10-7, 10-6, or 10-5 mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1 ± 1.4, 22.6 ± 1.8, 15.7 ± 2.1, HSP-60: 100 ± 12, 89 ± 6, 113 ± 11, HSP-70: 139 ± 9, 118 ± 7, 95 ± 9 and NSE: 1121 ± 80, 1019 ± 52, 731 ± 88), and improved cell viability (0.36 ± 0.03, 0.38 ± 0.04, 0.37 ± 0.03) in OGD-treated PC12 cells. DISCUSSION AND CONCLUSION: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.

Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp.

Inhibition of TLR4 signaling by Brucella TIR-containing protein TcpB-derived decoy peptides.

Brucella spp. avoid host immune recognition and thus, weaken the immune response to infection. The Toll/interleukin-1 receptor (TIR) domain-containing protein (TcpB/Btp1) of Brucella spp. is thought to be involved in blocking host innate immune responses by binding to adaptors downstream of Toll-like receptors. In this study, based on the observation that TcpB binds to the host target proteins, MAL, through the TIR domain, we examined decoy peptides from TcpB TIR domains and found that TB-8 and TB-9 substantially inhibit lipopolysaccharide (LPS)-induced signaling in vitro and in vivo. Both these peptides share a common loop, the DD loop, indicating a novel structural region mediating TIR interactions. The inhibition of LPS signaling by TB-8 and TB-9 shows no preference to MyD88-dependent cytokines, such as TNF-α and IL-1ß or TRIF-dependent cytokines including IFN-ß and IL-6. Furthermore, these two peptides rescue the virulence of Brucella ΔtcpB mutants at the cellular level, indicating key roles of the DD loop in Brucella pathogenesis. In conclusion, identification of inhibitors from the bacterial TIR domains is helpful not only for illustrating interacting mechanisms between TIR domains and bacterial pathogenesis, but also for developing novel signaling inhibitors and therapeutics for human inflammatory diseases.

Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells.

CONTEXT: Eucommia ulmoides Oliver (Eucommiaceae) leaf exhibits beneficial lipid-lowering and anti-obesity effects. However, the mechanisms remain unknown. OBJECTIVE: The objective of this study is to investigate the lipid-lowering effects of chlorogenic acid (CGA)-enriched extract from this plant (CAEF) in human hepatoma HepG2 cells, focusing on cholesterol metabolism. MATERIALS AND METHODS: HepG2 cells were treated with CAEF (10, 20, 25, 40, 60, and 80 mg/L), CGA (0.3, 3, 30, 300, and 600 µmol/L), and simvastatin (0.1, 1, 10, 50, and 100 µmol/L) for 24 or 48 h. The cytotoxicity, Oil red O staining, total cholesterol, and triacylglycerol in supernatants were determined. The mRNA expression of genes involved in cholesterol metabolism was determined with RT-PCR. The protein expression of HMG-CoA reductase (HMGCR) was examined by immunocytochemistry and western-blot. RESULTS: The IC50 values were 59.2 mg/L for CAEF, 335.9 µmol/L for CGA, and 10.5 µmol/L for simvastatin. By treating cells with CAEF (25 mg/L), CGA (30 µmol/L), or simvastatin (10 µmol/L) for 48 h, the efflux of total cholesterol and triacylglycerol was increased (CAEF, 4.06- and 31.00-folds; CGA, 2.94- and 2.17-folds; and simvastatin, 3.94- and 24.67-folds), and the cellular lipid droplets were reduced in Oil red O staining. CAEF and CGA increased mRNA expression of ABCA1, CYP7A1, and AMPKα2, while CAEF and simvastatin decreased SREBP2. However, their effects on LXRα mRNA expression were variable. Importantly, all drugs significantly inhibited protein expression of HMGCR at mRNA and protein levels. DISCUSSION AND CONCLUSION: CAEF is a promising dietary supplement to prevent obesity and dyslipidemia and the effects appear to be due, at least in part, to regulating cholesterol metabolism through inhibition of HMGCR in HepG2 cells.

The relationship between mean arterial pressure and decreased glomerular filtration rate in rural areas of Northeast China.

BACKGROUND: Low mean arterial pressure (MAP) can cause low renal blood flow and damage the kidneys. However, in the general population, it remains unclear whether or not decline in renal function is related to MAP. The present study examined the relationship between MAP and decreased glomerular filtration rate(GFR) in participants aged ≥ 35 years from the Liaoning province of China. METHODS: A total of 11345 representative individuals aged ≥ 5 years was selected and a cross-sectional survey was conducted from January 2012 to August 2013 to describe the gender-specific prevalence and factors associated with decreased GFR in rural areas of Liaoning Province. RESULTS: Men with decreased eGFR (eGFR < 60 ml/min per 1.73 m(2)) were older, and had higher meanWC, systolic and diastolic BP, PP, MAP, total fasting glucose, LDL-C ,glyceride and uric acid levels and were current drinker/smoker at the baseline (all P < 0.05). Those with low education level, low income, low physical activity, low hemoglobin and HDL-C level had decreased eGFR (all P < 0.05). In women, the results were similar to those of men, but DBP and drinking status had no associations with the eGFR at the baseline (all P < 0.05). After adjustment for age, men with MAP of > 112.2 mmHg versus ≤ 93.8 mmHg had ORs for decreased eGFR of 2.367 (95 % CI: 1.248 to 4.488) .After multivariable adjustment, an MAP of > 112.2 mmHg versus ≤ 93.8 mmHg had an OR for decreased eGFR of 3.249 (95 % CI:1.394 to 7.575) in men, whereas in women, MAP was not associated with decreased eGFR. CONCLUSIONS: MAP was associated with decreased eGFR in men, while in women MAP was not associated with decreased eGFR. These findings provide some evidence that a different adaptive response to renal regulation may exist in males and females.

Systematic separation and purification of iridoid glycosides and crocetin derivatives from Gardenia jasminoides Ellis by high-speed counter-current chromatography.

INTRODUCTION: Iridoid glycosides and crocetin derivatives are the main bioactive components of Gardenia. The processes of separation of these compounds reported in much of the literature are tedious, time consuming and require multiple chromatographic steps, which results in lower recovery and higher costs. OBJECTIVE: To develop a high-speed counter-current chromatography (HSCCC) method for the systematic separation and purification of iridoid glycosides and crocetin derivatives on a preparative scale from Gardenia. METHODS: After fractionation using HPD100 column chromatography, n-butanol:ethanol:water (10:1:10, v/v) was selected to purify gardenoside, 6ß-hydroxy geniposide and geniposidic acid from fraction A; ethyl acetate:n-butanol:water (2:1.5:3, v/v) was used to isolate geniposide from fraction B; crocin-1, crocin-2, crocin-3 and crocin-4 were purified by hexane:ethyl acetate:n-butanol:water (1:2:1:5, v/v) from fraction C. The head-to-tail elution mode was used with a flow rate of 8.0 mL/min and a rotary speed of 600 rpm. RESULTS: After HSCCC isolation, 151.1 mg of gardenoside, 52.2 mg of 6ß-hydroxy geniposide and 24.5 mg of geniposidic acid were obtained from 800 mg of fraction A; 587.2 mg of geniposide was obtained from 800 mg of Fraction B; 246.2 mg of crocin-1, 34.2 mg of crocin-2, 24.4 mg of crocin-3 and 24.7 mg of crocin-4 were obtained from 1000mg of fraction C. Their purities were found by UPLC analysis to be 91.7%, 93.4%, 92.5%, 98.2%, 94.1%, 96.3%, 94.1% and 98.9% respectively. CONCLUSION: The present results demonstrates that the main iridoid glycosides and crocetin derivatives in Gardenia can be obtained efficiently from extracts using HSCCC.

Cardioprotective effects of low-dose combination therapy with rosuvastatin and fasudil in the isolated rat heart.

The cardiovascular pleiotropic effects of statins and a Rho-kinase inhibitor (fasudil) could be of interest to prevent myocardial ischemia reperfusion injury (MIRI). In the present study, we investigated whether low-dose rosuvastatin and fasudil, separately not possessing cardioprotection, express cardioprotective effects when combined. The isolated rat hearts underwent 30 min global ischemia and 120 min reperfusion. Rosuvastatin (3 microM) and fasudil (1 microM) were administered 15 min before ischemia. NG-nitro-L-arginine methylester (30 microM) (L-NAME) was given at the onset of reperfusion. Myocardial infarct size, apoptosis, myocardial nitric oxide (NO) content and endothelial nitric oxide synthase (eNOS) expression were evaluated. The combination treatment significantly decreased infarct size and percentage of apoptosis and increased the content of NO and eNOS expression, whereas treatment with rosuvastatin and fasudil alone at the same doses did not lead to cardioprotection. Furthermore, L-NAME reversed the cardioprotective effect of rosuvastatin/fasudil combination treatment. In summary, rosuvastatin combined with fasudil treatment had synergistic protective effects against MIRI, which were mediated by increasing eNOS and NO production. This new concept could be valuable in MIRI prevention.

Acacetin inhibits activation of microglia to improve neuroinflammation after subarachnoid hemorrhage through the PERK signaling pathway mediated autophagy.

PURPOSE: To explore the effect of acacetin on subarachnoid hemorrhage (SAH) and its possible mechanism. METHODS: SAH model of rat was established, and intraperitoneally injected with three doses of acacetin. To verify the role of PERK pathway, we used the CCT020312 (PERK inhibitor) and Tunicamycin (activators of endoplasmic reticulum stress). The SAH score, neurological function score, brain edema content, and Evans blue (EB) exudate were evaluated. Western blot was used to determine the expression of inflammation-associated proteins and PERK pathway. The activation of microglia was also determined through Iba-1 detection. TEM and immunofluorescence staining of LC3B were performed to observe the autophagy degree of SAH rats after acacetin. Tunel/NeuN staining, HE and Nissl' staining were performed for neuronal damage. RESULTS: Acacetin increased the neurological function score, reduce brain water content, Evans blue exudation and SAH scores. The microglia in cerebral cortex were activated after SAH, while acacetin could inhibit its activation, and decreased the expression of TNF-α and IL-6 proteins. The pathological staining showed the severe neuronal damage and increased neuronal apoptosis after SAH, while acacetin could improve these pathological changes. We also visualized the alleviated autophagy after acacetin. The expression of Beclin1 and ATF4 proteins were increased, but acacetin could inhibit them. Acacetin also inactivated PERK pathway, which could improve the neuronal injury and neuroinflammation after SAH, inhibit the microglia activation and the overactivated autophagy through PERK pathway. CONCLUSION: Acacetin may alleviate neuroinflammation and neuronal damage through PERK pathway, thus having the protective effect on EBI after SAH.