Regulation of Mycobacterium biofilm development and novel measures against antibiotics resistance.

Currently, there are over 170 recognized species of Mycobacterium, the only genus in the family Mycobacteriaceae. Organisms belonging to this genus are quite diverse with respect to their ability to cause disease in humans. The Mycobacterium genus includes human pathogens (Mycobacterium tuberculosis complex and Mycobacterium leprae) and environmental microorganisms known as non-tuberculosis mycobacteria (NTM). A common pathogenic factor of Mycobacterium is the formation of biofilms. Bacterial biofilms are usually defined as bacterial communities attached to the surface, and are also considered as shared spaces of encapsulated microbial cells, including various extracellular polymeric substrates (EPS), such as polysaccharides, proteins, amyloid proteins, lipids, and extracellular DNA (EDNA), as well as membrane vesicles and humic like microorganisms derived refractory substances. The assembly and dynamics of the matrix are mainly coordinated by second messengers, signaling molecules, or small RNAs. Fully deciphering how bacteria provide structure for the matrix, thereby promoting extracellular reactions and benefiting from them, remains a challenge for future biofilm research. This review introduces a five step development model for biofilms and a new model for biofilm formation, analyses the pathogenicity of biofilms, their interactions with bacteriophages and host immune cells, and the key genes and regulatory networks of mycobacterial biofilms, as well as mycobacterial biofilms and drug resistance, in order to provide a basis for clinical treatment of diseases caused by biofilms.

USP18-mediated protein deISGylation and its role in tuberculosis and other infectious diseases.

The transcription of interferon-stimulated gene 15 (isg15) is induced by type I interferons. ISG15 can covalently modify target proteins through the sequential action of enzymesE1, E2, and E3, a process known as ISGylation. The ISGylation of host proteins is widely involved in immune responses, such as host antiviral defence. Ubiquitin-specific protease 18 (USP18), as a deubiquitinase (DUB), can remove ISG15 conjugated to target proteins and inhibit host immune responses by suppressing the type I interferon signaling. The dynamic balance between ISGylation and deISGylation mediated by ISG15 or USP18 respectively plays a significant role in the tuberculosis. Furthermore, similar to ISG15, USP18 is extensively involved in virus-host interaction. In this review, we summarize the roles of ISGylation and deISGylation in tuberculosis and other important diseases mediated by ISG15 and USP18 respectively, underlying regulator network. Further studies in this aspect will inspire new host-targeted strategies to control important diseases such as tuberculosis.

Protective effects of Naoxintong capsule alone and in combination with ticagrelor and atorvastatin in rats with Qi deficiency and blood stasis syndrome.

CONTEXT: Naoxintong Capsule (NXT), a Chinese medicine, has been widely used for the treatment of coronary heart disease (CHD) in clinics. OBJECTIVE: This study evaluated the cardioprotective effects of NXT alone and in combination with ticagrelor (TIC) and atorvastatin (ATO). MATERIALS AND METHODS: Qi deficiency and blood stasis rats were established by 8 weeks high fat diet feeding and 16 days exhaustive swimming and randomly divided into seven groups, that is, NXT (250, 500 and 1000 mg/kg/d), TIC (20 mg/kg/d), ATO (8 mg/kg/d), NXT (500 mg/kg/d)+TIC (20 mg/kg/d) and NXT (500 mg/kg/d)+ATO (8 mg/kg/d) group, with oral administration for 12 weeks. The contents of TC, TG, LDL-C, HDL-C, IL-1ß, IL-6, IL-8, TNF-α, AST, ALT, SOD, MDA, CK-MB, LDH, TXA2, PGI2, IgA, IgG, IgM and C3 in serum were measured. RESULTS: NXT + TIC group was significantly superior to the TIC group in decreasing the levels of TC (4.34 vs. 5.54), TG (3.37 vs. 4.66), LDL-C (1.21 vs. 1.35), LDH (4919.71vs. 5367.19) and elevating SOD level (248.54 vs. 192.04). NXT + ATO group was significantly superior to the ATO group in decreasing the levels of AST (195.931 vs. 241.63), ALT (71.26 vs. 83.16), LDH (4690.05 vs. 5285.82), TXA2 (133.73 vs. 158.67), IgG (8.08 vs. 9.80), C3 (2.03 vs. 2.35) and elevating the levels of HDL-C (1.19 vs. 0.91), SOD (241.91vs. 209.49). CONCLUSIONS: The present findings demonstrate that the combined use of NXT with TIC and ATO had better integrated regulating effects than TIC and ATO, respectively. The mechanism of action requires further research.

Antioxidant Activity and Hepatoprotective Potential of Quercetin 7-Rhamnoside In Vitro and In Vivo.

Hypericum japonicum is traditionally used as a folk medicine to treat cholestasis and hepatitis. Quercetin 7-rhamnoside (Q7R) is one of the main flavonoid components of Hypericum japonicum and has been rarely studied. The aim of the present study was to evaluate the antioxidant activity and hepatoprotective potential of Q7R. In the in vitro experiments, DPPH, ABTS and ferric reducing antioxidant power (FRAP) assays were first performed to assess the antioxidant properties of Q7R, and then a H2O2-induced oxidative damage cellular model was used to determine the cytoprotective and antioxidant properties of Q7R in human liver L-02 cells. In the in vivo experiment, the hepatoprotective activity of Q7R was evaluated by carbon tetrachloride (CCl4)-induced liver damage model in mice. The results of the three in vitro assays (DPPH, ABTS and FRAP) demonstrated that Q7R significantly exhibited antioxidant activity. The cell experiment results showed that Q7R possessed cytoprotective and antioxidant effects on H2O2-treated L-02 cells. In the in vivo experiments, Q7R suppressed the up-regulation of serum activities of ALT, AST, LDH and triglyceride (TG) levels with dose-dependency. Q7R down-regulated the production of MDA and increased the hepatic GSH content and antioxidant enzymes CAT activities. Hepatic morphological analysis was also performed to confirm the biochemical changes. In summary, these results suggested that Q7R could be considered as a potential source of natural antioxidants, and may become a promising candidate for the treatment of liver injury in the future.

Chromatogram-Bioactivity Correlation-Based Discovery and Identification of Three Bioactive Compounds Affecting Endothelial Function in Ginkgo Biloba Extract.

Discovery and identification of three bioactive compounds affecting endothelial function in Ginkgo biloba Extract (GBE) based on chromatogram-bioactivity correlation analysis. Three portions were separated from GBE via D101 macroporous resin and then re-combined to prepare nine GBE samples. 21 compounds in GBE samples were identified through UFLC-DAD-Q-TOF-MS/MS. Correlation analysis between compounds differences and endothelin-1 (ET-1) in vivo in nine GBE samples was conducted. The analysis results indicated that three bioactive compounds had close relevance to ET-1: Kaempferol-3-O-α-l-glucoside, 3-O-{2-O-{6-O-[P-OH-trans-cinnamoyl]-ß-d-glucosyl}-α-rhamnosyl} Quercetin isomers, and 3-O-{2-O-{6-O-[P-OH-trans-cinnamoyl]-ß-d-glucosyl}-α-rhamnosyl} Kaempferide. The discovery of bioactive compounds could provide references for the quality control and novel pharmaceuticals development of GRE. The present work proposes a feasible chromatogram-bioactivity correlation based approach to discover the compounds and define their bioactivities for the complex multi-component systems.

Naringenin Regulates CFTR Activation and Expression in Airway Epithelial Cells.

BACKGROUND/AIMS: Sputum symptoms are commonly seen in the elderly. This study aimed to identify an efficacious expectorant treatment stratagem through evaluating the secretion-promoting activation and cystic fibrosis transmembrane conductance regulator (CFTR) expression of the bioactive herbal monomer naringenin. METHODS: Vectorial Cl- transport was determined by measuring short-circuit current (ISC) in rat airway epithelium. cAMP content was measured by ELISA in primary cultured epithelial cells and Calu-3 cells. CFTR expression in Calu-3 cells was determined by qPCR. RESULTS: Addition of naringenin to the basolateral side of the rat airway led to a concentration-dependent sustained increase in ISC. The current was suppressed when exposed to Cl--free solution or by bumetanide, BaCl2, and DPC but not by DIDS and IBMX. Forskolin-induced ISC increase and CFTRinh-172/MDL-12330A-induced ISC inhibition were not altered by naringenin. Intracellular cAMP content was significantly increased by naringenin. With lipopolysaccharide stimulation, CFTR expression was significantly reduced, and naringenin dose-dependently enhanced CFTR mRNA expression. CONCLUSION: These results demonstrate that naringenin has the ability to stimulate Cl- secretion, which is mediated by CFTR through a signaling pathway by increasing cAMP content. Moreover, naringenin can increase CFTR expression when organism CFTR expression is seriously hampered. Our data suggest a potentially effective treatment strategy for sputum.

N16, a Nacreous Protein, Inhibits Osteoclast Differentiation and Enhances Osteogenesis.

N16 is a protein from the nacreous layer of Pinctada fucata, a pearl oyster. It has been found to promote biomineralization, and we hypothesized that it also plays a role in bone metabolism. The cDNA of N16 was cloned and expressed in Escherichia coli to produce N16 protein, which was purified to high homogeneity by ion-exchange and gel filtration columns. The effects of N16 on osteoclast differentiation and osteogenesis were clarified using the murine preosteoclast cell line RAW 264.7 and the preosteoblast cell line MC3T3-E1. Results on preosteoclasts showed that N16 only slightly inhibited cell survival but significantly inhibited differentiation induced by receptor activator of nuclear factor kappa-B ligand (RANKL). Apart from reduced formation of multinucleated osteoclasts, N16-treated cells exhibited lower gene expression and enzymatic activity typical of mature osteoclasts. Actin ring formation and intracellular acidification essential for osteoclastic function were also impaired upon N16 treatment. At concentrations nontoxic to preosteoblasts, N16 strongly up-regulated alkaline phosphatase activity and increased mineralized nodule formation, which are indicative of differentiation into osteoblasts. These effects coincided with an increase in mRNA expression of osteoblast markers osteopotin and osteocalcin. The present study demonstrated that N16 has both anabolic and antiresorptive effects on bone, which makes it potentially useful for treating osteoporosis.

Therapeutic effects of naringin in a guinea pig model of ovalbumin-induced cough-variant asthma.

Naringin, a well known component isolated from Exocarpium Citri Grandis, has significant antitussive effects. Recently, Naringin exhibited novel anti-inflammatory effect in chronic inflammatory diseases. In this work, we firstly evaluated the effects of naringin on enhanced cough, airway hyper-responsiveness (AHR), and airway inflammation in an ovalbumin-induced experimental cough-variant asthma (CVA) model in guinea pigs. We investigated the effect of naringin (18.4 mg/kg, per os, single dose or consecutively) on cough to inhaled capsaicin after challenge with an aerosolized antigen in actively sensitized guinea pigs. The effect of naringin on AHR to inhaled methacholine was evaluated 24 h after cough determination. Airway inflammation was assessed via bronchoalveolar lavage fluid (BALF) cytology and lung histopathology. Naringin, given consecutively, significantly reduced ovalbumin-induced enhanced cough and AHR, inhibited the increases in the leukocytes, interleukin-4 (IL-4), IL-5, and IL-13 in BALF compared with the model group. Moreover, the pathologic changes in lung tissues were clearly ameliorated by naringin treatment. These results suggest that naringin may be a beneficial agent for CVA treatment.

Protective effects of traditional Chinese medicine formula NaoShuanTong capsule on haemorheology and cerebral energy metabolism disorders in rats with blood stasis.

NaoShuanTong capsule (NSTC), an oral traditional Chinese medicine formula, is composed of Pollen Typhae, Radix Paeoniae Rubra, Rhizoma Gastrodiae, Radix Rhapontici and Radix Curcumae. It has been widely used to treat ischemic stroke in clinic for many years in China. In addition to neuronal apoptosis, haemorheology and cerebral energy metabolism disorders also play an important role in the pathogenesis and development of ischemic stroke. The present study was designed to evaluate the in vivo protective effects of NSTC on haemorheology and cerebral energy metabolism disorders in rats with blood stasis. Sixty specific pathogen-free sprague-dawley rats, male only, were randomly divided into six groups (control group, model group, aspirin (100 mg/kg/d) group, NSTC low-dose (400 mg/kg/d) group, NSTC intermediate-dose (800 mg/kg/d) group, NSTC high-dose (1600 mg/kg/d) group) with 10 animals in each. The rats except those in the control group were placed in ice-cold water (0-4 °C) for 5 min during the time interval (4 h) of two adrenaline hydrochloride injections (0.8 mg/kg) to induce blood stasis. After treatment, whole blood viscosity at three shear rates, plasma viscosity and erythrocyte sedimentation rate significantly decreased in NSTC intermediate- and high-dose groups; erythrocyte aggregation index and red corpuscle electrophoresis index significantly decreased in all the three dose NSTC groups. Moreover, treatment with high-dose NSTC could significantly improve Na+-K+ adenosine triphosphatase (ATPase) and Ca2+ ATPase activity, as well as lower lactic acid level in brain tissues. These results demonstrated the protective effects of NSTC on haemorheology and cerebral energy metabolism disorders, which may provide scientific information for the further understanding of mechanism(s) of NSTC as a clinical treatment for ischemic stroke. Furthermore, the protective effects of activating blood circulation as observed in this study might create valuable insight for the utilisation of NSTC to be a feasible alternative therapeutic agent for patients with blood stasis.

Effects of four antitussives on airway neurogenic inflammation in a guinea pig model of chronic cough induced by cigarette smoke exposure.

OBJECTIVE: The effects of four antitussives, including codeine phosphate (CP), moguisteine, levodropropizine (LVDP) and naringin, on airway neurogenic inflammation and enhanced cough were investigated in guinea pig model of chronic cough. METHODS: Guinea pigs were exposed to CS for 8 weeks. At the 7th and 8th week, the animals were treated with vehicle, CP (4.8 mg/kg), moguisteine (24 mg/kg), LVDP (14 mg/kg) and naringin (18.4 mg/kg) respectively. Then the cough and the time-enhanced pause area under the curve (Penh-AUC) during capsaicin challenge were recorded. The substance P (SP) content, NK-1 receptor expression and neutral endopeptidase (NEP) activity in lung were determined. RESULTS: Chronic CS exposure induced a bi-phase time course of cough responsiveness to capsaicin. Eight weeks of CS exposure significantly enhanced the airway neurogenic inflammation and cough response in guinea pigs. Two weeks of treatment with CP, moguisteine, LVDP or naringin effectively attenuated the chronic CS-exposure enhanced cough. Only naringin exerted significant effect on inhibiting Penh-AUC, SP content and NK-1 receptor expression, as well as preventing the declining of NEP activity in lung. CONCLUSIONS: Chronic CS-exposed guinea pig is suitable for studying chronic pathological cough, in which naringin is effective on inhibiting both airway neurogenic inflammation and enhanced cough.

Characteristic comparison of three rat models induced by cigarette smoke or combined with LPS: to establish a suitable model for study of airway mucus hypersecretion in chronic obstructive pulmonary disease.

There is a need of in vivo COPD models for mucus hypersecretion study. The current study compared three rat models induced by cigarette smoke (CS) exposure alone or combined with pre- or post-treatment with lipopolysaccharide (LPS). Forty rats were randomly divided into the four following groups: control group, LPS + CS group (CS exposure for 4-wk combined with LPS pretreatment), CS group (CS exposure for 6-wk), CS + LPS group (CS exposure for 6-wk combined with LPS post-treatment). The results showed that both CS and CS + LPS groups had more severe pro-inflammatory cytokines secretion, inflammatory cells infiltration, and emphysema as compared to that in LPS + CS group animals. From the PAS staining sections, we found a remarkable hyperplasia of goblet-cell in epitheliums of trachea, bronchi, and bronchiole of all of three modeling groups, especially in CS and CS + LPS groups. From the western-blotting results, there were significant increase in the activities of NF-κB, AP-1, EGFR, TLR4, and MAPKs in all of three modeling groups, while HDAC2 activity was remarkably repressed in CS group only. Moreover, the expression and secretion of MUC5AC were exhibited significant increase in all of three modeling groups, which correlated well with the total transcription activity integration of NF-κB, AP-1, and HDAC2 (r = 0.946, p < 0.01). These results indicated that MUC5AC hypersecretion is consistent with activation of EGFR-AP-1/NF-κB and TLR4-AP-1/NF-κB signaling pathways, as well as repression of HDAC2 activity. Based on these results, we speculated that the 6-wk CS exposure rat model is a reliable COPD rat model, while the 6-wk CS exposure combined with LPS post-treatment rat model is a suitable COPD exacerbation model for mucus hypersecretion study.

N16 suppresses RANKL-mediated osteoclatogenesis by down-regulating RANK expression.

N16 is an active protein existing in Pinctada martensi. Our previous studies have demonstrated that N16 inhibited osteoclast differentiation in vitro. To better understand how N16 regulates osteoclast differentiation, RAW264.7 cells, a murine monocytic cell line and murine bone marrow-derived macrophages (BMMs) were adopted. Treatment of RAW264.7 cells with RANKL activated osteoclastogenesis and N16 inhibited the formation of multinucleated osteoclasts and TRAP activity. The suppression occurred at the early stage of osteoclastogenesis. Moreover, we found that N16 inhibited PU.1 and MITF expressions, mirroring the inhibition of RANK expressions, indicating that N16 inhibited RANK expression by down-regulating the expressions of MITF and PU.1, thus preventing osteoclastogenesis.

Transcriptome analysis reveals functional roles of nacreous protein N16 in prednisolone-induced osteoporotic zebrafish.

N16 is an active protein isolated and screened from nacre proteins. Our previous studies have shown that N16 exerted a good effect on osteoporosis caused by dexamethasone on female rats. In order to further confirm the action of N16 against glucocorticoid-induced osteoporosis (GIOP) and clarify its possible mechanisms, prednisolone-treated larval zebrafish (Danio rerio) model was adopted. Administration of N16 resulted in a significant increase of vertebral bone density of osteoporotic zebrafish, indicating a good effect of N16 against GIOP. Transcriptomic sequencing analysis was then performed to investigate the mechanisms of the action of N16 against GIOP. It was observed that N16 modulated the osteoporotic phenotype by up-regulating the osteoblastic characteristic genes and down-regulating the osteoclastic characteristic genes in zebrafish.

Naoxintong Capsule Inhibits the Development of Cardiovascular Pathological Changes in Bama Minipig Through Improving Gut Microbiota.

Naoxintong capsule (NXT), a Chinese medicine, has performed excellent effects on the prevention and treatment against cardiovascular diseases. NXT is a fine powder mixture without any herb extraction, and there must be lots of ingredients hard to be absorbed. However, little is known about the correlation between the NXT's cardioprotective effects and gut microbiota. Herein, we report the effect of NXT on the development of cardiovascular diseases and clarify the correlation between NXT's cardioprotective effects and gut microbiota. In the current study, minipigs were selected and fed with high-fat diet and NXT daily for successive 8 months. During the process, up to 18 biomedical parameters were monthly determined to observe the dynamic changes after NXT treatment. At the end of experimental process, pathological examinations of heart, coronary artery, carotid artery, thoracic aorta, and abdominal aorta were conducted by HE staining and 16SrDNA sequencing, and analyzing of gut microbiota were conducted. Our results showed that NXT's effects against cardiovascular diseases were through regulating blood lipid profiles, inhibiting vascular inflammation, enhancing antioxidant capacity, and alleviating myocardial injury, without damages on liver and kidney particularly. Concurrently, we also found that long-term administration of NXT increased the diversity of gut microbiota, influenced the microbiome structure and composition stably, and revered the increase of the ratio of the Firmicutes to Bacteroidetes (F/B ratio) in relative abundance. Specifically, our results revealed some key bacterium of Caproiciproducens (enhanced), Sutterella (enhanced), Erysipelotrichaceae (enhanced), and Romboutsia (decreased) that were closely involved in NXT's effects. Taken together, our study demonstrates that NXT can inhibit the development of cardiovascular diseases by ameliorating high-fat diet-induced metabolic disorders and partly through improving gut microbiota.

Regulation effects of naringin on diesel particulate matter-induced abnormal airway surface liquid secretion.

BACKGROUND: PM2.5 is closely related to the incidence and mortality of respiratory diseases. Diesel particulate matter (DPM) is the main component of particulate air pollution and an important source of PM2.5. HYPOTHESIS/PURPOSE: This study mainly explored the effect of DPM on airway surface liquid (ASL) secretion and the regulation of naringin in this process, to evaluate therapeutic potentials of naringin for the treatment of abnormal secretion of the respiratory tract caused by PM2.5. METHODS: The concentration of lysozyme was measured by Lysozyme Assay Kit. Total protein content was determined by the BCA Protein Assay Kit. The concentration of cAMP and MUC5AC, expressions of CFTR, AQP1, and AQP5 proteins were measured by ELISA. Expressions of CFTR, AQP1 and AQP5 mRNA were determined by qPCR. Amount of CFTR on the cell membrane was determined by immunofluorescence. RESULTS: The in vitro and in vivo studies had indicated that DPM could inhibit ASL secretion and increased the viscosity of the liquid. Naringin had the functions to attenuate DPM-induced injury, reduce liquid viscosity by reducing MUC5AC and total protein secretion, increase DPM-induced CFTR, AQP1, and AQP5 mRNA and protein expression, positively regulate apical CFTR insertion and promote CFTR activation by increasing intracellular cAMP. CONCLUSION: These results demonstrated that naringin had regulating effects on the DPM-induced abnormal secretion of the respiratory tract.

Pharmacodynamic effects of Dan-hong injection in rats with blood stasis syndrome.

Dan-hong injection (DHI) is extracted from Salvia miltiorrhiza (SM) and Carthamus tinctorius (CT) and is widely used for the treatment of cardiovascular diseases. Our previous results showed DHI could improve hemorheology in rats. Since complex cellular interactions such as inflammation and oxidative stress are believed to be implicated in the pathogenesis of cardiovascular events, investigation of such pathological factors will contribute substantially to the understanding of the features and mechanisms of DHI. Therefore, in this study we used a rat model of blood stasis to explore the overall effects of DHI by detecting twenty three indexes, which were related to inflammation, immune response, vascular endothelial function, myocardial energy metabolism, oxidative stress, platelet aggregation, liver and renal function. Meanwhile, the interaction between SM and CT was discussed by comparing the effects of each single herb. DHI could significantly decrease the serum contents of IL-1ß, TNF-α, IL-6, IL-8, IgM, IgG, IgA, MPO, hs-CRP, MDA, LDH, CK-MB, PAF, ALP and Cr, while elevate NO, SOD, TP and UA levels, indicating that DHI could inhibit inflammation and platelet aggregation, thereby relieving immune response and peroxidation, protecting vascular endothelial and organ function, and then prevent and treat cardiovascular diseases. In terms of compatibility, SM and CT showed complementary effects on markers of inflammatory and oxidative status, vascular endothelial damage and myocardial energy metabolism. On the other hand, they counteracted each other and SM reduced the side effects of creatinine caused by CT. This study contributes to comprehensively understand the pharmacodynamics effects and mechanism of DHI.

Naringin and Naringenin Relax Rat Tracheal Smooth by Regulating BKCa Activation.

Naringin and its aglycone, naringenin, occur naturally in our regular diet and traditional Chinese medicines. This study aimed to detect an effective therapeutic approach for cough variant asthma (CVA) through evaluating the relaxant effect of these two bioactive herbal monomers as antitussive and antiasthmatic on rat tracheal smooth muscle. The relaxant effect was determined by measuring muscular tension with a mechanical recording system in rat tracheal rings. Cytosolic Ca2+ concentration was measured using a confocal imaging system in primary cultured tracheal smooth muscle cells. In rat tracheal rings, addition of both naringin and naringenin could concentration dependently relax carbachol (CCh)-evoked tonic contraction. This epithelium-independent relaxation could be suppressed by BaCl2, tetraethylammonium, and iberiotoxin (IbTX), but not by glibenclamide. After stimulating primary cultured tracheal smooth muscle cells by CCh or high KCl, the intracellular Ca2+ increase could be inhibited by both naringin and naringenin, respectively. This reaction was also suppressed by IbTX. These results demonstrate that both naringin and naringenin can relax tracheal smooth muscle through opening big conductance Ca2+-activated K+ channel, which mediates plasma membrane hyperpolarization and reduces Ca2+ influx. Our data indicate a potentially effective therapeutic approach of naringin and naringenin for CVA.

Evaluation of neuroactive effects of ethanol extract of Schisandra chinensis, Schisandrin, and Schisandrin B and determination of underlying mechanisms by zebrafish behavioral profiling.

Schisandra chinensis, a traditional Chinese medicine (TCM), has been used to treat sleep disorders. Zebrafish sleep/wake behavioral profiling provides a high-throughput platform to screen chemicals, but has never been used to study extracts and components from TCM. In the present study, the ethanol extract of Schisandra chinensis and its two main lignin components, schisandrin and schisandrin B, were studied in zebrafish. We found that the ethanol extract had bidirectional improvement in rest and activity in zebrafish. Schisandrin and schisandrin B were both sedative and active components. We predicted that schisandrin was related to serotonin pathway and the enthanol extract of Schisandra chinensis was related to seoronin and domapine pathways using a database of zebrafish behaviors. These predictions were confirmed in experiments using Caenorhabditis elegans. In conclusion, zebrafish behavior profiling could be used as a high-throughput platform to screen neuroactive effects and predict molecular pathways of extracts and components from TCM.

Preparative expression and purification of a nacreous protein N16 and testing its effect on osteoporosis rat model.

N16, a nacreous protein isolated from Pinctada martensii, is related to nacreous layer formation. Our previous study indicated that N16 showed dual regulatory effects by inducing osteoblast biomineralization as well as inhibiting osteoclast formation. In order to obtain large quantity of N16 for animal experiment and clinical trial, a fermentation and preparative purification method was established. The N16 cDNA was cloned to a BL21(DE3)plysE-pET32a vector and grown in a 20 L fermenter. The medium, temperature, pH and dissolved oxygen (DO) were optimized. N16 was expressed in inclusion bodies. It was denatured and refolded in 8 M urea buffer and purified to 97% purity by passing through a gel filtration column. The glucocorticoid induced osteoporosis (GIO) rat model was used to investigate the anti-osteoporosis activity of N16 in vivo. Results showed that the decrease of the bone mineral density (BMD) and the ultimate load was significantly relieved after N16 treatment. N16 displayed dual regulatory effects by promoting osteogenesis as well as inhibiting bone resorption in vivo. Our work will contribute to further clinical studies on N16 for osteoporosis treatment.

An experimental model for hypertensive crises emergencies: Long-term high-fat diet followed by acute vasoconstriction stress on spontaneously hypertensive rats.

Currently, the prevention and treatment of hypertensive crises especially when it occurs with serious adverse outcomes have led to worldwide controversy. Despite of clinical possibilities of multiple agents, clinical failures still occur frequently. Therefore, early evaluations and observations of different therapies on appropriate animals should be emphasized. In the present study, an animal model for hypertensive crises emergencies was firstly established and experimentally testified. Five-month-male spontaneously hypertensive rat was consecutively fed with 60%-Kcal fat diet for four, six, and eight weeks with body weight and blood pressure monitored every two weeks, and then followed by an acute vasoconstriction stress of 5-min ice-bath treatment in the 4-h time interval of two adrenaline injections (0.8 mg/kg). Forty-four biochemical parameters were detected, covering hepatic and renal function, blood glucose and lipid levels, myocardial enzymes and energy metabolisms, blood coagulative and anti-coagulative system, oxidative stress and anti-inflammatory cytokine, blood viscosity, and RAAS system. Six tissues including heart, brain, liver, kidney, coronary arteries, and mesenteries were removed for pathological observations with hematoxylin-eosin staining. As a result, multi-organ dysfunctions in the heart, brain, liver, kidney, vascular endothelium, and blood system were testified in the modeling rats at weeks 6 and 8. In conclusion, severe consequences of this animal model were highly similar to those in hypertensive crises emergencies, which could be further utilized in the early intervention of hypertensive crises emergencies including the possible risk factors control and efficient therapies assessment. Impact statement In the late 90s, numerous reports predicted that 1-2% of hypertensive individuals would undergo hypertensive crises (HPC) and figures reached as high as 7% when no antihypertensive therapies were administrated. Currently, clinical failures appear frequently due to the improper or excessive medication regimen instead of the illness itself. Therefore, early evaluations and observations of HPC on appropriate animal models ahead of patients should be discussed and emphasized more widely. In the present study, an appropriate animal model for HPC emergencies was firstly established, in which the consequences of long-term high-fat diet feeding followed by an acute vasoconstriction stress on the spontaneously hypertensive rats were experimentally testified. The proposed model would have a wide application prospects in early intervention of HPC emergencies including the controls of possible risk factors and assessments of efficient therapies.