1H-NMR-based metabolomics to dissect the traditional Chinese medicine promotes mesenchymal stem cell homing as intervention in liver fibrosis in mouse model of Wilson's disease.

BACKGROUND: We administered Bushen Huoxue Huazhuo Formula (BSHXHZF) and transplanted bone marrow mesenchymal stem cells (BMSCs) into mice with Wilson's disease (WD)-related liver fibrosis to evaluate the liver-protecting mechanism of this prescription. METHODS: Mice, randomly divided into different treatment groups, showed histopathological changes and degree of hepatocyte apoptosis. For hepatic hydroxyproline (Hyp) determination, transforming growth factor-ß1 (TGF-ß1) and bone morphogenetic protein-7 (BMP-7) mRNA and protein were measured. Chemical profiling of the extract of BSHXHZF using The liquid chromatography-mass spectrometry (LC-MS/MS) and revealing its antifibrosis mechanism using metabolomics. RESULTS: TCM+BMSC group livers exhibited few inflammatory cells. TUNEL revealed abundant brown apoptotic cells in model control groups, while the TCM+BMSC groups showed a significant increase in blue negative expression of liver cells. Hyp in toxic milk (TX) mice groups was significantly lower than that in model control groups (MG). Compared with MG, TGF-ß1 expression was significantly lower than all other groups, while BMP-7 expression was significantly higher. Metabolic analysis identified 20 potential biomarkers and 10 key pathways, indicating that BSHXHZF+BMSC intervention has a significant regulatory effect on metabolic disorders of these small molecule substances. CONCLUSION: BSHXHZF combined with BMSCs can inhibit liver fibrosis and hepatocyte apoptosis by improving related metabolic disorders, and achieving therapeutic effects in WD-related liver fibrosis.

Preclinical Investigations on Anti-fibrotic Potential of Long-Term Oral Therapy of Sodium Astragalosidate in Animal Models of Cardiac and Renal Fibrosis.

In traditional Chinese medicine, Radix Astragali has played a vital role in treating progressive fibrotic diseases. One of its main active components, astragaloside IV, is a promising anti-fibrotic treatment despite its extremely low bioavailability. Our study aimed to optimize sodium astragalosidate (SA) by salt formation to improve solubility and oral absorption for anti-fibrotic therapy in vivo. Isoproterenol-induced myocardial fibrosis rat models and obese BKS-db mice presenting diabetic kidney fibrosis were used in this study. Daily oral administration of SA (20 mg/kg) for 14 days ameliorated cardiac fibrosis by reducing collagen accumulation and fibrosis-related inflammatory signals, including TNF-α, IL-1ß, and IL-6. In db/db mice, SA (5,10, and 20 mg/kg per day for 8 weeks) dose-dependently alleviated lipid metabolism impairment and renal dysfunction when administered orally. Furthermore, Western blot and immunohistochemistry analyses demonstrated that SA treatment inhibited renal fibrosis by suppressing TGF-ß1/Smads signaling. Taken together, our findings provide the oral-route medication availability of SA, which thus might offer a novel lead compound in preclinical trial-enabling studies for developing a long-term therapy to treat and prevent fibrosis.

A Novel Air-Cooled Nd:YAG Laser for the Treatment of the Venous Lakes of the Lips.

Objective: To evaluate the therapeutic effect of a novel air-cooled Nd:YAG laser in the venous lakes of the lips (VLL). Background: The thermal injury is one of the most important issues during laser therapy for venous lakes. Methods: Six pieces of fresh pork livers were used to provide 30 regions with a diameter of 6 mm for experiment in vitro, among which 15 regions were treated by Nd:YAG laser with air cooling until the tissue turned gray-white, whereas the rest were treated without air cooling as control. The operation time of laser irradiation, the degree of temperature increase, and the depth of coagulation tissue were compared between two groups. Then, 60 VLL patients were selected for Nd:YAG laser treatment with or without air cooling. The operation time of laser irradiation, the degree of temperature increase, the postoperative pain visual analog scale (VAS) score, and the percentage of lesions removed within 1 month were compared. Results: In tissue studies, the treated group showed a longer operation time of laser irradiation (p < 0.01), a lower degree of temperature increase (p < 0.01), and there was no significant statistical difference in the depth of coagulation tissue (p = 0.624). In clinical studies, the treated group showed a longer operation time of laser irradiation (p < 0.01), a lower degree of temperature increase (p < 0.01), and a lower VAS score on the 1st and 2nd day, compared with the control group (p < 0.01). Conclusions: Air cooling during Nd:YAG laser for the treatment of VLL can prolong the surgical time, but lowered tissue temperature and reduced patient pain within 2 days under the premise of ensuring the treatment effect.

Regulation of TREM2 expression by transcription factor YY1 and its protective effect against Alzheimer's disease.

TREM2 encoding the transmembrane receptor protein TREM2 is a risk gene of Alzheimer's disease (AD), and the impairment of TREM2 functions in microglia due to mutations in TREM2 may significantly increase the risk of AD by promoting AD pathologies. However, how the expression of TREM2 is regulated and the transcription factors required for TREM2 expression are largely unknown. By luciferase assay, DNA pull-down, and in silico predictions, we identified Yin Yang 1(YY1) as a binding protein of the minimal promoter of the TREM2 gene, and the binding was further confirmed by EMSA and DNA pull-down assay. shRNA-mediated YY1 silencing significantly reduced the activity of the TREM2 minimal promoter and TREM2 protein levels in the microglial cell line BV2 and the neuroblastoma Neuro2A. Furthermore, we found that the levels of TREM2 and YY1 were both downregulated in lipopolysaccharide-treated BV2 cells and in the brain of AD model mice. These results demonstrated that YY1 plays a crucial role in the regulation of TREM2 expression. Our study suggests that microglial YY1 could be targeted to maintain TREM2 expression for AD prevention and therapy.

A novel method of bedside hyperthermic intraperitoneal chemotherapy as adjuvant therapy for stage-III gastric cancer.

OBJECTIVE: To investigate the efficacy and safety of a novel method of hyperthermic intraperitoneal chemotherapy (HIPEC) as adjuvant therapy for stage-III gastric cancer. METHODS: Patients with stage-III gastric cancer who underwent D2 radical gastrectomy were randomly assigned to the HIPEC or control group four weeks after surgery. The HIPEC group was treated with cisplatin (60 mg/m2) administered with a HIPEC device on days 1 and 3 (30 mg/m2 each time), along with oral S-1, 40-60 mg, twice daily, for 14 days. The control group was treated with cisplatin (60 mg/m2) administered intravenously plus oral S-1 (40-60 mg, 2/d for 14 days). The primary outcome of the study was disease-free survival (DFS). RESULTS: Total 114 patients were included in the study, with 57 patients in each group. The median DFS was 29.0 months in the HIPEC group, which was significantly longer than that in the control group (15.0 months, p = 0.006). The two-year DFS rate in the HIPEC group was higher than that in the control group (50.4% vs. 25.5%). Median OS was 42.0 month in the HIPEC group and 31.0 month in the control (p = 0.042). Peritoneal metastasis occurred in six patients in the HIPEC group (10.5%) and 12 patients in the control (21.1%, p = 0.198). No significant difference in the incidence of adverse event except for thrombocytopenia. CONCLUSION: HIPEC with cisplatin plus oral S-1 is a safe and effective adjuvant therapy for patients with advanced gastric cancer following D2 radical gastrectomy. Trial registration: This study was registered at ClinicalTrials.gov with the identifier (NCT number): NCT02396498.

Amino acids signatures of distance-related surgical margins of oral squamous cell carcinoma.

BACKGROUND: Histological assessment of resected margins has some drawbacks. We therefore aimed to identify a panel of metabolic markers for evaluating the surgical margins of oral squamous cell carcinoma during surgery. METHODS: A total of 28 case of OSCC samples were enrolled in the study. Gas chromatography-mass spectrometry based untargeted metabolic analysis was employed to acquire the metabolic perturbation of the distance-related surgical margins in the development group. The acquired MS data were then subjected to univariate and multivariate analysis by MetaboAnalyst. Ultra-high performance liquid chromatography-tandem mass spectrometerbased targeted metabolomics for quantitative analysis of the validation group was performed to verify the results of the development group. Another 60 OSCC patients with dysplastic surgical margins were used to further validate the results of the development group by immunohistochemical examination of key enzyme expression, and correlate them with clinicopathological parameters and clinical outcomes. FINDINGS: We finally identified 4 amino acids as negative margin markers, and 6 amino acids as dysplastic margin markers. IHC analysis showed that asparagine synthetase positive expression in dysplastic surgical margins and its higher expression was correlated with tumor recurrence and local relapse-free survival. INTERPRETATIONS: We developed a panel of metabolic molecular markers to supplement the evaluation of negative and dysplastic margins. FUND: This study was supported by Nanjing Municipal Key Medical Laboratory Constructional Project Funding (Since 2012); Center of Nanjing Clinical Medicine Tumor (Since 2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supranutritional dietary selenium depressed expression of selenoprotein genes in three immune organs of broilers.

The objective of this study was to investigate the effects of supranutritional dietary selenium (Se) on selenoproteins expression in three immune organs of chickens. A total of 160 1-day-old male Cobb broilers were randomly divided into two groups and fed a Se-deficient corn-soybean basal diet supplemented with 0.3 (adequate) and 3.0 (excess) mg/kg Se for 42 days. Immune organs were collected, and effects of supranutritional Se on messenger RNA abundance of 23 selenoprotein genes and eight inflammation-related genes were compared at day 42. Also enzyme activities were measured at days 14, 28 and 42. The results showed supranutritional dietary Se depressed growth performance of chicken and down-regulated nine and three selenoprotein genes in thymus and spleen, respectively, and only Sepp1 was up-regulated in the bursa of Fabricius. Also three, three and seven inflammation-related genes were up-regulated in three organs, respectively. Supranutritional Se elevated (P < 0.05) activities of superoxidase dismutase, total antioxidant capacity and glutathione peroxidase, mainly in early stages. In summary, supranutritional Se resulted in down-regulation of selenoprotein genes and up-regulation of inflammation-related genes in three immune organs of chicken, which indicated potential roles of those selenoprotein genes in immune organs of the chicken.

Inhibitory effects of emodin, baicalin, schizandrin and berberine on hefA gene: treatment of Helicobacter pylori-induced multidrug resistance.

AIM: To investigate the inhibitory effects of emodin, baicalin, etc. on the hefA gene of multidrug resistance (MDR) in Helicobacter pylori (H. pylori). METHODS: The double dilution method was used to screen MDR H. pylori strains and determine the minimum inhibitory concentrations (MICs) of emodin, baicalin, schizandrin, berberine, clarithromycin, metronidazole, tetracycline, amoxicillin and levofloxacin against H. pylori strains. After the screened MDR stains were treated with emodin, baicalin, schizandrin or berberine at a 1/2 MIC concentration for 48 h, changes in MICs of amoxicillin, tetracycline, levofloxacin, metronidazole and clarithromycin were determined. MDR strains with reduced MICs of amoxicillin were selected to detect the hefA mRNA expression by real-time quantitative PCR. RESULTS: A total of four MDR H. pylori strains were screened. Treatment with emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some strains, decreased by 1 to 2 times, but did not significantly change the MICs of clarithromycin, levofloxacin, and metronidazole against MDR strains. In the majority of strains with reduced MICs of amoxicillin, hefA mRNA expression was decreased; one-way ANOVA (SPSS 12.0) used for comparative analysis, P < 0.05. CONCLUSION: Emodin, baicalin, schizandrin and berberine significantly decreased the MICs of amoxicillin and tetracycline against some H. pylori strains, possibly by mechanisms associated with decreasing hefA mRNA expression.

Helicobacter pylori isolates from ethnic minority patients in Guangxi: resistance rates, mechanisms, and genotype.

AIM: To investigate the rate of Helicobacter pylori (H. pylori) resistance to clarithromycin among ethnic minority patients in Guangxi, explore the underlying mechanisms, and analyze factors influencing genotype distribution of H. pylori isolates. METHODS: H. pylori strains were isolated, cultured and subjected to drug sensitivity testing. The 23S rRNA gene of H. pylori isolates was amplified by PCR and analyzed by PCR-RFLP and direct sequencing to detect point mutations. REP-PCR was used for genotyping of H. pylori isolates, and NTsys_2 software was used for clustering analysis based on REP-PCR DNA fingerprints. Factors potentially influencing genotype distribution of H. pylori isolates were analyzed. RESULTS: The rate of clarithromycin resistance was 31.3%. A2143G and A2144G mutations were detected in the 23S rRNA gene of all clarithromycin-resistant H. pylori isolates. At a genetic distance of 78%, clarithromycin-resistant H. pylori isolates could be divided into six groups. Significant clustering was noted among H. pylori isolates from patients with peptic ulcer or gastritis. CONCLUSION: The rate of clarithromycin resistance is relatively high in ethnic minority patients in Guangxi. Main mechanisms of clarithromycin resistance are A2143G and A2144G mutations in the 23S rRNA gene. Clarithromycin-resistant H. pylori isolates can be divided into six groups based on REP-PCR DNA fingerprints. Several factors such as disease type may influence the genotype distribution of H. pylori isolates.

[Effects of acupuncture and "Tongxinluo" capsule on plasma lysophosphatidic acid level in patients with acute cerebral infarction].

OBJECTIVE: To observe the effect of acupuncture of Neiguan (PC 6), Sanyinjiao (SP 6), etc. on plasma lysophosphatidic acid (LPA) level in patients with acute cerebral infarction (ACI) so as to study its mechanism underlying improvement of ACI. METHODS: A total of 160 patients with ACI were randomly divided into four groups: medication (Aspirin and Panax Notoginseng Saponins for improving blood circulation), acupuncture, Tongxinluo, and acupuncture+ Tongxinluo groups, with 40 cases being in each group. Plasma LPA content was measured using biochemical method, and the therapeutic effects of the 4 groups were assessed by neurologic deficit score which were evaluated using the modified standards of the 4Th Session of Chinese National Cerebrovascular Conference issued in 1995. RESULTS: Following the treatment, plasma LPA levels and neurological deficit scores were significantly decreased in the medication, acupuncture, Tongxinluo and acupuncture+ Tongxinluo groups in com- parison with pre-treatment in each group (P<0.01), and LPA level and neurological deficit score of the acupuncture + Tongxinluo group were significantly lower than those of the other three groups (P<0. 01). Of the four 40 cases in the medication, acupuncture, Tongxinluo and acupuncture+Tongxinluo groups, 6, 7, 7 and 12 patients were basically cured, 9, 8, 9 and 18 experienced marked improvement in their symptoms, 14, 16, 15 and 6 were improved, 6, 5, 4 and 2 were invalid, 5, 4, 5 and 2 got worsened, with the effective rates(basically cured+ improvement) being 37.5%, 37.5%, 40.0% and 75.0%, respectively. The therapeutic effect of the acupuncture+Tongxinluo group was significantly superior to those of the other three groups (P<0. 01). CONCLUSION: Acupuncture can accelerate the recovery of neurological function and ameliorate clinical symptoms in ACI patients, which may be related to its effect in lowering plasma PLA. The therapeutic effect of acupuncture + Tongxinluo is relatively better than simple acupuncture and simple medication.

Proteomic screening of anaerobically regulated promoters from Salmonella and its antitumor applications.

Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.

A monoclonal-antibody-based ELISA for the detection of human FADD (Fas-associated death domain).

FADD (Fas-associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, measurement of FADD expression mainly depends on Western-blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli-expressed recombinant His(6)-FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin-conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His(6)-FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti-FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein.

Evaluation of kapok (Ceiba pentandra (L.) Gaertn.) as a natural hollow hydrophobic-oleophilic fibrous sorbent for oil spill cleanup.

Oil sorption capacity and hydrophobic-oleophilic characteristics of an agricultural product, kapok (Ceiba pentandra), was thoroughly examined. The kapok fiber has a hollow structure with large lumen. Its performance was compared with that of a polypropylene (PP), a widely used commercial oil sorbent for oil spill cleanup. The oils investigated were diesel, hydraulic oil (AWS46), and engine oil (HD40). Reusability of the kapok after application to various oils was also evaluated. Both loose (at its natural state) and densely packed kapok assemblies were examined. Sorption capacities of the packed kapok assemblies were very much dependent on their packing densities. At 0.02gcm(-3), its oil sorption capacities were 36, 43 and 45gg(-1) for diesel, ASW46 and HD40, respectively. The values decreased to 7.9, 8.1 and 8.6gg(-1) at 0.09gcm(-3). Its sorption capacities for the three oils were significantly higher than those of PP. When the oil-saturated kapok assemblies were allowed to drain, they exhibited high oil retention ability, with less than 8% of the absorbed diesel and HD40, and 12% of the absorbed AWS46 lost even after 1h of dripping. When applied on oil-over-water baths, the kapok exhibited high selectivity for the oils over the water; almost all oils spilled could be removed with the kapok, leaving an invisible oil slick on water. After the 4th cycle of reuse, the reused kapok assembly only lost 30% of its virgin sorption capacity if packed at 0.02gcm(-3), and the loss in sorption capacity was much less at higher packing densities. The hydrophobic-oleophilic characteristics of the kapok fiber could be attributed to its waxy surface, while its large lumen contributed to its excellent oil absorbency and retention capacity.

In situ oil/water separation using hydrophobic-oleophilic fibrous wall: a lab-scale feasibility study for groundwater cleanup.

Kapok, a natural plant fiber, possesses excellent hydrophobic-oleophilic characteristics. Its innovative use as hydrophobic-oleophilic wall that allows permeation of oil but not water into an oil recovery well is proposed. Its performance was investigated through laboratory experiments, in which diesel was used as the experimental oil. A two-dimensional hydraulic flume was setup to physically model the oil/water separation by the kapok wall. The influences of packing density, kapok wall thickness and oil thickness on the oil recovery rate were examined. The oil permeability of the packed kapok decreased from 0.0165 cm2 at 34 g/L packing density to 0.0038 cm2 at 70 g/L packing density. The kapok wall exhibited complete rejection of water while allowed oil to permeate through. The excellent oil/water separation by the kapok wall was due to surface interaction between the kapok fibers and the oil, which resulted in spontaneous penetration and permeation of the oil through the kapok wall. The oil recovery rate increased with thickness of the oil layer in the feed stream. When the oil thickness exceeded 60 mm, a constant flux of 3.8-5.0, 3.2-3.3 and 2.5-2.7 L/(m2 min) could be achieved by the kapok wall of 55-, 75- and 95-mm thick, respectively, under the natural pressure gradient. The kapok wall could be reused for several wetting/drying cycles, and only lost 27% of its initial oil permeability.