Objective To establish a simultaneous method for determination of 5 constituents from Panax quinquefolium stem leaf 20(S)-protopanaxdiol saponins,Ginsenoside-Rb1,Rc,Rb2,Rb3 and Rd by RP-HPLC.MethodsPanax quinquefolium stem leaf 20(S)-protopanaxdiol saponins was provided by the Ji'an Yisheng Pharmaceutical Stock Co.Ltd..The Agilent ODS column(5 ?m,4.6 mm?250 mm)was used,and eluted with acetonitrile-0.2% phosphoric acid water in gradient mode.The UV detection wavelength was 203 nm,and the flow rate was 1.0 ml/min,with the temperature of column at 40 ℃.The accuracy,stability and repeability of this method was evaluated.The yield rate was measured.ResultsFive compounds were baseline separated,with good linearity.A standard curve was plotted.Our method was proved to have sound accuracy,stability and repeability.The yield rate was 98.39%,97.36%,98.02%,97.70% and 97.01% respectively for Ginsenoside-Rb1,Rc,Rb2,Rb3 and Rd.ConclusionOur method of determination is rapid and accurate,and can be used for controlling the quality of Panax quinquefolium 20(S)-protopanaxdiol saponins.

Objective To examine loss of heterozygosity (LOH) and microsatellite instability (MSI) of locus D8S532 on chromosome 8 and their influence on the expression of sFRP1 in the hepatocellular carcinoma (HCCs), which may provide an experimental evidence for clarifying the mechanism of sFRP1 gene and tumor development. Methods DNA was extracted from formalin-fixed paraffin-embedded tissues. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and ordinary silver stain were used to study LOH and MSI of locus D8S532. Envision immunohistochemistry, Leica-Qwin computerized imaging system and Image-Pro PluS (IPP) version 4.5 professional imaging analysis software were used to assess the expression of sFRP1. Results The detection rates of LOH and MSI of locus D8S532 in the 36 specimens of HCC were 11.11% and 8.33% respectively. The down-regulation of sFRP1 was observed in 31 of 36 HCCs (86.11%) compared with non-carcinoma liver tissues, and the positive rate of sFRP1 protein of the HCCs was 52.78%( 19/36 ). The frequency of LOH was lower in the cases with positive expression of sFRP1 protein than those negative (0 vs 23.53%, P <0.05). Conclusion It was a common phenomenon that expression of sFRP1 protein is negative or low in Chinese with HCCs. The genetic instability of sFRP1 gene was one of causes, which lead to HCCs. LOH may play a major role in negative expression of sFRP1.

Objective:To investigate the effect of estrogen on the expression of P2X7 receptor ( P2X7R) in the cerebral cortex and neuroinflammation after subarachnoid hemorrhage ( SAH) in rats.Methods:The rat model of SAH was induced by modified mono-filament puncture method.Sixty male SD rats were divided randomly into three groups:sham group;SAH group and estrogen-treatment group.The local cerebral blood flow was detected with laser doppler blood flow meter.The content of tumor necrosis factor alpha ( TNF-α) and interleukin-6 (IL-6) were measured by ELISA analysis.The expression of P2X7R in the cerebral cortex was tested by immuno-histochemical and Western blot methods.Results:Compared with that in Sham group,cerebral blood flow was significantly decreased after SAH (P<0.05),the content of TNF-αand IL-6 in the cerebral cortex were significantly up-regulated at each time point after SAH (P<0.05),peaked at 24 h,and the expression of P2X7R significantly increased at 6 h,12 h and 24 h after SAH (P<0.05),peaked at 12 h.Compared with that in SAH group,cerebral blood flow was significantly increased in estrogen-treatment group (P<0.05),the levels of TNF-α,IL-6 and P2X7R were down-regulated in estrogen-treatment group ( P<0.05).Conclusion: Estrogen could attenuate neuroinflammation in the cerebral cortex after subarachnoid hemorrhage in rats, which may be associated with the down-regulation in P2X7R proteins.

BACKGROUND/AIMS: Early adverse life events (EALs) are relevant to irritable bowel syndrome in adulthood. Maternal separation (MS), as one of the EALs, has proved to induce visceral hypersensitivity in adult rats. However, the effect of MS on visceral hypersensitvity from the post-weaning period to adulthood remains unknown. METHODS: One hundred and ten neonatal Sprague-Dawley rats were randomly divided into 2 groups: rats in the MS group were exposed to 3 hours daily MS on postnatal day (PND) 2–14; the normal control (NC) group remained undisturbed. Visceral sensitivity was determined by measuring the visceromotor response to colorectal distention on PND21, 35, and 56. Anxiety-like behaviors were measured by the open field test. RESULTS: Compared with NC rats, MS rats showed significant visceral hypersensitivity from the post-weaning period to adult. The proportion of visceral hypersensitive rats decreased with age from 87.5% to 70.0% in the female MS group and from 90.0% to 66.7% in the male MS group. The relative VMR ratio of MS and NC on PND21 was higher than PND35 and PND56. MS rats showed decreased ability of movement and exploration to the novel environment in the post-weaning period, obesity in the prepubertal period, and more anxiety-like behaviors in adulthood. CONCLUSIONS: MS can significantly affect visceral sensitivity and behaviors of rats in different age stages, especially in the post-weaning period. Visceral hypersensitivity of MS rats is more pronounced in the post-weaning period and slightly restored in adults. Thus, visceral hypersensitivity in the post-weaning period might play a more meaningful pathophysiologic role in the formation of adult irritable bowel syndrome.
Adult ; Animals ; Female ; Humans ; Hypersensitivity* ; Irritable Bowel Syndrome ; Male ; Maternal Deprivation ; Obesity ; Rats ; Rats, Sprague-Dawley

Objective In comparison with Xpert C.difficile/Epi through detection of Clostridium difficile toxin genes from clinical stool , the performance of a laboratory-developed ( LD) assay was evaluated in detail.Methods A total of 176 stool specimens collected from patients with diarrhea in the First People′s Hospital of Yuhang District and the People′s Hospital of Yingzhou , Ningbo from August 1 to December 30 were detected by the two assays in parallel , and meanwhile the C.difficile strains will be isolated and identified for C.difficile toxin genes by a conventional PCR assay .The Cross-tabs Analysis was used for the results by using SPSS20.0 software.Results In comparison with the results of Xpert C.difficile/Epi as the standard, the LD assay had a sensitivity of 91.7％(22/24), a specificity of 100％(152/152), a positive predictive value (PPV) of 100％(22/22), and negative predictive value (NPV) 98.7％(152/154).The results of two assays were statistically coherent (Kappa=0.950, P<0.001).In comparison with culture and detection of toxin genes results , the LD assay had a sensitivity of 90.0％ ( 18/20 ) , a specificity of 97.0％(152/156), a PPV of 81.8％ (18/22), and NPV of 98.7％ (152/154)(Kappa=0.838, P<0.001), and the Xpert C.difficile/Epi assay had a sensitivity of 90.0％ (18/20), a specificity of 96.0％(150/156), a PPV of 75.0％(18/24), and NPV of 98.7％ (150/152)(Kappa=0.792, P<0.001). Conclusions The performance of the LD assay was similar to that of the Xpert C .difficile/Epi kit in detection of toxigenic C.difficile.The LD assay could be directly applied to detection of toxigenic C.difficile from clinical stool samples .The clinical application of this LD assay will also provide a domestic and promising diagnostic assay for diagnosis of C.difficile infection in China.

Aging-elevated DNMT3A R882H-driven clonal hematopoiesis (CH) is a risk factor for myeloid malignancies remission and overall survival. Although some studies were conducted to investigate this phenomenon, the exact mechanism is still under debate. In this study, we observed that DNMT3A R878H bone marrow cells (human allele: DNMT3A R882H) displayed enhanced reconstitution capacity in aged bone marrow milieu and upon inflammatory insult. DNMT3A R878H protects hematopoietic stem and progenitor cells from the damage induced by chronic inflammation, especially TNFα insults. Mechanistically, we identified that RIPK1-RIPK3-MLKL-mediated necroptosis signaling was compromised in R878H cells in response to proliferation stress and TNFα insults. Briefly, we elucidated the molecular mechanism driving DNMT3A R878H-based clonal hematopoiesis, which raises clinical value for treating DNMT3A R882H-driven clonal hematopoiesis and myeloid malignancies with aging.

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease (IBD) that has become a major gastroenterologic problem during recent decades. Numerous complicating factors are involved in UC development such as oxidative stress, inflammation, and microbiota disorder. These factors exacerbate damage to the intestinal mucosal barrier. Spirulina platensis is a commercial alga with various biological activity that is widely used as a functional ingredient in food and beverage products. However, there have been few studies on the treatment of UC using S. platensis aqueous extracts (SP), and the underlying mechanism of action of SP against UC has not yet been elucidated. Herein, we aimed to investigate the modulatory effect of SP on microbiota disorders in UC mice and clarify the underlying mechanisms by which SP alleviates damage to the intestinal mucosal barrier. Dextran sulfate sodium (DSS) was used to establish a normal human colonic epithelial cell (NCM460) injury model and UC animal model. The mitochondrial membrane potential assay 3-‍‍(4,5-dimethylthiazol-2-yl)-2,‍5-diphenyltetrazolium bromide (MTT) and staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and Hoechst 33258 were carried out to determine the effects of SP on the NCM460 cell injury model. Moreover, hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), western blot, and 16S ribosomal DNA (rDNA) sequencing were used to explore the effects and underlying mechanisms of action of SP on UC in C57BL/6 mice. In vitro studies showed that SP alleviated DSS-induced NCM460 cell injury. SP also significantly reduced the excessive generation of intracellular reactive oxygen species (ROS) and prevented mitochondrial membrane potential reduction after DSS challenge. In vivo studies indicated that SP administration could alleviate the severity of DSS-induced colonic mucosal damage compared with the control group. Inhibition of inflammation and oxidative stress was associated with increases in the activity of antioxidant enzymes and the expression of tight junction proteins (TJs) post-SP treatment. SP improved gut microbiota disorder mainly by increasing antioxidant enzyme activity and the expression of TJs in the colon. Our findings demonstrate that the protective effect of SP against UC is based on its inhibition of pro-inflammatory cytokine overproduction, inhibition of DSS-induced ROS production, and enhanced expression of antioxidant enzymes and TJs in the colonic mucosal barrier.
Animals ; Antioxidants/pharmacology* ; Colitis/prevention & control* ; Colitis, Ulcerative/metabolism* ; Colon/metabolism* ; Dextran Sulfate/toxicity* ; Disease Models, Animal ; Gastrointestinal Microbiome ; Inflammation/metabolism* ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Spirulina