Shexiang Tongxin Dropping Pill Allieviates Heart Failure via Extracellula Matrix-Receptor Interaction Pathways Based on RNA-Seq Transcriptomics and Experimental Studies.

OBJECTIVE: To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF). METHODS: Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting. RESULTS: The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs. CONCLUSIONS: STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.

The role of AMPK-Sirt1-autophagy pathway in the intestinal protection process by propofol against regional ischemia/reperfusion injury in rats.

Intestinal ischemia/reperfusion (II/R) is a clinical event associated with high morbidity and mortality. AMP-activated protein kinase (AMPK), a central cellular energy sensor, is associated with oxidative stress and inflammation. However, whether the AMPK is involved in the II/R-induced intestinal injury and the underlying mechanism is yet to be elucidated. Propofol has a protective effect on organs; yet, its specific mechanism of action remains unclear. This study explored the role of the AMPK-Sirt1-autophagy pathway in intestinal injury, and whether propofol could reduce intestinal injury and investigated the mechanisms in a rat model of II/R injury as well as a cell model (IEC-6 cells) of hypoxia/reoxygenation (H/R). Propofol, AMPK agonist (AICAR) and AMPK inhibitor (Compound C) were then administered, respectively. The histopathological changes, cell viability and apoptosis were detected. Furthermore, the levels of proinflammatory factors, the activities of oxidative stress, diamine oxidase, and signaling pathway were also analyzed. The results demonstrated that the AMPK-Sirt1-autophagy pathway of intestine was activated after II/R or H/R. Propofol could further activate the pathway, which reduced intestinal injury, inhibited apoptosis, reversed inflammation and oxidative stress, and improved the 24-hour survival rate in II/R rats in vivo, and attenuated H/R-induced IEC-6 cell injury, oxidative stress, and apoptosis in vitro, as fine as changes in AICAR treatment. Compound C abrogated the protective effect of propofol on II/R and H/R-induced injury. These results suggested a crucial effect of AMPK on the mechanism of intestinal injury and might provide a new insight into the mechanism of propofol reducing II/R injury.

Protective Mechanisms of Suxiao Jiuxin Pills () on Myocardial Ischemia-Reperfusion Injury in vivo and in vitro.

OBJECTIVE: To study the protective mechanism of Chinese medicine Suxiao Jiuxin Pills (, SXJ) on myocardial ischemia and reperfusion (I/R) injury. METHODS: Mouse myocardial I/R injury model was created by 30-min coronary artery occlusion followed by 24-h reperfusion, the mice were then divided into the sham group (n=7), the I/R group (n=13), the tirofiban group (TIR, positive drug treatment, n=9), and the SXJ group (n=11). Infarct size (IS), risk region (RR), and left ventricle (LV) were analyzed with double staining methods. In addition, H9C2 rat cardiomyocytes were cultured with Na2S2O4 to simulate I/R in vitro. The phosphorylation of extracellular regulated protein kinases1/2 (ERK1/2), protein kinase B (AKT), glycogen synthase kinase-3ß (GSK3ß), and protein expression of GATA4 in nucleus were detected with Western blot assay. RESULTS: The ratio of IS/RR in SXJ and TIR groups were lower than that in I/R group (SXJ, 22.4% ±6.6%; TIR, 20.8%±3.3%; vs. I/R, 35.4%±3.7%, P<0.05, respectively). In vitro experiments showed that SXJ increased the Na2S2O4-enhanced phosphorylation of AKT/GSK3ß and nuclear expression of GATA4. CONCLUSION: SXJ prevents myocardial I/R injury in mice by activating AKT/GSK3ß and GATA4 signaling pathways.

Correlation of diet, microbiota and metabolite networks in inflammatory bowel disease.

OBJECTIVES: Microbiota dysbiosis in inflammatory bowel disease (IBD) has been widely reported. The gut microbiota connect diet to the metabolism by producing small molecules via diverse metabolic pathways. In this study we aimed to investigate the dietary preferences of IBD patients, and to explore the interactions among gut microbiota composition, dietary components, and metabolites in relation to IBD. METHODS: Dietary preferences of IBD patients (including those with ulcerative colitis [UC] and Crohn's disease [CD]) and health controls were investigated, and their gut microbiota were analyzed using 16S rRNA gene sequencing and metagenomic analyses of fecal and biopsy samples. The metabolite profiles of the samples were then analyzed using gas and liquid chromatography-mass spectrometry analyses. RESULTS: The daily intake of folic acid, niacin, vitamins C and D, calcium, and selenium differed significantly between patients with IBD and healthy controls. A decrease in long-chain (such as arachidic, and oleic acid) and medium-chain fatty acids (sebacic acid and isocaproic acid) as well as bile acid was observed in patients with IBD. Compared with healthy controls, 22 microbial species (including Sulfolobus acidocaldarius, and Clostridium clostridioforme CAG132) in the UC group and 37 microbial species (such as Bacteroides fragilis and Fusobacterium nucleatum) in the CD group were found to be correlated to diet and metabolites. Bacteroides fragilis was enriched in patients with IBD and associated with multi-nutrients, and 21 metabolites including 25-hydroxyvitamin D3 and taurolithocholic acid. CONCLUSIONS: This study provides an interaction network to identify key micronutrients, microbiota components and metabolites that contribute to IBD.

Features of Ebola Virus Disease at the Late Outbreak Stage in Sierra Leone: Clinical, Virological, Immunological, and Evolutionary Analyses.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

An Interaction between the Inner Rod Protein YscI and the Needle Protein YscF Is Required to Assemble the Needle Structure of the Yersinia Type Three Secretion System.

The type III secretion system is a highly conserved virulence mechanism that is widely distributed in Gram-negative bacteria. It has a syringe-like structure composed of a multi-ring basal body that spans the bacterial envelope and a projecting needle that delivers virulence effectors into host cells. Here, we showed that the Yersinia inner rod protein YscI directly interacts with the needle protein YscF inside the bacterial cells and that this interaction depends on amino acid residues 83-102 in the carboxyl terminus of YscI. Alanine substitution of Trp-85 or Ser-86 abrogated the binding of YscI to YscF as well as needle assembly and the secretion of effectors (Yops) and the needle tip protein LcrV. However, yscI null mutants that were trans-complemented with YscI mutants that bind YscF still assembled the needle and secreted Yops, demonstrating that a direct interaction between YscF and YscI is critical for these processes. Consistently, YscI mutants that did not bind YscF resulted in greatly decreased HeLa cell cytotoxicity. Together, these results show that YscI participates in needle assembly by directly interacting with YscF.

Isolation and identification of polyphenols from Marsilea quadrifolia with antioxidant properties in vitro and in vivo.

Marsilea quadrifolia is an edible aquatic medicinal plant used as a traditional health food in Asia. Four new polyphenols including kaempferol 3-O-(2″-O-E-caffeoyl)-ß-d-glucopyranoside (1), kaempferol 3-O-(3″-O-E-caffeoyl)-α-l-arabinopyranoside (3), 4-methy-3'-hydroxypsilotinin (4) and (±)-(E)-4b-methoxy-3b,5b-dihydroxyscirpusin A (18) together with 14 known ones (2, 5-17) were isolated from the ethanol extract of M. quadrifolia. Structures of the new compounds were elucidated by extensive spectroscopic analyses. In DPPH and oxygen radical absorbance capacity antioxidant assays, some compounds showed stronger antioxidant activities and quercetin (9) was the most potent antioxidant in both assays. In a restraint-induced oxidative stress model in mice, quercetin significantly attenuated the increase in plasma ALT and AST levels as well as liver MDA content of restrained mice. Liver SOD activity was also significantly increased by quercetin, indicating a significant in vivo antioxidant activity. As a rich source of polyphenols with strong antioxidant activities, M. quadrifolia may be developed to a product for relieving oxidative stress.

Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica.

A new quassinoid, bruceene A (1) along with seventeen known quassinoids (2-18) was isolated from the fruits of Brucea javanica. The structure of 1 was elucidated by extensive spectroscopic methods, and was further confirmed by single-crystal X-ray diffraction analysis. Isolation of similar quassinoids 1-3 as those in genus Ailanthus from genus Brucea, indicated the close chemotaxonomic relationship between these two genera, which further supported the phylogenetic study by DNA analysis. Compounds 5, 7, 10 and 12 with a 3-hydroxy-3-en-2-one moiety showed potent inhibitory activities against the MCF-7 and MDA-MB-231 cells with IC50 values in the ranges 0.063-0.182 µM and 0.081-0.238 µM, respectively; while glycosidation at 3-OH significantly decreased the cytotoxicity. It was also found that the most potent compound 7 induced apoptosis in MCF-7 cells via the intrinsic mitochondrial apoptotic pathway.

Structures, chemotaxonomic significance, cytotoxic and Na(+),K(+)-ATPase inhibitory activities of new cardenolides from Asclepias curassavica.

Five new cardenolide lactates (1­5) and one new dioxane double linked cardenolide glycoside (17) along with 15 known compounds (6­16 and 18­21) were isolated from the ornamental milkweed Asclepias curassavica. Their structures were elucidated by extensive spectroscopic methods (IR, UV, MS, 1D- and 2D-NMR). The molecular structures and absolute configurations of 1­3 and 17 were further confirmed by single-crystal X-ray diffraction analysis. Simultaneous isolation of dioxane double linked cardenolide glycosides (17­21) and cardenolide lactates (1­5) provided unique chemotaxonomic markers for this genus. Compounds 1­21 were evaluated for the inhibitory activities against DU145 prostate cancer cells. The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates, while the C21 steroids were non-cytotoxic. Enzymatic assay established a correlation between the cytotoxic effects in DU145 cancer cells and the Ki for the inhibition of Na(+),K(+)-ATPase. Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds 18 or 20, and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na(+),K(+)-ATPase than the cardenolide lactate.

Two new bufadienolides from the rhizomes of Helleborus thibetanus with inhibitory activities against prostate cancer cells.

Two new bufadienolide glycosides (1 and 2) with an A/B trans ring fusion together with nine known compounds (3-11) were isolated from the rhizomes of Helleborus thibetanus. The structures of new compounds were elucidated by extensive spectroscopic analyses in combination with single-crystal X-ray diffraction. The bufadienolides 1 and 3-6 exhibited potent cytotoxic activities against the prostate cancer cells.

[Study on the functions of potential new genes of Yersinia pestis type three secretion system].

OBJECTIVE: To investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS). METHODS: Mutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A ß-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS. RESULTS: When grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The ß-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain. CONCLUSION: YpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Use of rich BHI medium instead of synthetic TMH medium for gene regulation study in Yersinia pestis.

OBJECTIVE: This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. METHODS: The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 °C, and with temperature shifting from 26 to 37 °C, the wild-type (WT) strain or its phoP or crp null mutant (ΔphoP or Δcrp, respectively) was subject to RNA isolation, and then the promoter activity of rovA or pla in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and ΔphoP to measure the promoter activity of rovA in these two strains with the ß-Galactosidase enzyme assay system. RESULTS: When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that of pla was stimulated by CRP. CONCLUSION: The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions (which are essential to triggering relevant gene regulatory cascades), can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.

Cellular fatty acids as chemical markers for differentiation of Acinetobacter baumannii and Acinetobacter calcoaceticus.

OBJECTIVE: Gas chromatography (GC) was used to investigate the cellular fatty acid (CFA) composition of 141 Acinetobacter baumannii and 32 A. calcoaceticus isolates from different locations in China and to find chemical markers to differentiate these two closely related bacteria. METHODS: Whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation, and extraction for GC analysis, followed by a standardized Microbial Identification System (MIS) analysis. RESULTS: All A. baumannii and A. calcoaceticus strains contained some major fatty acids, namely, 18:1 ω9c, 16:0, Sum In Feature 3, 12:0, 17:1ω8c, 3-OH-12:0, 17:0, Sum In Feature 2, 2-OH-12:0, and 18:0 compounds. Although most of the total CFAs are similar between A. baumannii and A. calcoaceticus strains, the ratios of two pairs of CFAs, i.e., Sum In Feature 3/18:1 ω9c versus 16:0/18:1 ω9c and Sum In Feature 3/18:1 ω9c versus unknown 12.484/18:1 ω9c fatty acids, could differentiate these two closely related bacteria. A. baumannii could be easily classified into two subgroups by plotting some ratios such as Sum In Feature 3/16:0 versus 17:0 and Sum In Feature 3/2-OH-12:0 versus 17:0 fatty acids. CONCLUSION: The ratios of some CFAs could be used as chemical markers to distinguish A. baumannii from A. calcoaceticus.

[Multi-locus sequence typing of multidrug-resistant of Acinetobacter baumannii from China and characterization of population structure of Acinetobacter baumannii].

OBJECTIVE: To characterize the genetic background of multidrug-resistant Acinetobacter baumannii (A. baumannii) from China, and the population structure of this pathogen. METHODS: A previously reported MLST scheme was applied to a collection of 33 multidrug-resistant strains of A. baumannii from China, and the data of all the strains in the A. baumannii MLST database were downloaded for the population structure analysis. The sequence types and clonal complexes were identified, the presence or absence of recombination was analyzed for each MLST locus, and the values of I(A)(S), and recombination/mutation ratio were calculated for the whole strain collection. A phylogenetic tree was constructed using all the allelic profiles in the database. RESULTS: A total of six sequence types were identified from the 33 Chinese strains tested, and 29 of these strains belonged to the CC92 clonal complex. Three (gdhB, gpi, and rpoD) of the seven MLST loci (gltA, gyrB, recA, cpn60, gdhB, gpi, rpoD) had undergone recombination with statistical evidence. For all allele profiles in the MLST database, the I(A)(S) value was 0.155 and the recombination/mutation ratio was 6.083. Sequence types from each clonal complex were grouped closely in the phylogenetic tree, which gave an overview of the microevolution of this pathogen. CONCLUSION: The spread of multidrug-resistant A. baumannii in China was closely related to the CC92 clonal complex. A. baumannii had an 'epidemic' population structure, i.e., a superficially clonal structure with high levels of recombination, in which successful epidemic clones arise especially including worldwide dissemination of the CC92 clonal complex to cause a widespread occurrence of multidrug-resistant infections.