This study reports the simplified carbapenem inactivation method (sCIM) to detect carbapenemase-producing gram-negative bacilli in a simple and accurate manner. This method is based on the modified carbapenem inactivation method (mCIM) with the improvement of experimental procedures. Instead of incubating the antibiotic disk in the organism culture media, the organism to be tested was smeared directly onto the antibiotic disk in the sCIM. For evaluating the sensitivity and specificity of the method, a total of 196 Enterobacteriaceae, 73 Acinetobacter baumannii, and 158 Pseudomonas aeruginosa isolates were collected. Polymerase chain reaction (PCR) was used to detect the carbapenemase genes. Phenotypic evaluations were performed using both the sCIM and the mCIM. PCR results showed that, of the 196 Enterobacteriaceae strains, 147 expressed the carbapenemase genes blaKPC−2 (58.5%), blaIMP−4 (21.8%), blaIMP−2 (2.0%), blaVIM−1 (6.1%), blaNDM−1 (10.2%), and blaOXA−48 (1.4%). sCIM results had high concordance with PCR results (99.5%) and mCIM results (100%) with the exception of one Klebsiella pneumoniae strain, which had an minimal inhibitory concentration (MIC) for imipenem of 0.25 mg/L. PCR demonstrated that 53 of the 73 A. baumannii isolates expressed the carbapenemase genes blaOXA−23 (98.1%) and blaVIM−2 (1.8%). sCIM and PCR results corresponded but all A. baumannii isolates were carbapenemase negative by the mCIM. PCR demonstrated that 25 of the 158 P. aeruginosa isolates expressed carbapenemase genes blaVIM−1 (52%), blaVIM−2 (8%), blaVIM−4 (36%), and blaIMP−4 (4%). sCIM results had high concordance with PCR results (100%) and the mCIM results (99.4%) with the exception of one P. aeruginosa isolate that expressed the blaVIM−4 gene. The sCIM offers specificity and sensitivity comparable to PCR but has the advantage of being more user-friendly. This method is suitable for routine use in most clinical microbiology laboratories for the detection of carbapenemase-producing gram-negative bacilli.
Background. Maren pills have been used to treat constipation. Aquaporin 3 (AQP3) plays a vital role in regulating water transfer in the colon. It has been reported that the downregulation of AQP3 can regulate liquid water metabolism and intestinal permeability in irritable bowel syndrome (IBS) rats’ colon via NF-κB pathway. In this study, we investigated whether the laxative effect of Maren pills is associated with the regulation of AQP3 and NF-κB signaling pathway in the colon. Methods. The compound diphenoxylate suspension-induced STC rats received Maren pills intragastrically for 1 consecutive week to evaluate the laxative effect of Maren pills involving the regulation of AQP3 and NF-κB signaling pathway. Moreover, human intestinal epithelial cells (HT-29) were treated with drug serum to obtain in vitro data. Results. Our results revealed that treatment with Maren pills increased the stool number, moisture content of feces, and intestinal transit rate in a dose-dependent manner. Maren pills significantly increased the AQP3, fibrosis transmembrane conductance regulator (CFTR), and protein kinase A (PKA) proteins in the colon of rats and in HT-29 cells. Mechanistically, Maren pills obviously inhibited the activation of NF-κB pathway in the colon of rats and in HT-29 cells. Conclusion. These results suggest that the laxative effect of Maren pills is associated with the increased expression of AQP3 by downregulating NF-κB signal pathway.
Background: Slow transit constipation (STC) is becoming a common and frequently occurring disease in today’s society, and it is necessary to explore the safe and effective treatment of STC.Method: Our study aimed to investigate whether the laxative effect of Maren pills (MRW) is associated with the regulation of intestinal microflora and intestinal metabolism in the colon. Loperamide hydrochloride-induced STC rats received MRW intragastrically for two consecutive weeks to evaluate the laxative effect of MRW involving the regulation of intestinal microflora, intestinal metabolism, and 5-HT signaling pathway. Intestinal microflora was detected by 16s rDNA sequencing, intestinal metabolism of short-chain fatty acids (SCFAs) was detected by HPLC, and the 5-HT signaling pathway was detected by WB, ELISA, immunofluorescence, and immunohistochemical analysis.Results: Our results revealed that the treatments with MRW increased not only the body weight, 24-h fecal number, 24-h wet fecal weight, 24-h dry fecal weight, fecal water content, and the intestinal propulsion rate but also the colonic goblet cell number, colonic Muc-2 protein expression, and colonic mucus layer thickness in the STC model rats. Moreover, MRW activated the 5-HT pathway by increasing the levels of 5-HT, 5-HIAA, 5-HT4R, CFTR, cAMP, and PKA in the colon tissue of STC rats. The 16S rDNA sequencing results showed that MRW improved the colonic microflora structure in colonic contents of STC rats, mainly by increasing Lactobacillus and decreasing Prevotella. Finally, we found that MRW regulated the SCFA metabolism in the colonic contents of the STC rats, mainly by increasing the contents of acetic acid, propionic acid, and butyric acid; the relative abundance of Lactobacillus was positively correlated with either contents of acetic acid, propionic acid, and butyric acid, and the relative abundance of Clostridium was negatively correlated.Conclusion: Our study further showed that MRW could improve constipation in STC rats, and the mechanism may be by regulating the intestinal microflora structure and improving the metabolism of SCFAs.
Slow transit constipation (STC) is a common type of constipation with a high incidence rate and a large number of patients. We aimed to investigate the therapeutic effects and potential mechanism of paeoniflorin (PAE) on loperamide-induced Sprague Dawley (SD) rat constipation models. Rats with loperamide-induced constipation were orally administered different concentrations of PAE (10, 20, or 40 mg/kg). In vitro, enterochromaffin (EC)-like RIN-14B cells were treated with 20, 40, or 80 μg/ml PAE. We found that PAE treatment significantly improved the symptoms of constipation and increased the intestinal transit rate. Hematoxylin and eosin (H&E) staining showed that PAE alleviated colonic tissue pathological damage. Besides, our results implied that PAE concentration-dependently promoted the content of 5-hydroxytryptamine (5-HT) catalyzed by tryptophan hydroxylase (Tph)-1 in the serum of loperamide-induced rats and in RIN-14B cells. Western blot and immunofluorescence (IF) stain indicated that PAE also promoted the expression of G protein-coupled BA receptor 1 (TGR5), transient receptor potential ankyrin 1 (TRPA1), phospholipase C (PLC)-γ1, and phosphatidylinositol 4,5-bisphosphate (PIP2) in vivo and in vitro. RIN-14B cells were cotreated with a TGR5 inhibitor (SBI-115) to explore the mechanism of PAE in regulating the 5-HT secretion. We observed inhibition of TGR5 reversed the increase of 5-HT secretion induced by PAE in RIN-14B cells. We provided evidence that PAE could promote 5-HT release from EC cells and improve constipation by activating the TRPA1 channel and PLC-γ1/PIP2 signaling. Thus, PAE may provide therapeutic effects for patients with STC.
Hypoxia-inducible factor (HIF) is identified to be a promising target to mediate the response to hypoxia. Its stability and activation are negatively controlled by prolyl hydroxylase 2 (PHD2). Thus, PHD2 inhibition has been perceived as a promising anti-anemia therapy. In this study, we carried out a structure-based virtual screening followed by in vitro and in vivo biological validation, with the goal to identify novel PHD2 inhibitors. As a result, a set of hits with new chemical scaffolds were revealed to be active in vitro for PHD2 inhibition. Compounds 2 and 3 were revealed to be capable of stabilizing HIF-α and stimulating erythropoietin (EPO) expression in cell-based assays. Notably, further in vivo assays revealed that 2 was capable of elevating the EPO plasma levels in C57BL/6 mice model. These findings provide new chemical scaffolds for further development of PHD2 inhibitors. K E Y W O R D Santi-anemia, EPO, HIF, PHD2 inhibitor, virtual screening SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section. How to cite this article: Yu Z, Li Z, Yu Q, et al. Discovery of prolyl hydroxylase 2 inhibitors with new chemical scaffolds as in vivo active erythropoietin inducers through a combined virtual screening strategy. Chem Biol Drug Des. 2020;95:270-278.
Background: Constipation is one of the most prevalent chronic gastrointestinal diseases. Notably, previous studies have demonstrated that Chinese herbal compounds may exert effects on constipation. The present study aimed to predict the mechanisms underlying the effects of Zhi Zhu Ma Ren Pill (ZZMRP), which includes Aurantii Fructus Immaturus, Atractylodis Macrocephalae Rhizoma, Fructus Cannabis, Paeonia lactiflora and Radix Asteris in the treatment of constipation, using network pharmacology and molecular docking. Methods: The components and target information of ZZMRP were accessed using the Traditional Chinese Medicine Systems Pharmacology database and analysis platform, and the associated targets of constipation were obtained from the GeneCards, Disgenet, Online Mendelian Inheritance in Man, DrugBANK and Therapeutic Target Database databases. The major targets were subsequently selected using a Venn diagram and network topology analysis, which was followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Molecular docking was performed to authenticate the binding activity between active components and core targets. Results: A total of 44 active components, 249 targets of ZZMRP and 1501 targets associated with constipation were acquired. A total of 122 intersection targets were discovered between ZZMRP and constipation. Subsequently, 18 key targets were authenticated, including tumor protein 53, RACalpha serine/threonine-protein kinase, JUN and caspase-3. GO and KEGG pathway enrichment analysis indicated that mitogenactivated protein kinase, tumor necrosis factor, and phosphoinositide 3-kinase/protein kinase B signaling pathways may be involved in the treatment of constipation using ZZMRP. Molecular docking suggested that quercetin, kaempferol, and luteolin exhibited high binding affinities with several of the primary targets. Conclusions: The active components, core targets, and signaling pathways of ZZMRP in the treatment of constipation were predicted, which may be applicable to the development of treatments for constipation and application of ZZMRP.
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