Dataset Information

Differential effects of Radix Paeoniae Rubra (Chishao) on cytokine and chemokine expression inducible by mycobacteria.

ABSTRACT:

Background

Upon initial infection with mycobacteria, macrophages secrete multiple cytokines and chemokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor-? (TNF-?), to mediate host immune responses against the pathogen. Mycobacteria also induce the production of IL-10 via PKR activation in primary human monocytes and macrophages. As an anti-inflammatory cytokine, over-expression of IL-10 may contribute to mycobacterial evasion of the host immunity. Radix Paeoniae Rubra (RPR, Chishao), a Chinese medicinal herb with potentials of anti-inflammatory, hepatoprotective and neuroprotective effects, is used to treat tuberculosis. This study investigates the immunoregulatory effects of RPR on primary human blood macrophages (PBMac) during mycobacterial infection.

Methods

The interaction of Bacillus Calmette-Guerin (BCG) with PBMac was used as an experimental model. A series of procedures involving solvent extraction and fractionation were used to isolate bioactive constituents in RPR. RPR-EA-S1, a fraction with potent immunoregulatory effects was obtained with a bioactivity guided fractionation scheme. PBMac were treated with crude RPR extracts or RPR-EA-S1 before BCG stimulation. The expression levels of IL-6, IL-8, IL-10 and TNF-? were measured by qPCR and ELISA. Western blotting was used to determine the effects of RPR-EA-S1 on signaling kinases and transcriptional factors in the BCG-activated PBMac.

Results

In BCG-stimulated macrophages, crude RPR extracts and fraction RPR-EA-S1 specifically inhibited IL-10 production while enhanced IL-8 expression at both mRNA and protein levels without affecting the expressions of IL-6 and TNF-?. Inhibition of BCG-induced IL-10 expression by RPR-EA-S1 occurred in a dose- and time-dependent manner. RPR-EA-S1 did not affect the phosphorylation of cellular protein kinases including MAPK, Akt and GSK3?. Instead, it suppressed the degradation of I?B? in the cytoplasm and inhibited the translocation of transcription factor NF-?B1 p50 to the nucleus.

Conclusion

RPR crude extracts and its fraction RPR-EA-S1 inhibited anti-inflammatory cytokine IL-10 and enhanced pro-inflammatory chemokine IL-8 expression in BCG-activated PBMac. The inhibitory effects of RPR-EA-S1 on IL-10 expression in BCG-activated PBMac may be due to the reduced nuclear translocation of NF-?B1 p50.

SUBMITTER: Wang L

PROVIDER: S-EPMC3076300 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Publications

Differential effects of Radix Paeoniae Rubra (Chishao) on cytokine and chemokine expression inducible by mycobacteria.

Wang Liangjie L Yang Cindy Lai Hung CL Or Terry Cho Tsun TC Chen Gang G Zhou Jian J Li James Chun Bong JC Lau Allan Sik Yin AS

Chinese medicine 20110330 1

<h4>Background</h4>Upon initial infection with mycobacteria, macrophages secrete multiple cytokines and chemokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α), to mediate host immune responses against the pathogen. Mycobacteria also induce the production of IL-10 via PKR activation in primary human monocytes and macrophages. As an anti-inflammatory cytokine, over-expression of IL-10 may contribute to mycobacterial evasion of the host immunity. Radix Paeoniae Rubra ( ...[more]

PMID: 21447195

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Project description:Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of ?-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression strain.

Project description:Background:Radix Paeoniae Alba (RPA) and other natural medicines have remarkable curative effects and are widely used in traditional Chinese Medicine (TCM). However, due to their multi-component and multi-target characteristics, it is difficult to study the detailed pharmacological mechanisms for those natural medicines in vivo. Therefore, their real effects on organisms is still uncertain. Methods:RPA was selected as research object, the present study was designed to study the complex mechanisms of RPA in vivo by integrating and interpreting the transcriptomic based RNA-seq and metabolomic based NMR spectrum after RPA administration in mice. A variety of dimension-reduction algorithms and classifier models were applied to the processing of high-throughput data. Results:Among serum metabolites, the contents of PC and glucose were significantly increased, while the contents of various amino acids, lipids and their metabolites were significantly decreased in mice after RPA administration. Based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, differential analysis showed that the liver was the site where RPA exerted a significant effect, which confirmed the rationality of "meridian tropism" in the theory in TCM. In addition, RPA played a role in lipid metabolism by regulating genes encoding enzymes of the glycerolipid metabolism pathway, such as 1-acyl-sn-glycerol-3-phosphate acyltransferase (Agpat), phosphatidate phosphatase (Lpin), phospholipid phosphatase (Plpp) and endothelial lipase (Lipg). We also found that RPA regulates several substance addiction pathways in the brain, such as the cocaine addiction pathway, and the related targets were predicted based on the sequencing data from pathological model in the GEO database. The overall effective pattern of RPA was intuitively presented with a multidimensional radar map through a self-designed model which found that liver and brain were mainly regulated by RPA compared with the traditional meridian tropism theory. Conclusions:Overall this study expanded the potential application of RPA and provided possible targets and directions for further mechanism study, meanwhile, it also established a multi-dimensional evaluation model to represent the overall effective pattern of TCM for the first time. In the future, such study based on the high-throughput data sets can be used to interpret the theory of TCM and to provide a valuable research model and clinical medication reference for the TCM researchers and doctors.

Dataset Information

Differential effects of Radix Paeoniae Rubra (Chishao) on cytokine and chemokine expression inducible by mycobacteria.

Background

Methods

Results

Conclusion

Publications

Differential effects of Radix Paeoniae Rubra (Chishao) on cytokine and chemokine expression inducible by mycobacteria.

Similar Datasets

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