Systems biology identifies that regulation of type I interferon signaling is involved in the anti-atherogenic effects of andrographis paniculata
Description
Andrographis paniculata (Burm. f.) Wall. ex Nees is a medicinal herb widely used in many Asian countries. Currently, there is little information about the effects and underlying mechanisms of whole andrographis paniculata plant on the development of atherosclerosis. To address this question, we carried out experiments in mouse primary peritoneal macrophages treated with crude serum preparations isolated from normal rats receiving vehicle or andrographis decoction treatment. Genome-wide mRNA sequencing revealed that ~70% of the genes regulated by lipopolysaccharides were reversed by andrographis. Further bioinformatics analysis indicated that andrographis inhibited type I interferon signaling. Given the established pathogenic effects of type I interferons in atherosclerosis, our results suggest that regulation of the type I interferon signaling in macrophage cells is partly involved in the anti-atherogenic effects of andrographis paniculata.
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Mouse primary peritoneal macrophages were induced with thioglycollate as previously described {Liu, 2016 #30}, and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Thermo Fisher, Waltham, MA, USA), in a humidified atmosphere with 5% CO2 at 37oC. Before treatment, cells were sub-cultured into 6-well plates (105 cells per well) using serum-free RPMI-1640. Then cells were incubated overnight with 10% rat serum from either vehicle control animals or andrographis-treated animals. Then the cells were challenged with lipopolysaccharides (LPS) (from Solarbio, Beijing, China) at 1 g/mL for 2 hr. PBS was used as vehicle control. Experimental design: 4 treatment groups with 3 independent replicates in each group 1. Control group (column title C1 to C3): treated with serum from vehicle control rats 2. Andrographis alone (column title A1 to A3): treated with serum from andrographis-treated rats 3. LPS alone (column title L1 to L3): treated with serum from vehicle control rats followed by LPS challenge 4. Andrographis+LPS (column title A+L1 to A+L3): treated with serum from andrographis-treated rats followed by LPS challenge For mRNA sequencing (RNAseq) studies, 3 independent samples were included in each experimental group. The RNA processing and sequencing services were provided by Sinotech Genomics (Shanghai, China). Total RNA was isolated using RNeasy mini kit (from QIAGEN, Hilden, Germany). RNA quantity was checked using Bioanalyzer 2100 (from Agilent, Santa Clara, CA, USA). Paired-end libraries were synthesized using the TruSeq RNA Sample Preparation Kit (from Illumina, San Diego, CA, USA). Briefly, poly-A mRNAs were purified using oligo dT-conjugated magnetic beads, and fragmented by treating with divalent cations under 94oC for 8 min. Cleaved RNA fragments were converted to first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using E. coli DNA Polymerase I and RNase H. The cDNA fragments were ligated to index adapters, and amplified by PCR to create the final cDNA library. Purified libraries were quantified using Qubit 2.0 Fluorometer (Thermo Fisher) and validated with Bioanalyzer 2100. Cluster generation was performed using cBot instrument (from Illumina). Paired-end sequencing was performed using a NovaSeq 6000 Sequencing System (Illumina). High quality clean reads were obtained by filtering with Cutadapt (version 1.9) (https://cutadapt.readthedocs.io/en/stable/) and verified with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Read alignment to the Mus musculus reference genome was performed using HISAT2 package (version 2.0.4) (https://daehwankimlab.github.io/hisat2). StringTie (version 1.3.4d) (http://ccb.jhu.edu/software/stringtie) was used for read assembly and estimation of the relative expression levels (expressed as fragment per kilobase per million reads/FPKM values).