The effects of CBPP-M on LPS-induced chronic bronchitis were further analyzed by RNA-Seq at the genetic level. The standards were set with a false discovery rate (FDR) ≤ 0.001 and |log2Ratio| ≥ 1. Differentially expressed genes were screened under these standards in Con
vs Mod, Con
vs CBPP-M and Mod
vs CBPP-M. A total of 40 genes were screened out and then clustered according to the log2Ratio. The detailed data of the log2Ratio in the cluster are shown in Additional file
3: Table S2. As shown in Fig.
4c, 34 genes were up-regulated and six genes were down-regulated in the Con
vs Mod. Most of differentially regulated genes were up-regulated in the Mod. However, in the CBPP-M, these were obviously down-regulated. Of the 34 up-regulated genes, 28 were associated with collagen formation, muscle contraction, inflammation or immunoregulation. Col1a1, Col1a2, Col3a1, Loxl1 and Serpinf1 are associated with collagen synthesis. Myh6, Myl7, Tnni, Scl4a1, Gbp4, Top2a and Tpx2 are associated with muscle contraction. Ten differentially expressed genes are associated with inflammation: S100a8, S100a9, Ngp, Rsad2, Clqtnf6, Wif1, Sfrp2, Grm3, Adamts17 and Serpinf1. Additionally, 12 genes are associated with immunity: Fcnb, Clec4d, Cpa3, Rectnlg, Igsf10, Scn3b, S100a8, S100a9, Ngp, Top2a, Tpx2 and Opcml.
Proline-glycine-proline (PGP) are extracellular peptides fragments found in collagen. Matrix metalloproteinase (MMP)-9 is the main enzyme involved in this procedure. The protein degradation products and enzymes were elevated in the sputum. Aerosolized LPS administered to mice has been shown to increase neutrophils and N-α-PGP levels in the airway [
18]. PGP are chemoattractants in chronic neutrophilic inflammation associated with IL-8 and with recruiting inflammatory cells to the lung. In addition, collagen could trigger the VEGF and TGF-beta pathways. Gene expression profiling of the lung from chronic obstructive pulmonary disease patients revealed that Col6a3 and Serpinf1 were associated with emphysema severity [
19]. Col1a1, Col1a2 and Col3a1 are involved in collagen I and III formation, and Loxl1 is usually active in collagen substrates. Our data show that these gene expression levels were noticeably decreased after CBPP-M treatment. Therefore, intervention with CBPP not only remitted airway inflammation but also reduced sputum.
Myh6 and Myl7 are components of myosin chains. Tnni confers calcium sensitivity to striated muscle actinomyosin. With increasing Ca
2+ concentration, the interaction of actin and myosin increases and results in a cough. Chronic airway bacterial infections with symptoms of cough and wheezing have been shown to be associated with neutrophilic inflammation and high levels of IL-8 [
20]. IL-8 could increase the phosphorylation of the myosin light chain and induce constriction in cystic fibrosis cells. In this paper, leukocyte numbers and IL-8 levels in BALF were increased in the Mod group. This result indicated that myosin-associated gene expression would be up-regulated. In muscle cells, the cytoskeleton and its binding proteins provide the power system for muscle contraction. Thus, the expression of the aforementioned five genes decreased in CBPP-M treatment indicates that CBPP could remit muscle contraction, arrest cough and relieve asthma.
In addition, S100a8 and S100a9 are calcium-binding proteins that have anti-microbial activity. NF-kappa-B could up-regulate the transcription of these genes. These genes promote tubulin polymerization, phagocyte migration and the infiltration of granulocytes at sites of wounding. Microtubule dynamics change with macrophage migration [
21]. Ngp is a neutrophilic granule protein that increases expression and is indicative of inflammation. Wif1 and Sfrp2 are related to the Wnt signaling pathway in the regulation of inflammation. Treatment with CBPP could down-regulate the expression of these genes, demonstrating anti-inflammatory and immune-regulatory function.