Qilongtian ameliorate bleomycin-induced pulmonary fibrosis in mice via inhibiting IL-17 signal pathway

Abstract

Pulmonary fibrosis (PF) is a special type of pulmonary parenchymal disease, with chronic, progressive, fibrosis, and high mortality. There is a lack of safe, effective, and affordable treatment methods. Qilongtian (QLT) is a traditional Chinese prescription that is composed of Panax notoginseng, Earthworm, and Rhodiola, and shows the remarkable clinical curative effect of PF. However, the mechanism of QLT remains to be clarified. Therefore, we studied the effectivity of QLT in treating Bleomycin (BLM) induced PF mice. 36 C57BL/6 J mice were randomized into the control group, the model group, the low-, medium- and high-dose QLT group, and Pirfenidone group. After establishing a model of pulmonary fibrosis in mice, the control and model groups were infused with a normal saline solution, and the delivery group was infused with QLT. Pulmonary function in the mice from each group was detected. Pulmonary tissue morphologies and collagen deposition were stained by HE and Masson. The content of hydroxyproline (HYP) was detected by alkaline hydrolysis and the mRNA and protein expression of related genes in pulmonary tissues were detected by using q-PCR, ELISA, and Western blot. Our studies have shown that QLT significantly reduced the inflammatory injury, hydroxy-proline content, and collagen deposition of pulmonary tissue in BLM-induced PF mice and down-regulated the cytokine related to inflammation and fibrosis and PF expression on the mRNA and protein level in PF mice. To identify the mechanism of QLT, the Transcriptome was measured and the IL-17 signal pathway was screened out for further research. Further studies indicated that QLT reduced the mRNAs and protein levels of interleukin 17 (IL-17), c-c motif chemokine ligand 12 (CCL12), c-x-c motif chemokine ligand 5 (CXCL5), fos-like antigen 1 (FOSL1), matrix metalloproteinase-9 (MMP9), and amphiregulin (AREG), which are inflammation and fibrosis-related genes in the IL-17 signal pathway. The results indicated that the potential mechanism for QLT in the prevention of PF progression was by inhibiting inflammation resulting in the IL-17 signal pathway. Our study provides the novel scientific basis of QLT and represents new therapeutics for PF in clinical.

Figure 1

Therapeutic effect of QLT in PF mice. (a) HE staining was used to indicate the morphology of pulmonary tissue (400 × magnification). (b) Masson staining was used to indicate collagen deposition in pulmonary tissue (400 × magnification). (c) The area percentage of blue collagen fibers in Masson pathological sections. (d) The level of hydroxyproline in pulmonary tissue. (n = 3; data are expressed as mean ± SD. ^∗P < 0.05, compared with Model group).

Figure 2

Effects of pulmonary function in each group. (a) Cchord: an index reflecting alveolar compliance and expansibility, representing the effect of changes in thoracic pressure on lung volume. It also reflects pulmonary tissue elasticity and airway resistance. (b) MMEF: It reflects the detection index of alveolar diffusion function. (c) PEF: it mainly reflects whether the large airway is blocked. (d) FEV 50/FVC: it is the volume of exhaled volume after maximum deep inhalation and maximum exhalation for 50 ms in mice, Obstructive or mixed type is slightly reduced to significantly reduced. (e) FRC: The decrease indicates a reduction in alveolar function. (f) TLC: The decrease is mostly related to restrictive ventilation disorder, suggesting that it is related to PF. (n = 3; data are expressed as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, compared with Model group).

Figure 3

Effects of inflammation and collagen-related indexes in each group. (a) The relative expression level of COL-I, COL-III, α-SMA, TGF-β, and TNF-α mRNA levels in pulmonary tissues. (b) The location and expression levels of COL-I and COL-III in pulmonary tissues were detected by using an immunofluorescence assay. (c) The protein levels of α-SMA, TGF-β, and TNF-α were detected by western blot. (d) The quantification and statistical analysis of (b–d). (n = 3; data are expressed as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, compared with Model group).

Figure 4

IL-17 signal pathway is prominent in the development of pulmonary fibrosis and the influence of QLT on IL-17 signal pathway. (a) Heat map of differential genes clustering in pulmonary tissues. (b) Volcano plot distribution of PM 2.5 differential genes cluster heat map in pulmonary tissues. (c) KEGG analysis bubble diagram of differential genes in pulmonary tissue (X-axis represents the P-value of the enrichment factor, and the Y-axis represents the names of the 12 pathways with significant differences. The size of the dots represents the number of target genes, and the color of the dots represents the range of the false discovery rate (FDR), which represents the ratio of target genes of a specific pathway to the number of all annotated genes in that pathway). (d) The 5 differential genes in pulmonary tissue were enriched into the genes table of IL-17 signal pathway.

Figure 5

The influence of QLT on IL-17 signal transduction pathway. qRT-PCR was used to verify the mRNA levels of (a) IL-17, (b) CCL12, (c) FOSL1, (d) MMP9, (e) CXCL5 and (f) AREG in pulmonary tissues. Elisa was used to verify the protein levels of (g) IL-17, (h) CCL12, (i) FOSL1, (j) MMP9, (k) CXCL5 and (l) AREG in pulmonary tissues. The data shown are Mean ± SEM of 3 independent experiments. (n = 3; data are expressed as mean ± SD. ^∗P < 0.05^∗∗P < 0.01, compared with Model group).