CpG oligodeoxynucleotides (ODNs) are synthetic DNA sequences containing unmethylated cytosine–guanine motifs, which activate TLR9, and CpG ODNs can activate many kinds of immune cells [
4]. CpG ODNs can cause the differentiation of B cells, stimulate B cells to secrete cytokines IL-6 and IL-10, and prevent apoptosis of B cells [
13]. In cooperation with IL-12, CpG ODNs can activate NK cells directly to secrete IFN-γ, which in turn, can further promote the activation of monocytes, macrophages and dendritic cells [
14]. At present, the research of the CpG as immune adjuvant is thorough. In the CpG ODN auxiliary hepatitis b vaccine clinical trials, researchers found that CpG ODN can effectively activate the body’s immune system to produce the cellular and humoral immune response with the hepatitis b vaccine, and found no obvious toxic effects [
15,
16]. It has been proven that CpG ODNs can treat infectious diseases, cancer and allergic diseases [
17‐
19]. For example, a study found that protamine nanoparticles with CpG-oligodeoxynucleotide prevent an allergen-induced Th2-response in BALB/c mice [
20]. Our previous experiments gave mice ODN1826 through intraperitoneal injection on day 1, 3, 5, 7 and 9, and found that CpG ODN1826 has obvious radio-sensitization effects to lung cancer, and no obvious cytotoxicity was found [
21]. Radiation-induced pulmonary fibrosis is the main pathological process of the late radiation induced lung injury [
2]. In the early damage, inflammatory mediators secretion increased continuously, causing alveolar exudates to increase, pulmonary interstitial hyperemia edema and inflammatory reaction cells infiltration. In the late damage, an exception repairing and tissue remodeling occurred, muscle fibroblasts increased, which resulted collagen deposition and fibrosis [
8,
22].
Recent studies suggest that the pathological morphological changes of radiation-induced lung injury mainly include pulmonary interstitial hyperemia and edema, an increase in alveolar exudates, inflammatory cells infiltration, late fiber connective tissue hyperplasia and alveolar atrophy [
23,
24]. The H&E staining and Masson staining of paraffin sections showed the radiation-induced pulmonary fibrosis mouse model was clearly established in this study. CpG ODN1826 could reduce fiber cell proliferation and collagen deposition. Hyp accounts for 13.4 % of collagen and is often used to reflect pulmonary fibrosis [
9]. Our study found that CpG ODN1826 could reduce the Hyp content of the exposed lung. From these results, we posit that CpG ODN1826 might ameliorate pulmonary fibrosis caused by irradiation.
TGF-β is the growth regulating factor for the epithelial origin cells in human, and has extensive biological effects in cell growth, differentiation, extracellular matrix deposition and the immune reaction of the body [
25]. TGF-β1, one of the subtypes of TGF-β, has been acknowledged as the most important cytokine to result the fibrosis in late injury, which can not only induce the fibroblast around the bronchioles branch and vascular into myofibroblasts, but also induce the normal lung epithelial cells into myofibroblasts, and thus play an important role in the process of fibrosis [
26,
27]. CpG ODNs may affect the secretion of various cytokines. However, the effect of CpG ODN1826 to TGF-β1 and radiation-induced pulmonary fibrosis is unknown. Our study found that the serum TGF-β1 concentrations in mice increased after irradiation, and the highest point we detected was on day 30. However, the serum TGF-β1 concentration of the RT + CpG group was lower than that of the RT group, suggesting CpG ODN1826 could reduce the serum concentration of TGF-β1 after irradiation. CD4+/CD25+ Treg, which has the function of regulating the immune system, is a T cell subgroup, and is an important factor for the secretion of TGF-β1 [
28]. FoxP3 is the most specific symbol of CD4+/CD25+ Treg, and the key regulatory gene in the development and functions to maintain CD4+/CD25+ Treg [
29]. One study has found that CpG ODNs could inhibit the expression of the FoxP3 gene, and reduce the proportion of CD4+/CD25+ Treg, and thus down-regulate the serum TGF-β concentration in lung cancers [
5]. Our study found that the expression of the FoxP3 in mice increased after irradiation, but the expression of the FoxP3 in RT + CpG group was lower than that of irradiation group. Therefore, CpG ODN1826 could reduce the serum concentration of TGF-β1 of radiation-induced pulmonary fibrosis mouse, which might be due to its effect on reduction of FoxP3 gene expression. Compared with the control group, FoxP3 gene was downregulated in the CpG group. However, as shown in Table
1, no significant difference was observed in the TGF-β1 expression of CpG group and the control group. One of the possible explanations is that CpG, as an immunopotentiator, exerts its effect on balancing T1/T2 immune cells. The mechanisms of how Foxp3 regulates TGF-β1 expression are still not fully understood. Peng et al. found that CD4 + CD25 + FoxP3+ Tregs attenuate TGF-β1 induced lung fibrosis and fibrocyte accumulation in part via suppression of FGF-9 [
11]. Sumitomo et al. found that transcription factor early growth response 3 is a key factor associated with TGF-β1 expression regulated by FoXp3 [
12]. Other than downregulation of FoxP3 gene expression, CpG might balance the secretion of TGF-β1 via some other mechanism. In addition, there are also many other factors may affect TGF-β1 expression. For example, Molteni et al. found that angiotensin had an important regulatory role on TGF-β1, and it promoted the secretion of TGF-β1 in the process of radiation-induced lung injury [
30]. Jain found that plasma endothelin-1 could stimulate the secretion of TGF-β1 in the study of idiopathic pulmonary fibrosis [
31]. We will therefore take further steps to elucidate the mechanism in our future studies. In conclusion, our study found that CpG ODN1826 could partly prevent radiation-induced pulmonary fibrosis, and reduce the serum concentration of TGF-β1 of radiation-induced pulmonary fibrosis, which might be related to the reduced expression of the FoxP3 gene. This finding indicatives a significant potential for the clinical application of CpG ODNs in treatment of the radiation-induced pulmonary fibrosis.