CXCL16 Induces the Progression of Pulmonary Fibrosis through Promoting the Phosphorylation of STAT3

Abstract

Aim: The transmembrane chemokine (C-X-C motif) ligand 16 (CXCL16) plays a vital role in the pathogenesis of organ fibrosis, including the liver and kidney. However, the detailed biological function of CXCL16 is still not fully understood in the progression of pulmonary fibrosis (PF). The aim of present study is to examine the function of CXCL16 in PF.

Materials and methods: In this study, we constructed the PF model on mouse by using bleomycin and analyzed the effect of the mouse recombinant protein CXCL16 on mouse lung fibroblast L929 (LF) as well. To further examine the connection between CXCL16 and STAT3 in mouse LF cells, the STAT3 inhibitor AG490 was utilized to inhibit the expression of STAT3. Meanwhile, lipopolysaccharide was used to enhance the phosphorylation of STAT3 (p-STAT3) in mouse LF cells.

Results: Our results indicated that the level of CXCL16/CXCR6 was significantly upregulated in the mouse PF model. Moreover, the level of p-STAT3 was also promoted. In addition, the mouse recombinant protein CXCL16 not only contributed to the proliferation of mouse LF cells but also induced the expression of p-STAT3 in LF cells. However, the effect of CXCL16 was deeply abolished by the STAT3 inhibitor AG490 in LF cells. Meanwhile, the antibody of CXCL16 deeply reduced the phosphorylation of STAT3 in lipopolysaccharide (LPS) cultured cells.

Conclusions: All these results demonstrated that CXCL16 promoted the phosphorylation of STAT3 and further demonstrated that STAT3 was a critical component in CXCL16/CXCR6 signaling pathway. This research not only enhanced the comprehension of CXCL16 but also indicated its potential value as a target in the treatment for human PF.

Figure 1

The serum level of CXCL16 was upregulated in PF sufferers (^∗∗ p < 0.01 vs Normal).

Figure 2

The PF model was successfully constructed in mice. (a) The lung tissues of normal and model mice were stained by hematoxylin and eosin (H&E) and Masson, respectively, 200x. (b) The production of HYP was increased in lung tissues of PF model mice (^∗∗∗ p < 0.001 vs Normal). (c) The mRNA level of CXCR6, CTGF, and α-SMA was upregulated in lung tissues of PF model mice (^∗∗∗ p < 0.001 vs Normal). (d) The protein level of CXCR6, CTGF, α-SMA, STAT3, and p-STAT3 was promoted in lung tissues of PF model mice (^∗∗∗ p < 0.001 vs Normal).

Figure 3

The level of CXCL16 was increased in mice PF model. (a) The serum level of CXCL16 was upregulated in the mouse PF model group (^∗∗∗ p < 0.001 vs Normal). (b, c) mRNA and protein levels of CXCL16 were upregulated in lung tissues of PF model mice, respectively (^∗∗∗ p < 0.001 vs Normal). M, the model mice that were treated by bleomycin; N, the normal mice treated by sterile saline.

Figure 4

The mouse recombinant protein of CXCL16 contributed to the growth of mouse LF cells. (a) Cell proliferation was detected at 0, 24, 48, and 72 hours after being cultured by mouse recombinant protein CXCL16 with different concentration as indicated (^∗ p < 0.05 vs Blank, ^∗∗ p < 0.01 vs Blank, ^∗∗∗ p < 0.001 vs Blank). (b) The production of HYP was promoted in mouse LF cells that were cultured by mouse recombinant protein of CXCL16 with different concentrations as indicated (^∗∗∗ p < 0.001 vs Blank). (c) The protein level of CXCR6, STAT3, and p-STAT3 was increased by the protein of CXCL16 in mouse LF cells as indicated (^∗∗∗ p < 0.001 vs Blank).

Figure 5

The effect of CXCL16 was inhibited by the STAT3 inhibitor AG490 on mouse LF cells. (a) The inhibitor AG490 suppressed the production of HYP in CXCL16 treated cells (^∗∗∗ p < 0.001 vs Blank; ###p < 0.001 vs CXCL16). (b) The serum level of TNF-α, IL-6, collagen I, and collagen III detected in different cells as indicated. (c) The protein level of CTGF, α-SMA, STAT3, and p-STAT3 was inhibited by AG490 in CXCL16 cultured cells (^∗ p < 0.05 vs Blank, ^∗∗ p < 0.01 vs Blank, ^∗∗∗ p < 0.001 vs Blank; ###p < 0.001 vs CXCL16).

Figure 6

The antibody of CXCL16 reduced the effect of LPS on mouse LF cells. (a) The antibody of CXCL16 deeply suppressed the production of HYP in LPS cultured cells (^∗∗∗ p < 0.001 vs Blank; ###p < 0.001 vs L100). (b) The phosphorylation of STAT3 was inhibited by the antibody of CXCL16 in LPS cultured cells (^∗∗ p < 0.01 vs Blank, ^∗∗∗ p < 0.001 vs Blank; ##p < 0.01 vs L100, ###p < 0.001 vs L100).