Polydatin alleviated radiation-induced lung injury through activation of Sirt3 and inhibition of epithelial-mesenchymal transition

Abstract

Radiation-induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy. It is characterized with two main features including early radiation pneumonitis and fibrosis in later phase. This study was to investigate the potential radioprotective effects of polydatin (PD), which was shown to exert anti-inflammation and anti-oxidative capacities in other diseases. In this study, we demonstrated that PD-mitigated acute inflammation and late fibrosis caused by irradiation. PD treatment inhibited TGF-β1-Smad3 signalling pathway and epithelial-mesenchymal transition. Moreover, radiation-induced imbalance of Th1/Th2 was also alleviated by PD treatment. Besides its free radical scavenging capacity, PD induced a huge increase of Sirt3 in culture cells and lung tissues. The level of Nrf2 and PGC1α in lung tissues was also elevated. In conclusion, our data showed that PD attenuated radiation-induced lung injury through inhibiting epithelial-mesenchymal transition and increased the expression of Sirt3, suggesting PD as a novel potential radioprotector for RILI.

Keywords: epithelial-mesenchymal transition (EMT); free radicals; polydatin (PD); radiation-induced lung injury.

Figure 1

Polydatin reduced radiation‐caused hyperaemia oedema in pulmonary tissue. (A) Representative images of lung tissues in control group, single irradiation group, polydatin group and WR2721 group from 1 to 16 weeks. (B) A line graph of lung coefficient in different groups following local irradiation. Values are given as mean ± SEM (n = 30), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 2

Polydatin mitigated radiation pneumonia and collagen deposition in lung tissues. Representative images of HE (A) and Masson staining (C) of lung tissue sections at 1–16 week post‐irradiation. (B) A bar graph of Ashcroft scoring of HE slides of lung tissues. (D) A quantification of collagen deposition area in slides from lung tissues. Values are given as mean ± SEM (n = 30), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 3

Polydatin inhibited radiation‐induced epithelial–mesenchymal transition. Representative images of EMT markers, E‐cadherin (A, B), Vimentin (C, D) and α‐SMA (E, F) IF staining in different groups at 8‐ and 16‐week post‐irradiation.

Figure 4

Polydatin reversed radiation‐induced EMT process evaluated by a Western blotting assay. (A) Western blot analysis of EMT markers in lung tissues from different groups. (B–E) Qualification of protein expression levels of E‐cadherin, Vimentin and α‐SMA in different groups. Values are given as mean ± SEM (n = 10), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 5

Polydatin regulated Th1/Th2‐related cytokines in acute lung injury. (A–H) Cytokines concentration in serum of irradiated mice with different treatments. Values are given as mean ± SEM (n = 10), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 6

Polydatin reduced the expression levels of TGF‐β1, Smad3 and Snail. (A, C, E) Representative images of IHC staining of TGF‐β1, Smad3 and Snail in lung tissues from different groups at 1 and 16 week post‐irradiation. (B–D) Bar graphs showing quantification analysis of TGF‐β1, Smad3 and Snail‐positive cells in slide from lung tissues. Values are given as mean ± SEM (n = 10), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 7

Polydatin scavenged free radicals and exerted an anti‐oxidative capacity. (A) Representative images of free radical signals from groups treated with different concentration of PD. (B) quantification analysis of second peak of free radicals in different groups. (C, D) The concentration of SOD and MDA in lung homogenate from mice with different treatments. (E) Representative images of TUNEL staining of lung tissues at 1‐ and 18‐week post‐irradiation. Values are given as mean ± SEM (n = 10), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 8

Polydatin protected normal alveolar epithelial cells from radiation induced cell death and EMT. (A) A bar graph of cell survival after treatments with different concentration PD. (B, C) Cell viability from different groups assayed by CCK8 assay and clone formation assay. (D) Western blot analysis of EMT markers in BEAS‐2B cells with/without PD treatment. (E–H) Quantification analysis of raw density of Western blot figures after in different groups. Values are given as mean ± SEM (n = 10), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 9

Polydatin elevated the level of Sirt3 and its related genes Nrf2, PGC1α. (A) Western blot analysis of Sirt family members including, Sirt1, Sirt2, Sirt3 and Sirt6. (B) Cell survival assay of cells transfected with Sirt3 siRNA and negative control after different treatments. (C, E, G) Representative images of Sirt3, Nrf2 and PGC1α IHC staining in lung tissues at 1 and 8 week after irradiation. (D, F, H) Bar graphs showing quantification analysis of Sirt3, Nrf2 and PGC1α‐positive cells in lung tissues from different groups. Values are given as mean ± SEM (n = 10), *P < 0.05 and **P < 0.01 versus single radiation group.

Figure 10

Modelling of PD in radiation‐induced lung injury. Irradiation of lung tissues induces pneumonitis and later chronic pulmonary fibrosis. PD alleviated acute pneumonitis through activating of Sirt3 and scavenging free radicals. PD also suppressed inflammatory cytokines and reversed Th1/Th2 imbalance. Pulmonary fibrosis was also inhibited by PD through inhibiting of EMT.