Blueberry Juice Attenuates Pulmonary Fibrosis via Blocking the TGF-β1/Smad Signaling Pathway

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal, and chronic lung disease, lacking a validated and effective therapy. Blueberry has demonstrated multiple pharmacological activities including anti-inflammatory, antioxidant, and anticancer. Therefore, the objective of this study was to investigate whether blueberry juice (BBJ) could ameliorate IPF. Experiments in vitro revealed that BBJ could significantly reduce the expressions of TGF-β1 modulated fibrotic protein, which were involved in the cascade of fibrosis in NIH/3T3 cells and human pulmonary fibroblasts. In addition, for rat primary lung fibroblasts (RPLFs), BBJ promoted the cell apoptosis along with reducing the expressions of α-SMA, vimentin, and collagen I, while increasing the E-cadherin level. Furthermore, BBJ could reverse epithelial-mesenchymal transition (EMT) phenotypic changes and inhibit cell migration, along with inducing the upregulation of E-cadherin in A549 cells. Compared with the vehicle group, BBJ treatment alleviated fibrotic pathological changes and collagen deposition in both bleomycin-induced prevention and treatment pulmonary fibrosis models. In fibrotic lung tissues, BBJ remarkably suppressed the expressions of collagen I, α-SMA, and vimentin and improved E-cadherin, which may be related to its inhibition of the TGF-β1/Smad pathway and anti-inflammation efficacy. Taken together, these findings comprehensively proved that BBJ could effectively prevent and attenuate idiopathic pulmonary fibrosis via suppressing EMT and the TGF-β1/Smad signaling pathway.

Keywords: TGF-β1/Smad signaling pathway; blueberry juice; epithelial–mesenchymal transition; idiopathic pulmonary fibrosis; reactive oxygen species.

FIGURE 1

BBJ induced apoptosis in NIH/3T3 and HPF. (A) NIH/3T3 was treated with BBJ or NS for 24, 48, and 72 h. The cell viability was disclosed by MTT assay. (B) Apoptotic rate, ROS, and ΔΨm of NIH/3T3 treated with BBJ for 24 h were analyzed and expressed as bar histograms. (C) Expression of cleaved caspase-3 in NIH/3T3 was detected through a western blot assay and calculated by ImageJ after incubation with BBJ for 24 h. (D) Under treatment with various doses of BBJ for 24 h, the viability of HPF was determined by MTT tests, and cell morphology was observed. (E) HPF was dealt with BBJ for 24 h, and apoptosis and ROS were analyzed by flow cytometry. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control group.

FIGURE 2

BBJ restrained activation and differentiation of NIH/3T3 and HPF induced by TGF-β1. NIH/3T3 or HPF was planted, starved for 6 h, stimulated by 5 ng/ml TGF-β1 for 1 h, and then incubated with 50 μl/ml BBJ for an extra 24 h. (A) Expressions of α-SMA, vimentin, collagen-I, Smad2/3, p-Smad2/3, and β-actin in NIH/3T3 were presented by western blotting. (B) The expression of α-SMA in NIH/3T3 was visualized by a confocal microscope (×40). (C) Expressions of α-SMA, E-cadherin, vimentin, collagen-I, Smad2/3, p-Smad2/3, and β-actin in HPF were tested by western blotting. (D) The expression of α-SMA in HPF was visualized by a confocal microscope (×40). *p < 0.05; **p < 0.01; ***p < 0.001 versus the control group or the TGF-β1 group.

FIGURE 3

The inhibitory effect of BBJ in RPLF. (A) Cell morphology of RPLF was photographed by an inverted microscope (×10). (B) RPLFs were treated with BBJ or normal saline for 24, 48, and 72 h. Cell viability was detected by MTT tests. (C) ROS in RPLF was analyzed after 24 h treatment of BBJ or NS. (D) Expressions of α-SMA, E-cadherin, vimentin, collagen-I, and β-actin in RPLF were determined by western blot assay. Protein expressions were treated statistically by ImageJ. *p < 0.05; **p < 0.01; ***p < 0.001 versus the control group.

FIGURE 4

BBJ could reverse EMT progress in A549. (A) The cytotoxicity of BBJ on A549 was determined by an MTT test. A549 cells were treated with different doses of BBJ and 200 μl/ml normal saline for 24, 48, and 72 h. Data were presented as mean ± SD from three experiments. (B) ROS of A549 treated with BBJ for 24 h were detected by flow cytometry and counted. (C) After starvation for 6 h with serum-free medium, fresh medium with 5 ng/ml TGF-β1 was replaced. One hour later, 50 μl/ml BBJ was added. After 24 h, cell morphology of A549 was observed by a microscope (×10). (D) A549 cells were seeded in six-well plates for 24 h and starved for 6 h with serum-free medium. Then, a scratch was made by 100 μl pipette tips, and fresh medium with 5 ng/ml TGF-β1 was added. One hour later, 50 μl/ml BBJ was added. The scratches were photographed at 0 and 24 h by an inverted microscope (×10). The scratch areas were treated statistically by ImageJ. (E) After incubation with 5 ng/ml TGF-β1 for 1 h and 50 μl/ml BBJ for an extra 24 h, expressions of E-cadherin, vimentin, p-Smad2/3, Smad2/3, and β-actin in A549 were analyzed by western blotting. (F) After stimulation by TGF-β1 for 1 h and treatment with BBJ for 24 h, expressions of α-SMA and E-cadherin were visualized by a confocal fluorescence microscope (×40). *p < 0.05; **p < 0.01; ***p < 0.001 versus the control group or TGF-β1 group.

FIGURE 5

The BBJ could prevent fibrosis formation induced by BLM in mice. (A) Arrangement of animal study in the prevention model. The fibrosis model was built by tracheal injection of BLM on day 0. Next day, vehicle and BBJ-fed groups were administrated by saline and BBJ for 4 weeks, respectively. (B) Lung sections of each group were stained with H&E (×10). Modified Ashcroft scores were applied to evaluate the degree of pulmonary fibrosis. (C) Lung sections of each group were stained by Masson trichrome (×10). Collagen volume fractions were analyzed from the results of Masson-stained lung sections by ImageJ. (D) Hydroxyproline contents were tested by the hydroxyproline assay kit. (E) The expressions of collagen-I and α-SMA in lung sections were detected by IHC staining, and these proteins’ expressions were calculated by ImageJ. (F) Expressions of collagen-I, α-SMA, vimentin, and β-actin in lung tissues of each group were analyzed by western blotting. ImageJ software was used to calculate these proteins’ expressions. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the vehicle group.

FIGURE 6

BBJ could alleviate pulmonary fibrosis caused by BLM. (A) Arrangement of animal study in the treatment model. The fibrosis model was built by tracheal injection of BLM on day 0. On day 7, vehicle, BBJ-fed, and NTB-fed groups were administrated with saline, BBJ, and NTB for 3 weeks, respectively. (B) Lung sections of each group were stained with H&E (×10). Modified Ashcroft scores were applied to evaluate the degree of pulmonary fibrosis. (C) Lung sections of each group were stained with Masson trichrome (×10). Collagen volume fractions were analyzed from the results of Masson-stained lung sections by ImageJ. (D) Hydroxyproline contents were tested by the hydroxyproline assay kit. (E) The expressions of collagen-I, α-SMA, p-Stat3, and p-Smad3 in lung sections were detected by IHC staining, and these proteins’ expressions were calculated by ImageJ. (F) Expressions of α-SMA, E-cadherin, vimentin, collagen-I, Smad2/3, p-Smad2/3, Stat3, p-Stat3, and β-actin in lung tissues of each group were analyzed by western blotting. ImageJ software was used to calculate these proteins’ expressions. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the vehicle group.

FIGURE 7

BBJ could inhibit lung fibrosis in vivo and in vitro. Followed by a series of processing, BBJ was utilized in assays in vitro and in vivo. BBJ was proven to inhibit EMT in A549. BBJ could also suppress differentiation of fibroblasts to myofibroblasts induced by TGF-β1 proved by the changes of related proteins. In addition, in lung fibrosis, the C57BL/6 mouse model built by BLM, BBJ could prevent and ameliorate lung fibrosis by suppressing the Smad2/3 pathway and Stat3.