Grape seed proanthocyanidins prevent irradiation-induced differentiation of human lung fibroblasts by ameliorating mitochondrial dysfunction

Radiation-induced lung fibrosis (RILF) is a long-term adverse effect of curative radiotherapy. The accumulation of myofibroblasts in fibroblastic foci is a pivotal feature of RILF. In the study, we found the inhibitory effect of grape seed proanthocyanidins (GSPs) on irradiation-induced differentiation of human fetal lung fibroblasts (HFL1). To explore the mechanism by which GSPs inhibit fibroblast differentiation, we measured the reactive oxygen species (ROS) levels, mitochondrial function, mitochondrial dynamics, glycolysis and the signaling molecules involved in fibroblast transdifferentiation. GSPs significantly reduced the production of cellular and mitochondrial ROS after radiation. The increases in mitochondrial respiration, proton leak, mitochondrial ATP production, lactate release and glucose consumption that occurred in response to irradiation were ameliorated by GSPs. Furthermore, GSPs increased the activity of complex I and improved the mitochondrial dynamics, which were disturbed by irradiation. In addition, the elevation of phosphorylation of p38MAPK and Akt, and Nox4 expression induced by irradiation were attenuated by GSPs. Blocking Nox4 attenuated irradiation-mediated fibroblast differentiation. Taken together, these results indicate that GSPs have the ability to inhibit irradiation-induced fibroblast-to-myofibroblast differentiation by ameliorating mitochondrial dynamics and mitochondrial complex I activity, regulating mitochondrial ROS production, ATP production, lactate release, glucose consumption and thereby inhibiting p38MAPK-Akt-Nox4 pathway.


GSPs inhibited irradiation-induced myofibroblast differentiation.
To investigate the effect of GSPs on irradiated HFL1 cells, we pre-treated HFL1 cells with GSPs (2.5, 5 or 10 μg/ml) or MitoQ (200 nmol/L) for 24 h before irradiation and examined the expression of α-SMA and fibronectin in HFL1 cells 72 h after irradiation by qRT-PCR, Western blot and immunofluorescent assays. As shown in Fig. 1A, after irradiation, the mRNA expression of α-SMA and fibronectin was significantly increased (P < 0.01) compared with the control HFL1 cells. When cells were pre-treated with GSPs (2.5, 5.0 or 10.0 μg/ml) for 24 h before irradiation, the expression of α-SMA and fibronectin was reduced (P < 0.01). To verify the effect of GSPs on the expression of α-SMA and fibronectin, we measured the expression levels of these two proteins by Western blot and immunofluorescent assays. As shown in Fig. 1B and C, GSPs also significantly inhibited α-SMA and fibronectin protein expression (P < 0.01), similar to the mRNA expression levels. Similar results were obtained using the mitochondria-targeted antioxidant MitoQ, which accumulates within mitochondria and is reduced to the antioxidant ubiquinol.

GSPs decreased intracellular and mitochondrial ROS production in irradiated HFL1 cells.
To determine whether the effect of GSPs inhibition on irradiation-induced HFL1 cell differentiation was associated with ROS production, we measured cellular ROS levels in irradiated cells pretreated with GSPs or MitoQ by flow cytometry using the fluorescent probe DCFDA. As shown in Fig. 2A, DCFDA fluorescence intensity in HFL1 cells gradually increased after irradiation, peaked at 3 h and declined at 6 h. Interestingly, when HFL1 cells were pretreated with GSPs for 24 h before γ-ray irradiation, the cellular ROS production was significantly reduced compared with the model group (P < 0.01). The mitochondria-targeted drug MitoQ had a similar effect to GSPs.
To determine whether irradiation-induced cellular ROS comes from mitochondria and GSPs affect mitochondrial ROS production in HFL1 cells, we analyzed mitochondrial superoxide generation production by flow cytometry and confocal microscopy using the fluorescent probe MitoSOX. After γ-ray irradiation, the MSR fluorescence intensity in cells peaked at 1 h and declined at 3 h, compared with the control group. Moreover, both GSPs (2.5, 5.0 or 10 μg/ml) and MitoQ (200 nm/L) suppressed irradiation-induced mitochondrial ROS production ( Fig. 2B and C). MitoSOX is a reagent that enables us to specifically measure superoxide generation in the mitochondria of live cells. The specificity of mitochondrial ROS staining by MitoSOX was confirmed by double staining with MitoTracker. The MitoSOX signal was localized in MitoTracker-stained organelles, indicating that the MitoSOX staining was specific to mitochondria. The fluorescence assay also revealed that mitochondrial ROS increased markedly after irradiation, and pre-treatment with GSPs (10 μg/ml) or MitoQ (200 nm/L) significantly reduced the generation of mitochondrial ROS (Fig. 2C). To explore the signaling pathways involved in fibroblast transdifferentiation in response to mitochondrial ROS, the level of phosphorylation of p38MAPK and Akt were determined. As shown in Fig. 2D, phosphorylation of p38MAPK and Akt increased at 1 h after irradiation, and pretreatment with GSPs could decrease the level of phosphorylation of p38MAPK and Akt. In addition, the elevation of NADPH oxidase 4 (NOX4) expression induced by irradiation was attenuated by use of GSPs (Fig. S1A).
The effect of GSPs on mitochondrial transmembrane potential in irradiated HFL1 cells. To determine whether the effect of GSPs on mitochondrial ROS production was associated with changes in mitochondrial transmembrane potential, we next examined changes in irradiated cells pretreated with GSPs or MitoQ by flow cytometry using JC-1. As shown in Fig. 3A and B, irradiation led to a significant decrease in mitochondrial membrane potential compared to control cells, as indicated by the increase in green fluorescence. When cells were pre-treated with GSPs (10 µg/ml) or MitoQ (200 nm) for 24 h, the percentage of cells showing red fluorescence increased significantly (P < 0.01). These results imply that irradiation-induced ROS may be associated with Scientific RepoRTs | 7: 62 | DOI:10.1038/s41598-017-00108-9 a decrease in mitochondrial membrane potential (MMP), and that GSPs and MitoQ can ameliorate the reduction of MMP induced by irradiation.
The effect of GSPs on mitochondrial dynamics in irradiated HFL1 cells. Mitochondrial fission and fusion are known to play an important role in maintaining mitochondrial integrity and function. To characterize the effect of GSPs on changes in mitochondrial dynamics, we first monitored mitochondrial morphological changes by confocal microscopy. As shown in Fig. 4A and B, most of the control cells (>95%) showed normal, Figure 1. Expression of α-SMA and fibronectin in irradiation-induced HFL1 cells. HFL1 cells were pre-treated with GSPs (2.5, 5, 10 μg/ml) or MitoQ (200 nmol/L) for 24 hours before irradiation (γ-ray, 8 Gy) and were then cultured for another 72 h. The expression of the indicated genes was analyzed in the cells by qRT-PCR (A) and Western blotting (B). (C) Cells were immunostained for α-SMA and fibronectin. Confocal microscopy was performed. A representative image is shown from three replicates. Scale bars: 50 µM. Data are expressed as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus control group. # P < 0.05 and ## P < 0.01 versus model group. Abbreviations: G2.5, 2.5 μg/ml GSPs; G5, 0.5 μg/ml GSPs; G10, 10 μg/ml GSPs. tubular mitochondria. However, after irradiation with 8 Gy, approximately 40% of cells contained normal tubular mitochondria like those seen in control cells, and >60% of irradiated HFL1 cells contained mitochondria with a fragmented, punctiform morphology. Moreover, pretreatment with GSPs or MitoQ protected mitochondria from fragmentation. We also measured the expression of mitochondrial fusion-related genes (mfn1, mfn2 and opa1) and fission-related genes (drp1 and fis1) by qRT-PCR. Figure 4C shows the effect of irradiation on the expression of the main fission-and fusion-related genes. Specifically, there was a statistically significant increase in drp-1 expression and decrease in mfn-1/2 expression after irradiation. GSPs or MitoQ pretreatment reduced the expression of drp-1 and enhanced the expression of mfn-1/2 (Fig. 4C).  The mRNA levels of the mitochondrial fusion-related genes (mfn1, mfn2 and opa1) and fission-related genes (drp1 and fis1) were measured using qRT-PCR. Data are expressed as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus control group. # P < 0.05 and ## P < 0.01 versus model group.

The effect of GSPs on ATP levels and glycolysis in irradiated HFL1 cells. Mitochondrial ATP con-
tent is a classic indicator of mitochondrial respiration. To investigate whether GSPs alter mitochondrial ATP production in irradiated HFL1 cells, the cellular ATP content of HFL1 cells was determined after treatment with GSPs. As shown in Fig. 5, ATP content was significantly increased in irradiated cells at 1 h, 24 h and 72 h (P < 0.01) after treatment with GSPs. Treatment with GSPs at doses of 2.5, 5.0 or 10 μg/ml significantly decreased ATP production in a dose-dependent manner (P < 0.01). MitoQ treatment also reduced ATP content at 1 h and 24 h. Furthermore, to identify the effect of GSPs on glycometabolism, lactate release and glucose consumption were examined in irradiated HFL1 at 72 h. As shown in Fig. 5B, irradiation significantly increased glucose consumption and pretreatment with GSPs or MitoQ could attenuate glucose uptake in irradiated cells (P < 0.05). Concomitantly, lactate release of irradiated HFL1 was increased (P < 0.05), whereas pretreatment with GSPs attenuated the lactic acid accumulation caused by irradiation (P < 0.05) (Fig. 5C).

The effect of GSPs on mitochondrial respiration in irradiated HFL1 cells.
To explore the possible mechanisms by which GSPs alter mitochondrial ROS production, we next tested whether irradiation affected cellular respiration. Cellular oxygen consumption (OCR) was monitored by XF96 using ETC inhibitors. As shown in Fig. 6A, irradiation significantly increased the rate of oxygen consumption compared with control cells. The presence of GSPs (10 μg/ml) or MitoQ inhibited the basal stimulation of OCR by irradiation. Furthermore, the maximal respiratory capacity induced by FCCP was approximately 1-fold higher in irradiated cells compared with control cells, and GSPs or MitoQ decreased the maximal respiratory capacity of irradiated cells to levels comparable with control cells (Fig. 6A and B). Similarly, irradiation increased the proton leak of HLF1 cells compared with control cells, and treatment with GSPs or MitoQ decreased the proton leak (P < 0.01).

The effect of GSPs on mitochondrial respiratory chain complexes I and III in irradiated HFL1 cells.
It is well known that ROS production occurs predominantly at mitochondrial ETC complexes I and III.
We next hypothesized that the mitochondrial dysfunction and generation of ROS described above in irradiated HFL1 cells could be due to adaptations in mitochondrial respiratory ETC complexes. To investigate this hypothesis, we measured the expression of several genes encoding proteins involved in the mitochondrial respiratory chain by qRT-PCR. Figure 7A shows that the expression of NDUFC2 and NDUFV1, which encode the subunits of oxidative phosphorylation (OXPHOS) complex I, were significantly decreased in irradiated HFL1 cells (P < 0.05) compared with the control group. When cells were treated with GSPs (10.0 μg/ml) for 24 h before irradiation, the expression of NDUFC2 and NDUFV1 was increased. MitoQ (200 nm/L) increased NDUFV1 mRNA expression. Nevertheless, none of the genes encoding OXPHOS complex III subunits that were measured showed any change in expression (Fig. 7B). These results suggest that the inhibitory effect of GSPs on ROS production might be mediated by regulating complex I function. Treatment with GSPs improved the expression and enzyme activity of complex I. Based upon the results described above, we chose to further explore the protein expression and enzyme activity of complex I in irradiated HFL1 cells. Immunofluorescence analysis confirmed that the expression of complex I was reduced 72 h after irradiation (Fig. 8A). Concurrently, the enzyme activity of complex I in irradiated HFL cells was approximately 4-fold lower than that of control cells. Nevertheless, GSPs or MitoQ increased complex I expression and enzyme activity (P < 0.01) (Fig. 8B) in irradiated cells.

Discussion
To the best of our knowledge, this study is the first report regarding the inhibitory effects of GSPs on irradiation-induced cell differentiation in the human lung fibroblast cell line HFL1 in vitro. We clarify that irradiation leads to increases in cellular and mitochondrial ROS levels, up-regulation of mitochondria respiration and ATP production, increased proton leak, increased glycometabolism, decreased mitochondrial membrane potential, disturbance of mitochondrial fission and fusion homeostasis, impaired mitochondria complex I activity, activated p38MAPK-Akt-Nox4 pathway and lung fibroblast cell differentiation. Furthermore, we report that GSPs can significantly reduce irradiation-induced cellular and mitochondria ROS production, improve mitochondrial dysfunction and dynamics, glycometabolism and inhibit p38MAPK-Akt-Nox4 pathway in irradiated HFL1 cells.
It is well known that both radiotherapy and accidental irradiation result in fibrosis in many tissues including the lungs, liver, skin and kidneys 19 . Fibrosis is the marker of many pathological organizational reconstructions and causes clinical disease. In our previous study, radiation pneumonitis and pulmonary fibrosis occurred in the lung after irradiation in rats (data not shown), which is consistent with other reports 2,20 . During pulmonary fibrogenesis, excessive amounts of extracellular matrix components, such as α-SMA, fibronectin and collagen, are deposited and may lead to scarring and destruction of the lung architecture. Myofibroblasts are the mainly cells responsible for matrix secretion and are primarily derived from fibroblast differentiation 21 . Our present work shows that the mRNA and protein expression of α-SMA and fibronectin was significantly increased after irradiation in the human lung fibroblast cell line HFL1. In addition, irradiation significantly increased wound healing/ migration of HFL1 cells (data was shown in Fig. S2A and S2B) whereas it did not affected cell proliferation, which further demonstrated that irradiation caused lung fibroblasts differentiation (Fig. S2C). Pretreatment of the cells with GSPs prevented the increase in expression of these two proteins and wound healing/migration of HFL1 cells (data was shown in Fig. S2A and S2B). GSPs also did not affect cell proliferation (Fig. S2C). These results indicate that GSPs have an inhibitory effect on irradiation-induced cell differentiation.
Previous data showed that irradiation-induced cellular ROS generation could increase profibrotic gene expression in normal human lung fibroblasts and that the cellular ROS level was associated with fibroblast  Complex I enzyme activity was measured using spectrophotometry and expressed as ΔmOD/min/mg protein. The data were expressed as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus control group. # P < 0.05 and ## P < 0.01 versus model group. differentiation 22 . In addition, it has been reported that ionizing radiation induces cellular ROS production in various cell types such as normal human lung fibroblasts, non-small cell lung cancer cells and brain microvascular endothelial cells [23][24][25] . Consistent with these previous reports, we found that cellular ROS were significantly increased after irradiation in HFL1 cells. Interestingly, in our work, GSPs could decrease irradiation-induced ROS production, which suggests that the inhibitory effect of GSPs on irradiation-induced fibroblast differentiation may be related to the reduced production of ROS.
More importantly, we found that mitochondrial ROS increased instantaneously in HFL1 cells after irradiation, which indicates that mitochondria may be an important source of increased cellular ROS. Blocking ROS generation in mitochondria with MitoQ, a mitochondria-targeted antioxidant, markedly reduced irradiation-induced increases in the of mRNA and protein expression of α-SMA, a well characterized marker of myofibroblast differentiation 26 . The results indicate that mitochondrial ROS are required for irradiation-induced myofibroblast differentiation. In our work, GSPs had an inhibitory effect on mitochondrial ROS production that is similar to the effect of MitoQ. Collectively, our results suggest that GSPs attenuate fibroblast differentiation, presumably by targeting mitochondria. Therefore, we focused our studies on the effect of GSPs on mitochondria.
Because it has been reported that mitochondrial ROS production is primarily related to mitochondrial ETC dysfunction 12 , we tested whether mitochondrial ETC functions, such as mitochondrial respiration and ATP content, were changed in irradiated HFL1 cells. After irradiation for 24 h, we found that irradiation promoted mitochondrial respiration with increased basal respiration and maximum respiration. In addition, ATP production was also markedly increased 1 h and 24 h after irradiation. These results are similar to the previous report in irradiated A549 cells 12 . Higher mitochondrial respiration has been linked to higher cellular energy production, so it is possible that the up-regulation of mitochondrial respiration by irradiation resulted in the up-regulation of ATP production at the early stage of irradiation. These changes in mitochondrial respiration have been identified during fibroblast differentiation recently 27 , which indicate a metabolic remodeling of the activated fibroblasts to meet the increased energetic demands associated with the enhanced secretory, synthetic and contractile function of myofibroblasts essential for fibrosis processes. In the ETC, electrons are passed through a series of proteins via oxidation-reduction reactions and the last destination for an electron along the ETC is an oxygen molecule. Under normal conditions, the oxygen is reduced to produce water; however, approximately 0.12-2% of O 2 incorporated for respiration is estimated to be turned into ROS, and this has been particularly well documented for complex I and complex III 12 . According to these data, it is possible that the upregulated ETC functional state contributes to mitochondrial ROS production because more O 2 is incorporated for respiration. In our present work, we found that the levels of mitochondrial respiration and ATP production did not change significantly between cells that had been pre-treated with GSPs or MitoQ and the control cells. Because the regulatory effect of GSPs on the mitochondrial ETC is similar to that of the mitochondria-targeted antioxidant MitoQ, we propose that GSPs could be used to improve the mitochondria ETC and reduce mitochondrial ROS production.
Furthermore, increased proton leak was also observed for 24 h after irradiation in our work, which demonstrates that irradiation caused abnormalities in the mitochondrial ETC. It is well documented that mitochondrial proton leak plays an important role in mitochondrial coupling efficiency and ROS production 28 . The proton leak, which includes basal leak and inducible leak, dissipates the mitochondrial membrane potential through the re-entry of protons into the mitochondrial matrix without generating ATP 28 . Basal proton leak contributes significantly to the basal metabolic rate of a resting mammal. The inducible leak can be activated by superoxide or peroxidation products and contributes to heat production. Previous studies have reported that there is a protective feedback loop between ROS and proton leak under which increased ROS production can increase mitochondrial proton leak and decreased proton leak can reduce ROS production 29,30 . Thus, we can conclude that the increased proton leak is due to an increase in ROS production in the irradiated HFL1 cells, and the reduced proton leak in GSPs-treated cells can bring about the inhibition of ROS production. However, excessive proton leak may result in inhibition of the respiratory chain complexes, which has been demonstrated in intact C2C12 myoblasts treated with a lipophilic positively charged moiety of triphenylphosphonium 31 . Therefore, a plausible explanation of our results may be that in the early stages, ROS-induced proton leak protects mitochondria from oxidative damage to some extent but subsequently a large amount of proton leak negatively affects the activity of the ETC complexes.
Given that ROS generation in the mitochondria primarily occurs at complexes I and III of the respiratory chain, we further investigated the changes in complexes I and III in irradiated HFL1 cells. Our study demonstrated down-regulation of the nuclear DNA-encoded complex I subunit genes NDUFC2 and NDUFV1, whereas the mitochondria encoded genes ND2 and ND6 showed no significant change after exposure to irradiation in human lung fibroblast cells. Additionally, complex III subunit genes showed no significant change compared to the control group. This suggests that irradiation causes complex I dysfunction and nuclear genomic instability in HFL1 cells. It has been demonstrated that NDUFV1 gene mutation leads to complex I deficiency in muscle and cultured fibroblasts from pediatric patients 32 . The NDUFC2 gene has higher mutation rates and has been recommended as a target candidate for treating colorectal carcinoma tumorigenesis 33 . The NDUFC2 gene also shows lower expression in papillary thyroid carcinoma patients 34 . Additionally, complex I deficiency is mainly caused by nuclear-encoded subunit gene mutations 35 , and our results showed that both the protein expression level and enzyme activity of complex I were decreased after irradiation. Interestingly, GSPs could elevate the mRNA expression of NDUFC2 and NDUFV1 in irradiated cells and increase complex I activity, which further indicates that the regulatory effect of GSPs on mitochondrial ETC might target complex I in the mitochondria.
Complex I, a multi-subunit enzyme, serves as the main electron entry point in the respiratory chain and is important for respiration in many aerobic organisms. Additionally, complex I has been identified as the main source of cellular ROS in a previous study 36 . Yashida et al. has reported that irradiation-induced decreased activity of complex I, results in the release of ROS from the mitochondrial electron transport chain (ETC), and causes persistent oxidative stress 37 . On the other side, excess mitochondrial ROS would leads to oxidative damage in the mitochondria and affects the activity of complex I and complex III 38,39 . Our study showed that the expression level and enzyme activity of complex I in the ETC were reduced in HFL1 cells 72 h after irradiation. In addition, mitochondrial ROS levels peaked at 1 h and declined at 3 h after irradiation, but at 72 h, the ROS levels of irradiated cells were still higher than the normal group, which indicates that the cells were continuing to suffer from ROS attack. Therefore, it is conceivable that irradiation damages complex I, which bring about mitochondrial dysfunction and ROS production, and then a sustained increase in mitochondrial ROS further leads to complex I deficiency. Finally excessive ROS levels induce cell differentiation.
As the balance of mitochondrial fusion and fission are involved in complex I dysfunction 40 , we further studied the changes in mitochondrial dynamics after irradiation. After irradiation, mitochondrial fragmentation was elevated in HFL1 cells. Additionally, a decrease in the mitochondrial membrane potential was observed in irradiated HFL1 cells. Increased drp1 and decreased mfn1/2 mRNA expression was also observed in the irradiated cells. Drp1 is an important mitochondrial fission-related gene and mfn1/2 is the main factor that regulates mitochondrial fusion 41 . These results indicate that irradiation leads to impaired mitochondrial morphology and an imbalance in mitochondrial dynamics in HFL1 cells. It has been reported that the disturbance in mitochondrial homeostasis results in an increase in ROS production after irradiation with ionizing radiation 42 . Furthermore, Huang et al. reported that aberrant mitochondrial homeostasis impairs mitochondrial complex I activity 40 . In addition, increased mitochondrial fission after ionizing radiation in normal human fibroblast-like cells involves delayed mitochondrial ROS production 41 . According to the discussion above, complex I dysfunction might be the primary source of mitochondrial ROS production. Additionally, irradiation disturbs mitochondrial homeostasis (fission and fusion), which might lead to complex I dysfunction. Our results demonstrated that GSPs could modulate mitochondrial fission and fusion and improve mitochondria morphology and mitochondrial membrane potential in irradiated HFL1 cells. Therefore, we can propose that the inhibitory effect of GSPs on mitochondria ROS production might be due to improvements in complex I activity induced by regulating mitochondria dynamics.
The above experiments suggested that irradiation increased mitochondrial respiration at the early stage in HFL1 cells, whereas it impaired mitochondrial activity with decreased Complex I activity, reduced mitochondrial potential and disrupted mitochondrial dynamics, at the later stage. In general, mitochondrial dysfunction could impair oxidative phosphorylation and the citric acid cycle, and then lead to decreased ATP production. However, ATP production still significantly increased at 72 h in irradiated HFL1 cells in our study. It is speculated that increased ATP production was mainly from enormous activation of glycolysis at the later stage of irradiation. Therefore, to explore whether the mitochondrial dysfunction leads to increased glycolysis and enormous activation of glycolysis contributed to increased ATP production, we further investigated the levels of glycolysis (indicated by lactate assay and glucose uptake assay). Our results showed that irradiation significantly increased glucose consumption and lactate release in HFL1 cells (p < 0.05), which indicated that irradiation increased the activation of glycolysis at the later stage and increased ATP was mainly from glycolysis. Pretreatment with GSPs or MitoQ could attenuate glucose uptake and lactic acid accumulation in irradiated cells (p < 0.05).
In addition, to test the changes of the plasma signaling pathway involved in fibroblast transdifferentiation in response to mitochondrial ROS, the phosphorylation levels of p38MAPK and Akt were determined. We found that phosphorylation of p38MAPK and Akt increased after irradiation, and GSPs could significantly decrease the expression of p-p38MAPK and p-Akt. We also found that irradiation increased Nox4 expression (Fig. S1A) in irradiated HFL1 cells. Treatment with siNox4 significantly decreased α-SMA and Fibronectin expression levels in irradiated cells (Fig. S1C), suggesting that Nox4 is involved in radiation-induced fibroblast differentiation, which is similar to the previous report 22 . Furthermore, GSPs could significantly reduced Nox4 expression in irradiated HFL1 cells. Additionally, after siNOX4 interference, HFL1 cells were pre-treated with GSPs (5 μg/ml) for 24 hours before irradiation. Expression of α -SMA and FN was detected by Western blotting after 72 hours. Interestingly, we found that GSPs reduced NOX4 expression (Fig. S1A). Previous data demonstrated that radiation can activate p38MAPK-Akt-Nox4 pathway directly or indirectly via ROS 22 . Therefore, GSPs might inhibit lung fbroblast cell differentiation by inhibiting p38MAPK-Akt-Nox4 pathway via decreasing ROS production.
In conclusion, our results indicate that irradiation promotes mitochondrial respiration, disrupts mitochondrial balance (fission and fusion), causes complex I dysfunction, increases glycometabolism, leads to accelerated ROS production and finally results in fibroblast differentiation by activating p38MAPK-Akt-Nox4 pathway. GSPs exhibited an inhibitory effect on cellular and mitochondrial ROS production, the regulation of ETC function, and p38MAPK-Akt-Nox4 pathway in irradiated HFL1 cells. According to these results, we conclude that the inhibitory effect of GSPs on HFL1 cell differentiation involves improved mitochondrial complex I activity induced by regulating mitochondrial homeostasis, which reduces ROS production and then lead to inhibition of p38MAPK-Akt-Nox4 pathway.

Methods
Cell culture and treatment. Human fetal lung fibroblasts (HFL1) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in F12K medium (Ginuo Biomedical Technology Company, Hangzhou, China) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin at 37 °C in a 5% CO 2 humidified atmosphere. The GSPs were dissolved in a small amount of dimethylsulfoxide (DMSO) and diluted with complete cell culture medium [maximum concentration of DMSO, 0.1% (v/v) in medium] before being added to sub-confluent cells (60-70% confluence). After being serum-starved in serum-free medium for 24 h, HFL1 cells were incubated for 24 h with various concentrations of GSPs or MitoQ (200 nM) in F12K medium with 10% FBS, exposed to 8 Gy γ-radiation, and then cultured for an additional 1-72 h without changing the medium. Cells treated with DMSO [0.1% (v/v)] only in F12K medium with 10% FBS for the indicated time served as the vehicle control. The model cells were also treated with vehicle (0.1% DMSO) in F12K medium with 10% FBS. The cells were used to assess the effects of GSPs and MitoQ on radiation-induced differentiation.