Introduction
Mitochondrial oxidative stress is a critical player in idiopathic pulmonary fibrosis (IPF) [
1] and TGF-β-induced lung fibroblast (LF) activation [
2]. Targeting mitochondrial reactive oxygen species (mtROS) can alleviate CS-related lung fibrosis in vivo [
3]. Lung fibroblasts (LF) are effector cells in the pathogenesis of IPF. It can be activated by CS [
4]. However, whether mtROS of LF was induced by CS and how mtROS activated LF is uncertain. NADPH oxidases (NOXs) is one of the main sources of ROS. In LF isolated from IPF patients, NOX4 is elevated while NOX1, 2 and 5 have no significant change [
5]. In addition, NOX4 can be upregulated by CS [
4]. Several lines of evidences manifested that NOX4 can be located in mitochondria since it contains a mitochondrial targeting signal [
1]. Thus, we aim to investigate whether and how CS increased mitochondrial NOX4 (mtNOX4) and thereby produced more mtROS.
Mitochondria is the powerhouse and center of metabolism in eukaryotic cells. Evidences indicated that mitochondrial oxidative stress is closely associated with metabolic disorders [
6]. In fibrotic lungs, fatty acid (FA) and total lipid content [
7] are increased. And high fat diet can aggravate experimental pulmonary fibrosis [
8], implying lipid metabolism participates in lung fibrosis. In addition, lipid metabolism can be affected by CS [
9]. However, whether CS-induced mitochondrial oxidative stress dysregulated lipid metabolism and subsequentially activated LF and its underlying mechanisms are unclear. Fatty acid oxidation (FAO), for which mitochondria is an important place, is a process closely associated with lipid homeostasis. CPT1a is a key rate-limiting enzyme of mitochondrial FAO and is reported to be involved in fibrotic diseases, such as kidney [
10] and liver fibrosis [
11]. Moreover, its upstream regulator, PPARα, also exhibits anti-fibrotic effect in liver [
12], kidney [
13], heart [
14] and lung [
15]. Therefore, its noteworthy to find out whether CS-induced mtROS activated LF through dysregulating PPARα/CPT1a-mediated FAO and lipid metabolism, which may be targets for lung fibrosis therapy.
SIRT1, a lysine deacetylase, is a modulator of mitochondrial oxidative stress [
3] and NOX4 expression [
16]. It has been reported that its anti-fibrotic effect is associated with decreased mtROS [
3,
17]. It can also inhibit fibroblasts activation [
3,
17] and modulate lipid metabolism [
19]. However, whether SIRT1 protect against LF activation by regulating mtNOX4- related mtROS and lipid metabolism and its mechanisms need to be further investigated.
In the present study, we explored how mitochondrial redox balance was disrupted by CS and how mtROS activated LF. We found imbalance of mtNOX4 and SOD2 resulting from decreased SIRT1 was responsible for CS-induced mtROS. mtROS impaired autophagy flux to activate LF by inhibiting PPARα/CPT1-related FAO and lipophagy.
Materials and methods
Animals
Six-week-old male C57 mice were randomly divided into four groups: Control, CS, CS + MitoQ (1.5 mg/kg, HY-100116, MCE), CS + SRT1720 (20 mg/kg, S1129, Selleck). 10 mice in each group. In the three CS groups, mice were placed in an 80 × 35 × 33 cm chamber and exposed to 5 commercial cigarettes for 30 min each time, and two times a day. For MitoQ and SRT1720 group, MitoQ and SRT1720 were injected intraperitoneally into mice every two days or each day, respectively. 4 weeks later, lungs were harvested. All mice were obtained from Southern Medical University Animal Center (Guangzhou, China) and housed in standard environment. All experimental procedures on mice were approved by Committee on the Ethics of Animal Experiments of Southern Medical University (Permit No. SYXK 2015-0056).
Cell culture and treatment
Primary LF were isolated from 6-week-old mice as previously described [
20] and cultured with DMEM containing 15% FBS at 37 °C. Passage 2 cells were treated with MitoQ (50 nM, HY-100116, MCE), fenofibrate (10 μM, T1149, Topscience), oleic acid (10 μM, S4707, Selleck), etomoxir (50 μM, S8244, Selleck), bafilomycin (5 nM, S1413, Selleck) and SRT1720 (4 μM, S1129, Selleck) (Additional file
1). The dosages of these compounds were based on published papers. And MTT test for them was performed (Additional file
2).
Preparation of cigarette smoke extract (CSE)
Firstly, smoke of 1 cigarette was collected by a 20 ml syringe which contained 2 ml PBS. Then, the absorbance of the solution was detected at the wavelength of 490 nm. The concentration was considered as 100% when the absorbance was 0.1. Next, adjusted its pH to 7.4 and filtered it with 0.2 μm membrane. The obtained CSE was kept in 4 °C and applied within 20 min.
MitoSOX red, lysotracker red, nile red and BODIPY 493/503 staining
Living cells were incubated with MitoSOX™ Red (2.5 μM, M36008, Invitrogen), Lysotracker Red DND-99 (50 nM, L7528, Invitrogen), Nile Red (1 μM, HY-D0718, MCE) or BODIPY staining solution (2 μM, GC42959, GLPBIO) for 15 min at 37 °C in dark. Then, cells were washed with HBSS/Ca/Mg and analyzed by fluorescence microscopy (IX73, Olympus or Imager D2, Carl Zeiss).
Immunofluorescence staining
Lung sections or cells treated with 4% paraformaldehyde for 15 min and 0.2% triton for 10 min were blocked with 5% goat serum for 60 min at room temperature. Then, they were incubated with primary antibodies at 4 °C for overnight and stained with FITC- (A0562, beyotime) and Coralite594-conjugated secondary antibody (SA00013-4, Proteintech) at room temperature for 1 h, after which nuclear were stained with DAPI (F6057, sigma). Pictures were captured with confocal microscopy (LSM880, Carl Zeiss) or fluorescence microscopy (Imager D2, Carl Zeiss). Primary antibodies used here were as follows: anti-NOX4 (ab154244, abcam), anti-COX IV (200147, ZENBIO), anti-LC3 II/I (A5179, bimake), anti-SOD2 (A5377, bimake), anti-collagen I (ABM40379, Abbkine), anti-CPT1a (15184-1-AP, proteintech) and anti-PPARα (Abp55667, Abbkine).
Western blot analysis
The relative expression of total protein or mitochondrial protein were detected by western blot. Antibodies used here were as follows: Collagen I (ab260043, abcam), α-SMA (ab7817, abcam), VDAC1 (A5224, Bimake), p62 (18420-1-AP, proteintech), LC3 II/I (A5179, bimake), SIRT1 (13161-1-AP, Proteintech), GAPDH (RM2001; Ray Antibody Biotech), and secondary antibodies (92632210, 92632211, Licor). The bands were visualized by Odyssey System (LI-COR).
Statistical analysis
Results were shown as mean ± SD. Data analysis were performed by SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Intergroup comparison of the mean values was analyzed by one-way analysis of variance (ANOVA). Statistical significance was defined as p < 0.05.
Discussion
In the present study, we centered on mtROS to explore how it was regulated by CS and how it contributed to LF activation. Our results showed that CS-induced mtROS was due to the imbalance of mtNOX4 and SOD2 caused by decreased SIRT1. And it activated LF by dysregulating PPARα/CPT1a-mediated FAO and lipophagy, both of which resulted from blocked autophagy flux (Fig.
6D).
mtROS, a critical player in IPF development and LF activation [
25], can be induced by CS in a variety of cells [
3,
26‐
28]. Our previously study has suggested mtROS may be a therapeutic target for CS-related pulmonary fibrosis [
3]. However, how it worked in LF is incompletely known. In the present study, we explored how CS regulated mtROS and how mtROS participated in CS-induced LF activation. We previously proved increased NOX4 was a contributor of LF activation [
4]. Studies reported that NOX4 can be localized in mitochondria and the elevation of mtNOX4 was related with LF activation [
4]. Here we demonstrated CSE increased mtNOX4. And consistent with previous researches [
27], we also evidenced the expression of SOD2, the main antioxidant enzyme of mitochondria, was reduced. Therefore, CSE disrupted mitochondrial redox balance. Furthermore, we first unveiled the inhibitory effect of MitoQ on LF activation. Similarly, studies also demonstrated MitoQ can prevent the activation of cardiac and nasal fibroblast [
29,
30]. In addition, the antifibrotic effect of MitoQ has been evidenced in lung [
3], liver [
31] and kidney [
32]. Moreover, the safety of MitoQ has been confirmed by Phase II clinical trials [
33]. Altogether, MitoQ may be an effective and safety treatment for IPF or other smoking-related disease.
Lipid metabolism has been reported to be disrupted in fibrotic lungs [
7,
34] and participate in fibroblast activation [
35]. It can also be dysregulated by CS [
9]. As lipid metabolism is closely modulated by mitochondria, we explored whether CS-induced mtROS activated LF by dysregulating lipid metabolism. Firstly, we examined the expression of CPT1a, since it is a key rate-limiting enzyme of mitochondrial FAO and is involved in kidney [
10] and liver fibrosis [
11]. And we found CSE downregulated CPT1a of LF. In addition, we proved that PPARα, the upstream regulator of CPT1a, was also inhibited. Consistent with previous study that PPARα activators exert anti-fibrotic effects in liver [
12], kidney [
13], heart [
14] and lung [
15], we revealed that PPARα activator prevented CSE-induced LF activation by elevating CPT1a. Furthermore, we proved that decreased FAO was due to mtROS. CPT1a inhibitor ETO as well as oleic acid, a fatty acid that is upregulated in the plasm of IPF patients [
36] and has pro-fibrotic effect [
37], can inhibit the anti-fibrotic effect of mitoQ, demonstrating that mtROS activated LF by dysregulating PPARα/CPT1a-related FAO. These results implying that pulmonary lipid metabolic state may have diagnosis potential for IPF. And interfering it may be effective for IPF.
To make clear how mtROS dysregulated lipid metabolism, we paid our attention to autophagy due to following reasons. Firstly, autophagy can prevent LF activation and fibrosis through multiple pathways [
38,
39], but how it worked on lipid metabolism in CSE-treated LF is rarely explored. Secondly, autophagy is critical for mitochondrial homeostasis [
40] which plays an important role in lipid metabolism. And our previous studies showed autophagy flux was impaired by CSE in an oxidative stress-related pathway [
4]. Thirdly, compelling evidences indicated lipophagy, a process of autophagy-mediated LDs degradation, is necessary for lipid homeostasis [
22]. Abnormal lipophagy has also been reported to be involved in fibrotic diseases [
41]. Therefore, it is reasonable to postulate that CSE-induced mtROS disrupt lipid metabolism in an autophagy dependent pathway. As we expected, we found autophagy flux can be blocked by mtROS. And it played an essential role in lipid metabolism not only by regulating PPARα and CPT1a but also inhibiting lipophagy. In addition, our results indicated that mtROS did not influence the transfer of LDs to autophagosomes, but inhibited the transfer of LDs to lysosomes. This result was consistent with our previous finding that CS-induced dysfunction of lysosome contributed to impaired autophagy flux [
4]. We supposed the effect of autophagy on PPARα/CPT1a-mediated FAO was associated with mitophagy, a process essential for mitochondrial homeostasis. However, the regulatory effect of lipophagy on FAO has also been reported [
22]. Therefore, it still needs to be explored whether autophagy regulated FAO by mitophagy or lipophagy. The importance of autophagy in lipid metabolism was further confirmed by results that CSE-untreated LF has compensatory capacity for lipid homeostasis maintenance in the presence of ETO or OA. However, in BA-treated LF, the capacity was lost even in the absence of CSE. Thus, CSE-induced mtROS blocked autophagy flux to dysregulated lipid metabolism by inhibiting PPARα/CPT1a and lipophagy. The study further revealed the mechanism of autophagy in pulmonary fibrosis.
As we mentioned above, mtNOX4/SOD2-mediated mtROS played a critical role in CSE-induced LF activation. Hence, it is noteworthy to find the mechanism by which CSE disrupt the balance of mtNOX4 and SOD2. In the present study, we focused on SIRT1. For one thing, the negative effect of SIRT1 on mitochondrial oxidative stress has been confirmed in multiple organs, such as kidney [
42], liver [
43] and lung [
3]. Moreover, it has been reported that NOX4 and SOD2 can be modulated by SIRT1 [
16,
44]. For another, its anti-fibrotic effect has been confirmed in pulmonary fibrosis [
18,
45]. And studies showed SIRT1 can protect against TGF-β-induced LF activation [
18]. It also modulates lipid metabolism [
19] and autophagy [
3]. However, whether it can rebalance mtNOX4 and SOD2 and thereby regulate autophagy and lipid metabolism to protect against CSE-induced LF activation is uncertain. Here, we uncovered that SIRT1 rescued autophagy flux by rebalancing mtNOX4 and SOD2. And it promoted lipophagy and PPARα/CPT1a expression in an autophagy-dependent pathway. Therefore, activating SIRT1 may be a valuable treatment against pulmonary fibrosis or CS-related disorders. However, there are still some challenges, since changes of SIRT1 is complex. For example, CSE inhibited SIRT1 activity in alveolar epithelial type II cells [
3] but decreased SIRT1 expression in LF. This reminded us that it would be more rational to perform different interventions in different cell types because of the complexity of the human body, although the anti-fibrotic effect of whole body SRT1720 stimulation has been confirmed in mice.
The utility of α-SMA as the marker of activated LF was challenged recent years as it was only upregulated in a subset of these cells [
23]. Moreover, in lungs of control mice, α-SMA-positive LF is rarely detected, which make it difficult to compare the difference of indicators between control and pro-fibrotic LF in vivo. A present unbiased single-cell RNA sequencing study revealed that col1a1 is expressed in 99.8% activated LF, 80.4% nonactivated LF and 4.7% non-LF. Although not a perfect marker, it is better than others such as α-SMA, which is expressed in 63.6% activated LF, 11% nonactivated LF and 4.1% non-LF [
23]. The superiority and rationality of col1a1 as the marker of LF was further confirmed by another single-cell RNA sequencing study [
24]. So, we chose col1a1 as the marker of LF to detect the different expression of above indicator in LF in vivo.
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