Oxidative Medicine and Cellular Longevity
Volume 2022 (2022), Article ID 9041914, 19 pages
https://doi.org/10.1155/2022/9041914
Downregulation of SIRT3 Aggravates Lung Ischemia Reperfusion Injury by Increasing Mitochondrial Fission and Oxidative Stress through HIF-1α-Dependent Mechanisms
Correspondence should be addressed to Fei Lin
Received 9 January 2022; Revised 11 April 2022; Accepted 27 May 2022; Published 29 September 2022
Academic Editor: Gerardo Garcia-Rivas
Copyright © 2022 Chunxia Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Lung ischemia-reperfusion injury (LIRI) is a severe multifaceted pathological condition that can lead to poor patient outcome where oxidative stress and the resulting inflammatory response can trigger and exacerbate tissue damage in LIRI patients. Sirtuin3 (SIRT3), a member of the sirtuin family, protects against oxidative stress-related diseases. However, it remains unclear if and how SIRT3 alleviates lung injury induced by ischemia/reperfusion (I/R). Our previous study showed that lung tissue structures were severely damaged at 6 h after lung I/R in mice, however, repair of the injured lung tissue was significant at 24 h. In this study, we found that both SIRT3 mRNA and protein levels were markedly increased at 24 h after lung I/R in vivo. Meanwhile, inhibition of SIRT3 aggravated lung injury and inflammation, augmented mitochondrial fission and oxidative stress and increased Hypoxia-inducible factor-1α (HIF-1α) expression in vivo. The results suggest that SIRT3 may be an upstream regulator of HIF-1α expression. Knockdown of SIRT3 resulted in excessive mitochondrial fission and increased oxidative stress in vitro, and we found that knocking down the expression of HIF-1α alleviated these changes. This suggests that the SIRT3-HIF-1α signaling pathway is involved in regulating mitochondrial function and oxidative stress. Furthermore, inhibition of dynamin-related protein 1 (Drp-1) by the inhibitor of mitophagy, Mdivi-1, blocked mitochondrial fission and alleviated oxidative stress in vitro. Taken together, our results demonstrated that downregulation of SIRT3 aggravates LIRI by increasing mitochondrial fission and oxidative stress. Activation of SIRT3 inhibits mitochondrial fission and this mechanism may serve as a new therapeutic strategy to treat LIRI.
1. Introduction
Lung ischemia-reperfusion injury (LIRI) is a common and severe complication that imposes a significant threat to graft and recipient survival, leading to increased morbidity and mortality among patients undergoing lung transplantation [1]. Organ ischemia and subsequent reperfusion is unavoidable in lung transplantation, which usually leads to acute, sterile inflammation after transplantation called I/R injury [2]. The pathogenesis of LIRI is complex, involving many pathophysiological processes such as oxidative stress injury, calcium overload, endoplasmic reticulum stress injury, inflammatory injury, autophagy, and apoptosis [3–5].
Oxidative stress plays a crucial role in the pathophysiologic processes of I/R injury [6]. Any mismatch between pulmonary oxygen demand and supply can lead to oxidative stress and the accumulation of high amounts of reactive oxygen species (ROS) [7]. It is worth noting that because mitochondria are the main target organs and source of ROS, mitochondrial dysfunction is recognized as a key factor contributing to I/R injury [8]. Therefore, strategies to target the sources of ROS, reduce mitochondrial oxidative stress and preserve mitochondrial function are attractive approaches to ameliorate LIRI.
Under physiological conditions, mitochondria have to maintain a dynamic balance between mitochondrial fission and fusion to regulate its morphology, number and size, which are essential for mitochondrial homeostasis [9, 10]. However, in some pathological conditions, such as acute kidney injury and diabetic nephropathy [11, 12], excessive mitochondrial fission results in increased mitochondrial fragmentation which inhibits the cellular respiratory chain, leading to cellular dysfunction and aggravating tissue damage [13, 14]. Mitochondrial fission is primarily mediated by dynamin-related protein 1 (Drp-1) [15], a member of the dynamin family of large GTPases, mostly localized in the cytoplasm [16]. Drp-1 is recruited to the outer membrane of mitochondria by a variety of adaptor proteins and then aggregates along the site of mitochondrial fission in the future [17, 18]. Active Drp-1 oligomerizes in a ring-like structure and assists the constriction of outer mitochondrial membrane to divide the mitochondria into two separate mitochondria when they contract. Mdivi-1 is a cell permeable quinazolinone originally described as a selective inhibitor of Drp-1 and reported to inhibit mitochondrial fission [15]. Wu et al. proposed that Mdivi-1 prevents Drp-1 from self-assembly into the ring-like structure, thus limiting its association with mitochondria. A different study also proposed that Mdivi-1 blocks the oligomerization of Drp-1 which is necessary for its GTPase activity [19]. Other studies have demonstrated that Mdivi-1 alleviates both myocardial [20] and cerebral I/R injuries by dampening cell apoptosis [21]. However, the relationship between mitochondrial dysfunction and oxidative stress in LIRI remains unclear.
Sirtuin3 (SIRT3) is primarily localized in the mitochondria and plays an important role in the regulation of mitochondrial function and ROS production [22]. Previous studies have demonstrated that increased SIRT3 expression protects cells against oxidative stress through isocitrate dehydrogenase 2 activation [23]. In addition, SIRT3 overexpression protected from doxorubicin-induced cardiomyopathy in mice [24]. Hypoxia-inducible factors (HIFs) are key factors that control the hypoxia-inducible pathways by regulating expression of multiple genes. Hypoxia-inducible factor-1α (HIF-1α) is a major transcription factor of oxygen homeostasis-related genes and has been described as a key regulator of hypoxia induced diseases [25]. A previous study showed that SIRT3 exerts tumor suppressive effects by suppressing ROS and regulating HIF-1α [26]. However, the specific roles of SIRT3 and HIF-1α in LIRI remain largely unclear.
Given the importance of SIRT3 in regulating mitochondrial function [27], we hypothesized that SIRT3 suppression will lead to excessive mitochondrial fission and increase oxidative stress by altering expression of HIF-1α. Furthermore, we speculated that Mdivi-1 could reduce mitochondrial oxidative stress after inhibiting mitochondrial fission.
2. Materials and Methods
2.1. Experimental Animals
Experiments were approved by the Institutional Animal Care and Use Committee of Guangxi Medical University Cancer Hospital, and strictly followed the Institute’s guidelines for the care and use of laboratory animals. Sixty male wild-type C57BL/6 J mice (6–8 weeks, 20–22 g) were purchased from the Animal Center of Guangxi Medical University (Nanning, China) and were adapted to the laboratory environment for 2 weeks before the experiments were conducted. The mice were housed in specific pathogen-free conditions and ventilated polycarbonate cages with dimensions of 325x210x150 mm (less than 5 mice per cage, 23°C, 50% humidity, 12 h light/dark cycle) with free access to sterilized food and water.
2.2. Mouse Model of Lung I/R and Experimental Design
Mice were randomly divided into five groups: Sham, DMSO- (dimethyl sulfoxide-) treated, a group treated with the SIRT3 selective inhibitor 3-TYP (3-(1H-1,2,3-triazol-4-yl) pyridine) dissolved in DMSO (Selleck Chemicals, USA), I/R, and I/R+3-TYP groups ( per group for all experiments). The mice were anaesthetized with 50 mg/kg intraperitoneal injection of pentobarbital and then, subjected to the procedure to occlude the left pulmonary hilum with a microvascular clamp. After 60 min of ischemia, the clamp was removed to allow recovery of the blood flow for 24 h before the mice were killed by carotid artery bloodletting for the I/R and I/R+3-TYP groups, while the mice in the Sham, DMSO, and 3-TYP groups only underwent a thoracotomy, and the left lung was collected for subsequent experiments after modeling. The mice in the 3-TYP and I/R +3-TYP groups received 50 mg/kg of 3-TYP by intraperitoneal injection every 2 days for a total of three times before the surgery [28–30]. The DMSO group was given an equal volume of DMSO at the same time points as the 3-TYP-treated group.
2.3. Cell Culture and Hypoxia/Reoxygenation (H/R)
The RAW 264.7 murine macrophages cell line was purchased from Procell Life Science&Technology Co.,Ltd. The cultured cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% O2. The H/R model of cells was established in vitro as previously described [13, 31]. Briefly, RAW 264.7 cells were cultured in serum-fed and glucose-free medium (Gibco, USA) and exposed to hypoxic conditions (94% N2, 5% CO2, 1% O2) at 37°C for 1 h. Then, the medium was replaced with a glucose-containing medium and cells were allowed to grow in 5% CO2 and 95% O2 for 24 h according to the requirements of the experiment.
2.4. Histological Analysis
Lung tissues harvested from the different groups of C57BL/6 J mice were fixed in 4% paraformaldehyde and embedded in paraffin, sectioned at a thickness of 4 μm and stained with hematoxylin and eosin (H&E) and the tissue morphology was observed. The histological changes were assessed blind using a light microscope by two independent researchers. The degree of lung injury was estimated and acute lung injury scores were evaluated as previously described [32].
2.5. Lung Wet-to-Dry Ratio
The lung wet/dry (W/D) ratio was measured as an indicator of pulmonary edema and congestion. As we described previously [33], the lower lobe of the left lung was immediately weighed after the mice were sacrificed, incubated at 60°C for 96 h, and then weighed again. The weights were used to calculate the wet-to-dry ratio.
2.6. Enzyme-Linked Immunosorbent Assays (ELISA)
The concentrations of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in left lung tissue homogenate or in bronchoalveolar lavage fluid (BALF) of left lung after modeling were estimated using specific ELISA kits (Elabscience Biotechnology, Wuhan, China) for mice according to the manufacturer’s instructions.
2.7. Transmission Electron Microscopy (TEM)
Left lung tissues from different groups were taken within 1 to 3 minutes after modeling, cut into pieces of about a millimeter, and fixed in 2.5% glutaraldehyde for at least 2 h. After embedding in resin, the samples were cut into ultrathin slices by an ultramicrotome and observed with an H-7560 transmission electron microscope (H7560, Tokyo, Japan).
2.8. Immunofluorescence Staining
The left lung tissues from different groups were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned at a thickness of 4 μm. After dewaxing and rehydration treatment, the slices were immersed in 0.01 M citrate buffer (pH 6.0) and boiled in a pressure cooker for 10 minutes. The slides were incubated with primary antibodies for SIRT3 (ab246522, 1 : 200; Abcam, USA), HIF-1α (ab179483, 1 : 500; Abcam, USA), and F4/80 (a monoclonal antibody specific for alveolar macrophages, ab6640, 1 : 200; Abcam, USA) overnight at 4°C in a humidifying box, followed by washing with PBS three times (5 min each), the slides were exposed to the secondary antibody Donkey anti-Rabbit IgG H&L (Alexa Fluor 594) (ab150076, 1 : 500; Abcam, USA) for 1 h at room temperature in the dark. Nuclei were visualized by staining with DAPI (P0131, Beyotime Biotechnology, Shanghai, China) for 5 min. After washing with PBS, the sections were observed with a fluorescence microscope (EVOS FL AutoLife Technologies), and representative fields were chosen for application.
2.9. Immunohistochemistry
The paraffin-embedded sections of mouse left lung tissues were blocked with 3% hydrogen peroxide for 15 min to inactivate endogenous peroxidases and then incubated overnight at 4°C with rabbit monoclonal anti-Drp-1 (ab184247, 1 : 1000; Abcam, USA) and rabbit polyclonal anti-Fis-1 (ab229969, 1 : 750; Abcam, USA). PBS instead of primary antibody was used as a negative control for immunofluorescence staining. The sections were washed and subsequently incubated with a secondary antibody (PV-0023-2, Bioss, China) for 1 h at room temperature. Then the sections were stained with DAB until the stain developed. Six fields of immunohistochemical images were randomly selected and semi-quantitative analysis of Drp-1 and Fis-1 was performed using the ImageJ software as previously described [34].
2.10. Measurement of Oxidative Stress
The Malondialdehyde (MDA) ELISA Kit (E-EL-0060c, Elabscience Biotechnology, Wuhan, China), Glutathione (GSH) ELISA Kit (E-EL-0026c, Elabscience Biotechnology, Wuhan, China), and Superoxide Dismutase (SOD2) ELISA Kit (CSB-EL022398MO, Wuhan, China) were used for measuring the levels of oxidative stress. The lung tissues after modeling from different groups were placed in cold saline (1: 9, w/v, one gram of tissue to 9 mL of PBS), homogenized on ice, and centrifuged at 12,000 rpm for 15 min. The supernatant from lung tissue or cell culture for detecting the levels of MDA, GSH, and SOD2 according to the manufacturer’s instructions.
2.11. Measurement of ATP Content
ATP was measured using a bioluminescence assay kit (S0026, Beyotime Biotechnology, Shanghai, China). Briefly, the mouse left lung tissues or RAW264.7 cells after modeling from different groups were immediately lysed in the lysis buffer provided with the kit. The supernatant was collected by centrifugation at 12,000 rpm for 15 min at 4°C. The concentration of ATP in the samples was determined by mixing 20 μL supernatant with 100 μL luciferase reagent. A standard curve was prepared using a series of standards at known concentrations. The luminescence of each sample was measured on a microporous plate photometer (BioTek Instruments Inc, USA).
2.12. Measurement of Mitochondrial ROS (mROS)
mROS was measured using the MitoSOX™ Red mitochondrial superoxide indicator (M36008, Thermo Fisher, USA). MitoSOX is a novel fluorogenic probe for sensitive selective detection of superoxide (instead of reactive nitrogen species) in the mitochondria of live cells. Once in the mitochondria, the MitoSOX reagent is oxidized by superoxide and exhibits red fluorescence. The left lung tissues after modeling from different groups were immediately cut and digested with collagenase type I (17018029, Thermo Fisher, USA) on a shaker at 37°C for 30 min. The samples were filtered two times using a cell mesh, and then the red blood cells were lysed with red blood cell lysis buffer (R1010, Solarbio, China), and followed by centrifugation to obtain the single cells. The obtained mouse single cells or RAW264.7 cells after modeling in vitro were seeded into 24-well plates at a density of cells/mL. After cells had adhered to the wells, the cells were incubated with MitoSOX (5 μm) for 10 min in the dark and nuclei were visualized by staining with Hoechst 3342 (C0030, Solarbio, China) for 20 min at room temperature as previously described [35]. The stained cells were then washed with DMEM and observed using a fluorescence microscope (EVOS FL AutoLife Technologies).
2.13. Mitochondrial Membrane Potential (MMP) Assay
The JC-10 assay (Solarbio, Beijing, China) was used to detect the MMP of RAW264.7 cells from different groups in vitro or the single-cell suspension extracted from mouse lung tissues. JC-10 is a lipophilic, cyanocyanine cationic dye that can selectively penetrate mitochondria and reversibly emit red fluorescence to green fluorescence in the case of reduced membrane potential. The healthy cells have a high membrane potential and the aggregated JC-10 exhibits red fluorescence, when the MMP was depolarized like apoptosis, the JC-10 monomers exhibited green fluorescence, and simultaneously, red fluorescence was reduced. The cells were seeded into 6-well plates at a density of cells/mL. After the cells had adhered, the cells were incubated with JC-10 staining working solution at 37°C in 5% CO2 for 20 minutes. The stained cells were then washed with dyeing buffer three times. The fluorescence intensity for both aggregated and monomeric forms of JC-10 was measured by fluorescence microscopy (EVOS FL AutoLife Technologies).
2.14. Western Blotting
The left lung tissues or RAW264.7 cells in vitro after modeling from different groups were lysed in radio immunoprecipitation assay lysis buffer on ice, and protein concentrations were measured using a Bicinchoninic acid protein assay kit (P0011, Beyotime Biotechnology, Shanghai, China). Subsequently, equal amounts of protein from each group were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes and the membranes were blocked with 5% nonfat milk in TBS-Tween for 1 h at room temperature. Then, the membranes were incubated overnight at 4°C with the primary antibodies against loading control β-actin (4970S, 1 : 1000; Cell Signaling Technology, USA) and rabbit monoclonal anti-SIRT3 (ab246522, 1 : 1000; Abcam, USA), rabbit monoclonal anti-HIF-1α (ab179483, 1 : 1000; Abcam, USA), rabbit monoclonal anti-DRP-1 (ab184247, 1 : 1000; Abcam, USA), rabbit polyclonal anti-Fis-1 (ab229969, 1 : 2500; Abcam, USA), rabbit monoclonal anti-Mfn-2 (ab124773, 1 : 5000; Abcam, USA). The membranes were washed three times with TBST, and incubated for 1 h with the secondary antibody (goat anti-rabbit H&L IRDye@800 CW, ab216723,1 : 10000; Abcam, USA) at room temperature. Proteins were detected using the Alpha Innotech System (BioRad), and quantified using the ImageJ software.
2.15. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)
Total RNA was extracted from the left lung tissue of mice after modeling using the RNAiso Plus Kit (9109, TAKARA, Japan) following the manufacturer’s instructions. After that, 1 μg of total RNA was used for cDNA synthesis with the PrimeScript™ RT Reagent Kit (RR047A, TAKARA, Japan). Primers for the specific genes of interest were synthesized by Sangon Biotech (Shanghai) as follows: GAPDH, forward: 5-TGTGTCCGTCGTGGATCTGA-3, reverse: 5-TTGCTGTTGAAGTCGCAGGAG-3; SIRT3, forward: 5-CTACATGCACGGTCTGTCGAA-3, reverse: 5- GCCAAAGCGAAGTCAGCCATA-3; HIF-1α, forward: 5-ACCTTCATCGGAAACTCCAAAG -3, reverse: 5- CTGTTAGGCTGGGAAAAGTTAGG-3. Comparative method was used to calculate the relative quantification of the target gene, with GAPDH as an internal reference.
2.16. Lentivirus Infection
RAW264.7 cells in normal culture with good growth condition were infected with lentiviruses (Genechem, Shanghai, China) expressing short hairpin (sh) RNA ( TU/mL, MOI of 10 : 1) to knockdown SIRT3 (5-GCAAGGTTCCTACTCCATA-3) and HIF-1α (5-CTGATAACGTGAACAAATA-3) expression in the presence of polybrene (2 μg/mL) according to the manufacturer’s instructions. After infection for 72 h, levels of SIRT3 and HIF-1α were determined by western blotting, and the data were used to construct the H/R model. The cells were randomly divided into six groups: Control, H/R, H/R+shRNA (negative control for lentiviruses), H/R+S (H/R+SIRT3 shRNA, knockdown of SIRT3 expression after lentivirus infection), H/R+H (H/R+HIF-1α shRNA, knockdown of HIF-1α expression after lentivirus infection), and H/R+H+S (H/R+SIRT3 shRNA+HIF-1α shRNA, knockdown of SIRT3 and HIF-1α expression after lentivirus infection).
2.17. Cell Viability
The cell counting kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China) assay was used to evaluate cell viability. RAW264.7 cells after modeling from different groups were cultured in 96-well plates at a density of cells/mL in the presence of a complete medium. CCK-8 solution was added to each well and the plates were incubated at 37°C for 1 h. Absorbance at 450 nm was then measured using a microplate reader (BioTek Instruments Inc, USA).
2.18. Statistical Analysis
All the quantitative data were expressed as the (SEM). Comparisons among three and more groups were analyzed using one-way analysis of variance. The statistical analysis was conducted using SPSS 25.0 (IBM, USA). A value of less than 0.05 () was considered statistically significant.
3. Results
3.1. Expression of SIRT3 Increased after Lung I/R
We had previously shown that the injured lung tissue was more significantly repaired at 24 h after lung I/R compared to 6 h in vivo. Therefore, we chose the 24 h time point to investigate the possible role of SIRT3 in LIRI. The function of SIRT3 was blocked using 3-TYP prior to surgery. As shown in Figures 1(a)–1(c), DMSO and 3-TYP treatment exerted no significant changes for both protein and mRNA levels of SIRT3 compared with the Sham group. However, the level of SIRT3 increased significantly in the I/R group while the addition of 3-TYP to the I/R group of mice significantly inhibited the expression of SIRT3 (). This trend in SIRT3 expression was validated through immunofluorescence analysis (Figure 1(d)). In addition, the SIRT3 staining was overlaid with localization of the F4/80, suggesting that SIRT3 protein is expressed in macrophage cells.