|
| |
 |
|
| ORIGINAL ARTICLE |
|
| Year : 2023 | Volume
: 66
| Issue : 5 | Page : 335-344 |
|
Klotho inhibits the activation of NLRP3 inflammasome to alleviate lipopolysaccharide-induced inflammatory injury in A549 cells and restore mitochondrial function through SIRT1/Nrf2 signaling pathway
Yanjun Zeng, Gang Xu, Congrui Feng, Danyan Cai, Sizhi Wu, Yuanling Liu, Yuluo Chen, Wei Ma
Department of Geriatric Medicine, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China
| Date of Submission | 28-Feb-2023 |
| Date of Decision | 31-Mar-2023 |
| Date of Acceptance | 20-Apr-2023 |
| Date of Web Publication | 31-Jul-2023 |
Correspondence Address:
Dr. Wei Ma
Guangzhou First People's Hospital, Panfu Road No. 1, Yuexiu District, Guangzhou, Guangdong 510000
China
Dr. Yanjun Zeng
Guangzhou First People's Hospital, Panfu Road No. 1, Yuexiu District, Guangzhou, Guangdong 510000
China
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-23-00029
Acute lung injury is a severe clinical condition constituting a major cause of mortality in intensive care units. This study aimed to investigate the role of klotho in alleviating lipopolysaccharide (LPS)-induced acute lung injury. LPS-induced acute lung injury was used to simulate the acute lung injury caused by severe pneumonia in vitro. The viability and apoptosis of A549 cells were detected by cell counting kit-8 assay and flow cytometry. The inflammatory response, oxidative stress, and mitochondrial function in A549 cells were analyzed by commercial assay kits and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1) staining. The expression of apoptosis-related proteins, Sirtuin 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway-related proteins, and NOD-like receptor family pyrin domain containing 3 (NLRP3) expression in A549 cells was detected by western blot. The mtDNA synthase level in A549 cells was analyzed by reverse transcription-quantitative polymerase chain reaction. The results showed that, klotho had no cytotoxic effect on A549 cells. The viability and mitochondrial function were inhibited and apoptosis, inflammatory response, and oxidative stress were aggravated in LPS-induced A549 cells, which were all reversed by klotho. Klotho activated the SIRT1/Nrf2 signaling pathway to inhibit the LPS-induced NLRP3 inflammasome activation in A549 cells. However, EX527, a SIRT1 inhibitor, attenuated the klotho effect to suppress viability and mitochondrial function and promoted apoptosis, inflammatory response, and oxidative stress of A549 cells. In conclusion, klotho inhibited the activation of NLRP3 inflammasome to alleviate LPS-induced inflammatory injury of A549 cells and restore mitochondrial function through activating the SIRT1/Nrf2 signaling pathway.
Keywords: Inflammatory injury, klotho, mitochondrial function, NLRP3 inflammasome, SIRT1/Nrf2 signaling pathway
How to cite this article:
Zeng Y, Xu G, Feng C, Cai D, Wu S, Liu Y, Chen Y, Ma W. Klotho inhibits the activation of NLRP3 inflammasome to alleviate lipopolysaccharide-induced inflammatory injury in A549 cells and restore mitochondrial function through SIRT1/Nrf2 signaling pathway. Chin J Physiol 2023;66:335-44 |
How to cite this URL:
Zeng Y, Xu G, Feng C, Cai D, Wu S, Liu Y, Chen Y, Ma W. Klotho inhibits the activation of NLRP3 inflammasome to alleviate lipopolysaccharide-induced inflammatory injury in A549 cells and restore mitochondrial function through SIRT1/Nrf2 signaling pathway. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:335-44. Available from: https://www.cjphysiology.org/text.asp?2023/66/5/335/382563 |
| Introduction | |  |
Acute lung injury represents a serious heterogeneous pulmonary disorder featured by a multitude of lung changes arising from a wide variety of lung injuries.[1] It is commonly acknowledged that acute lung injury remains a life-threatening clinical syndrome contributing to overall mortality and morbidity.[1] Acute lung injury is manifested by dyspnea and may evolve into acute respiratory distress syndrome attributed to overdue treatment.[2] Mechanical ventilation is regarded a potentially life-saving therapy for patients with acute lung injury.[3] Nonetheless, lung injury has been reported to be aggravated following high airway pressures and tidal volumes.[3] Therefore, it is of great significance to strengthen the study on the pathogenesis of acute lung injury and seek new therapeutic targets for the control and prognosis of patients.
Klotho protein is the encoding product of klotho gene, which consists of two extracellular domains (KL1 and KL2), one transmembrane domain and one short cytoplasmic tail.[4] Previous studies have shown that klotho is mainly expressed in the brain, kidney, reproductive organs, pituitary gland, and parathyroid gland.[5],[6] Klotho is also expressed in lung tissue and downregulated in lung tissues of chronic obstructive pulmonary disease.[7] In vivo, lipopolysaccharide (LPS) significantly decreased the expression of anti-aging klotho, suggesting that klotho expression was affected by acute inflammatory stress.[8] Klotho could alleviate paraquat-induced acute lung injury through reducing inflammatory responses and mitochondria-dependent apoptosis.[9] Klotho could maintain lung alveolar and vascular structure to reduce pulmonary vascular remodeling in neonatal hyperoxia-exposed rodents.[10] Klotho was lowly expressed in the lungs of smokers, which promoted inflammation and oxidative stress-induced cell damage induced by cigarette smoke.[11] Klotho was an anti-inflammatory regulatory protein in the kidney. In a diabetic mouse model, decreased klotho promotes the development of inflammation.[12] Alpha-klotho could protect against oxygen-induced acute lung injury.[13],[14] Acute lung injury caused by severe pneumonia can lead to respiratory failure, prolonged hospitalization, and even death.[15] The role of klotho in LPS-induced acute lung injury has not been reported.
Studies indicated that klotho expression was upregulated to attenuate LPS-induced acute kidney injury and klotho protected neuronal cells from LPS-mediated neuroinflammation. Therefore, it was speculated that klotho might also alleviate LPS-induced acute lung injury by reducing inflammatory responses.
| Materials and Methods | | |
Cell culture and cell model
Human alveolar epithelial type II cells (A549) were provided by iCell Bioscience Inc (China). A549 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Corning, NY, USA) with 10% fetal bovine serum (Gibco, LifeTech, USA) at 37°C with 5% CO2. To investigate whether klotho had cytotoxicity on A549 cells, A549 cells were pretreated with various doses (100, 200, and 400 pM) of recombinant human klotho protein (R&D Systems, MN, USA) for 1 h followed by exposure to 50 μg/mL LPS for 24 h. A549 cells were pretreated with 10 μM EX527, a Sirtuin 1 (SIRT1) inhibitor for 10 min before exposure to klotho.
Cell counting kit-8 assay
Cell viability was evaluated by a CCK-8 kit provided by Beyotime Institute of Biotechnology (Cat. No. C0038). After indicated treatment, A549 cells were seeded into a 96-well plate at a density of 3 × 104 cells/well and cultured for 24 h. Then, each well was incubated with 10 μL CCK-8 solution for 1 h at 37°C, which was detected by a microplate reader at a wavelength of 450 nm.
Flow cytometry
After indicated treatment, A549 cells were obtained and resuspended in 1× binding buffer. Then, 100 μL of the cell suspension was transferred to a tube, which was added with 5 μL Annexin V-fluorescein isothiocyanate and propidium iodide (Cat. No. 6592, Cell Signaling Technology, Boston, MA, USA). The mixture was incubated at room temperature for 15 min in the dark. Finally, treated cells were analyzed by a FACScalibur flow cytometer.
Western blot
After indicated treatment, A549 cells were lysed in radioimmunoprecipitation assay buffer at 4°C and centrifuged to isolate total proteins. The concentration of total proteins was quantified using a bicinchoninic acid protein assay kit (Cat. No. P0010s, Beyotime Institute of Biotechnology, China). Equal protein amounts were loaded to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred onto polyvinylidene fluoride membranes. Blocked with 5% nonfat milk for 2 h at room temperature, membranes were incubated with primary antibodies against B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), SIRT1, nuclear factor erythroid 2-related factor 2 (Nrf2), NOD-like receptor family pyrin domain containing 3 (NLRP3), gasdermin D N-terminal (GSDMD-N), interleukin-18 (IL-18), IL-1β, Caspase-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at 4°C overnight. Then, membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody at room temperature for 1 h. Finally, an enhanced chemiluminescence reagent was added to membranes to present the immunoreactive bands, which were quantified by ImageJ software.
Enzyme-linked immunosorbent assay
The concentrations of tumor necrosis factor-alpha (TNF-α), IL-6, and IL-1β in the culture supernatant of treated A549 cells were detected using TNF-α (Cat. No. F02810), IL-6 (Cat. No. F01310), and IL-1β (Cat. No. F01220). ELISA kits were provided by Shanghai Westang Biotechnology Co., Ltd. (China). All experimental procedures were performed according to manufacturer instructions.
Detection of oxidative stress levels, adenosine triphosphate level, and nicotinamide adenine dinucleotide phosphate level
The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), adenosine triphosphate (ATP) level, and nicotinamide adenine dinucleotide phosphate (NADPH) level in treated A549 cells were detected by SOD (Cat. No. A001-3-2), GSH-Px (Cat. No. A005-1-2), MDA (Cat. No. A003-4-1), ATP (Cat. No. A016-1-1), and NADPH (Cat. No. A127-1-1) assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer instructions.
5,5',6 ,6'-tetrachloro-1,1',3 ,3'-tetraethyl-benzimidazolyl carbocyanine iodide staining
After indicated treatment, A549 cells were stained with JC-1 solution (Cat. No. M34152, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C for 20 min and washed by JC-1 staining buffer for two times. The green and red fluorescence was detected by a laser confocal microscope. The transition from red fluorescence to green fluorescence reflected the decrease of mitochondrial membrane potential.
Reverse transcription quantitative polymerase chain reaction
Total RNA was extracted from treated A549 cells using Trizol® reagent (Cat. No. 9109, Takara, Japan). cDNA was synthesized from RNA using First Strand cDNA Synthesis Kit (Cat. No. 6110B, Takara, Japan). The gene expression was detected by SYBR Green one-step qRT-PCR Kit (Cat. No. 638317, Takara, Japan) and analyzed by a 7500 Fast Real-Time PCR System. Relative mtDNA expression was calculated using the 2-ΔΔcq method and normalized to GAPDH.[16]
Statistical analysis
Statistical analyses were conducted using GraphPad Prism 1.8.0. (San Diego, CA, USA). Data from three independent replicates were presented as mean ± standard deviation. Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) test. P < 0.05 was considered to be statistically significant.
| Results | | |
Klotho inhibited LPS-induced apoptosis of A549 cells
To evaluate the role of Klotho in acute lung injury, the cytotoxicity of klotho alone on A549 cells was firstly detected. Through CCK-8 assay, it was observed that different concentrations of klotho had no effect on the viability of A549 cells [Figure 1]a. The LPS-induced inflammatory response has been recognized as the main cause of acute lung injury. Thereafter, LPS was utilized to induce an in vitro acute injury model in A549 cells. As depicted in [Figure 1]b, LPS suppressed the viability of A549 cells, which was concentration-dependently improved by klotho. Further, cell apoptosis was estimated by flow cytometry analysis and the results illuminated that the apoptosis of A549 cells was promoted by LPS and was inhibited by klotho in a concentration-dependent manner [Figure 1]c and the representative images are displayed in [Figure 1]d. Bcl-2 and Bax are pivotal regulators of apoptosis. Western blot analyzed that LPS decreased the anti-apoptotic Bcl-2 expression and increased pro-apoptotic Bax expression, which were reversed by klotho [Figure 1]e. Overall, klotho enhanced the viability, whereas suppressed the apoptosis of LPS-challenged A549 cells. | Figure 1: Klotho inhibited LPS-induced apoptosis of A549 cells. (a) The viability of A549 cells treated by different concentrations of klotho was detected by CCK-8 assay. (b) The viability of LPS-induced A549 cells treated by different concentrations of klotho was detected by CCK-8 assay. (c) Quantification of the percentage of apoptotic cells was detected by flow cytometry. (d) Representative images of flow cytometry analysis for the apoptosis of LPS-induced A549 cells treated by different concentrations of klotho. (e) The expression of apoptosis-related proteins in LPS-induced A549 cells treated by different concentrations of Klotho was detected by western blot. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05 and ***P < 0.001. The experiments were performed for three times. LPS: Lipopolysaccharide, CCK-8: Cell counting kit-8.
Click here to view |
Klotho alleviated LPS-induced inflammatory response and oxidative stress in A549 cells
Inflammatory response and oxidative stress are key factors in the mechanism of acute lung injury. To assess the impacts of Klotho on inflammatory response in LPS-exposed A549 cells, the levels of inflammatory cytokines including TNF-α, IL-6, and IL-1β were examined. Through ELISA, it was discovered that the levels of TNF-α [Figure 2]a, IL-1β [Figure 2]b, and IL-6 [Figure 2]c in A549 cells were increased by LPS induction and klotho reduced the levels of TNF-α [Figure 2]a, IL-1β [Figure 2]b, and IL-6 [Figure 2]c in a dose-dependent manner in LPS-induced A549 cells. SOD, GSH-Px, and MDA are significant markers of oxidative stress. The experimental results presented that LPS downregulated the levels of SOD [Figure 2]d and GSH-Px [Figure 2]e and upregulated the level of MDA [Figure 2]f in A549 cells. However, klotho upregulated the levels of SOD [Figure 2]d and GSH-Px [Figure 2]e and downregulated the level of MDA [Figure 2]f in LPS-induced A549 cells. In summary, klotho protected against inflammatory response and oxidative stress in LPS-treated A549 cells. | Figure 2: Klotho alleviated LPS-induced inflammatory response and oxidative stress in A549 cells. The levels of (a) TNF-α, (b) IL-1β, and (c) IL-6 in the culture supernatant of LPS-induced A549 cells treated by different concentrations of klotho were detected by TNF-α, IL-6, and IL-1β ELISA kits. The levels of (d) SOD, (e) GSH-Px, and (f) MDA in LPS-induced A549 cells treated by different concentrations of Klotho were detected by SOD, GSH-Px, MDA assay kits. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001. The experiments were performed for three times. LPS: Lipopolysaccharide, TNF-α: Tumor necrosis factor-alpha, IL: Interleukin, SOD: Superoxide dismutase, MDA: Malondialdehyde, ELISA: Enzyme-linked immunosorbent assay, GSH-Px: Glutathione peroxidase.
Click here to view |
Klotho restored mitochondrial function in LPS-induced A549 cells
Loss of mitochondrial function has been reported to be associated with acute lung injury.[17],[18] As demonstrated by JC-1 staining, LPS increased green fluorescence in A549 cells, which indicating that the mitochondrial membrane potential was decreased, and klotho increased red fluorescence in LPS-induced A549 cells, which indicating that the mitochondrial membrane potential was increased [Figure 3]a. ATP is mainly produced by mitochondria and NADPH widely existing in mitochondria plays an important role in antioxidant defense and reductive biosynthesis. The ATP content was decreased [Figure 3]b, and NADPH content was increased [Figure 3]c in LPS-induced A549 cells, which were reversed by klotho. mtDNA is the genetic material in mitochondria. LPS reduced the mtDNA synthase level in A549 cells and klotho increased the mtDNA synthase level in LPS-induced A549 cells [Figure 3]d. Accordingly, Klotho alleviated LPS-induced damage to mitochondrial function in A549 cells. | Figure 3: Klotho restored mitochondrial function in lipopolysaccharide-induced A549 cells. (a) The mitochondrial membrane potential in LPS-induced A549 cells treated by different concentrations of klotho was determined by JC-1 staining. Magnification, ×200. The content of (b) ATP and (c) NADPH in LPS-induced A549 cells treated by different concentrations of klotho was detected by ATP and NADPH assay kits. (d) The mtDNA expression level in LPS-induced A549 cells treated by different concentrations of klotho was detected by RT-qPCR. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001. The experiments were performed for three times. LPS: Lipopolysaccharide, ATP: Adenosine triphosphate, NADPH: Nicotinamide adenine dinucleotide phosphate, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction.
Click here to view |
Klotho regulated the SIRT1/Nrf2 signaling pathway to inhibit LPS-induced activation of NLRP3 inflammasomes in A549 cells
Previous studies have confirmed that NLRP3 inflammasome plays a pivotal role in the development of acute lung injury and SIRT1/Nrf2 signaling is involved in the process of acute lung injury.[19],[20] LPS was noticed to downregulate the expression of SIRT1 and Nrf2 while upregulated the expression of NLRP3, GSDMD-N, IL-18, IL-1β, and Caspase-1 in A549 cells. However, klotho promoted the expression of SIRT1 and Nrf2 and suppressed the expression of NLRP3, GSDMD-N, IL-18, IL-1β, and Caspase 1 in LPS-induced A549 cells [Figure 4], suggesting that klotho led to NLRP3 inflammasome inactivation and SIRT1/Nrf2 signaling activation in A549 cells exposed to LPS. | Figure 4: Klotho regulated the SIRT1/Nrf2 signaling pathway to inhibit LPS-induced activation of NLRP3 inflammasome in A549 cells. The expression of SIRT1/Nrf2 signaling pathway-related proteins expression in LPS-induced A549 cells treated by different concentrations of klotho was detected by western blot. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05 and ***P < 0.001. The experiments were performed for three times. SIRT1: Sirtuin 1, Nrf2: Nuclear factor erythroid 2-related factor 2, LPS: Lipopolysaccharide.
Click here to view |
SIRT1 inhibitor partially reversed the protective effect of klotho on LPS-induced A549 cells
To corroborate that klotho participated in the progression of acute lung injury through modulating SIRT1/Nrf2 signaling, SIRT1 inhibitor EX527 was applied. As expected, klotho promoted the expression of SIRT1 and Nrf2 and suppressed the expression of NLRP3, GSDMD-N, IL-18, IL-1β, and Caspase-1 in LPS-induced A549 cells, which were all partially reversed by SIRT1 inhibitor EX527 [Figure 5]a. CCK-8 assay proved that EX527 suppressed the viability of LPS-induced A549 cells treated by klotho [Figure 5]b. On the contrary, as displayed by flow cytometry [Figure 5]c, klotho weakened the apoptotic capacity of LPS-treated A549 cells, which was then abrogated by the addition of EX527 [Figure 5]d. This finding was also further validated by the results that EX527 downregulated the Bcl-2 expression and upregulated Bax expression in LPS-induced A549 cells treated by klotho [Figure 5]e. Concurrently, EX527 increased the levels of TNF-α [Figure 6]a, IL-1β [Figure 6]b, and IL-6 [Figure 6]c in LPS-induced A549 cells treated by klotho. Furthermore, EX527 decreased the levels of SOD [Figure 6]d and GSH-Px [Figure 6]e and raised the level of MDA [Figure 6]f in LPS-induced A549 cells treated by klotho. Besides, EX527 increased green fluorescence and decreased red fluorescence to reduce the mitochondrial membrane potential in klotho-treated A549 cells exposed to LPS [Figure 6]g. Further, EX527 reduced ATP [Figure 6]h and mtDNA synthase levels [Figure 6]j and increased NADPH levels [Figure 6]i in LPS-induced A549 cells treated by klotho. Taken together, klotho activated SIRT1/Nrf2 signaling to reduce apoptosis, inflammatory response, oxidative stress, mitochondrial dysfunction as well as NLRP3 inflammasome activation in LPS-induced A549 cells. | Figure 5: SIRT1 inhibitor partially suppressed the SIRT1/Nrf2 signaling pathway and reversed the protective effect of klotho on the apoptosis of LPS-induced A549 cells. (a) The expression of SIRT1/Nrf2 signaling pathway-related proteins expression in LPS-induced A549 cells treated by klotho and EX527 was detected by western blot. (b) The viability of LPS-induced A549 cells treated by klotho and EX527 was detected by CCK-8 assay. (c) Representative images of flow cytometry analysis for the apoptosis of LPS-induced A549 cells treated by klotho and EX527. (d) Quantification of the percentage of apoptotic cells by flow cytometry. (e) The expression of apoptosis-related proteins in LPS-induced A549 cells treated by klotho and EX527 was detected by western blot. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001. The experiments were performed for three times. SIRT1: Sirtuin 1, Nrf2: Nuclear factor erythroid 2-related factor 2, LPS: Lipopolysaccharide, CCK-8: Cell counting kit-8.
Click here to view |
 | Figure 6: SIRT1 inhibitor partially reversed the protective effect of klotho on the inflammatory response, oxidative stress and mitochondrial function of LPS-induced A549 cells. The levels of (a) TNF-α, (b) IL-1β and (c) IL-6 in the culture supernatant of LPS-induced A549 cells treated by Klotho and EX527 were detected by TNF-α, IL-6 and IL-1β ELISA kits. The levels of (d) SOD, (e) GSH-Px, (f) MDA in LPS-induced A549 cells treated by klotho and EX527 were detected by SOD, GSH-Px, MDA assay kits. (g) The mitochondrial membrane potential in LPS-induced A549 cells treated by klotho and EX527 was determined by JC-1 staining. Magnification, x200. The content of (h) ATP and (i) NADPH in LPS-induced A549 cells treated by Klotho and EX527 was detected by ATP and NADPH assay kits. (j) The mtDNA expression level in LPS-induced A549 cells treated by klotho and EX527 was detected by RT-qPCR. Data from three independent replicates were presented as mean ± standard deviation. *P < 0.05, **P < 0.01 and ***P < 0.001. The experiments were performed for three times. SIRT1: Sirtuin 1, TNF-α: Tumor necrosis factor-alpha, IL: Interleukin, SOD: Superoxide dismutase, MDA: Malondialdehyde, ELISA: Enzyme-linked immunosorbent assay, GSH-Px: Glutathione peroxidase, ATP: Adenosine triphosphate, NADPH: Nicotinamide adenine dinucleotide phosphate, LPS: Lipopolysaccharide, RT-qPCR: Reverse transcription quantitative polymerase chain reaction.
Click here to view |
| Discussion | | |
At present, the specific pathogenesis of acute lung injury has not been fully elucidated, but it has been established that inflammatory response and oxidative stress response play an important role in the progression of the disease.[21],[22] When the body is invaded by pathogenic bacteria, it can directly cause damage to respiratory epithelial cells and mucosa, and at the same time, it can lead to the release of a variety of inflammatory factors and oxygen free radicals, leading to the increase of local inflammatory reaction, lung inflammatory reaction, and systemic inflammatory reaction, aggravating lung injury. At the same time, the increase of oxygen free radicals can lead to an oxidation/antioxidant imbalance in the body. Oxidative stress can promote airway mucosal reaction, and oxidative stress can also lead to cell DNA damage, thus aggravating lung tissue damage induced by severe pneumonia.[23],[24] Factors such as oxidative stress and inflammation can cause an imbalance in mitochondrial function. Inflammation and oxidative stress can induce organ damage, and pro-inflammatory mediators such as IL-6, IL-1β, TNF-α, and oxidative stress indexes such as SOD, GSH-Px, and MDA caused lung tissue damage.[25],[26] In this study, LPS-induced A549 cells were used to simulate acute lung injury induced by severe pneumonia. LPS induction promoted inflammatory response and oxidative stress by increasing the levels of TNF-α, IL-6, IL-1β, and MDA and decreasing the levels of SOD and GSH-Px in A549 cells.
Mitochondria are important for many cellular functions, such as apoptosis, calcium signaling, metabolism, and energy production.[27],[28] Mitochondrial dysfunction can lead to any tissue damage, and studies have shown that impaired mitochondrial function is associated with lung injury-related diseases.[17],[29] This study indicated that mitochondrial dysfunction was occurred in LPS-induced A549 cells. The mitochondrial membrane potential, ATP, and mtDNA synthase levels were decreased, and NADPH level was increased in LPS-induced A549 cells.
Research showed that klotho could significantly inhibit the release of LPS-induced myocardial inflammatory cytokines (TNF-α, IL-1β, and IL-6), decreased the content of MDA, and increased the activity of SOD and GSH-Px in the antioxidant system to enhance the scavenging of oxygen free radicals. In addition, the apoptosis rate of cells was significantly reduced after pretreatment with klotho.[30] Another study indicated that klotho obviously decreased levels of reactive oxygen species, TNF-α, and IL-6 in LPS-induced HK-2 cells, and also reduced apoptosis of LPS-induced HK-2 cells.[31] The present study also indicated that klotho suppressed inflammation and oxidative stress and improved mitochondrial dysfunction to inhibit the apoptosis of LPS-induced A549 cells.
SIRT1 is a newly discovered target of acute lung injury, which has become a new approach for the prevention and treatment of acute lung injury. SIRT1 activation suppressed inflammation, apoptosis, and endothelial tight junction permeability, thereby protecting against lung injury.[32],[33] However, the effect of klotho on the SIRT1 signaling pathway in acute lung injury induced by severe pneumonia remains unknown. The present study revealed that klotho obviously upregulated the expression of SIRT1 in LPS-induced A549 cells. Nrf2, a key antioxidant sensor, is identified as an important downstream target of SIRT1 and resists oxidative stress damage; it is also closely related to acute lung injury.[19] Research reported that activating SIRT1/Nrf2 signaling pathway may reduce NLRP3 inflammasome activation.[34] NLRP3 inflammasome increases the levels of mature IL-1β and IL-18 and cleaves procaspase-1 in acute lung injury to regulate the inflammatory response.[35] Klotho has been reported to regulate the SIRT1 signaling pathway, Nrf2 signaling pathway, and NLRP3 inflammasome pathway. Klotho exerted antioxidant and anti-apoptotic effects and alleviated mitochondrial dysfunction through the SIRT1 signaling pathway.[36] Klotho protected against H2O2-induced injury of human retinal pigment epithelial cells through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)-Nrf2/heme oxygenase-1 signaling pathway.[37] Klotho protected diabetic cardiomyopathy by inactivating the NLRP3 inflammasome pathway.[38] This study indicated that klotho suppressed NLRP3 inflammasome activation through enhancing SIRT1/Nrf2 signaling in LPS-induced A549 cells. The expression of GSDMD-N, IL-18, IL-1β, and Caspase-1 in LPS-induced A549 cells was also decreased by klotho treatment. Of course, EX527, a SIRT1 inhibitor, could partially weaken the protective effect of klotho on LPS-induced A549 cells to promote inflammation, oxidative stress, and mitochondrial function. In addition, there may be another pathway involved in the mechanism of klotho in LPS-induced A549 cell injury, and we will further explore this possibility in future experiments.
| Conclusion | | |
Taken together, klotho inhibited the activation of NLRP3 inflammasome to alleviate LPS-induced inflammatory injury and oxidative stress of A549 cells and restored mitochondrial function through SIRT1/Nrf2 signaling pathway. This finding might provide a new sight for the treatment of acute lung injury. The effects of klotho on the LPS-induced acute lung injury animal model and the regulation of SIRT1/Nrf2 signaling pathway will be performed in our next experiments.
Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Financial support and sponsorship
This work was supported by Science and Technology Projects in Guangzhou (No. 202201010012, No. 20221020262 and No. 202203010099) and Guangzhou Key Discipline of Medicine (Geriatric Medicine, 2021-2023, ZDXK202103).
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Butt Y, Kurdowska A, Allen TC. Acute lung injury: A clinical and molecular review. Arch Pathol Lab Med 2016;140:345-50.  |
| 2. | Bellani G, Laffey JG, Pham T, Fan E, Brochard L, Esteban A, et al. Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. JAMA 2016;315:788-800. |
| 3. | Nieman GF, Gatto LA, Habashi NM. Impact of mechanical ventilation on the pathophysiology of progressive acute lung injury. J Appl Physiol (1985) 2015;119:1245-61. |
| 4. | Neyra JA, Hu MC, Moe OW. Klotho in clinical nephrology: Diagnostic and therapeutic implications. Clin J Am Soc Nephrol 2020;16:162-76. |
| 5. | Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45-51. |
| 6. | Li SA, Watanabe M, Yamada H, Nagai A, Kinuta M, Takei K. Immunohistochemical localization of klotho protein in brain, kidney, and reproductive organs of mice. Cell Struct Funct 2004;29:91-9. |
| 7. | Li L, Zhang M, Zhang L, Cheng Y, Tu X, Lu Z. Klotho regulates cigarette smoke-induced autophagy: Implication in pathogenesis of COPD. Lung 2017;195:295-301. |
| 8. | Ohyama Y, Kurabayashi M, Masuda H, Nakamura T, Aihara Y, Kaname T, et al. Molecular cloning of rat klotho cDNA: Markedly decreased expression of klotho by acute inflammatory stress. Biochem Biophys Res Commun 1998;251:920-5. |
| 9. | Zhang Z, Nian Q, Chen G, Cui S, Han Y, Zhang J. Klotho alleviates lung injury caused by paraquat via suppressing ROS/P38 MAPK-regulated inflammatory responses and apoptosis. Oxid Med Cell Longev 2020;2020:1854206. |
| 10. | Batlahally S, Franklin A, Damianos A, Huang J, Chen P, Sharma M, et al. Soluble klotho, a biomarker and therapeutic strategy to reduce bronchopulmonary dysplasia and pulmonary hypertension in preterm infants. Sci Rep 2020;10:12368. |
| 11. | Gao W, Yuan C, Zhang J, Li L, Yu L, Wiegman CH, et al. Klotho expression is reduced in COPD airway epithelial cells: Effects on inflammation and oxidant injury. Clin Sci (Lond) 2015;129:1011-23. |
| 12. | Zhao Y, Banerjee S, Dey N, LeJeune WS, Sarkar PS, Brobey R, et al. Klotho depletion contributes to increased inflammation in kidney of the db/db mouse model of diabetes via RelA (serine) 536 phosphorylation. Diabetes 2011;60:1907-16. |
| 13. | Gazdhar A, Ravikumar P, Pastor J, Heller M, Ye J, Zhang J, et al. Alpha-klotho enrichment in induced pluripotent stem cell secretome contributes to antioxidative protection in acute lung injury. Stem Cells 2018;36:616-25. |
| 14. | Gagan JM, Cao K, Zhang YA, Zhang J, Davidson TL, Pastor JV, et al. Constitutive transgenic α-klotho overexpression enhances resilience to and recovery from murine acute lung injury. Am J Physiol Lung Cell Mol Physiol 2021;321:L736-49. |
| 15. | Matthay MA, Zimmerman GA, Esmon C, Bhattacharya J, Coller B, Doerschuk CM, et al. Future research directions in acute lung injury: Summary of a National Heart, Lung, and Blood Institute working group. Am J Respir Crit Care Med 2003;167:1027-35. |
| 16. | Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-8. |
| 17. | Shi J, Yu J, Zhang Y, Wu L, Dong S, Wu L, et al. PI3K/Akt pathway-mediated HO-1 induction regulates mitochondrial quality control and attenuates endotoxin-induced acute lung injury. Lab Invest 2019;99:1795-809. |
| 18. | Aggarwal S, Dimitropoulou C, Lu Q, Black SM, Sharma S. Glutathione supplementation attenuates lipopolysaccharide-induced mitochondrial dysfunction and apoptosis in a mouse model of acute lung injury. Front Physiol 2012;3:161. |
| 19. | Mohamed GA, Ibrahim SR, El-Agamy DS, Elsaed WM, Sirwi A, Asfour HZ, et al. Terretonin as a new protective agent against sepsis-induced acute lung injury: Impact on SIRT1/Nrf2/NF-κBp65/NLRP3 signaling. Biology (Basel) 2021;10:1219. |
| 20. | Zhang Y, Li X, Grailer JJ, Wang N, Wang M, Yao J, et al. Melatonin alleviates acute lung injury through inhibiting the NLRP3 inflammasome. J Pineal Res 2016;60:405-14. |
| 21. | Zhou H, Fan EK, Fan J. Cell-cell interaction mechanisms in acute lung injury. Shock 2021;55:167-76. |
| 22. | Zhang Y, Cui Y, Feng Y, Jiao F, Jia L. Lentinus edodes polysaccharides alleviate acute lung injury by inhibiting oxidative stress and inflammation. Molecules 2022;27:7328. |
| 23. | da Cunha LG Jr., Ferreira MF, de Moraes JA, Reis PA, Castro-Faria-Neto HC, Barja-Fidalgo C, et al. ExoU-induced redox imbalance and oxidative stress in airway epithelial cells during Pseudomonas aeruginosa pneumosepsis. Med Microbiol Immunol 2015;204:673-80. |
| 24. | Payus AO, Rajah R, Febriany DC, Mustafa N. Pulmonary embolism masquerading as severe pneumonia: A case report. Open Access Maced J Med Sci 2019;7:396-9. |
| 25. | Ye C, Xu B, Yang J, Liu Y, Zeng Z, Xia L, et al. Mucin1 relieves acute lung injury by inhibiting inflammation and oxidative stress. Eur J Histochem 2021;65:3331. |
| 26. | Li T, Wu YN, Wang H, Ma JY, Zhai SS, Duan J. Dapk1 improves inflammation, oxidative stress and autophagy in LPS-induced acute lung injury via p38MAPK/NF-κB signaling pathway. Mol Immunol 2020;120:13-22. |
| 27. | Morrison TJ, Jackson MV, Cunningham EK, Kissenpfennig A, McAuley DF, O'Kane CM, et al. Mesenchymal stromal cells modulate macrophages in clinically relevant lung injury models by extracellular vesicle mitochondrial transfer. Am J Respir Crit Care Med 2017;196:1275-86. |
| 28. | Aghapour M, Remels AH, Pouwels SD, Bruder D, Hiemstra PS, Cloonan SM, et al. Mitochondria: At the crossroads of regulating lung epithelial cell function in chronic obstructive pulmonary disease. Am J Physiol Lung Cell Mol Physiol 2020;318:L149-64. |
| 29. | Peng FF, Shao Q, Zhao N, Shi-Lin HU, Qian KJ, Liu F. Protective effect of MCC950 against lung injury induced by mitochondrial damage-associated molecular patterns. J Med Postgrad 2017;30:1166-71. |
| 30. | Liu P, Yang L. Effect of klotho on LPS-induced cardiomyocyte damage and its mechanism. Chin J Arterioscler 2017;25:688-92. |
| 31. | Zhou Y, Kuang Y, Zhou J. Klotho protects against LPS-induced inflammation injury by inhibiting Wnt and NF-κB pathways in HK-2 cells. Pharmazie 2017;72:227-31. |
| 32. | Liu Y, Guan H, Zhang JL, Zheng Z, Wang HT, Tao K, et al. Acute downregulation of miR-199a attenuates sepsis-induced acute lung injury by targeting SIRT1. Am J Physiol Cell Physiol 2018;314:C449-55. |
| 33. | Fu C, Hao S, Xu X, Zhou J, Liu Z, Lu H, et al. Activation of SIRT1 ameliorates LPS-induced lung injury in mice via decreasing endothelial tight junction permeability. Acta Pharmacol Sin 2019;40:630-41. |
| 34. | Arioz BI, Tastan B, Tarakcioglu E, Tufekci KU, Olcum M, Ersoy N, et al. Melatonin attenuates LPS-induced acute depressive-like behaviors and microglial NLRP3 inflammasome activation through the SIRT1/Nrf2 pathway. Front Immunol 2019;10:1511. |
| 35. | Zhang L, Zhu XZ, Badamjav R, Zhang JZ, Kou JP, Yu BY, et al. Isoorientin protects lipopolysaccharide-induced acute lung injury in mice via modulating Keap1/Nrf2-HO-1 and NLRP3 inflammasome pathways. Eur J Pharmacol 2022;917:174748. |
| 36. | Li C, Jiang S, Wang H, Wang Y, Han Y, Jiang J. Berberine exerts protective effects on cardiac senescence by regulating the Klotho/SIRT1 signaling pathway. Biomed Pharmacother 2022;151:113097. |
| 37. | Wen X, Li S, Zhang Y, Zhu L, Xi X, Zhang S, et al. Recombinant human klotho protects against hydrogen peroxide-mediated injury in human retinal pigment epithelial cells via the PI3K/Akt-Nrf2/HO-1 signaling pathway. Bioengineered 2022;13:11767-81. |
| 38. | Li X, Li Z, Li B, Zhu X, Lai X. Klotho improves diabetic cardiomyopathy by suppressing the NLRP3 inflammasome pathway. Life Sci 2019;234:116773. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
|
Search Pubmed for