Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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Advances in Clinical and Experimental Medicine

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doi: 10.17219/acem/184639

Publication type: original article

Language: English

License: Creative Commons Attribution 3.0 Unported (CC BY 3.0)

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Li XB, Liu JL, Zhao S. Recombinant Klotho protein protects pulmonary alveolar epithelial cells against sepsis-induced apoptosis by inhibiting the Bcl-2/Bax/caspase-3 pathway [published online as ahead of print on May 14, 2024]. Adv Clin Exp Med. 2025. doi:10.17219/acem/184639

Recombinant Klotho protein protects pulmonary alveolar epithelial cells against sepsis-induced apoptosis by inhibiting the Bcl-2/Bax/caspase-3 pathway

Xiao Bo Li1,2,A,B,C,E,F, Jia Li Liu1,2,B,C, Shuang Zhao1,2,C, Jing Li1,2,B,D, Guang-Yan Zhang1,2,C,D, Qing Tang1,2,B, Wei Yong Chen1,2,A,C

1 Respiratory Department, Chengdu Seventh People’s Hospital, China

2 Respiratory Department, Tumor Hospital Affiliated to Chengdu Medical College, China

Graphical abstract


Graphical abstracts

Abstract

Background. Inflammation-induced apoptosis of alveolar type II epithelial cells is a primary contributor to sepsis-induced acute respiratory distress syndrome (ARDS). Klotho is a single-pass transmembrane protein with anti-inflammatory and anti-apoptotic effects. However, the role and mechanism of Klotho in the development of ARDS remains unknown.

Objectives. This study aimed to investigate the effect of Klotho on sepsis-induced apoptosis in human pulmonary alveolar epithelial cells (HPAEpiCs) together with the potential mechanism.

Materials and methods. Cecal ligation and puncture (CLP) were performed to generate an in vivo sepsis model, and HPAEpiCs were treated with lipopolysaccharide (LPS) to mimic sepsis in vitro. Both models were administered recombinant Klotho protein. The morphology of the lung tissue was observed, and apoptotic cells and cell viability were detected. Interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA), while the expression of Bcl-2, Bax and cleaved caspase-3 was detected with western blotting.

Results. Klotho reversed the CLP-induced decrease in mouse survival in vivo (p < 0.001) and increased inflammatory cell infiltration and inflammatory substance exudation in the lung tissue of mice with sepsis (both p < 0.001). Klotho also suppressed apoptosis (p < 0.001) as demonstrated by IL-1β, IL-6 and TNF-α expression (all p < 0.001), and Bcl-2/Bax/caspase-3 pathway activation (p < 0.001). Klotho pretreatment significantly prevented LPS-induced apoptosis in vitro (p < 0.001), as demonstrated by IL-1β, IL-6 and TNF-α upregulation (all p < 0.001); and Bcl-2/Bax/caspase-3 pathway activation in HPAEpiCs (p < 0.001).

Conclusions. This study demonstrated that Klotho can ameliorate acute lung injury (ALI) induced by sepsis by inhibiting inflammatory responses and exerting anti-apoptotic effects by suppressing Bcl-2/Bax/caspase-3 pathway activation.

Key words: sepsis, ARDS, apoptosis, Klotho, HPAEpiC

Background

Sepsis is a life-threatening inflammatory response, with approx. 30% mortality rate.1 The lungs are the most vulnerable organ attacked by inflammatory factors during sepsis, and sepsis progresses to acute respiratory distress syndrome (ARDS) in approx. 50% of patients.2 Although great progress has been made recently in the treatment of ARDS, its morbidity and mortality remain high.3 Therefore, understanding the complex pathogenesis of ARDS is of great significance. Acute respiratory distress syndrome is a complex clinical syndrome with a heterogeneous clinical phenotype, and it is characterized by pulmonary edema in the interstitium and air spaces of the lungs. The accumulation of fluid increases the work of breathing and impairs gas exchange, resulting in hypoxemia, reduced CO₂ excretion and, ultimately, acute respiratory failure.4 Acute respiratory failure ultimately leads to hypoxia, which has a poor prognosis.5, 6 Furthermore, acute respiratory failure is among the sequela of complications that can develop in response to sepsis, which can induce severe inflammatory responses.7

Sepsis is facilitated by bacterial infections inducing the excessive release of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1 and IL-6, causing cellular injury and multiple organ dysfunction syndrome.8 Lipopolysaccharide (LPS) is a bacterial endotoxin that can induce a severe inflammatory response.9 Lipopolysaccharide is commonly used to establish murine lung injury models to mimic sepsis-induced ARDS in humans, and such models help investigate the pathogenesis of ARDS.10 Alveolar epithelial cell apoptosis complicates the pathogenesis of ARDS.11 Apoptosis can be activated by 2 pathways, namely, the mitochondrial pathway and the external death receptor pathway. When cells encounter direct or indirect DNA damage, reactive oxygen species (ROS), hypoxia, or inflammation, the mitochondrial apoptosis pathway is activated.12, 13 These stimuli ultimately disrupt mitochondrial function by inducing the expression of proapoptotic Bcl-2 family members, such as Bcl-2, Bax and Bak.14 The imbalance of Bax and Bcl-2 can induce the activation of caspase-3,15 thereby promoting apoptosis.16 Many studies have revealed that LPS induces apoptosis in HPAEpiCs.17, 18 However, the mechanism of LPS-induced HPAEpiCs apoptosis has not yet fully elucidated.

It has been reported that acute inflammation/sepsis suppresses the activation of α-Klotho, and α-Klotho upregulation during acute sepsis may participate in the counter-regulatory response to severe inflammation in patients with sepsis.19 Klotho deficiency aggravates sepsis-related multiple organ dysfunction.20 Klotho has been recognized as a gene involved in the aging process in mammals for more than 30 years,21 and this single-pass transmembrane protein counteracts oxidative stress22 and cell senescence23 and promotes autophagy.24 In addition, Klotho has anti-apoptotic effects.25 Klotho suppresses diabetes-induced podocyte apoptosis26 and inhibits H2O2-induced apoptosis in periodontal ligament stem cells.27 Normal lungs do not express Klotho protein, but they can obtain cell protection from soluble circulating Klotho.28 However, the effect of Klotho on sepsis-induced apoptosis in alveolar epithelial cells has not been reported. Chen et al. revealed that Klotho can inhibit proliferation and increase apoptosis in A549 cells by regulating the expression of the apoptosis-related genes Bcl-2 and Bax.29 Therefore, we hypothesized that Klotho protects alveolar epithelial cells against sepsis or LPS-induced apoptosis by inhibiting the Bcl-2/Bax/caspase-3 pathway.

Objectives

The objective of this study was to explore the effects of Klotho on sepsis-induced inflammatory cytokine release and LPS-induced HPAEpiC apoptosis and demonstrate the potential mechanism of Klotho protects HPAEpiC against sepsis or LPS-induced apoptosis.

Materials and methods

Cecal ligation and puncture (CLP) were performed to generate a mouse sepsis model, and HPAEpiCs were treated with LPS to create an in vitro model of sepsis-induced ARDS. Both models were treated with recombinant Klotho protein to explore its effects on sepsis-induced alveolar type II epithelial cells and its potential mechanism.

Cell culture

The HPAEpiC was brought from the American Type Culture Collection (ATCC, Manassas, USA). The cells were cultured in a high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and incubated at 37°C under 5% CO2 and 95% humidity; then, cells were passaged when confluence reached 90%. The use of HPAEpiC was approved by the Human Ethics Committee of Chengdu Seventh People’s Hospital on January 26, 2022 (Human Experimental Inspection Form of Chengdu Seventh People’s Hospital No. 2200647).

Animals and treatment

The C57BL/6 mice (male, 8 weeks old, weight = 20–25 g) were purchased from ENSIWEIER Biotechnology Co., LTD (Chongqing, China). The mice were supplied sterile water and food and kept in a specific pathogen-free environment (temperature: 22°C ±2°C; humidity: 40–60%). The animal experiment was approved by the Animal Ethics Committee of Chengdu Seventh People’s Hospital on January 13, 2022 (Animal Experimental Ethical Inspection Form of Chengdu Seventh People’s Hospital No. 2200638). The mice were randomly divided into 4 groups, namely, the sham, Klotho, CLP, and CLP+Klotho groups (n = 10/group). Mice in the sham group underwent sham operation without CLP, whereas mice in the Klotho group were intraperitoneally injected with recombinant mouse Klotho protein (10 μg/kg; R&D Systems, Minneapolis, USA). Mice in the CLP group underwent CLP, and those in the CLP+Klotho group underwent CLP, followed by intraperitoneal treatment with recombinant mouse Klotho protein. Them, 24 h after CLP, 2% isoflurane was used to anesthetize the mice, which were euthanized via cervical dislocation, and their lungs were collected for hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated biotinylated UTP labeling (TUNEL) assay and western blotting.

Cecal ligation and puncture mouse model

Cecal ligation and puncture mouse model was created as described previously.30 In brief, after anesthesia was induced with 2% isoflurane, the cecum of each mouse was completely exposed through an abdominal surface incision. Then, 70% of the total length of the cecum was ligated with a 4-0 silk suture and a penetrating puncture was performed with a No. 22 needle (BD Biosciences, Franklin Lakes, USA). Sham-operated mice underwent the same procedure without ligation and puncture of the cecum.

Humane endpoints of surgery/treatment in mice

Several evaluation items are often used as the endpoints of animal experiments. First, experiments may cause pain and discomfort to animals. If drugs or other methods cannot be used to relieve their pain and distress, euthanasia is performed. Second, animals should be euthanized in response to the rapid weight loss of 15–20% or cachexia and long-term muscle catabolism. Third, animals should be euthanized if they do not eat for 24–48 h or if they ingest only a small amount of food for 3 days. Lastly, animals should be euthanized when they cannot drink or eat by themselves.

Survival studies

Survival rates were assessed as described by Alves et al.31 To evaluate the effect of Klotho on the survival of mice with sepsis, 80 mice were randomly divided into the sham, Klotho, CLP, and CLP+Klotho groups (n = 20/group). The mice in the CLP+Klotho and Klotho groups received intraperitoneal injections of 10 μg/kg Klotho once daily. Survival curves were plotted every 6–12 h for 7 consecutive days. After 7 days, 2% isoflurane was used to anesthetize the surviving mice, which were euthanized via cervical dislocation.

Collection of bronchoalveolar lavage fluid and cell culture medium for IL-1β, IL-6 and TNF-α detection

Bronchoalveolar lavage fluid (BALF) was collected as previously described.32 In brief, all mice were anesthetized with 2% isoflurane and immediately subjected to thoracotomy, and the right great bronchus below the tracheal bifurcation was ligated. Then, the left bronchial tube was intubated and rinsed 3 times with ice-cold phosphate-buffered saline (PBS) (0.5 mL). The mice were sacrificed via cervical dislocation under anesthesia. We also collected the culture medium of HPAEpiCs. In brief, the cells were treated with 0.1 mg/L, 1 mg/L or 10 mg/L LPS or 10 mg/L LPS combined with 50 mg/L recombinant Klotho for 24 h; then, the conditioned culture media was collected. The collected BALF and cell culture medium were centrifuged at 1000 × g and 4°C for 10 min. The supernatant was stored at −80°C for further analysis. The total protein level was detected using the bicinchoninic acid (BCA) assay kit (Beyotime Biotechnology, Shanghai, China), and the protein concentration was standardized before enzyme-linked immunosorbent assay (ELISA) detection (Beyotime Biotechnology) of IL-1β, IL-6 and TNF-α in BALF and cell culture medium according to the manufacturer’s protocol.

Hematoxylin and eosin staining

Morphological changes in the lungs of mice were evaluated with H&E staining.33 In brief, paraffin sections of lung tissue were soaked in xylene I and xylene II for 10 min. Then, the sections were successively incubated in 100% (I, II), 90%, 80%, and 70% alcohol for 5 min each and rinsed 3 times with running water for 5 min each. Finally, the sections were stained with hematoxylin for 5 min and eosin (1%) staining for 2 min, then rinsed with water. Images of the lung tissues were captured (magnification: ×400) using an optical microscope (Leica DFC550 DM4 B; Leica Camera AG, Wetzlar, Germany).

TUNEL assay

To detect apoptosis in lung tissue in situ, we used a TUNEL kit.34 In brief, 5-µm-thick paraffin sections of lung tissue were dewaxed, rehydrated, and treated with protease K working solution for 30 min. The slides were then incubated in the TUNEL reaction mixture at 37°C for 1 h. The slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, and the tissue was mounted in an Anti-Fade Mounting Medium. Images of the lung tissues were captured using an optical microscope (magnification ×200; Leica DFC550 DM4 B ).

Cell Counting Kit-8 cell viability assay

Changes in cell viability were determined using the Cell Counting Kit-8 (CCK-8) assay.35 In brief, HPAEpiCs (5 × 103/well) were plated in 96-well plates. The cells were exposed to LPS (0.1 mg/L, 1 mg/L or 10 mg/L) for 24 h or 10 mg/L LPS for 12 h, 24 h or 48 h. Then, 10 µL of CCK-8 was added to each well. The absorption of each well was read at 450 nm using a microplate reader (Bio-Rad 680; Bio-Rad Hercules, USA). In addition, cells were pretreated with 10 mg/L LPS for 1 h and then incubated with various concentrations (0 mg/L, 50 mg/L and 100 mg/L) of recombinant Klotho protein for 24 h or 50 mg/L recombinant Klotho protein for 12 h, 24 h or 48 h. Cell Counting Kit-8 was added to each well as previously described, and the plate was incubated at 37°C for 1 h. The absorption of each well was read at 450 nm using a microplate reader (Bio-Rad 680; Bio-Rad).

Annexin/propidium iodide (PI) staining

Annexin V/propidium iodide (PI) staining was used to investigate cell apoptosis.36 In brief, cells were exposed to 0.1 mg/L, 1 mg/L or 10 mg/L LPS or 10 mg/L LPS combined with 50 mg/L recombinant Klotho for 24 h. Untreated cells served as the control group. The cells were then harvested and washed with PBS to remove the medium. At least 1 × 105 cells were resuspended in 100 µL of binding buffer containing Annexin V–FITC and PI and incubated for another 15 min at room temperature in the dark. Fluorescence from 1 × 104 cells in the Annexin V–FITC and PI binding channels FL-1 (Annexin V–FITC) and FL-3 (PI) was quantified using FACScan and analyzed using Cellquest Pro (BD Biosciences, Franklin Lakes, USA).

Western blotting

Protein expression was detected with western blotting as previously described.37 The cells and lung tissue of mice were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors and centrifuged for 15 min at 12,700 × g and 4°C. The supernatant was collected, the protein concentration was measured, and the samples were boiled for 5 min. Then, 30 μg of protein was electrophoresed on an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skim milk at room temperature for 2 h. The membrane was incubated overnight with primary antibodies against GAPDH (ab8245; 1:5000), Bcl-2 (ab182858; 1:1000), Bax (ab32503; 1:1000), pro-caspase-3 (ab32499; 1:1000), and cleaved caspase-3 (ab214430; ab2302; 1:1000; all from: Abcam, Cambridge, UK) at 4°C, then washed 3 times with Tris-buffered saline containing 0.1% Tween-20, then incubated with HRP Anti-Rabbit IgG antibody (ab288151; 1:10000; Abcam) for 2 h. The protein bands were visualized using chemiluminescence and analyzed using Quantity One software v. 6.0 (Bio-Rad).

Statistical analyses

All experiments were repeated at least 3 times, and data were expressed as the mean ± standard deviation (M ± SD). All the data were analyzed using GraphPad Prism 8.0 (GraphPad Software, San Diego, USA). Statistical significance was indicated by p < 0.05. Quantitative data were presented as dots and medians. The survival analysis between groups used the log-rank (Mantel–Cox) method, and due to the limited sample size, other data were assessed with non-parametric tests. The Kruskal–Wallis test was used for a comparison of 3 or more groups, followed by Dunn’s post hoc test. The medians and quartiles were presented in supplement tables. The test levels of α = 0.05 and p < 0.05 were considered significant.

Results

Klotho increased mouse survival and decreased IL-1β, IL-6 and TNF-α levels

To determine the effects of Klotho on mice with sepsis, mice that underwent CLP were intraperitoneally injected with recombinant Klotho protein. All mice in the CLP group died on day 4, whereas 40% of those in the CLP+Klotho group survived (p < 0.002, Figure 1A). Hematoxylin and eosin staining of mouse lung tissue in the CLP group revealed swelling, cell edema, inflammatory cell infiltration, and inflammatory mediator exudation, and these effects were ameliorated by Klotho treatment (Figure 1B). We also detected IL-1β, IL-6 and TNF-α in the BALF of mice. The results showed that IL-1β (p < 0.001), IL-6 (p < 0.001) and TNF-α (p < 0.001) levels were significantly higher in the CLP group compared to the sham group, and these increases were significantly reduced by Klotho treatment (all p < 0.05; Figure 1C–E).

Effects of Klotho on alveolar epithelial cell apoptosis and Bcl-2/Bax/caspase-3 signaling in mice with sepsis

To detect the effect of Klotho on alveolar epithelial cell apoptosis in mice with sepsis, apoptotic cells were detected using TUNEL staining. The number of TUNEL-positive cells was significantly higher in the CLP group compared to sham mice (p < 0.001) and CLP+Klotho groups (p < 0.05; Figure 2A,B). We also detected the protein levels of Bcl-2, Bax and caspase-3 in the lung tissue of mice, finding that CLP decreased the Bcl-2/GAPDH ratio (p < 0.001) and increased the Bax/GAPDH (p < 0.001) and cleaved caspase-3/caspase-3 ratios (p < 0.001); and these effects were reversed by Klotho treatment (all p < 0.05; Figure 2C,D).

Lipopolysaccharide-induced apoptosis, inflammatory factor release and Bcl-2/Bax/caspase-3 pathway activation in HPAEpiCs

Human pulmonary alveolar epithelial cells were exposed to 0.1 mg/L, 1 mg/L or 10 mg/L LPS for 24 h or 10 mg/L LPS for 12 h, 24 h and 48 h, followed by cell viability assessment. The results showed that LPS reduced cell viability in a concentration- (p < 0.05; Figure 3A) and time-dependent manner (p < 0.05; Figure 3B). The apoptotic rate of HPAEpiCs was tested using Annexin/PI staining. Lipopolysaccharide significantly increased the percentage of apoptotic cells compared with that in the control group (p < 0.05; Figure 3C–D). In addition, 10 mg/L LPS significantly increased the release of IL-1β (p < 0.05), IL-6 (p < 0.05) and TNF-α (p < 0.05; Figure 3E–G).

Because LPS induced HPAEpiC apoptosis, we detected the protein expression of Bcl-2, Bax and cleaved caspase-3 in LPS-treated cells. The results illustrated that LPS decreased the Bcl-2/GAPDH ratio (p < 0.01) and increased the Bax/GAPDH and cleaved caspase-3/pro-caspase-3 ratios (both p < 0.05; Figure 3H–K). These results indicated that LPS induced apoptosis and Bcl-2/Bax/caspase-3 pathway activation in HPAEpiCs.

Effect of recombinant Klotho protein on viability, apoptosis and inflammatory factor release in LPS-exposed HPAEpiCs

To explore the effect of Klotho protein on the viability of LPS-exposed HPAEpiCs, we treated HPAEpiCs with LPS (10  mg/L) for 1 h followed by incubation with 10 mg/L, 50 mg/L or 100 mg/L recombinant Klotho for 24 h or 50 mg/L recombinant Klotho protein for 12 h, 24 h or 48 h. The results indicated that recombinant Klotho protein reversed the LPS-induced decrease in cell viability in a concentration- (p < 0.05; Figure 4A) and time-dependent manner (p < 0.001; Figure 4B). The apoptotic rate of HPAEpiCs was tested using Annexin/PI staining (Figure 4C). As previously observed, 10 mg/L LPS induced significant apoptosis in HPAEpiCs (p < 0.01), and this effect was reversed by 50 mg/L recombinant Klotho protein treatment (p < 0.05; Figure 4D). In addition, recombinant Klotho protein treatment reversed the LPS-induced release of IL-1β, IL-6 and TNF-α (all p < 0.05; Figure 4E–G).

Recombinant Klotho protein blocked Bcl-2/Bax/caspase-3 signaling in LPS-exposed HPAEpiCs

The cells were treated with LPS (10 mg/L) for 1 h and then incubated with 50 mg/L recombinant Klotho protein, after which we detected the protein expression of Bcl-2, Bax, pro-caspase-3, and cleaved caspase-3. The results demonstrated that LPS decreased the Bcl-2/GAPDH ratio and increased the Bax/GAPDH and cleaved caspase-3/pro-caspase-3 ratios (all p < 0.01), and these effects were partially reversed by recombinant Klotho protein administration (all p < 0.05; Figure 5A–D).

Discussion

In vivo, we found that recombinant Klotho protein significantly reduced the inflammatory response and percentage of apoptotic cells in lung tissue, increased the survival rate of mice with sepsis and prevented the activation of BCL-2/Bax/caspase-3 signaling. Moreover, we observed that Klotho increased the viability and decreased the apoptosis of HPAEpiCs exposed to LPS in vitro. Finally, we demonstrated that LPS activated the Bcl-2/Bax/caspase-3 pathway, which was inhibited by Klotho.

The lungs represent the initial organ affected by inflammation in sepsis.38 Sepsis causes severe systemic inflammatory response syndrome, leading to ARDS/acute lung injury (ALI) and high mortality and morbidity rates.39 In ARDS, severe inflammation caused by bacterial infection disrupts the endothelial barrier, increasing the permeability of the pulmonary vasculature to circulating fluids, macromolecules and leukocytes, and resulting in alveolar flooding and neutrophil influx, which is responsible for the high mortality rate.40 One of the pathophysiological characteristics of sepsis and subsequent ARDS/ALI are hyperactive and dysregulated endogenous inflammatory cytokines, such as IL-6, IL-1β and TNF-α.41, 42, 43, 44 In our study, we observed that CLP-induced sepsis decreased survival in mice and increased alveolar flooding, neutrophil influx and inflammatory factor release in the lungs. Human pulmonary alveolar epithelial cells apoptosis contributes to the pathogenesis of ALI and ARDS, which are common complications of sepsis.45, 46 Upon apoptosis in alveolar epithelial cells, DNA breaks are induced, and the cell decreases in size, resulting in its destruction by nearby phagocytes. These events lead to the destruction of the alveolar epithelium and serious damage to the alveolar–capillary barrier, which in turn promotes the pathogenesis of ALI and ARDS.47 Our study found that CLP induced apoptosis in alveolar epithelial cells in mouse lung tissue.

Sepsis is mostly caused by endotoxin, which is released from Gram-negative bacteria.48 Lipopolysaccharide is an important component of endotoxin that can cause a cascade of immune stimulation and toxic pathophysiological activities in the body, including ALI induced by sepsis.49 Lipopolysaccharide is often used to generate animal models of diseases involving dysregulated inflammatory responses, such as ALI.50, 51 Accumulating evidence indicates that inflammation is involved in apoptosis,52 and it has been reported that LPS can induce apoptosis in neurons,53 cardiomyocytes,54 renal tubular cells,55 macrophages,56 and HPAEpiCs.57 Our research also found that LPS decreased the viability and increased the apoptosis rate in HPAEpiCs.

An imbalance between Bax and Bcl-2 plays an important role in the mitochondria-mediated caspase cascade, which was confirmed by an increased Bax/Bcl-2 ratio, release of cytochrome C from the mitochondria to the cytoplasm, cleavage of caspase-3, and subsequent induction of apoptosis.58, 59 Yang et al. reported that LPS induced apoptosis in alveolar macrophage cells by increasing the Bax/Bcl-2 ratio,60 while Chopra et al. demonstrated that CLP-induced sepsis resulted in myocardial apoptosis through upregulation of the ratio of Bax/Bcl-2.61 Herein, we demonstrated that CLP and LPS treatment resulted in decreased Bcl-2 expression and increased Bax and cleaved caspase-3 expression.

Klotho is expressed primarily in renal cells, and it has anti-sepsis properties.20 It has been reported that renal Klotho expression was significantly reduced in patients with sepsis-induced acute kidney injury (AKI) and in mice with sepsis.62 Recombinant Klotho ameliorates sepsis-induced multiple organ dysfunction,63, 64, 65 whereas Klotho deficiency has been reported to increase the production of TNF-α, IL-1β and IL-6, which aggravate sepsis-induced multiple organ dysfunction syndrome.20 In a CLP model, Klotho knockout mice exhibited significantly higher mortality, higher bacterial loads, and higher TNF-α, IL-6 and IL-10 concentrations.66 In our study, Klotho increased the survival of mice with sepsis and decreased alveolar flooding, neutrophil influx and inflammatory factor release in the lungs of mice.

Accumulating evidence indicates that Klotho exerts anti-apoptotic effects; it has been shown to suppress endothelial apoptosis via a mitogen-activated kinase pathway.67 Genetic Klotho deficiency increases cardiomyocyte apoptotic activity, whereas Klotho supplementation can reverse changes in apoptotic activity caused by d-galactose.68 Klotho pretreatment inhibited apoptosis in retinal pigment epithelial cells by increasing Bcl-2 levels and decreasing Bax and cleaved caspase-3 levels. Moreover, it has been reported that Klotho protects lung epithelial cells from hyperoxia-induced apoptosis.69 However, the effect of Klotho on CLP- or LPS-induced apoptosis in alveolar type II epithelial cells has never been explored. Our study has provided the first evidence that recombinant Klotho protein inhibited CLP- and LPS-induced apoptosis in HPAEpiCs. We also found that recombinant Klotho protein blocked the CLP- and LPS-induced activation of the Bcl-2/Bax/caspase-3 pathway in HPAEpiCs.

Limitations

We did not perform gene intervention in mouse lung tissue and HPAEpiCs. We also did not further explore the effect of Klotho on the Bcl-2/Bax/caspase-3 pathway, which are future research goals.

Conclusions

Our research indicated that recombinant Klotho protein protected alveolar type II epithelial cells against sepsis-induced apoptosis and increased their survival by blocking the Bcl-2/Bax/caspase-3 pathway. Considering the protective effect of Klotho on sepsis-induced apoptosis in alveolar type II epithelial cells, it could represent a promising agent for treating sepsis-induced ALI.

Supplementary data

The Supplementary materials are available at https://doi.org/10.5281/zenodo.10975284.The package includes the following files:

Supplementary Table 1. Statistical methods, results and sample size for Figure 1.

Supplementary Table 2. Statistical methods, results and sample size for Figure 2.

Supplementary Table 3. Statistical methods, results and sample size for Figure 3.

Supplementary Table 4. Statistical methods, results and sample size for Figure 4.

Supplementary Table 5. Statistical methods, results and sample size for Figure 5.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication

Not applicable.

Figures


Fig. 1. Klotho increased the survival of mice with sepsis and decreased the release of interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α). A. The survival rates of mice in the sham, Klotho, cecal ligation and puncture (CLP), and Klotho+CLP groups (n = 20/group) were analyzed. Data were analyzed with the log-rank (Mantel–Cox) test; **p < 0.002 compared with the CLP group; ***p < 0.001 compared with the CLP group or CLP+Klotho group. B. Hematoxylin and eosin (H&E) staining was performed to detect the morphological structure of the lung tissue of mice in the 4 groups (n = 10/group). C–E. IL-1β, IL-6, and TNF-α levels were measured (n = 10/group). The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test, and then Dunn’s multiple comparison test was used for post hoc analysis; ***p < 0.001 compared with the CLP group; *p < 0.05 and **p < 0.01 compared with the CLP+Klotho group
Fig. 2. Effects of Klotho on alveolar epithelial cell apoptosis and Bcl-2/Bax/caspase-3 signaling in mice with sepsis. A,B. The terminal deoxynucleotidyl transferase-mediated biotinylated UTP labeling (TUNEL) assay was performed to detect apoptotic cells in the lung tissues of mice in the sham, cecal ligation and puncture (CLP), Klotho, and CLP+Klotho groups (n = 10/group). TUNEL-positive cells were counted and compared among the groups. The graph presents the results of data dots and medians. The data were analyzed with the Kruskal–Wallis test, and then Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 and ***p < 0.001 compared with the CLP group. C–F. Western blotting was performed to detect the expression of Bcl-2, Bax, pro-caspase-3, and cleaved caspase-3. The graph presents the results of data dots and medians. The data were analyzed with the Kruskal–Wallis test, and then the Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 and ***p < 0.001 compared with the CLP group
Fig. 3. Lipopolysaccharide induced apoptosis and activated the Bcl-2/Bax/caspase-3 pathway in human pulmonary alveolar epithelial cells (HPAEpiCs). A. Human pulmonary alveolar epithelial cells were exposed to 0.1 mg/L, 1 mg/L or 10 mg/L LPS for 24 h, and then the Cell Counting Kit-8 (CCK-8) assay was performed to detect cell viability. The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. The Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 compared with the control group. B. Human pulmonary alveolar epithelial cells were exposed to 10 mg/L LPS, and the CCK-8 assay was performed to detect cell viability after 12 h, 24 h and 48 h. The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. A Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 compared with the control (0 h) group. C–D. Human pulmonary alveolar epithelial cells were treated with LPS (0.1 mg/L, 1 mg/L or 10 mg/L) for 24 h, and the apoptosis rate was determined with flow cytometry. The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. The Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 compared with the control group. E–G. Interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA). The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. The Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 compared with the control group. H. Human pulmonary alveolar epithelial cells were treated with 0.1 mg/L, 1 mg/L or 10 mg/L LPS for 24 h, and the expression of Bcl-2, Bax, pro-caspase-3, and cleaved caspase-3 was detected with western blotting. I–K. The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. The Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 and **p < 0.01 compared with the control group
Fig. 4. Effects of recombinant Klotho protein on cell viability, apoptosis and inflammatory factor expression in lipopolysaccharide-treated human pulmonary alveolar epithelial cells. A. Human pulmonary alveolar epithelial cells were pretreated with 10 mg/L lipopolysaccharide (LPS) for 1 h and then incubated with various concentrations of recombinant Klotho (10 mg/L. 50 mg/L and and 100 mg/L) for 24 h. The Cell Counting Kit-8 (CCK-8) assay was performed to detect cell viability. The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. The Dunn’s multiple comparison test was used for post hoc analysis; *p < 0.05 compared with the control group; B. Human pulmonary alveolar epithelial cells were pretreated with 10 mg/L LPS for 1 h, followed by treatment with 50 mg/L recombinant Klotho protein for different periods (12 h, 24 h and 48 h). Cell viability was assessed using the CCK-8 assay. The graph presents the results of data dots and medians. Data were analyzed with Robust 2-way mixed analysis of variance (ANOVA) using WRS2 package; ***p < 0.001 compared with the control group. C,D. Cells were pretreated with 10 mg/L LPS for 1 h followed by incubation with 50 mg/L recombinant Klotho protein for 24 h. The apoptosis rate was determined with flow cytometry. The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. The Dunn’s multiple comparison test was used for post hoc analysis; **p < 0.01 compared with the 10 mg/L LPS group; #p < 0.05 compared with the 10 mg/L LPS+50 mg/L Klotho group; E–G. Interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) levels in the cell culture medium were detected using enzyme-linked immunosorbent assay (ELISA). The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. Dunn’s multiple comparison test was used for post hoc analysis; **p < 0.01 compared with the 10 mg/L LPS group; #p < 0.05 compared with the 10 mg/L LPS + 50 mg/L Klotho group
Fig. 5. Recombinant Klotho protein blocked the lipopolysaccharide (LPS)-induced activation of the Bcl-2/Bax/caspase-3 pathway in human pulmonary alveolar epithelial cells. A–D. Human pulmonary alveolar epithelial cells were pretreated with 10 mg/L LPS for 1 h and then incubated with 50 mg/L recombinant Klotho protein for 24 h. Bcl-2, Bax, pro-caspase-3, and cleaved caspase-3 expression were detected with western blotting. The graph presents the results of data dots and medians. Data were analyzed with the Kruskal–Wallis test. The Dunn’s multiple comparison test was used for post hoc analysis; **p < 0.01 compared with the 10 mg/L LPS group; #p < 0.05 compared with the 10 mg/L LPS + 50 mg/L Klotho group

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