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. 2025 Oct 17;30:933–945. doi: 10.1016/j.reth.2025.10.004

Targeting ER stress-mediated apoptosis by MSC-derived exosomes: A novel therapeutic strategy against pulmonary fibrosis

Ruixi Luo 1,⁎, Yaqiong Wei 1, La Wang 1, Peng Chen 1, Didong Lou 1, Weiyi Tian 1,⁎⁎
PMCID: PMC12554144  PMID: 41147010

Abstract

Introduction

Idiopathic pulmonary fibrosis (IPF) is marked by a gradual decline in pulmonary function over time and is associated with a grim prognosis. In the pathogenesis of IPF, persistent endoplasmic reticulum (ER) stress plays a significant role in promoting fibrosis through pathways involving apoptosis. Mesenchymal stem cell-derived exosomes (MSC-Ex) have shown promise in mitigating pulmonary fibrosis by inhibiting apoptosis. Nonetheless, the precise mechanisms underlying this effect remain unclear. In our previous findings, we demonstrated that MSCs alleviate pulmonary fibrosis by regulating ER stress. Building upon this, we sought to investigate whether MSC-Ex could mitigate alveolar epithelial cell apoptosis through the ER stress pathway. We posited that targeting ER stress could represent a crucial mechanism by which MSC-Ex alleviate apoptosis in IPF models.

Methods and results

In this study, bleomycin (BLM) induced apoptosis in A549 cells, and MSC-Ex treatment reduced apoptotic cells and the Bax/Bcl-2 ratio. ER stress is involved in BLM-induced apoptosis in A549 cells, and MSC-Ex reduced ER stress-related protein (Bip and CHOP) expression and reversed the morphological changes of the ER in A549 cells. Moreover, blockade of ER stress with ER stress inhibitor TUDCA contributed to the amelioration of apoptosis in A549 cells, indicating that MSC-Ex reduced BLM-induced apoptosis at least partly by modulating ER stress. In vivo, MSC-Ex injection decreased BLM-induced pulmonary fibrosis in mice, as well as ER stress and apoptosis in the lung tissues.

Conclusions

In conclusion, ER stress induced apoptosis in BLM-treated A549 cells, and MSC-Ex treatment mitigated apoptosis via inhibiting ER stress. This study provides a novel mechanism for MSC-Ex-mediated protection on apoptosis in an IPF model and suggests that MSC-Ex could be a promising therapeutic strategy for IPF.

Keywords: Exosome, Mesenchymal stem cell, Endoplasmic reticulum stress, Apoptosis, Pulmonary fibrosis

1. Introduction

Idiopathic pulmonary fibrosis (IPF), the most prevalent interstitial lung disease, is characterized by a gradual and progressive decline in pulmonary function over time. Due to the absence of effective diagnostic methods and treatments, patients with IPF often face a bleak prognosis [1]. In Asia, the incidence of IPF is 0.35–1.30 per 10,000 people, and carries a heavy economic burden on families and society [2]. Additionally, IPF is not curable at present. Hence, it is imperative to unveil the molecular mechanisms underlying the pathogenesis of IPF and to explore effective therapeutic interventions aimed at slowing or reversing its progression.

According to previous studies, excessive alveolar epithelial cells (AECs) apoptosis contribute to fibrosis, and inhibiting apoptosis was reported to relieve IPF [3]. Endoplasmic reticulum (ER) stress is frequently found in lung tissues in patients with IPF [4] and continuous ER stress contributes to fibrosis via apoptosis-mediated pathways [5]. Modulating ER stress might be a method to alleviate apoptosis and slow down the process of IPF.

Due to the capacity of self-renew and multi-differentiating potential, Mesenchymal stem cells (MSCs) have emerged as a dependable source in biotherapy, renowned for their robust therapeutic potential across a spectrum of diseases due to their paracrine properties [6]. Several studies revealed that MSCs primarily mitigate lung fibrosis through their paracrine effects [7,8]. We previously found MSCs alleviated lung fibrosis by modulating ER stress in a bleomycin-treated mouse IPF model [9]. Notably, although MSCs exert several amazing therapeutic effects, some limitations, such as cell senescence, malignant transformation, tumor formation and strict transportation conditions, restrict their application in clinical cell therapy. Thus, how to maximize the beneficial effects of MSCs while avoiding their disadvantages is a research hotspot.

Interestingly, MSC-derived exosomes (MSC-Ex) are considered as a key mediator for the paracrine effects of MSCs. MSC-Ex refers to the small bilayer vesicles containing small RNAs and proteins. MSC-Ex have similar biological characteristics and functions to MSCs [10]. Data from numerous studies suggest that MSC-Ex might be a promising treatment modality for cell-free MSC-based therapies with less risk [11]. MSC-Ex could alleviate lung injury and pulmonary fibrosis by inhibiting ER stress, inflammation, ROS [12] and apoptosis [13,14]. However, the detailed molecular mechanisms of MSC-Ex on reducing alveolar epithelial cell apoptosis remains unstudied. Therefore, we questioned whether MSC-Ex could reduce AEC apoptosis via the ER stress pathway and hypothesized that ER stress is an important target in MSC-Ex-mediated alleviation of apoptosis in IPF models.

2. Materials and methods

2.1. Cell culture

A549 cells were cultured in DMEM/F-12 medium (L1041-500, BDBIO, Hangzhou, China) supplemented with 10 % fetal bovine serum (FBS, SA301, Cellmax, Beijing, China) at 37 °C. Human umbilical cord-derived MSCs were purchased from Zhongqiao Xinzhou Co., Ltd. (DF-GMP-ZB09BA, Shanghai, China). MSCs were cultured in MSC Complete Medium (ZQ-1320, Zhongqiao Xinzhou) supplemented with 5 % serum substitute (ZQ-1320S, Zhongqiao Xinzhou) and antibiotics (AC03L332, Life-iLab, Shanghai, China).

2.2. Preparation and Identification of MSC-Ex

MSC-Ex were isolated using Exosome Isolation Kit (UR52121, Umibio, China). The purified Ex was resuspended, passed through a 0.22-μm pore filter (Jet Biofil, China) and kept at −80 °C. The morphology of Ex was observed using transmission electron microscopy (TEM), and the size was analyzed by nanoparticle trafficking analysis (NTA, ZetaVIEW, Particle Metrix, Meerbusch, Germany). Meanwhile, Ex markers CD63 (A19023, ABclonal, Wuhan, China) and TSG101 (GTX70255, GeneTex, Irvine, CA, USA) were verified using Western blotting.

2.3. Experiment Grouping

Bleomycin (BLM, B107423, Aladdin, Shanghai, China), Thapsigargin (TG, abs44076907, Absin, Shanghai, China) and TUDCA (abs816166, Absin) were dissolved in PBS and stored at −80 °C. Based on our preliminary findings [9], A549 lung adenocarcinoma cell lines were treated with BLM or TG (100 μM) for 24 h with or without pretreatment with MSC-Ex (100 μg/ml) or TUDCA (100 μM) for 1 h.

2.4. CCK8 assay

Cell viability was measured using Cell Counting Kit-8 (CCK-8, C6005, New Cell & Molecular Biotech, Suzhou, China) assay as previously described [9].

2.5. Apoptosis analysis

Cells were stained with the Annexin V-FITC/propidium iodide (PI) fluorescence probes (MK1028, Boster, Wuhan, China), and the proportion of apoptotic cells was detected using flow cytometry.

2.6. Q-PCR

Q-PCR was conducted in a CFX96 Real-Time PCR System (BioRad, Hercules, USA). In brief, cells and tissues were subjected to total RNA extraction using a Total RNA Extract Reagent (RE600, Coolaber, Beijing, China) and reversed transcription to cDNA using a Reverse Transcription Kit (AG11705, Accurate Biotechnology Co., Ltd., ChangSha, China). q-PCR is performed using StarLighter HP SYBR Green qPCR Mix (FS-Q1006, Foreverstar Biotech, Beijing, China) and primers (Table S1) specific to the target gene. The 2-△△CT method was used to quantify relative gene expression.

2.7. Western blotting

Cell and tissue proteins were extracted using a protein extraction kit (E1WP1011, EnoGene, Nanjing, China). After SDS-PAGE and transfer, the bands were blocked with 5 % skim milk. Subsequently, they were incubated with corresponding primary and secondary antibodies. The antibody information is as follows: CHOP (R23316, Zen-bio, Chengdu, China), BiP (102056-T46/40, Sino Biological Inc., Beijing, China), Vimentin (BF8006, Affinity, USA), Bax (ET1603, HuaBio, Hangzhou, China), Bcl-2 (ET1702, HuaBio),β-Actin (bs-0061R, Bioss, Beijing, China), Goat anti-rabbit IgG (H + L) (AS014, ABclonal, Wuhan, China).

2.8. Animal experiments

SPF-grade lineC57BL/6 mice were kept at the Guizhou University of Traditional Chinese Medicine animal center, following the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The mice were kept individually in single cages within the animal care facility at a suitable temperature range of 21–25 °C and humidity levels of 50–70 %, with a 12-h light/dark cycle. According to the principle of random allocation based on body weight, 15 C57BL/6 mice (male, 8w old) were randomly divided into 3 groups. The IPF model was established by intratracheal injection of BLM (3.5 mg/kg) in mice (n = 10). On the second day after surgery, 5 mice were randomly selected from the model group and administered MSC-Ex (400 μg/mouse, once every 2 weeks for 4 weeks) via tail vein injection according to the results of prior studies [15]. The remaining mice were injected with an equal volume of PBS (Fig. 7A). The anesthesia and euthanasia procedures for the mice were conducted in accordance with the ethical standards and approved by the Institutional Animal Care and Use Committee (IACUC) at University of Traditional Chinese Medicine. Mice were euthanized with an overdose of sodium pentobarbital (150 mg/kg i.p.). Each experimental result in the animal study included data values from all mice in the group (n = 5).

Fig. 7.

Fig. 7

Fig. 7

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MSC-Ex decreased BLM-induced pulmonary fibrosis in mice. (A) Diagram of animal experiment procedure. (B) Body weight curves of mice in different groups (n = 5, Student's t-test). (C) The overall appearances of the anterior views of lung. (D) Representative HE stained images of mouse lung tissues. Scale bar, 250 μm. (E) and (F) The alveolar number and percentage of alveolar space were analyzed by ImageJ software (n = 10, one-way ANOVA with Bonferroni post hoc test). (G)–(I) Serum levels of IL-6, IL-1β and TNF-α in different groups (n = 5, Student's t-test). (J) Immunofluorescence double-staining of α-SMA (red) and Collagen I (green) in different groups. Scale bar, 100 μm. Data are shown as the means ± SEM (∗P < 0.05, ∗∗∗P < 0.001 vs. the Cont group; #P < 0.05 vs. the BLM group).

2.9. Hydroxyproline content

The lung hydroxyproline (HYP) content (μg/mg, wet weight) was measured using a HYP assay kit (A030-2-1, NNJCBIO, Nanjing, China).

2.10. ELISA

The serum levels of IL-1β (SEKM-0002, Solarbio, Beijing, China), IL-6 (EMC004.96, NeoBioscience Technology, Shenzhen, China), TNF-α (MM-0132M1, Meimian, Yancheng, China) and TGF-β1 (U96-1615E, YOBIBIO, Shanghai, China) were measured using ELISA kits.

2.11. Immunofluorescence

After embedding and sectioning of the lung tissue, α-SMA (ET1607-53, Huabio, Hangzhou, China), Collagen I (343277, Zen-bio, Chengdu, China), BiP and Vimentin (BF8006, Affinity, USA) expressions were observed under a fluorescence microscope following incubation with primary antibody, secondary antibody, and DAPI.

2.12. TUNEL staining

TUNEL staining was conducted with a TUNEL Apoptosis Assay kit (T6013S, UElandy, Suzhou, China) as previously reported [9].

2.13. Statistical analysis

The experiments were conducted independently on a minimum of three occasions. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Duncan's multiple-range test, Student's t-test, or Bonferroni post hoc test, as appropriate. For relative quantification experiments, we first normalize the control group in each individual experiment to a value of 1, and express the values of other groups as fold changes relative to the control. The final results are obtained by averaging the values from three or more independent experiments. The results are expressed as means ± SD, with statistical significance set at P < 0.05.

3. Results

3.1. BLM induced apoptosis in A549 cells

A549 cells were treated with different concentrations of BLM for 24h (Fig. 1A). The results revealed a concentration-dependent decrease in cell viability with increasing concentrations of BLM (Fig. 1B). The apoptosis assay showed the apoptotic cells were increased after BLM (100 μg/mL) addition (Fig. 1C). Furthermore, the Western blot analysis demonstrated a reduction in Bcl-2, an anti-apoptotic protein, and an elevation in Bax, a pro-apoptotic protein, following 24 h of BLM stimulation (Fig. 1D).

Fig. 1.

Fig. 1

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BLM induced apoptosis in A549 cells. (A) Diagram of cell experiment. (B) Effects of BLM (25, 50, 100, 200 μg/ml) on the viability of A549 cells after 24 h. Cell viability was measured by CCK8 assay (n = 4, one-way ANOVA with Dunnett's test). (C) A549 cells were treated with BLM (100 μg/ml) for 24 h and 48 h. Percentage of total apoptotic cells were assessed with Annexin V/PI staining (n = 3, one-way ANOVA with Duncan's post hoc test). (D) A549 cells were treated with BLM (50, 100 μg/ml) for 24 h. The protein expression of Bcl-2 and Bax were measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). Data are shown as the means ± SEM (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the Cont group).

3.2. Identification of MSC-Ex

We isolated MSC-Ex from the cell culture medium using an efficient assay kit (Fig. 2A). The result of NTA showed that the mean diameter of MSC-Ex was 181.1 nm (Fig. 2B). TEM showed the MSC-Ex exhibit a discoidal or vesicular morphology (Fig. 2C). Furthermore, Western blotting revealed exosomal markers TSG101 and CD63 were positive in MSC-Ex, while Vimentin is not detectable, confirming the absence of MSC contamination in the MSC-Ex preparation. (Fig. 2D).

Fig. 2.

Fig. 2

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Identification of MSC-Ex. (A) Exosome isolation diagram. (B) The size distribution of MSC-Ex was analyzed by NTA. (C) Representative image of MSC-Ex under TEM. (D) The expression of typical MSC-Ex markers (TSG101 and CD63) and Vimentin were detected by Western blotting.

3.3. MSC-Ex reduced BLM-induced apoptosis in A549 cells

Next, we designed in vitro experiments to observe the effects of MSC-Ex on BLM-treated A549 cells (Fig. 3A). We observed that MSC-Ex clearly restored the decrease in cell viability induced by BLM, and the addition of MSC-Ex alone (without BLM) did not affect the cell viability (Fig. 3B). The apoptotic cells were decreased by MSC-Ex treatment compared with BLM group (Fig. 3C). In addition, MSC-Ex treatment reversed the reduction of Bcl-2 and the increase of Bax observed in the BLM group (Fig. 3D). Meanwhile, MSC-Ex treatment without BLM did not influence the protein levels of Bcl-2 or Bax (Fig. 3E).

Fig. 3.

Fig. 3

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MSC-Ex reduced BLM-induced apoptosis in A549 cells. (A) A549 cells were treated with BLM (100 μg/ml) in the presence or absence of MSCs-Ex (100 μg/ml) for 24 h. (B) Cell viability was measured by CCK8 assay (n = 4, one-way ANOVA with Dunnett's test). (C) Percentage of total apoptotic cells was assessed with Annexin V/PI staining (n = 3, one-way ANOVA with Duncan's post hoc test). (D) and (E) The protein expression of Bcl-2 and Bax was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). Data are shown as the means ± SEM (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the Cont group; #P < 0.05, ##P < 0.01 vs. the BLM group).

3.4. MSC-Ex alleviated BLM-induced ER stress in A549 cells

After BLM treatment, the levels of two typical ER stress related proteins, BiP and CHOP, were increased after BLM stimulation for 24 h and 48 h (Fig. 4A). Furthermore, TEM showed that the ER in BLM group was robustly swollen and expanded, confirming that ER stress occurred (Fig. 4E). Interestingly, MSC-Ex reduced the protein expression of BiP and CHOP after 24 h (Fig. 4B), and the addition of MSC-Ex alone did not affect BiP and CHOP expressions (Fig. 4C). Similarly, q-PCR revealed that MSC-Ex could reduce the mRNA levels of BiP and CHOP (Fig. 4D). Moreover, MSC-Ex reversed the BLM-induced morphological changes of ER, and the ER became ordered and tubular after MSC-Ex addition (Fig. 4E).

Fig. 4.

Fig. 4

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MSC-Ex alleviated BLM-induced ER stress in A549 cells. (A) A549 cells were treated with BLM (100 μg/ml) for 24 h and 48 h. The protein expression of BiP and CHOP was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). (B) and (C) The protein expression of BiP and CHOP in different groups was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). (D) The mRNA expression levels of Bip and Chop was measured using q-PCR (N. = 3, one-way ANOVA with Duncan's post hoc test). (E) Cellular ultrastructure in different groups. The arrows indicate the endoplasmic reticulum. Scale bar, 500 nm. Data are shown as the means ± SEM (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the Cont group; #P < 0.05 vs. the BLM group).

3.5. ER stress contributes to BLM-induced apoptosis in A549 cells

To test whether ER stress contributes to apoptosis in BLM-treated A549 cells, we introduced thapsigargin (TG), an ER stress activator. The results of CCK8 showed that a concentration-dependent decrease in cell viability with increasing concentrations of TG after 24 h (Fig. 5A). Then, we found TG (100 μM) increased the ratio of apoptosis cells after 24h (Fig. 5B). Meanwhile, Bcl-2 level was decreased and Bax was increased after TG addition (Fig. 5C). These results indicated that ER stress contributes to BLM-induced apoptosis in A549 cells.

Fig. 5.

Fig. 5

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ER stress is involved in BLM-induced apoptosis in A549 cells. (A) Effects of TG (25, 50, 100, 200 μM) on the viability of A549 cells after 24 h. Cell viability was measured by CCK8 assay (n = 3, one-way ANOVA with Dunnett's test). (B) A549 cells were treated with TG (100 μM) for 24 h. Percentage of total apoptotic cells were assessed with Annexin V/PI staining (n = 3, one-way ANOVA with Duncan's post hoc test). (C) A549 cells were treated with TG (50 and 100 μM) for 24 h. The protein expression of Bcl-2 and Bax was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). Data are shown as the means ± SEM (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. the Cont group).

3.6. ER stress inhibition contributes to the anti-apoptotic effect of MSC-Ex

To further investigate whether the inhibitory effect of MSC-Ex on apoptosis is associated with the modulation of ER stress, we introduced tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. TUDCA (100 μM) alleviated ER stress, as evidenced by decreased BiP and CHOP expression compared with the BLM group (Fig. 6A). Interestingly, TUDCA restored cell viability (Fig. 6B), reduced the ratio of apoptotic cells and Bax protein level while increasing Bcl-2 protein expression (Fig. 6C and D), indicating that the amelioration of BLM-induced apoptosis in A549 cells is associated with the inhibition of ER stress. Next, we assessed the regulatory effect of MSC-Ex on ER stress using the ER stress inducer thapsigargin (TG). The results demonstrated that MSC-Ex markedly attenuated TG (100 μM)-induced ER stress in A549 cells (Fig. 6E), highlighting the potent capacity of MSC-Ex to modulate ER stress. Furthermore, MSC-Ex reduced the ratio of apoptotic cells compared to BLM group, whereas TG attenuated this effect (Fig. 6F). Taken together, these findings suggest that ER stress inhibition contributes to the anti-apoptotic effect of MSC-Ex.

Fig. 6.

Fig. 6

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ER stress inhibition contributes to the anti-apoptotic effect of MSC-Ex. A549 cells were treated with BLM (100 μg/ml) in the presence or absence of TUDCA (100 μM) for 24 h. (A) The protein expression of BiP and CHOP was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). (B) Cell viability was measured by CCK8 assay (n = 4, one-way ANOVA with Dunnett's test). (C) Percentage of total apoptotic cells was assessed with Annexin V/PI staining (n = 3, one-way ANOVA with Duncan's post hoc test). (D) The protein expression of Bcl-2 and Bax was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). (E) The protein expression of BiP and CHOP in different groups was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). (F) Percentage of total apoptotic cells was assessed with Annexin V/PI staining (n = 3, one-way ANOVA with Duncan's post hoc test). Data are shown as the means ± SEM (∗P < 0.05, ∗∗P < 0.01, vs. the Cont group; #P < 0.05, ##P < 0.01 vs. the BLM/TG group).

3.7. MSC-Ex mitigated pulmonary fibrosis in mice

We next conducted in vivo experiments to further confirm the results in vitro (Fig. 7A). During the experiment, no deaths occurred in the three groups until 28 days. BLM caused a decrease in body weight, and MSC-Ex infusion reversed this effect 4 weeks after surgery (Fig. 7B). However, the lung morphology did not differ between the three groups of mice (Fig. 7C). In mice receiving BLM, HE staining revealed decreased alveolar space and numbers, increased connective tissue, and thickened alveolar walls, and this finding was reversed by MSC-Ex infusion (Fig. 7D–F). Meanwhile, MSC-Ex decreased the serum levels of IL-6 (Fig. 7G), IL-1β (Fig. 7H), and TNF-α (Fig. 7I), indicating that MSC-Ex reduced inflammatory factors in BLM-induced mice. In addition, immunofluorescence staining showed that fibrosis maker α-SMA and Collagen I were upregulated in lung tissues of BLM-treated mice, and MSC-Ex treatment decreased the protein levels (Fig. 7J).

We then assessed pulmonary fibrosis. BLM significantly increased HYP levels, while MSC-Ex reversed it (Fig. 8A). Masson staining showed BLM increased collagen deposition in the alveolar and interstitial regions, whereas a notable decline was noted after MSC-Ex treatment (Fig. 8B and C). Moreover, serum TGF-β1 was significantly elevated in IPF mice models but MSC-Ex application reversed it (Fig. 8D). In addition, immunofluorescence staining showed that fibrosis maker vimentin was upregulated after BLM treatment, and MSC-Ex treatment decreased the protein level (Fig. 8E).

Fig. 8.

Fig. 8

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MSC-Ex decreased BLM-induced pulmonary fibrosis in mice. (A) HYP content in lung tissue was assessed (n = 5, one-way ANOVA with Bonferroni post hoc test). (B) Representative images of Masson's staining of lung tissues. Scale bar, 100 μm. (C) Fibrosis area was analyzed by ImageJ software (n = 10, one-way ANOVA with Bonferroni post hoc test). (D) Serum levels of TGF-β in different groups (n = 5, Student's t-test). (E) Immunofluorescence double-staining of ER stress-related protein (BiP, green) and fibrosis-related protein (vimentin, red) in different groups. Scale bar, 100 μm. Data are shown as the means ± SEM (∗P < 0.05, ∗∗P < 0.01 vs. the Cont group; #P < 0.05 vs. the BLM group).

3.8. MSC-Ex ameliorated ER stress and apoptosis in fibrotic mouse lungs

Initially, we tested whether MSC-Ex injection decreased ER stress in lung tissues. Q-PCR showed that in BLM-treated mice, both Bip and Chop were increased, which were decreased by MSC-Ex treatment (Fig. 9A). In addition, similar results were obtained by Western blot (Fig. 9B). Moreover, immunofluorescence staining showed that MSC-Ex decreased the BiP protein expression (Fig. 8E). Meanwhile, TUNEL staining revealed that apoptotic cells were increased in BLM-treated mice but MSC-Ex treatment reduced apoptotic cells (Fig. 9C). Furthermore, the protein levels of Bcl-2 was decreased and Bax was increased in lungs of mice in the BLM group, while MSC-Ex infusion reversed this phenomenon (Fig. 9D). In summary, the findings above suggest that MSC-Ex ameliorated ER stress and apoptosis in fibrotic mouse lungs.

Fig. 9.

Fig. 9

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MSC-Ex ameliorated ER stress and apoptosis in fibrotic mouse lungs. (A) The mRNA expression levels of Bip and Chop in lung tissues were measured using q-PCR (n = 3, one-way ANOVA with Duncan's post hoc test). (B) The protein expression of BiP and CHOP in lung tissues was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). (C) Representative images of TUNEL staining of lung tissues. Scale bar, 50 μm (200 × ), 20 μm (600 × ). (D) The protein expression of Bcl-2 and Bax in lung tissues was measured via Western blotting, and the results were quantified using densitometry with ImageJ software (n = 3, one-way ANOVA with Duncan's post hoc test). Data are shown as the means ± SEM (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.01 vs. the Cont group; #P < 0.05 vs. the BLM group).

4. Discussion

The current study investigated the impacts and molecular mechanisms of MSC-Ex on in vivo and in vitro fibrosis models. BLM increased apoptosis in A549 cells by activating ER stress, and MSC-Ex can alleviate apoptosis via blockade of ER stress. Meanwhile, MSC-Ex could ameliorate apoptosis in lungs and improve pulmonary fibrosis in BLM-treated mice. ER stress might be the target in MSC-Ex-mediated alleviation of apoptosis in pulmonary fibrosis.

Though the pathogenesis is unclear, the development of IPF is likely influenced by multiple factors. Continuous AECs injury and apoptosis, especially in type II alveolar epithelial cells (AECIIs), is a critical determinant of the development of IPF [16]. Apoptosis refers to “gene-directed cellular self-destruction”, however, the increased apoptosis in AECs leads to fibrotic tissue repair due to the lack of effective re-epithelialization for the damaged alveolar wall. Clinical data showed that apoptotic AECs were persistently present [17], and in the lung tissue of patients with IPF, there was an increase in the expression of the apoptotic protein Bax and a decrease in the antiapoptotic protein Bcl-2 [18]. Furthermore, inhibiting the apoptosis of AECs could help to alleviate fibrosis [19,20]. Therefore, blockade of apoptosis or its inducer and mediators could be a potential therapeutic strategy for IPF.

MSCs displayed therapeutic potential for various diseases due to their powerful paracrine properties [21]. We demonstrated that MSCs could ameliorate lung fibrosis in mice IPF models [9]. Shangya Chen et al. showed that MSCs increased the Bcl-2/Bax ratio, and then pulmonary fibrosis was attenuated in rat models [22]. Qinqin Shen et al. showed conditioned medium from MSCs inhibited BLM-caused apoptosis in A549 cells [23], indicating that MSCs perform their anti-fibrotic functions using a paracrine mechanism. In fact, in addition to paracrine cytokines and other protein factors, MSC-Ex are known as a conveyor to deliver bioactive factors and nucleic acids to the target cells [24]. MSC-Ex have similar biological characteristics and functions to MSCs. Some studies indicated that MSC-Ex can mitigate pulmonary fibrosis in vivo [25,26]. In a recent study, MSC-Ex could alleviate lung ischemia/reperfusion injury via transporting anti-apoptotic miRNA [27], but few studies have investigated the anti-apoptotic capacity of MSC-Ex in an IPF model. In the present study, we treated A549 cells and mice with BLM to construct IPF models, and we found BLM induced apoptosis under fibrotic settings. Furthermore, the apoptotic cells and Bax level were decreased, and the cell viability and Bcl-2 level were restored after MSC-Ex treatment. Notably, compared with MSC-based therapy, Ex-based therapy has no immunogenicity and could be applied in different species [28]. In the current study, we treated mice with human umbilical cord-derived Ex and promising effects were observed. Regrettably, the metabolism and distribution of exosomes in mice were not assessed in the study, and further investigation is warranted.

Apoptosis can be generated and associated with several biological events, including inflammation, oxidative stress, ER stress, DNA damage, mitochondrial injury and infection [29]. Blockade of these events might alleviate apoptosis. Shulamit B showed that ROS antagonist glutathione decreased ROS levels with subsequent apoptosis. Additionally, there is growing evidence indicating that ER stress contributes significantly to the pathogenesis of IPF [30], and ER stress could directly induce apoptosis in different cells [31,32]. To confirm this, we introduced an ER stress activator, TG. As expected, TG increased the apoptotic cells and Bax/Bcl-2 ratio, indicating that apoptosis occurred. Lola Buono et al. showed that MSC-Ex inhibiting apoptosis by modulating ER stress in cells [33]. Moreover, MSC-Ex could inhibit excessive nucleus pulposus cell apoptosis during intervertebral disc degeneration by regulating ER stress [34]. Therefore, we speculated that ER stress might be the target in MSC-Ex-mediated alleviation of apoptosis in an IPF model. As expected, we observed that MSC-Ex reduced ER stress-related protein (Bip and CHOP) expression and reversed the BLM-induced morphological changes of the ER in A549 cells and lung tissues of an IPF mouse model. We further investigated whether inhibiting ER stress alleviated apoptosis in this setting. Consistent with our hypothesis, ER stress inhibitor TUDCA mitigated BLM-induced apoptosis, indicating ER stress might be a potent target by which MSC-Ex exert their anti-apoptotic effect. To our knowledge, this is the first evidence suggesting that MSC-Ex could reduce BLM-induced apoptosis at least partly via modulating ER stress in an IPF model.

However, ER stress involves three signaling pathways: the PERK/ATF4, ATF6, and IRE1α/XBP1 pathways. We selected BiP, an early-stage ER stress protein, and CHOP, a late-stage protein, as markers of ER stress. When BiP expression increases without a corresponding change in CHOP, it indicates that ER stress is in an early and reversible phase. However, if both BiP and CHOP levels are elevated, it suggests that ER stress is sustained and the cell has initiated an apoptotic program [35]. Our study found that both BiP and CHOP were significantly upregulated following BLM treatment, indicating that ER stress may contribute to apoptosis. The use of the ER stress agonist TG further confirmed our hypothesis, showing that ER stress can induce apoptosis in A549 cells. Unfortunately, we did not assess the three canonical ER stress pathways in detail, which would help determine which pathway MSC-Ex regulates more effectively. However, our previous study showed that MSCs exert a more pronounced regulatory effect on the IRE1α branch in a pulmonary fibrosis model [9]. Therefore, we speculate that the IRE1α pathway may also be a key target of MSC-Ex, and this remains to be further verified in future studies.

Consistent with the results of in vitro studies, MSC-Ex could ameliorate ER stress and apoptosis in mice IPF model. We found MSC-Ex decreased Bip and CHOP gene and protein expressions. TUNEL staining revealed that MSC-Ex treatment clearly reduced the number of apoptotic cells. Furthermore, MSC-Ex increased the Bcl2/Bax ratio. Collectively, our study indicated that ER stress-mediated apoptosis is at least partially associated with the protective effects of MSC-Ex on lung fibrosis. However, The upstream underlying mechanism remains to be investigated. Unlike single agents or cytokines, MSC-Ex contains variable amounts of protein and nucleic acids, and identifying the specific protein or nucleic acids involved in the protective effects of MSC-Ex is critical. In addition, in vivo settings differ from in vitro settings regarding their complex environments. Although MSC-Ex exhibited a significant ability to counteract ER stress, its antifibrotic effect was not particularly ideal. This is mainly because the development of pulmonary fibrosis involves a series of complex pathological changes with multiple contributing factors. While the inhibition of the ER stress–apoptosis pathway by MSC-Ex played an important role in the antifibrotic process, other factors, such as inflammation, mitochondrial damage, and oxidative stress, continued to promote fibrosis progression. The potential modulatory effects of MSC-Ex on these factors were not addressed in the present study. Therefore, the underlying molecule mechanisms might be more complicated, and whether there are other pathways involved in MSC-Ex-mediated protection from IPF still needs to be explored.

In conclusion, the present study indicated ER stress mediated apoptosis in BLM-treated A549 cells, and ER stress may be a key target for the anti-fibrotic effect of MSC-Ex. This study provides a novel mechanism for MSC-Ex-mediated protection from apoptosis in an IPF model and suggests that human MSC-Ex presents itself as a promising therapeutic strategy for IPF.

Authors' contributions

The study was conceived by Ruixi Luo, and Weiyi Tian. Ruixi Luo performed the experiments and data collection with help from Yaqiong Wei, Peng Chen and La Wang. Ruixi Luo was aware of the experimental details and group allocation at different stages of the animal study. Peng Chen and Didong Lou supervised and managed the implementation of the experiment to ensure its rationalization, and also verified and guided the details of the experimental content. The paper was written by Ruixi Luo and edited by Didong Lou and Weiyi Tian.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

This study was approved by the Institutional Animal Care and Use Committee of Guizhou University of Traditional Chinese Medicine (Ethic approval number: 20210041).

Funding

This study was supported by General Program of the Guizhou Provincial Basic Research Program (Natural Science, QKHJCMS[2025]151) and the project of Key Laboratory of Microbio and Infectious Disease Prevention & Control of Guizhou Province (ZDSYS[2023]004).

Declaration of competing interest

The authors declare that they have no Conflict of Interest.

Acknowledgements

We thank Home for Researchers (www.home-for-researchers.com) for their help with language. We thank Figdraw (www.figdraw.com) for providing the platform for creating illustrations.

Footnotes

Peer review under responsibility of the Japanese Society for Regenerative Medicine.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.reth.2025.10.004.

Contributor Information

Ruixi Luo, Email: luoruixi058@gzy.edu.cn.

Weiyi Tian, Email: tianweiyi@gzy.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (12.2KB, docx)

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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