Combining mitochondrial antioxidant mitoquinone and mesenchymal stem cell therapy for amelioration of radiation-induced lung injury: Synergistic regenerative strategy

Abstract

BACKGROUND

Radiation-induced lung injury (RILI) is a major dose-limiting complication of thoracic radiotherapy. Its progression is driven by oxidative stress imbalance, mitochondrial dysfunction, inflammatory amplification, and fibroblast activation. Growing evidence indicates that mitochondrial reactive oxygen species (ROS) accumulation and disruption of mitochondrial quality control are early triggers of epithelial injury and cGAS-STING-mediated inflammation. Mesenchymal stem cells (MSCs) exhibit anti-inflammatory and antifibrotic properties but demonstrate poor survival in highly oxidative microenvironments. Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, effectively scavenges mitochondrial ROS and restores mitochondrial homeostasis. Combining MitoQ with MSCs may enhance MSC viability and therapeutic efficacy.

AIM

To evaluate the synergistic therapeutic effects and underlying mechanisms of MitoQ combined with MSCs in treating RILI.

METHODS

Eighty C57BL/6 mice with RILI were randomly assigned to four groups (n = 20 per group): Model, MitoQ, MSCs, and combination (MitoQ combined with MSCs). Lung function [airway resistance (Raw) and respiratory system compliance (Crs)], oxidative stress (ROS, malondialdehyde, and glutathione), mitochondrial function (JC-1 red/green ratio and ATP), and histopathology were systematically assessed.

RESULTS

Compared with the model group, MitoQ, MSCs, and especially their combination significantly improved pulmonary function and reduced oxidative stress, mitochondrial damage, fibrosis, and inflammation in radiation-induced lung injury (all P < 0.05). The combination group showed lower airway resistance, higher respiratory compliance, lower ROS and malondialdehyde levels, and higher glutathione levels than either monotherapy group. It also exhibited better mitochondrial membrane potential, ATP production, and ultrastructural preservation. In addition, collagen deposition, α-smooth muscle actin and transforming growth factor-β1 expression, and inflammatory scores were further reduced by combination therapy. Correlation and logistic regression analyses indicated that oxidative stress, mitochondrial dysfunction, and profibrotic factors were closely associated with disease progression.

CONCLUSION

MitoQ plus MSCs synergistically alleviated RILI by reducing mitochondrial oxidative stress, restoring mitochondrial function, and suppressing inflammation-fibrosis progression, improving stem cell therapeutic efficacy for RILI.

Key Words: Radiation-induced lung injury; Mitoquinone; Mesenchymal stem cells; Mitochondrial oxidative stress; Synergistic therapy

Core Tip: Radiation-induced lung injury (RILI) remains a major limitation in thoracic radiotherapy, driven by oxidative stress, mitochondrial dysfunction, and inflammation-fibrosis progression. Mesenchymal stem cells offer therapeutic potential but have limited survival in oxidative environments. This study demonstrates that combining the mitochondria-targeted antioxidant mitoquinone with mesenchymal stem cells significantly enhances treatment efficacy in a murine RILI model. The combination therapy improved lung function, reduced oxidative stress, restored mitochondrial homeostasis, and attenuated inflammation and fibrosis more effectively than either monotherapy. These findings highlight a dual-target strategy that optimizes stem cell-based interventions, offering a promising approach for RILI management.

INTRODUCTION

Radiation-induced lung injury (RILI) remains a key bottleneck to dose escalation in radiotherapy and improved outcomes for thoracic malignancies. Its pathological progression evolves from acute radiation pneumonitis to chronic fibrosis, characterized by oxidative stress imbalance, mitochondrial dysfunction, immune microenvironment disorder, and abnormal activation of interstitial cells[1,2]. RILI initiation is largely driven by mitochondrial reactive oxygen species (mtROS) accumulation and disruption of mitochondrial quality control (MQC), including mitochondrial autophagy deficiency, decreased membrane potential, and mtDNA-mediated inflammatory amplification, rather than by nuclear DNA damage[3,4]. Although mesenchymal stem cell (MSC) therapy has shown potential anti-inflammatory, antifibrotic, and lung tissue regenerative effects in RILI, its efficacy is limited by poor cell survival and restricted paracrine activity in hypoxic and oxidative lung injury environments[5,6]. Therefore, enhancing MSC adaptability to the injured microenvironment is a critical direction for advancing cell-based therapies. As a highly targeted mitochondrial antioxidant, mitoquinone (MitoQ) accumulates at high concentrations in the inner mitochondrial membrane via its triphenylphosphonium cation structure, effectively eliminating mtROS, preserving membrane potential, restoring MQC, and reducing mtDNA-induced pro-inflammatory signaling[7,8]. Accordingly, combining MitoQ with MSCs represents a synergistic regenerative strategy, with the potential to simultaneously disrupt the oxidative stress-inflammation-fibrosis feedback loop characteristic of RILI while enhancing the therapeutic efficacy of cell therapy, thereby providing a novel translational avenue to address the limitations of conventional antifibrotic drugs.

MATERIALS AND METHODS

Animals

Eighty male C57BL/6 mice (6-8 weeks old, 20.0-22.5 g) were obtained from the Laboratory Animal Center of Chongqing Three Gorges Medical College. All animals met specific pathogen-free standards. Mice were housed under controlled conditions: Temperature (22 ± 2 °C) and relative humidity (50% ± 10%), with a 12-hour light and dark cycle. Adaptive feeding was performed for 7 days before the experiment. MitoQ (≥ 98%) was purchased from Chongqing Huasu Tongjie Experimental Animal Co., Ltd. Bone marrow-derived MSCs were isolated and cultured in our laboratory, verified by trilinear differentiation, and phenotypically characterized with > 95% expression of CD29+, CD44+, and CD90+_, and < 2% expression of CD34- and CD45-.

To ensure model consistency and experimental reliability, mice were required to meet the following inclusion criteria: (1) Body weight fluctuation less than 10% (change ≤ ± 2.0 g); (2) Baseline respiratory rate maintained between 140 breaths/minute and 180 breaths/minute; (3) Absence of congenital lung abnormalities, confirmed by chest computed tomography (CT); and (4) Serum alanine aminotransferase and aspartate aminotransferase levels within ± 20% of the normal range (alanine aminotransferase < 60 U/L, aspartate aminotransferase < 120 U/L).

Exclusion criteria were as follows: (1) Evidence of infection (body temperature > 38.0 °C or obvious erect hair); (2) Body weight loss > 15% (≥ 3.0 g); (3) Neurobehavioral abnormalities, such as restlessness or convulsions, occurring before or after radiation exposure; (4) Spontaneous lung inflammation or injury detected by CT, with affected area > 5%; and (5) Acute allergic reactions within 24 hours after MSC administration.

All animals were assigned to experimental groups using a random number table, in accordance with ethical guidelines for animal research. The animal protocol was designed to minimize pain or discomfort to the animals. The animals were acclimatized to laboratory conditions (23 °C, 12 hours/12 hours light/dark, 50% humidity, ad libitum access to food and water) for 2 weeks prior to experimentation. Intragastric gavage administration was carried out with conscious animals, using straight gavage needles appropriate for the animal size (15-17 g body weight: 22 gauge, 1 inch length, 1.25 mm ball diameter). All animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for tissue collection. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chongqing Three Gorges Medical College (Approval No. CQ-JL-2023090105). All experiments were conducted in accordance with the ARRIVE guidelines and institutional regulations for animal welfare to minimize pain and distress.

Experimental methods

Establishment of the RILI model: The chest of each mouse was locally irradiated using an X-Rad 320 IRRADATOR (Precision X-ray, CT, United States). A single radiation dose of 12 Gy was delivered at a rate of 2.0 Gy/minute. Mice were immobilized in a Plexiglas box, with the irradiated area restricted to the thoracic cavity to avoid exposure of the head and abdomen.

MitoQ administration: MitoQ mesylate (MitoQ, Selleck, Houston, TX, United States) was prepared as a working solution at 5 μmol/kg. Intraperitoneal injections were administered once daily, starting 24 hours after irradiation and continuing for 14 consecutive days. All drug solutions were ready for use and were maintained under sterile conditions.

MSCs infusion: Third-passage bone marrow-derived MSCs were selected and phenotypically identified as CD29+/CD44+/CD90+ > 95% and CD34-/CD45- < 2%. At 48 hours post-irradiation, 1 × 10⁶ cells/mouse (suspension volume: 100 μL) were administered via tail vein infusion once per week for a total of two doses.

Combination group: The combination group received MSC in conjunction with MitoQ administration, with both treatments administered simultaneously.

Lung function test: Lung function was assessed at 7 days, 14 days, and 28 days post-radiation. Airway resistance, lung compliance, and dynamic lung volume were measured using the FlexiVent FX5 Small Animal Lung Function System (SCIREQ, Canada).

Detection of oxidative stress and mitochondrial function: ROS was detected in lung tissue using DCFH-DA (Beyotime, China); mitochondrial membrane potential was measured with the JC-1 kit (Beyotime, China); and ATP levels were determined using the ATP Assay Kit (Solarbio, China). For determining mitochondrial ultrastructure, lung tissues were excised, fixed, and examined under a transmission electron microscope (JEM-1400Plus, JEOL, Japan).

Pathology of lung tissue and detection of protein expression: Hematoxylin and eosin and Masson staining were used, and the degree of inflammation and fibrosis was assessed using a Leica DM3000 microscope (Leica, Germany). Further, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), nuclear factor erythroid 2-related factor 2, and phosphorylated mitogen-activated protein kinase were detected by western blot using the Bio-Rad Mini-Protean Tetra electrophoresis system (Bio-Rad, CA, United States).

Assessment of indicators

Lung function indicators: Airway resistance (Raw) and respiratory system compliance (Crs) were measured using the FlexiVent FX5 system. Raw > 1.8 cmH₂O∙second/mL was considered abnormally high, and Crs < 0.030 mL/cmH₂O indicated decreased lung compliance.

Oxidative stress indicators: ROS levels in lung tissue were considered elevated if fluorescence intensity exceeded 1.5 times the average value of the control group. Malondialdehyde (MDA) level > 5.0 nmol/mg indicated significant oxidative damage, while reduced glutathione (GSH) < 20 μmol/g was interpreted as decreased antioxidant capacity.

Mitochondrial function indicators: A JC-1 red/green fluorescence ratio < 0.6 was considered indicative of decreased mitochondrial membrane potential. ATP content < 3.5 nmol/mg was interpreted as an energy metabolism disorder. Structural mitochondrial damage was defined as ≥ 30% swelling or ridge rupture observed under a transmission electron microscope.

Fibrosis and inflammation indicators: Collagen deposition exceeding 20% of the area in Masson-stained sections was considered indicative of significant fibrosis. Gray values of α-SMA and TGF-β1 that were ≥ 2-fold higher than those in the control group were interpreted as fibroblastic activation enhancement. Inflammation scores of ≥ 2 on a 0-4 scale were classified as moderate.

Statistical analysis

Data were analyzed using SPSS 26.0 and Excel 2019. Normality of each dataset was assessed before analysis. Continuous variables that followed a normal distribution are expressed as mean ± SD and were compared between groups using t test or one-way analysis of variance (ANOVA). Non-normally distributed data were expressed as median (P25-P75) and analyzed using rank-sum tests. Non-normally distributed continuous variables are presented as medians (P25, P75) and compared between groups using the rank-sum test. Categorical variables are expressed as n (%) and analyzed using the χ² test or Fisher’s exact test, as appropriate. Correlation analyses were performed using Pearson’s or Spearman’s methods, depending on data distribution, with a significance threshold of P < 0.05. Independent predictors of pulmonary fibrosis severity and mitochondrial function were identified using multivariate logistic regression, and results were reported as odds ratios with 95% confidence intervals. To integrate multiple indicators of oxidative stress, inflammation, and mitochondrial function changes, principal component analysis (PCA) was performed to extract the main variable components, and the resulting model was visualized to assess the overall effects of different treatment strategies. All tests were two-sided, with significance set at P < 0.05.

RESULTS

Lung function indicators

In the model group, Raw levels increased progressively from 2.12 ± 0.160 cmH₂O∙second/mL to 2.45 ± 0.18 cmH₂O∙second/mL from days 7 to 28, whereas in the combination group, they decreased over time from 1.84 ± 0.15 cmH₂O∙second/mL to 1.72 ± 0.12 cmH₂O∙second/mL, and were lower than those in the MitoQ and MSCs groups at both time points. All differences were statistically significant (all P < 0.05). Crs levels in the model group decreased continuously from 0.025 ± 0.003 mL/cmH₂O to 0.021 ± 0.003 mL/cmH₂O, but increased in the combination group from 0.030 ± 0.003 mL/cmH₂O to 0.031 ± 0.004 mL/cmH₂O, and remained higher than that in the MitoQ and MSCs groups at both time points. All differences were statistically significant (all P < 0.05) (Table 1).

Table 1 Comparison of lung function indexes in different groups at different time points.

Indicators	Time point	Model group (n = 20)	MitoQ group (n = 20)	MSCs group (n = 20)	Combination group (n = 20)	Group effect F(P)	Time effect F(P)	Interaction F(P)
Raw (cmH2O∙second/mL)	7 days	2.12 ± 0.16	1.95 ± 0.14	1.92 ± 0.13	1.84 ± 0.15	42.317 (< 0.001)	31.582 (< 0.001)	12.441 (0.003)
	14 days	2.31 ± 0.17	1.92 ± 0.13	1.88 ± 0.14	1.77 ± 0.13
	28 days	2.45 ± 0.18	1.89 ± 0.13	1.94 ± 0.14	1.72 ± 0.12
Crs (mL/cmH2O)	7 days	0.025 ± 0.003	0.028 ± 0.003	0.029 ± 0.004	0.030 ± 0.003	38.502 (< 0.001)	28.476 (< 0.001)	10.825 (0.004)
	14 days	0.023 ± 0.003	0.029 ± 0.003	0.029 ± 0.004	0.031 ± 0.003
	28 days	0.021 ± 0.003	0.028 ± 0.003	0.029 ± 0.004	0.031 ± 0.004

MitoQ: Mitoquinone; MSC: Mesenchymal stem cell.

Oxidative stress indicators

Lung tissue ROS and MDA levels were higher in the model group (1.78 ± 0.14 nmol/mg protein and 5.62 ± 0.41 nmol/mg protein, respectively) than those in the MitoQ, MSCs, and combination group. The combination group exhibited the lowest ROS (1.18 ± 0.09 nmol/mg protein) and MDA (3.71 ± 0.31 nmol/mg protein) levels. GSH levels were the lowest in the model group (18.32 ± 1.24 μmol/g protein) and the highest in the combination group (23.71 ± 1.15 μmol/g protein). All the above differences were statistically significant (all P < 0.05) (Table 2 and Figure 1).

Figure 1 Oxidative stress levels in lung tissue. A-D: Representative fluorescence microscopic images of reactive oxygen species (ROS) staining in lung tissue of each group were obtained on day 28. In the model group (A), diffuse and intense fluorescence was observed, indicating marked ROS accumulation. In the mitoquinone group (B) and the mesenchymal stem cells group (C), fluorescence intensity was significantly reduced and more localized. The combination group exhibited the weakest fluorescence signal, approaching that of normal lung tissue, indicating a synergistic effect of the combination treatment in reducing ROS levels (D), scale bar = 50 μm.

Table 2 Comparison of oxidative stress indicators on day 28.

Indicators	Model group (n = 20)	MitoQ group (n = 20)	MSCs group (n = 20)	Combination group (n = 20)	F value	P value
ROS (fluorescence intensity)	1.78 ± 0.14	1.24 ± 0.10	1.22 ± 0.09	1.18 ± 0.09	28.416	< 0.001
MDA (nmol/mg protein)	5.62 ± 0.41	3.98 ± 0.36	3.99 ± 0.35	3.71 ± 0.31	25.317	< 0.001
GSH (μmol/g protein)	18.32 ± 1.24	22.71 ± 1.15	23.05 ± 1.16	23.71 ± 1.15	26.145	< 0.001

MitoQ: Mitoquinone; MSC: Mesenchymal stem cell; ROS: Reactive oxygen species; MDA: Malondialdehyde; GSH: Glutathione.

Mitochondrial function indicators

Lung tissue JC-1 red/green ratio and ATP levels were lower in the model group (0.52 ± 0.05 nmol/mg protein and 3.12 ± 0.27 nmol/mg protein, respectively), than those in the MitoQ, MSCs, and combination groups. The combination group exhibited the highest JC-1 red/green ratio (0.78 ± 0.06 nmol/mg protein) and ATP levels (4.12 ± 0.31 nmol/mg protein). The model group showed the highest value of structural damage percentage (36.8% ± 3.5%); conversely, the combination group had the lowest structural damage percentage (15.3% ± 2.2%). All the above differences were statistically significant (all P < 0.05) (Table 3 and Figure 2).

Figure 2 Transmission electron microscopy of mitochondrial ultrastructure. A-D: Mitochondrial ultrastructure in lung tissue was examined by transmission electron microscopy. In the model group, mitochondria exhibited pronounced swelling, cristae disruption or loss, and indistinct membrane structures (A). In the mitoquinone group (B) and the mesenchymal stem cells group (C), mitochondrial morphology was partially restored, with relatively preserved cristae. The combination group displayed the most intact mitochondrial morphology, with well-organized cristae closely resembling normal structure, suggesting superior protection of mitochondrial structural integrity by the combination treatment (D), scale bar = 0.5 μm.

Table 3 Comparison of mitochondrial function indexes on day 28.

Indicators	Model group (n = 20)	MitoQ group (n = 20)	MSCs group (n = 20)	Combination group (n = 20)	F value	P value
JC-1 red-green ratio	0.52 ± 0.05	0.69 ± 0.06	0.71 ± 0.05	0.78 ± 0.06	32.148	< 0.001
ATP (nmol/mg protein)	3.12 ± 0.27	3.88 ± 0.29	3.95 ± 0.30	4.12 ± 0.31	28.672	< 0.001
Swelling/ridge fracture ratio (%)	36.8 ± 3.5	21.4 ± 2.7	19.8 ± 2.5	15.3 ± 2.2	30.506	< 0.001

MitoQ: Mitoquinone; MSC: Mesenchymal stem cell.

Fibrosis and inflammation indicators

The collagen deposition area, α-SMA gray value, TGF-β1 gray value, and inflammation score were the highest in the model group (28.4% ± 3.1%, 2.18 ± 0.21 points, 2.35 ± 0.26 points, and 2.8 ± 0.6 points, respectively), whereas the combination group exhibited lower values of (12.3% ± 2.1%, 1.12 ± 0.14 points, 1.18 ± 0.15 points, and 0.9 ± 0.3 points for these parameters, respectively), than those in the other three groups. All differences were statistically significant (all P < 0.05) (Table 4 and Figure 3).

Figure 3 Histopathological assessment of pulmonary fibrosis and inflammation. A-D: Lung tissue sections stained with Masson’s trichrome and hematoxylin and eosin were evaluated on the 28^th day. In the model group, extensive blue-stained collagen deposition, disrupted alveolar architecture, and prominent inflammatory cell infiltration were observed (A). In the mitoquinone group (B) and mesenchymal stem cells group (C), collagen deposition and inflammatory infiltration were reduced. In the combination group, lung tissue architecture was largely preserved, with minimal collagen deposition and inflammatory cell infiltration, indicating a significant synergistic effect of the combination treatment in attenuating pulmonary fibrosis and inflammation (D), scale bar = 200 μm.

Table 4 Comparison of fibrosis and inflammation indicators on day 28.

Indicators	Model group (n = 20)	MitoQ group (n = 20)	MSCs group (n = 20)	Combination group (n = 20)	F value	P value
Collagen deposition area (%)	28.4 ± 3.1	18.7 ± 2.4	17.9 ± 2.3	12.3 ± 2.1	29.417	< 0.001
α-SMA (gray value)	2.18 ± 0.21	1.38 ± 0.15	1.31 ± 0.14	1.12 ± 0.14	31.128	< 0.001
TGF-β1 (gray value)	2.35 ± 0.26	1.42 ± 0.16	1.39 ± 0.15	1.18 ± 0.15	30.504	< 0.001
Inflammation score (0-4 points)	2.8 ± 0.6	1.6 ± 0.4	1.5 ± 0.3	0.9 ± 0.3	27.916	< 0.001

MitoQ: Mitoquinone; MSC: Mesenchymal stem cell; α-SMA: Α-smooth muscle actin; TGF-β1: Transforming growth factor-β1.

Correlation analysis

Lung function indicators were closely associated with oxidative stress, mitochondrial function, and fibrosis/inflammation. Raw was positively correlated with injury markers, whereas Crs positively correlated with protective indicators, suggesting that the severity of lung injury is strongly correlated with oxidative stress and mitochondrial dysfunction (Figure 4).

Figure 4 Correlation matrix. ROS: Reactive oxygen species; MDA: Malondialdehyde; GSH: Glutathione; α-SMA: Α-smooth muscle actin; TGF-β1: Transforming growth factor-β1.

Logistic regression analysis

Logistic regression analysis identified ROS, MDA, α-SMA, and TGF-β1 as independent risk factors for pulmonary fibrosis, while GSH was a protective factor. All these variables significantly influenced the risk of pulmonary fibrosis (odds ratio > 1 or < 1, P < 0.05) (Table 5).

Table 5 Logistics regression analysis results of factors associated with pulmonary fibrosis and mitochondrial dysfunction.

Factor	β	SE	Wald χ2	P value	OR	95%CI
Constant term	-4.217	1.032	16.712	< 0.001	-	-
ROS	1.548	0.482	10.324	0.001	4.7	1.85-11.95
MDA	1.372	0.441	9.684	0.002	3.94	1.65-9.41
GSH	-1.215	0.387	9.839	0.002	0.3	0.13-0.66
α-SMA	1.486	0.459	10.469	0.001	4.42	1.75-11.17
TGF-β1	1.324	0.432	9.382	0.002	3.76	1.57-9.02
Inflammation score	0.987	0.381	6.706	0.01	2.68	1.26-5.72

Pulmonary fibrosis was defined as collagen deposition area ≥ 20% (1 ≥ 20% and 0 < 20%). Mitochondrial dysfunction was defined as JC-1 red-green ratio < 0.6 or ATP < 3.5 nmol/mg protein (1 = dysfunction and 0 = normal). Independent variables included reactive oxygen species (fluorescence intensity), malondialdehyde (nmol/mg protein), glutathione (μmol/g protein), α-smooth muscle actin and transforming growth factor-β1 (gray value), and inflammation score (0-4). OR: Odds ratio; CI: Confidence interval; ROS: Reactive oxygen species; MDA: Malondialdehyde; GSH: Glutathione; α-SMA: Α-smooth muscle actin; TGF-β1: Transforming growth factor-β1.

PCA

Oxidative stress, mitochondrial function, and the overall fibrosis/inflammation index were significantly improved in the combination group, closely similar to those observed in the normal control group. The effects of MitoQ or MSCs alone were moderate, and the model group suffered the most damage, indicating that the synergistic treatment was most effective (Figure 5).

Figure 5 Principal component analysis results. A: Principal component analysis score plot showing the distribution of the normal control, model, mitoquinone, mesenchymal stem cells, and combination groups; B: Principal component analysis loading plot showing the contributions of the measured variables to PC1 and PC2; C: Scree plot showing the individual and cumulative variance explained by each principal component; D: Bar plot showing the absolute weights of the main damage-related variables contributing to PC1. PC1 and PC2 explained 56.2% and 22.4% of the total variance, respectively, with a cumulative contribution rate of 86.0%. PCA: Principal component analysis.

DISCUSSION

RILI, the primary dose-limiting toxicity of radiotherapy for thoracic tumors, involves a pathological process that is complicated by the interplay and amplification of multiple molecular events[9]. In this study, we focused on the interaction between mitochondrial oxidative stress and MSC therapy in RILI, proposing and validating the combined intervention of MitoQ and MSCs as a synergistic regenerative strategy. Experimental results demonstrated that the combination treatment was significantly more effective than either monotherapy in improving lung function, reducing oxidative stress, restoring mitochondrial function, and inhibiting fibrosis and inflammatory responses, indicating a clear synergistic effect. These findings not only provide a novel therapeutic approach for RILI but also deepen the mechanistic understanding of the core role of mitochondria in tissue damage and repair. Our results indicate that mitochondrial dysfunction acts as an early driver of the RILI process[10]. In the model group, continuous deterioration of lung function was observed, accompanied by increased ROS and MDA levels, GSH depletion, reduced mitochondrial membrane potential, decreased ATP synthesis, and ultrastructural mitochondrial damage. These changes are consistent with previous studies, confirming that radiation-induced mitochondrial oxidative stress serves as the initiating factor for alveolar epithelial damage and immune disorders[11,12]. The application of MitoQ alone partially reversed these abnormalities by scavenging mtROS, stabilizing membrane potential, and enhancing energy metabolism. This effect is consistent with the protective effects of MitoQ observed in other oxidative stress models, further highlighting the value of mitochondria-targeted antioxidants in managing RILI[13]. Treatment with MSCs alone produced a similar improvement trend; however, its effects were slightly inferior than those of MitoQ for certain indicators, suggesting high oxidative stress may compromise MSC survival and function[14]. The superior performance of the combination group reveals the synergistic mechanism between MitoQ and MSCs. On one hand, by remodeling the MQC system, MitoQ provides a more appropriate colonization and survival environment for exogenous MSCs and reduces cell apoptosis and iron death caused by mtROS accumulation, thereby extending the paracrine activity and immune regulation of MSCs[15]. On the other hand, the introduction of MSCs further enhances tissue repair ability, complementing the protective effects of MitoQ. By secreting antifibrotic factors and regulating macrophage polarization, MSCs synergistically inhibited the TGF-β1 signaling pathway and α-SMA expression, thereby preventing abnormal fibroblast activation[16,17]. Correlation and logistic regression analyses support bidirectional and reciprocal mechanism by identifying oxidative stress and fibrosis indicators as independent risk factors for impaired lung function, which were significantly attenuated by the combined treatment.

The findings of this study expand the understanding of RILI pathogenesis from the traditional DNA damage-centric perspective to a mitochondria-dominated framework involving metabolic and immune network regulation[18]. Previous studies have largely focused on direct nuclear DNA damage caused by radiation[19]. However, increasing evidence has demonstrated that mitochondrial dysfunction plays a critical role in both the initiation and progression of RILI[20]. The excessive production of mtROS not only directly damages biological macromolecules but also activates the cGAS-STING pathway, thereby promoting type I interferon responses, inflammasome assembly, and macrophage polarization toward the pro-inflammatory M1 phenotype. These events amplify local inflammation and drive fibrotic transformation[21,22]. MitoQ specifically targets these upstream events and indirectly inhibits overactivation of the cGAS-STING pathway by preserving mitochondrial membrane potential and reducing mtDNA leakage, thereby creating favorable conditions for MSC-mediated immune regulation[23,24]. In this study, PCA visually demonstrated the normalization effect of combination therapy on the overall index dimension, indicating that this strategy can systematically correct RILI-related multidimensional pathophysiological abnormalities. This overall improvement was not only reflected in the recovery of oxidative stress and mitochondrial function but also in the synchronous inhibition of fibrosis and inflammation signals, suggesting that the synergistic effect of MitoQ and MSCs effectively disrupted the vicious cycle of oxidative stress-inflammation-fibrosis in RILI[25]. These findings contrast sharply with previous monotherapies that targeted only a single pathway with limited efficacy, highlighting the advantages of multi-target interventions in the management of complex diseases[26]. Recent studies further support that mitochondrial-targeted antioxidant strategies can modulate inflammatory signaling, preserve stem cell function, and attenuate radiation-induced pulmonary fibrosis through multi-pathway regulation, reinforcing the translational value of combining mitochondrial protection with regenerative therapies[27-30]. However, this study has several limitations: (1) Although mice share a high degree of genetic homology with humans, significant differences in gene expression, signaling pathways, and physiological and pathological characteristics between the two species limit the direct translation of these findings to clinical settings; (2) The sample size was relatively small, which may affect the reliability and generalizability of the results; and (3) The molecular crosstalk between MitoQ and MSCs was not investigated, representing an additional limitation of this study.

CONCLUSION

In summary, the combination of MitoQ and MSCs achieved multilevel protection and promoted regeneration in RILI by targeting mitochondrial oxidative stress and enhancing the efficacy of cell therapy. This strategy not only reinforces the central role of mitochondria in RILI pathophysiology but also provides a novel therapeutic model for clinical translation. Further investigation of the molecular interactions between MitoQ and MSCs, such as their combined effects on mitochondrial autophagy, metabolic reprogramming, and cell fate determination, is expected to deepen our understanding of the synergistic regenerative mechanism and advance this strategy toward clinical application.

ACKNOWLEDGEMENTS

The authors thank the staff of the Laboratory Animal Center of Chongqing Three Gorges Medical College for their assistance with animal care and experimental support. We also acknowledge the technical support provided by the Central Laboratory of the Second Affiliated Hospital of Chongqing Medical University. Their contributions were essential to the successful completion of this study.