Introduction

ALI is a severe disease characterised by an excessive inflammatory response.ARDS is an acute inflammatory lung disease with more severe clinical manifestations than ALI, usually accompanied by increased alveolar permeability, causing severe alveolar oedema and further exacerbation of hypoxia, with high annual morbidity and mortality rates worldwide [1]. A worldwide observational investigation carried out from 2016 to 2017 across 145 Pediatric Intensive Care Units (PICUs) in 27 nations delineated a morbidity rate of 17% and a mortality rate of 3.2% among 23,280 patients [2]. Notwithstanding strides in therapeutic approaches, the absence of efficacious pharmaceutical interventions capable of substantially mitigating mortality and enhancing patient prognoses persists as a critical challenge [3, 4]. Currently, the diagnosis and treatment of a number of diseases, including stem cells and their related derivatives, have attracted the attention of researchers [5]. For example, a study has shown that serum EV-derived ASS1 levels predicted the progression of HEV-ALF and were strongly correlated with the severity of patients with HEV infection. In addition, serum levels of exosome-derived CPS1 have been reported as a diagnostic and prognostic biomarker for patients with HEV-ALF [23]. This novel classification approach based on the different cellular origins of EVs enables a more accurate description of their distinct characteristics and functions.

We conducted a meta-analysis based on data collected from preclinical animal models of ALI to systematically assess the efficacy of EVs as a potential treatment.

Materials and methods

We use the PRISMA statement to conduct this meta-analysis [24]. The study protocol was registered on the International Prospective Register of Systematic Reviews (PROSPERO): (CRD42022368159).

Study selection

Two investigators (ZXF and CZY) searched and screened the applicable literature independently and then assessed the titles and abstracts of every retrieved article to decide which required further assessment. Disagreements between the investigators were resolved with a discussion or adjudicated by another reviewer (ZMH).

The inclusion criteria were as follows: (1) any animal models with ALI; (2) any studies intervened with various cell-derived EVs; (3) negative control (treatment without EVs). The primary outcome was the lung injury score. Secondary outcomes included inflammatory factors IL-1β, IL-6 and TNF-α, anti-inflammatory factor IL-10, W/D ratio of the lung, total protein in BALF, white blood cell counts in BALF, and neutrophil counts in BALF.

The exclusion criteria were as follows: (1) the animal models without ALI; (2) the data were repeated; (3) incomplete information; (4) review, letter, commentary, correspondence, case report, conference abstract, expert opinion, or editorial.

Data extraction

The data were extracted with the aid of two unbiased reviewers (ZXF and CZY) in a standardized way. For discrepancies, a third reviewer (ZMH) extracted them again.

The following data were collected: first author, country or region, type of ALI model, species, treatment time, measurement time and EV cell origins, diameter, and dose. We extracted data from graphics based on Engauge Digitizer version 4.1 software [25, 26].

To extract data from the study, we saved all relevant screenshots from the results as images and uploaded these images to the program. The first step was to determine what type of graph we were analyzing. Secondly, three known values were assigned to three points on the axis to calibrate it. Then, we directly clicked on each point on the graph to get its exact coordinates and used those coordinates to calculate its mean and standard deviation.

Quality assessment

Two independent authors (ZXF and ZMH) evaluated each study's methodological quality with a Collaborative Approach to Meta-Analysis and a Review of Animal Data from Experimental Studies (CAMARADES) 10-item checklist [27]. (Table 2).

Statistical analysis

All statistical analyses were conducted using RevMan version 5.3 and State 15.1 statistical software. It was considered statistically significant when the P < 0.05 (two-tailed). Continuous outcomes were expressed as standardized mean differences (SMD) with a 95% confidence interval (95%CI). Using the I2 statistic, heterogeneity was assessed among studies. An I2 > 50% indicates significant heterogeneity [28]. In order to inspect conceivable between-study heterogeneity and to discover different viable confounding factors, subgroup, sensitivity, and meta-regression analyses were carried out. The publication bias was detected through funnel plots and Egger's test. If publication bias was indicated, we recalculated pooled risk estimates by including those missing studies from the Trimfill method.

Results

Search results and study characteristics

Figure 1 is the diagram of the literature search process; 31 studies conform to the inclusion criteria [14, 29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57]. The main characteristics of these studies are presented in Table 1. All of these studies were published between 2010 and 2022. Among these studies, the number of bone marrow mesenchymal stromal cell (BMSC)-EVs is 16 [13, 14, 33, 36,37,38, 40,41,42,43, 45, 46, 49, 50, 53, 54]. 5 used adipose-derived mesenchymal stromal cell (ADMSC)-EVs [30, 34, 35, 44, 52]. 3 used human umbilical Wharton’s jelly mesenchymal stromal cell (WJ-MSC)-EVs [31, 32, 57]. 3 used alveolar macrophages (AMs)-EVs [47, 55, 56]. 1 used human umbilical cord mesenchymal stromal cell (UCMSC)-EVs [49]. 1 used human umbilical vein endothelial (UVLC)-EVs [29]. 1 used bone endothelial progenitor cells (EPC)-EVs [51]. 1 used Alveolar Epithelial Cell [39].

Fig. 1
figure 1

The diagram of the literature search process

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Table 1 Abbreviations: ADMSC adipose derived mesenchymal stromal cells, BMSC bone marrow mesenchymal stromal cells, C control group, CLP caecal ligation and puncture,EPC endothelial progenitor cell, EVs extracellular vesicles, MVs microvesicles, NR not reported, SD Sprague–Dawley, T treatment group, UCMSC umbilical cord mesenchymal stromal cells, UVEC umbilical vein endothelial cells, WJMSC Wharton’s jelly mesenchymal stromal cells BLM: bleomycin LPS: lipopolysaccharide SwIV: swine/MN/08; H1N1 MDR-P: multidrug-resistant PA
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The main animal models were mice and rats; in one study the animal model was a pig. ALI induced by intratracheal injection of lipopolysaccharide or E.coli was the most common method in these studies. At the same time, there are other research methods, such as intratracheal injection of Bleomycin, H1N1 virus, pseudomonas Aeruginosa, cecal ligation and perforation, and trauma. Different studies had different administration methods, intervention times, and intervention doses. In addition, there was a significant difference in the time point at which the results were assessed, with most studies completed within two days and a few lasted one week. All the included studies were peer-reviewed publications. All the experimental animals were randomly assigned to the intervention group and the control. The quality assessment of all studies is shown in Table 2.

Table 2 Abbreviation: 1,peer-reviewed journal; 2,temperature control; 3,animals were randomly allocated; 4,blind established model; 5, blinded outcome assessment; 6,use of anesthetic without significant intrinsic vascular protection activity; 7, appropriate animal model (diabetic, advanced age or hypertensive); 8,calculation of sample size; 9,statement of compliance with animal welfare regulations; 10,statement of potential conflict of interests
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Meta-analysis: ALI with EVs group versus ALI without EVs group (control group)

Heterogeneity text

There were 14 articles in this study. After the Heterogeneity test, I2 = 90% > 50%, and Q test P < 0.1, the articles were considered to have a strong heterogeneity. The reasons for heterogeneity should be investigated. Based on the data of this study, a subgroup analysis was carried out from the aspects of the origin of extracellular vesicles, the species of experimental animals, intervention time, administration methods, and different scoring system of lung injury score. For the overall 14 articles, random effects were selected for meta-analysis, and the results were as follows (Fig. 2).

Fig. 2
figure 2

Main outcome of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group. Main outcome is lung injury score. The size of each square represents the proportion of information given by each trial. Crossing with the vertical line suggests no diference between the two groups. ALI acute lung injury, IV inverse variance, CI confdence interval, df degree of freedom

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The results of the meta-analysis showed that the lung injury score of the experimental group was 1.93 lower than that of the control group, and the degree was statistically significant (P < 0.05).

Sensitivity analysis

A sensitivity analysis of 14 articles was carried out, and the results were as follows (Fig. 3). As can be clearly seen from the above figure, none of the studies significantly caused heterogeneity, and the stability of the overall study was high. Further subgroup analysis was carried out.

Fig. 3
figure 3

Sensitivity analysis of Lung Injury Score 14 studies were included in the sensitivity analysis, and after excluding each study, the combined effects of the remaining studies were within 95% confidence interval of the total effect

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Subgroup analysis

The 14 articles were divided into two groups according to the source of extracellular vesicles. The results of the meta-analysis were as follows (Fig. 4). Based on the subgroup analysis above, the heterogeneity between the two groups was extreme, reaching a high degree of heterogeneity. Among them, the efficacy of stem-progenitor cell extracellular vesicles was -3.63, which was significant (Z = 5.34, P < 0.05). This means that stem progenitor cell extracellular vesicles significantly reduce the severity of acute lung injury to a large extent. Secondly, there was no heterogeneity in the group of non-stem progenitor cell extracellular vesicles, and the efficacy amount was 4.34 (Z = 6.55, P < 0.05). These results suggest that the extracellular vesicles of non-stem progenitor cells significantly increase the severity of acute lung injury. However, that the different sources of extracellular vesicles are the cause of heterogeneity cannot be explained.

Fig. 4
figure 4

subgroup analysis The subgroup meta-analysis of lung injury score that compares different origins of extracellular vesicles. The analysis didn’t detect any statistically significant difference among the stem-progenitor cell extracellular vesicles(1), non-stem progenitor cell extracellular vesicles(2)

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Then, the subgroup analysis was carried out from the aspects of experimental animal species, intervention time, intervention mode and different scoring system of lung injury score. The results are shown in Figs. 5, 6, 7, and 8. Each subgroup shows a high degree of heterogeneity, and the combined results also show high heterogeneity. The results of subgroup analysis do not support that the type of experimental animals, intervention time, intervention mode and different scoring system of lung injury score are the causes of heterogeneity. Next, meta-regression was used to investigate the source of heterogeneity.

Fig. 5
figure 5

subgroup analysis The subgroup meta-analysis of lung injury score that compares different species of animals. The analysis didn’t detect any statistically significant difference among the SD rats(1), C57BL/6J mice(2)

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Fig. 6
figure 6

subgroup analysis The subgroup meta-analysis of lung injury score that compares different intervention time. The analysis didn’t detect any statistically significant difference among the intervention time less than 48 h (1), intervention time more than 48 h (2)

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Fig. 7
figure 7

subgroup analysis The subgroup meta-analysis of lung injury score that compares different administrations. The analysis didn’t detect any statistically significant difference among the intravenous administration (1), intratracheal administration(2)

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Fig. 8
figure 8

subgroup analysis The subgroup meta-analysis of lung injury score that compares different scoring system. The analysis didn’t detect any statistically significant difference among the semi-quantitative grading system(1), the lung injury scoring system(2)

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Multivariable meta-regression analysis

From the above table (Fig. 9), we can see that the independent variable "different sources of extracellular vesicles "and" the species of experimental animals" can significantly affect the efficacy (P < 0.05), while the other independent variables—the mode of administration, the time of intervention and the different scoring system of the lung injury score—have no statistical effect. Overall, we believed that the different sources of extracellular vesicles and the species of experimental animals are the cause of greater heterogeneity. Var9 extracellular vesicles are primarily derived from stem and non-stem progenitor cells; var10 experimental animals are mainly divided into mice and rats; var11 administration is divided into intratracheal administration and intravenous administration; var12 intervention time is divided into whether the intervention time is more than 48 h or not; var13 different scoring system of the lung injury score is divided into the semi-quantitative grading system [58] and the lung injury scoring system [59].

Fig. 9
figure 9

multivariable meta-regression analysis P < 0.05 Considering this variable is the cause of heterogeneity.(var9: extracellular vesicles are primarily derived from stem progenitor cells and non-stem progenitor cells; var10: experimental animals are mainly divided into mice and rats; var11: administration is divided into intratracheal administration and intravenous administration; var12: intervention time is divided into whether the intervention time is more than 48 h or not; var13: different scoring system of the lung injury score is divided into the semi-quantitative grading system and the lung injury scoring system)

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Publication bias

The results were as follows (Fig. 10). From the figure, it can be seen clearly that the funnel diagram of this study is basically symmetrical, and the Egger bias test and the Begg bias test were carried out at the same time. The Egger test showed that there was no significant publication bias (P = 0.390 > 0.05). The Begg test results are consistent with the Egger test results (P = 0.08 > 0.05). Therefore, it can be said that there is no publication bias in the literature of this study.

Fig. 10
figure 10

Funnel plots

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Primary outcome

Lung injury score is considered the primary outcome. Secondary outcomes included IL-1β, IL-6, TNF-α, IL-10, W/D ratio, total protein in BALF, BALF total cell counts, white blood cell counts, and BALF neutrophil counts.

The results showed that compared with the negative control group, stem progenitor cell extracellular vesicle therapy could significantly reduce the lung injury score. The standardized mean difference (SMD) = -3.63 95%CI [- 4.97, 2.30], p < 0.05 I2 = 86.6%, while the non-stem progenitor extracellular vesicle (alveolar epithelial cells and alveolar macrophages) remedy considerably increased the lung injury score. (SMD) = 4.34 95%CI [3.04, 5.63], p < 0.05 I2 = 0%. (Fig. 4).

Secondary outcomes

A total of 16 studies reported BALF neutrophil counts. Compared with the control group, the number of neutrophils in the alveoli of the experimental group decreased (SMD = -2.67, 95% CI[-3.65,-1.69], p < 0.00001), I2 = 84% (Fig. 11A). There were 12 BALF cell count studies total. The results showed that compared with the control group, the total number of alveolar cells in the experimental group decreased (SMD = -3.55, 95% CI[-4.94,-2.15], p < 0.05, I2 = 87%) (Fig. 11B). The comprehensive results of six studies showed that extracellular vesicles could reduce the number of alveolar leukocytes compared with the control group (SMD = -1.24, 95% CI[-2.21,-0.27], p < 0.05) I2 = 76% (Fig. 11C).

Fig. 11
figure 11

Secondary outcomes of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group. Secondary outcomes are BALF neutrophil count(A), total number of alveolar cells(B), alveolar leukocytes count(C). The size of each square represents the proportion of information given by each trial. Crossing with the vertical line suggests no diference between the two groups. ALI acute lung injury, IV inverse variance, CI confdence interval, df degree of freedom

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A total of 10 studies investigated IL-10 in lung tissue. The results showed that compared with the ALI control group, EVs treatment could increase the level of IL-10. (SMD = 1.74, 95% CI [0.42, 3.06], p < 0.05, I2 = 86%) (Fig. 12A) A total of 13 studies reported IL-1β where their aggregate results showed that EVs could reduce the level of IL-1β compared with the control group. (SMD = -2.72, 95% CI [-4.70,-0.74], p < 0.05, I2 = 91%) (Fig. 12B) The comprehensive results of 10 studies show that EV can reduce the level of IL-6. (SMD = -3.90, 95% CI [-6.01,-1.78], p < 0.05, I2 = 90%) (Fig. 12C) In addition, 22 studies provided data on TNF-a, and the combined results show that EV can reduce the level of TNF-a (SMD = -3.69, 95% CI [-5.203,-2.35], p < 0.05, I2 = 91%) (Fig. 13). 23 studies investigated the level of total protein in BALF. The results showed that compared with the control group, EV ameliorated protein exudation. (SMD = -2.22, 95% CI [-2.91,-1.53], p < 0.05, I2 = 83%) (Fig. 14A) Compared with the control group, EV treatment can reduce W/D ratio (SMD = -2.74, 95% CI [-4.15,-1.30], p < 0.00001, I2 = 83%) (Fig. 14B).

Fig. 12
figure 12

Secondary outcomes of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group. Secondary outcomes are IL-10(A), IL-1β(B), IL-6(C).The size of each square represents the proportion of information given by each trial. Crossing with the vertical line suggests no diference between the two groups. ALI acute lung injury, IV inverse variance, CI confdence interval, df degree of freedom

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Fig. 13
figure 13

Secondary outcomes of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group. Secondary outcomes is TNF-a. The size of each square represents the proportion of information given by each trial. Crossing with the vertical line suggests no diference between the two groups. IV inverse variance, CI confdence interval, df degree of freedom

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Fig. 14
figure 14

Secondary outcomes of the meta-analyses of the ALI with EVs group compared with the ALI without EVs control group. Secondary outcomes are total protein in BALF(A), W/D ratio(B).The size of each square represents the proportion of information given by each trial. Crossing with the vertical line suggests no diference between the two groups. ALI acute lung injury, IV inverse variance, CI confdence interval, df degree of freedom

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Discussion

This meta-analysis of 31 studies provides a comprehensive summary of the impact of EVs on preclinical animal models of ALI. Analysis of the combined data suggests that the attenuation of the inflammatory response and the improvement in lung function depend on the type of EVs involved.

Previous meta-analyses have shown that MSC-derived EVs reduce the inflammatory response of inflammatory cells, decrease lung permeability, decrease the activity of inflammatory mediators, and increase the activity of anti-inflammatory mediators in an animal model of ARDS, thereby attenuating lung injury and improving survival in an animal model of ARDS [60]. However, previous studies have not considered the effects of other sources of EVs on ALI/ARDS. In our meta-analysis, we incorporated extracellular vesicles (EVs) from diverse cellular origins to investigate their impact on lung injury scores, cell, and inflammatory factors in bronchoalveolar lavage fluid (BALF). Our findings offer valuable insights for future studies. Multiple regression analyses revealed that the cell source independently influenced the efficacy of EVs. Additionally, the choice of experimental animal introduced notable heterogeneity, while no statistically significant differences were observed among modes of administration, duration of intervention, and lung injury scoring systems. This meta-analysis demonstrates the mitigating effects of stem progenitor cell-derived extracellular vesicles (EVs) on ALI/ARDS severity in an animal model. The Lung Injury Score (LIS), a pivotal pathophysiological metric in clinical trials for assessing lung injury severity, was significantly reduced by stem progenitor cell-derived EVs, indicative of a diminished overall severity of lung injury. In addition, our study found that stem cell-derived EVs down-regulated the levels of inflammatory factors such as IL-1 β, IL-6, and TNF-a, and up-regulated the levels of IL-10, a traditional anti-inflammatory cytokine. Modulation of the balance of pro-inflammatory and anti-inflammatory cytokines by stem cell-derived EVs may be important for ameliorating lung injury and improving survival. In contrast, EVs from alveolar epithelial cells and macrophages exacerbate the extent of lung injury. The lung W/D ratio is a widely used index to assess pulmonary vascular permeability in animal experiments. In the present meta-analysis, the lung W/D ratio decreased, suggesting that stem cell-derived EVs increase lung water clearance. Also, our meta-analysis showed that neutrophils, leukocytes, and total protein in bronchoalveolar lavage fluid were reduced after intervention with stem cell EVs. This suggests that stem cell-derived EV therapy reduces the effects of lung tissue infection, vascular permeability and lung tissue damage. Numerous research have proven that RNAs carried by means of EVs are integral for their therapeutic characteristics [61, 62], and the proteins contained in EVs are also additionally associated with various biological functions in the human body. EVs are membrane-bound vesicles launched by way of all cell types, which are necessary data carriers for controlling angiogenesis, extracellular matrix remodelling, gene expression, inflammation, cell proliferation, cell migration, and morphogenetic elements [63,64,65,66].

As surface molecules, EVs can target receptors, facilitating signal transduction via receptor-ligand interactions. They undergo internalization through endocytosis, phagocytosis, and fusion with target cell membranes. With a composition akin to normal cell membrane and surface proteins, EVs are readily taken up by target cells, delivering their contents to the cytoplasm and thereby modulating the physiological state of recipient cells. This characteristic raises optimism regarding their potential as the next generation of drug transporters [67,68,69,70]. EV derived from stem cells reduces immunogenicity compared to stem cells and also reduces the dangers associated with cellular therapies such as cytokine release syndrome [63]. Currently, there are two main therapeutic approaches to ALI/ARDS: supportive therapy and pharmacological interventions. Despite increased understanding of the pathophysiology of ARDS, the efficacy of preferred treatments such as lung-protective ventilation, prone positioning, and neuromuscular blockers is often limited [71]. Currently, there are no successful pharmacological therapies for ARDS [72]. T herefore, there is a need to study the effects of EVs on ALI/ARDS.

Extracellular vesicles are emerging as a promising therapeutic and diagnostic tool, with studies demonstrating their potential role in the treatment and diagnosis of digestive system diseases, cancer, and other areas of research [6, 73, 74]. Additionally, a meta-analysis have shown that stem cell-derived EVs improve cardiac function and reduce infarct size in myocardial infarction animals [75]. Another meta-analysis have found that MSC-EVs have therapeutic potential for acute and chronic liver diseases [76]. Furthermore, several meta-analysis studies have indicated that EVs are involved in the treatment of acute kidney injury and osteoporosis [77, 78]. However, some studies have suggested that EVs may not only have a therapeutic effect in disease development but may also contribute to disease progression.A study found that the increase of peripheral blood miR-1298-5p in sepsis-associated ALI patients triggered lung inflammation by inhibiting the proliferation of human bronchial epithelial cells and inducing epithelial permeability changes [79]. Another study revealed that EVs in the plasma of sepsis patients are rich in miR-210-3p, which can enhance various responses associated with sepsis-induced ALI by regulating autophagy and inflammation. These responses include macrophage inflammatory responses, bronchial epithelial cell apoptosis, and changes in lung microvascular endothelial cell permeability [80].

Recent studies have shown that there may be differences in the membrane lipid composition and capsule contents released by the same cells [81, 82]. In our meta-analysis, EVs of various sizes were included. the large heterogeneity among EVs is a major obstacle to understanding the composition and functional characteristics of the different secreted components. For further studies, the isolation, identification, and compositional analysis of EVs of different sizes are key determinants of ALI treatment. In addition, more efficient and safer methods of EVs preparation, isolation, characterisation and stockpiling skills are also important factors in determining whether EVs can be used on a large scale in the clinic.

Limitations

Firstly, the overall sample size we analysed was very small due to the small sample size of the preclinical trial. Secondly, extracting data from drawings using Engauge Digitizer software in the absence of raw data may have altered the raw data and thus affected the results. This is despite the fact that we used a method where data were extracted separately by multiple reviewers. Finally, due to the lack of large animal tests and routine clinical parameters (e.g., respiratory mechanics), small tests may miss important information that is not conducive to guiding clinical applications.

Conclusion

This meta-analysis demonstrated that stem cell-derived EV therapy could improve lung function and inflammatory response in preclinical ALI animal models, while non-stem cell-derived EVs aggravate lung injury. This result provides an essential clue for human clinical trials of EVs.