2.1. Animal Ethics

Seventy-two adult male Sprague-Dawley rats weighing ~200 ± 20 g were purchased from Zhuhai Bestest Bio-Tech (https://www.bestest100.com). They were kept in a specific pathogen-free (SPF) environment at the International Healthcare Innovation Institute (Jiangmen) at a constant temperature of 25°C, humidity range of 60%–70%, and 12 h of light per day, drinking and eating freely. All animal experiments were approved by the Experimental Animal Welfare and Ethics Committee of the department (N2022032). At the end of the experiment, blood was collected from the abdominal aorta under anesthesia with sodium pentobarbital (30 mg/kg), and the rats were exsanguinated under anesthesia from the abdominal aorta to euthanasia. All methods were performed in accordance with the relevant regulations and guidelines.

2.2. Plasmid Construction and Viral Solution Production

Using the commercial vector GV208, Genechem (https://www.genechem.com.cn/ Shanghai, China) was contracted to produce an overexpression plasmid for DCN (NM_001920). Next, 293T cells were transfected with a plasmid using polyethylenmine to produce a lentiviral (LV)-DCN viral solution at a titer of 3.5 + E9 transduction units/mL. The viral broth produced from the blank vector was designated as Con.

2.3. Cell Line Construction

Con. and LV-DCN virus solutions were mixed 1:100 (20 μL : 1980 μL) with stem cell serum-free medium (#NC0103+NC0103, Yocon, Beijing, China) and, used to infect HUC-MSCs (P1 cells, resuscitated 48 h prior and inoculated at 1 × 10⁵ cells/2 mL in 6-well plates), with three wells per group. HUC-MSCs were provided by CD Science, Ltd. (Guangzhou, China). After 10 h, the medium was replaced with fresh medium, and the cells were passaged when the degree of cell fusion reached 95%. The passage (P2) cells were cultured in a complete medium supplemented with 2 µL/mL puromycin (#HY-K1057, MCE, America). Cells that were not successfully transfected were screened for apoptosis. After one amplification passage, P4 MSC-Con. and MSC-DCN cell lines were established. A serum-free cell cryopreservation solution (#C40100, NCM Biotech. Suzhou, China) was used to cryopreserve the cells for further research.

2.4. Identification of DCN Expression

2.5. Flow Cytometry Identification of Stem Cell Surface Markers

MSC-Con. and MSC-CN cultures were subjected to cell digestion, and the following antibodies were incubated according to the manufacturer’s instructions: anti-CD11b (CoraLite Plus 488 #CL488-65116), anti-CD19 (CoraLite Plus 488 #CL488-65197), CD34 (CoraLite Plus 488 #CL488−60180), CD73 (CoraLite Plus 405 #CL405-65162), CD90 (#66766-1-Ig), and CD105 (#10862-1-AP), with CD90 and CD105 reincubated with the secondary antibody Alexa Fluor405 (#ab175652, Abcam, Britain). Subsequently, flow cytometry was performed according to the standard procedures. Surface markers were selected based on the MSC minimum standards, and six were selected for detection [21, 22]. All antibodies were purchased from Proteintech (Wuhan, China) except for Alexa Fluor405. The machine used was Attune N-×T (Thermo, Massachusetts, America).

2.6. Apoptosis Assay

MSC-Con. and MSC-DCN cultures were subjected to cell digestion and incubated according to the AnnexinV-APC/PI Double-Staining Apoptosis Detection Kit (#KGA1107−100, Nanjing Jancheng Biotech, Nanjing, China) [20], and then detected using flow cytometry.

2.7. CCK-8 Assay

Transfected P6 MSC-Con. and MSC-DCN cell lines were inoculated into 96-well plates at 2 × 10³ cells/100 μL/well in 36 wells each. Next, 10 μL CCK-8 working solution (#FD3788-3000, Ford Biotech, Hangzhou, China) was added to six wells of each group on days 0, 1, 2, 3, 4, and 5. Cell proliferation was detected after incubation at 37°C for 1 h, and the absorbance was measured at 450 nm (Multiskan Fc, Thermo, America). The medium was replaced on day 3. Optical density values were measured daily and compared with those on day 0 to calculate the cell proliferation rate.

2.8. MSC Differentiation Assay

Transfected MSC-Con. and MSC-DCN were inoculated into 6 wells each of a 12-well plate at 8 × 10⁴ cells/1 mL/well. After 24 h, the two types of cells were replaced by an osteoblast differentiation induction culture system (#05465, Stemcell, Toronto, Canada) and an adipogenic differentiation induction culture system (#05412, Stemcell, Toronto, Canada), with three wells of each. The culture medium was changed every 3 days for the next 21 days. Subsequently, osteogenic differentiation, alizarin red staining, and adipogenic differentiation oil red O staining were performed as per standard procedures, and the stained images were captured under a microscope.

2.9. Cell Preparation

Established P4 MSC-Con. and MSC-DCN cell lines were resuscitated and cultured in a serum-free medium for 3 days. Cells were passaged 1:6 at ≥95% cell fusion, digested, collected, washed twice with saline, and counted after 3 days of culture. The cells were resuspended in 1 × 10⁶ or 5 × 10⁶ cells/500 μL/1.5 Eppendorf tube in saline and transported on ice to the SPF animal room within 1 h for rat tail vein transplantation.

2.10. Establishment and Treatment of BLM-Induced Pulmonary Fibrosis Model

The rats were fed for 3 days, anesthetized with sodium pentobarbital (30 mg/kg, i.p.), and the trachea was separated under aseptic conditions. BLM (Hanhui Pharmaceuticals Co., Shanghai, China) was injected into the trachea at 5 U/mL/kg, and the rats were rotated vertically from left to right for 1 min to ensure the BLM was evenly dispersed in the lungs. Finally, the incision was closed using sutures. About 24 h after modeling, the rats were randomly divided into the model, positive drug treatment (PA), MSC-Con., MSC-DCN Low and MSC-DCN High groups. The model group was administered 500 µL of saline via tail vein injection, and the PA group was treated with prednisone acetate (Zhongsheng Pharma, Dongguan, China) (3 mg/kg) by gavage for seven consecutive days. Our previous study found that comparing a high dose (10⁷) and low dose (10⁶) of BM-MSCs in the treatment of IPF model rats, the lung histopathology, degree of alveolitis, and degree of pulmonary fibrosis in the high-dose group were significantly better than those in the low-dose group. According to the official website of the China Drug Clinical Trial Registry (http://www.chinadrugtrials.org.cn/), the optimal dose for intravenous transplantation of MSCs for the treatment of IPF is between 3 × 10⁷ and 2 × 10⁸ cells. On this basis, two doses were chosen for the experiments. The MSC-Con. group was injected with 5 × 10⁶ cells of MSC-Con. The MSC-DCN Low group received 1 × 10⁶, and the MSC-DCN High group received 5 × 10⁶ MSC-DCN through the tail vein. The cells used for the injections were described as cell preparation contents. Half of the rats were euthanized on days 14 and 28 after treatment [23, 24], which was 15 and 29 days after modeling, to obtain the lung, spleen, kidney, liver, and heart tissues, as well as serum.

2.11. Blood Biochemical Assays

The sera obtained were subjected to serum MMP9 (#CSB-E08008r, CUSABIO, Wuhan, China), TIMP1 (#CSB-E08005r, CUSABIO, Wuhan, China), interleukin (IL) 1β (#ml028514, mlbio, Shanghai, China), IL10 (#ml028497, mlbio, Shanghai, China), superoxide dismutase (SOD) (#ml059387, mlbio, Shanghai, China) and malondialdehyde (MDA) (#ml077384, mlbio, Shanghai, China) tests, using enzyme-linked immunosorbent assays. Biochemical kits for serum lactate dehydrogenase (LDH) (#A020-2-2), creatinine (CRE) (#C011-2-1), alanine aminotransferase (ALT) (C009-2-1), and aspartate aminotransferase (AST) (C10-2-1) were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The experimental procedures were performed according to the manual and published literature [25].

2.12. Histological Staining

After euthanasia, the lung, heart, spleen, kidney, and liver tissues of the rats were removed, paraffin-embedded, serially sectioned, and stained with hematoxylin and eosin (HE) and Masson staining according to standard pathology procedures. Images were obtained using the digital scanning microscopy imaging system on a microscope slide (PreciPoint M8, Germany). Referring to a previously published article [8], scores of 1, 2, 3, and 4 were assigned according to no, mild, moderate, and severe alveolar inflammation, respectively. Mild alveolitis is characterized by mononuclear cell infiltration, widened alveolar septa with normal structure, and infiltration confined to the proximal pleural area, involving <20% of the lungs. Moderate alveolitis involves 20%–50% of the lungs, and severe alveolitis involves >50% of the lung, with occasional mononuclear cells and hemorrhages in the alveolar cavities causing solid lesions. The degree of pulmonary fibrosis on Masson staining was scored according to the Ashcroft Score criteria [26]. Data are presented as median and interquartile range (M, [P25, P75]).

2.13. Immunohistochemical (IHC) Staining

IHC staining of the lung and spleen tissues was performed according to the same method used for histological staining and standard procedures to detect the expression of lung CD68 (#28058-1-AP), CD206 (#18704-1-AP), interferon-gamma (IFN-γ) (#15365-1-AP), tumor necrosis factor-alpha (TNFα) (#60291-1-Ig), TIMP1 (#16644-1-AP), MMP9 (#10375-2-AP), fibronectin-1 (FN1) (#66042-1-Ig), collagen type III (COL III) (#22734-1-AP), serum nuclear factor NF-E2-related factor (NRF2) (#80593-1-RR), quinone-forming reductase 1 (NQO1) (#67240-1-Ig), heme oxygenase-1 (HO1) (#66743-1-Ig), TGF-β1 (#21898-1-AP), and SMAD7 (#25840-1-AP), as well as the expression of spleen CD68 and CD206. All the antibodies were purchased from Proteintech (Wuhan, China). The experimental steps and expression scores for IHC were as described previously [27, 28]. The percentage of positive cells was graded as follows: 0, no positive cells; 1, <10% positive cells; 2, 10%–35% positive cells; 3, 35%–75% positive cells; and 4, >75% positive cells. Staining intensity was graded as follows: 0, no staining; 1, weak staining (yellowish); 2, moderate staining (yellowish-brown); 3, strong staining (brown) and 4, super strong staining (brown-black). The staining index was the product of the two scores. Staining indices in the different groups are presented as the median and interquartile range (M, [P25, P75]).

2.14. Cell Model Intervention Experiment In Vitro

In vitro experiments were performed to confirm the effects of MSC-DCN on macrophages and lung fibroblasts. The THP-1 and MRC-5 cell lines were purchased from Procell (Wuhan, China). TPH-1 cells were cultured in RPMI 1640 (#PM150110, Procell, Wuhan, Chian) supplemented with 10% fetal bovine serum (FBS) (#F102, Vazyme, Nanjing, China) and 1% penicillin–streptomycin solution (P/S) (#PB180120, Procell, Wuhan, China). MRC-5 cells were cultured in minimum essential medium (MEM) (#PM150410, Procell, Wuhan, China) supplemented with 10%FBS and 1% P/S. THP-1 and MRC-5 cells in the exponential growth phase were seeded in 12-well plates on glass coverslips. THP-1 cells were cultured with an additional 20 µg/L phorbol 12-myristate 13-acetate (PMA) (#P1585, Sigma–Aldrich, America) for 12 h to induce the M0 macrophage state, followed by 20 µg/L IL4 (#UA040026, UA. Bio, Hangzhou, Chian) and 20 µg/L IL13 (#UA040113, UA. Bio, Hangzhou, Chian) for 48 h to induce M2 macrophage polarization [29]. MRC-5 cells were cultured with 5 µg/L TGF-β1(#UA040085, UA. Bio, Hangzhou, Chian) and, after 24 h, seeded in 12-well plates for 48 h to induce the fibrogenic model. P4 MSC-Con. and MSC-DCN were cultured in a Transwell upper chamber in advance to 95% fusion and then transferred to the 12-well plates with THP-1 or MRC-5 cells at the bottom at the beginning of the cell model induction (Figure S7B,D). After 48 h of coculture, the expression of the target protein was detected by immunofluorescence according to the standard procedure and as previously described [30]. CD68 (#66231-2-Ig) and CD206 (#18704-1-AP) were detected in THP-1 cells whereas FN1 (#66042-1-Ig) and COL III (#22734-1-AP) were detected in MRC-5 cells. CD68 and FN1 were labeled with goat–anti-mouse secondary antibody (Alexa Fluor 488, #ab150113, Abcam), and CD206 and COL III were labeled with 647-conjugated goat–anti-rabbit secondary antibody (#RGAR005, Proteintech, Wuhan, China). DAPI was used to label the nuclei with an antifade mounting medium (#P0131, Beyotime, Shanghai, China). Images were acquired using a fluorescence microscope (Axio Imager M2, Ziss, Germany). The fluorescence intensity and number of nuclei were identified using ImageJ software. The ratio of fluorescence intensity to the number of nuclei represents the relative fluorescence intensity.

2.15. Data Analysis

All results are presented as the mean ± standard deviation or median and interquartile range (M, [P25, P75]). The corresponding data were statistically analyzed using GraphPad Prism 10 software. More than three groups were tested using Tukey’s multiple comparisons test, and differences between the two groups were analyzed using an unpaired t-test. The log-rank (Mantel–Cox) test was used for survival analysis. Statistical significance was set at p ≤ 0.05.

3.1. MSC-DCN Cell Line Was Successfully Generated Without Altering Its Stem Cell Properties

After LV infection and screening with puromycin (2 μg/mL), the MSC-Con. and MSC-DCN cell lines were generated. Compared to MSC-Con. the expression of the DCN gene and protein was significantly upregulated in MSC-DCN (Figure 1A,B). There were no surface markers changed of transfected MSCs, and they met the criteria for the identification of MSCs, with CD11b, CD19, and CD34 expressed <2%, whereas CD73, CD90, and CD105 were expressed >98% (Figure 1E). After the induction of osteogenic and adipogenic differentiation, the two groups of HUC-MSCs showed differentiation potential. After 21 days of osteogenic differentiation, alizarin red staining was positive, and calcified nodules were visible (Figure 1F). After 21 days of adipogenic differentiation, fat vesicle formation was observed using oil red O staining (Figure 1F). The CCK8 results showed that there was no difference in the proliferative ability of the two groups of cells, and they entered the exponential growth period when cultured for 48 h (Figure 1C). Flow cytometry after Annexin V-APC/PI staining showed that DCN overexpression did not promote apoptosis (Figure 1D). In conclusion, the MSC-DCN cell line was successfully constructed, and overexpression of the DCN protein did not affect the properties of MSCs.

3.2. Establishment of BLM-Induced Pulmonary Fibrosis Rat Model

On days 15 and 29 after the establishment of the model, half of the mice were anesthetized, and the corresponding organs were collected. About 4 days after BLM administration, all groups, except for the control group, gradually showed symptoms such as arched back, hard breathing, and mental burnout. The scatter plot of animal weight showed that the body weight of the model group was significantly lower than that of the control and PA groups (Figure S1A). Compared to the control group, the survival rates of the model and PA groups decreased significantly (Figure S1B), indicating that BLM led to the death of rats in the IPF model. On days 15 and 29, the lungs of rats in the model group showed severe alveolar inflammation and fibrosis, destruction of the alveolar structure, formation of cystic cavities, and other parenchymal lesions. Prednisone acetate showed therapeutic effects on day 29 of modeling after continuous gavage treatment and partially alleviated the symptoms of pulmonary fibrosis (Figure S1D,G). The scores for alveolar inflammation and fibrosis in the model group significantly increased, and PA treatment showed some efficacy in reducing the score 29 days after modeling (Figure S1E,F,H,I). Intratracheal administration had no effect on the rat heart, liver, kidney, and spleen structures (Figure S2A). There were no significant differences in the serum LDH, ALT, AST, or CRE levels; however, LDH, ALT, and AST levels increased slightly in the model group (Figure S2B–E). In addition, intragastric administration of prednisone acetate for 7 days increased the serum ALT levels in rats. Thus, we successfully established a rat model of BLM-induced pulmonary fibrosis. Prednisone acetate plays a role in alleviating the progression of pulmonary fibrosis; however, it generated side effects while exerting its action.

3.3. DCN Overexpression in HUC-MSC Can Be Used for Protecting BLM-Induced Pulmonary Fibrosis and Is Safe

The day after the rats were intratracheally injected with BLM, they were subjected to tail vein transplantation of the corresponding cells, according to the MSC-Con., MSC-DCN Low, and MSC-DCN High groups. About 4 days after modeling, the rats in each group gradually showed symptoms, such as back-arching and hard breathing. Compared to the model group, MSC treatment gradually improved the symptoms. Death occurred in all groups, with the highest number of survivors in the MSC-DCN group; however, there was no statistically significant difference in the survival rate (Figure S1C). Compared with the model group, the mental state of rats recovered, and their body weights increased significantly after MSC-DCN and MSC-Con treatment, especially in the MSC-DCN High group. There was no difference in body weight between the MSC-Con. and MSC-DCN Low groups (Figure 2B). HE and Masson staining showed that HUC-MSC therapy reduced BLM-induced pulmonary fibrosis, including alveolar inflammation and pulmonary interstitial fibrosis (Figure 2C–H). No structural abnormalities were observed in the heart, liver, spleen, or kidneys (Figure S3A), and no differences were observed in serum LDH, AST, or CRE (Figure S3B,C,E) in each group treated with MSC. After 28 days of MSC treatment, the serum ALT levels in the other groups were lower than those in the model group, especially in the MSC-DCN High group (p = 0.038, Figure S3D). As seen from the above results, DCN-overexpressing MSCs demonstrated better therapeutic potential in pulmonary fibrosis, as reflected by the fact that a smaller dose achieved a therapeutic effect roughly comparable to that of high-dose MSC-Con. The therapeutic effect of the same dose of MSC-DCN was superior to that of MSC-Con. Thus, HUC-MSC transplantation is a safe and effective treatment for pulmonary fibrosis.

3.4. HUC-MSC Overexpressing DCN Inhibits Inflammation and Fibrous Deposition in Lung Tissue

Based on these results, we concluded that MSC-Con. or MSC-DCN was effective in treating pulmonary fibrosis in rats. NQO1, HO1, and NRF2 are potent active components against tissue damage. We originally thought that MSC therapy could treat fibrosis by altering the local expression of NQO1, HO1, and NRF2 in lung tissues to play an anti-inflammatory and anticellular damage role. IHC results showed no difference in the expression of NQO1 among the three groups (Figure S4A,D). Compared with the control group, NRF2 in the model group was significantly upregulated after 29 days (Figure S4C), but there was no difference in expression between the PA and MSC transplantation groups compared to the model group (Figure S4F). Compared to the control group, the expression of HO1 in the model group was significantly upregulated at 15 and 29 days after modeling, and NRF2 in the lung tissue was significantly downregulated on the 29 days after PA treatment (Figure S4B,C). Compared with the model group, HO1 expression was significantly downregulated in the lungs of all groups treated with MSC transplantation, whereas there was no difference in the MSC-Con. and MSC-DCN Low groups (Figure S4E). There were no changes in the serum levels of SOD, a factor associated with antioxidant and anti-inflammatory damage, and subtle changes in MDA levels. There was no difference in the serum SOD content between the groups of rats on days 15 and 29 after modeling (Figure S4H,J). In contrast to the control group, the serum MDA content in the model group was significantly upregulated on day 15 after modeling, significantly decreased after PA treatment, and did not differ on day 29 after modeling (Figure S4G). Serum MDA was significantly downregulated in the MSC transplantation treatment groups on day 15 after modeling compared to the model group, while there was no difference in expression on day 29 (Figure S4I).

TNFα and IFN-γ respond to inflammatory levels in local tissues, whereas serum IL1β and IL10 respond to systemic inflammatory responses. Therefore, we detected serum IL1β and IL10 levels in rats on days 15 and 29 after modeling, respectively, as well as TNFα and IFN-γ levels in lung tissue using IHC. On days 15 and 29 after modeling, lung tissue TNFα expression was significantly upregulated in the model group compared to that in the control group, with a decreasing trend but no statistically significant difference after PA treatment (Figure S5A). IFN-γ was significantly upregulated in the model group at day 15 and tended to decrease in the PA group after treatment but was not statistically different, whereas there was no difference between all groups at day 29 (Figure S5B). Compared to the model group, both MSC-Con. and MSC-DCN treatments significantly downregulated the expression of tissue TNFα at both assay time points, and the results were almost the same between the MSC-DCN Low and the MSC-Con. groups (Figure 3A–C). However, IFN-γ levels were significantly different between the model and treatment groups (Figure 3D). Serum IL1β levels in rats in the model group increased significantly on day 15, whereas PA and normal levels appeared to be the same; however, there was no difference between the groups on day 29 (Figure S5C). Compared with the model group, the MSC treatment groups showed significantly reduced levels of IL1β (Figure 3E) on days 15 and 29. However, regardless of time, there was no significant difference in the serum IL10 levels among the groups (Figure 3F, Figure S5D). These results suggest that BLM can cause changes in the local and systemic inflammatory levels in rats, especially in the levels of the pro-inflammatory factor TNFα and serum IL1β. Stem cell therapy can significantly alleviate these symptoms. However, modeling and treatment had no effect on the anti-inflammatory factor IL10.

MMP9 and TIMP1 are the main factors that maintain the balance between exudation and absorption of the local ECM. In the early stages of fibrosis, an increase in MMP9 levels leads to increased exudation of the local ECM and the production of raw materials for the subsequent formation of collagen. Compared to the control group, the expression of MMP9 and TIMP1 in the lung tissue of the model group increased significantly on days 15 and 29, and the expression of MMP9 decreased on 15 days after PA treatment (Figure S5E,F). High TIMP1 expression is likely due to self-repair feedback in rats. The expression of MMP9 was stable in the serum of rats in the control, model, and PA groups (Figure S5G) and was not affected by any experiment or treatment. The expression of serum TIMP1 was consistent with the normal levels found in lung tissue (Figure S5H). Ideal results could still be obtained. Compared with the model group, after 15 days of modeling, the MMP9 levels in the lung tissue of each cell treatment group were significantly reduced, whereas, at the 29-day time point, there was a downward trend without a statistically significant difference (Figure 4A–C). This may be because 29 days after modeling, the local tissue was already in the repair and collagen formation stages; therefore, the expression of MMP9 had stabilized, and the serum MMP9 level did not change significantly (Figure 4G). However, we still found that, compared with the model group, the tissue and serum levels of TIMP1 were significantly downregulated after MSC transplantation (Figure 4D–F,H), probably because rat fibrosis is significantly reversed after MSC transplantation, and thus, there is no need to produce excessive TIMP1 repair factor.

The local expression levels of FN1 and COL III directly reflect the number of tissue’ myofibroblasts and the degree of fiber deposition. IHC staining of FN1 and COL III in lung tissue showed that the expression of FN1 in the lung tissue of the model group increased significantly on days 15 and 29 compared with that in the control group, and PA treatment significantly improved this condition (Figure S5I). The expression of COL III in the model group also increased significantly, which significantly decreased in the early stage of PA treatment but did not show an effect on day 29 (Figure S5J). This further demonstrated that our experimental model was successful. Compared with the model group, FN1 and COL III in the lung tissue of the MSC treatment group were significantly reduced at both time points, and the downregulation effect in the MSC-DCN High group was more apparent (Figure 5A–F).

In summary, stem cells overexpressing DCN can improve the local cellular microenvironment in pulmonary fibrosis by regulating local and systemic inflammation levels and ultimately reducing fibrous deposition to achieve the effect of treating pulmonary fibrosis.

3.5. DCN Overexpression by MSCs Can Inhibit the Recruitment of Macrophages in Lung Tissue and Reduce M2 Polarization In Vitro

Based on these results, we conclude that MSCs can effectively treat pulmonary fibrosis by improving systemic inflammation and suppressing the local inflammatory environment in lung tissue. We believe that this is related to macrophages. Macrophages are not only involved in local inflammation, but their phagocytic and secretory functions are also important for the formation of local fibrosis. As markers of macrophages and M2 macrophages, CD68 and CD206 not only reflect the recruitment of local macrophages but also distinguish their polarization. The IHC results showed that more macrophages were recruited to the lung tissue in the model group than in the normal group. Significantly more CD68 and CD206 positive macrophages were found in the lung tissues of the model group on days 15 and 29 after modeling (Figure S6A,B). PA treatment reduced the local recruitment of CD68 and CD206 positive cells in the later stages (Figure S6A,B). The immunomodulatory functions of MSCs are satisfactory. Intravenous transplantation has a lung-priming effect, with the majority of MSCs sequestered in the lungs. We found that local CD68-positive cells were significantly reduced in rat lungs in all cell treatment groups and that the MSC-DCN group was superior to the MSC-Con. group in reducing CD68-positive macrophage recruitment 15 days after modeling. The MSC-DCN High group showed the lowest CD68 positivity (Figure 6A–C). Conversely, CD206 expression was significantly reduced in the MSC-DCN High group, whereas no significant changes were observed in the MSC-Con. and MSC-DCN Low groups (Figure 6D–F). This suggests that DCN-overexpressing MSCs play a significant role in regulating macrophage polarization, which may be a key pathway for MSC treatment of pulmonary fibrosis.

To further demonstrate the direct effect of MSCs on macrophage M2 polarization, we performed in vitro cell experiments. THP-1 cells were treated with PMA (185 µg/L) for 12 h to induce the M0 state and further treated with IL4 (20 µg/L) and IL13 (20 µg/L) for 48 h to induce the polarized M2 state (Figure S7A). Immunofluorescence of CD68 and CD206 showed that the coculture of MSCs and THP-1-induced M0 macrophages did not promote spontaneous M2 polarization (Figure 7A–C). However, upon progression to the M2 state, MSC coculture significantly reduced polarization, especially MSC with DCNs, which showed a greater effect (Figure 7D–F). IL10 levels in cell supernatants were also tested by flow cytometry. IL10 levels were noticeably increased after M2 induction and significantly decreased with MSC coculture (Figure S7C).

The spleen is the main immune regulatory organ, and most macrophages originate from it. The number and polarization of splenic macrophages are feedback indices of systemic inflammation. After the establishment of the BLM model, the expression of CD68 and CD206 in the spleen showed an upward trend, but there was no statistically significant difference (Figure S6C,D). Although we observed a decrease in the expression of CD68 and CD206 in the spleens of rats treated with MSC transplantation, but this difference was not significant (Figure 8A–F). This may be due to an insufficient number of rats and an increase in the number of experimental animals show a difference.

3.6. Effect on TGF-β Signaling of MSC-DCN

The TGF-β/Smad signaling pathway is a key regulator of many fibrotic diseases. As described above, we successfully established a pulmonary fibrosis model. We found that TGF-β1 was significantly upregulated in all lung tissues of rats in the model group, and PA treatment significantly reduced TGF-β1 expression on day 15 after modeling; however, there was no statistically significant difference on day 29 (Figure S6E). In the cell transplantation treatment group, MSC-Con. and MSC-DCN showed a strong effect on reducing TGF-β1. TGF-β1 protein expression was downregulated in all cell transplantation groups, with the same effect observed in the MSC-Con. and MSC-DCN Low groups but more significantly in the MSC-DCN High group (Figure 9A–C). Smad7 acts as a negative regulator of TGF-β and was significantly downregulated in lung tissues in the model and PA groups (Figure S6F). After MSC treatment, local Smad7 expression was significantly increased in lung tissue on day 29 of modeling (Figure 9D–F). Thus, we demonstrated that MSCs play a role in the treatment of BLM-induced pulmonary fibrosis through the TGF-β1/Smad7 pathway and that MSCs overexpressing DCN showed better efficacy.

3.7. MSC-DCN Reduced TGF-β1-Induced Fibrosis in MRC-5 Cells

To further demonstrate the effect of MSCs in reducing lung fibrosis, MRC-5 cells were treated with TGF-β1 to induce fibrosis. After TGF-β1 treatment for 48 h, the levels of the fibrosis-associated proteins FN1 and COL III in MRC-5 cells were considerably increased (Figure 10A–C). Coculture with MSCs significantly reduced the expression of FN1 and COL III in MRC-5 cells, with MSC-DCNs exhibiting a better effect (Figure 10A–C). Compared with the TGF-β1 group, COL III in the MSC-DCN cocultured group was significantly decreased, whereas the MSC-Con. group showed no statistical difference (Figure 10C).