Human Umbilical Cord Mesenchymal Stem Cells Combined with Pirfenidone upregulates the Expression of RGS2 in the Pulmonary Fibrosis in Mice

Human Umbilical Cord Mesenchymal Stem Cells Combined with Pirfenidone upregulates the Expression of RGS2 in the Pulmonary Fibrosis in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Human Umbilical Cord Mesenchymal Stem Cells Combined with Pirfenidone upregulates the Expression of RGS2 in the Pulmonary Fibrosis in Mice Xian Wu, Hao Gou, Huijun Qiu, Hanmin Liu, Zhou Fu, Lina Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1507756/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Objective The therapeutic effect of umbilical cord-derived mesenchymal stem cells(hUC-MSCs) in combination with pirfenidone(PFD) on pulmonary fibrosis in mice and its possible mechanism were investigated. Methods C57BL/6 mice were randomly divided into six groups: control group, model group, P 10 group, P 30 group, P 100 group and P 300 group. Modeled by tracheal intubation with 3 mg/kg bleomycin drip, each dose of PFD was given daily by gavage from day 7 onwards. Mice were observed continuously for 21 days and survival was recorded. Lung tissues were collected on day 21, and HE and Masson staining were performed to assess morphological changes and collagen deposition in the lungs. Collagen content was measured by Sircol method, and fibrosis marker levels were detected by PCR and Western Blot. Another batch of C57BL/6 mice was then randomly divided into five groups: hUC-MSCs control group, model group, P 100 group, hUC-MSCs treatment group and hUC-MSCs + P 30 group. 5×10 5 hUC-MSCs were injected into the tail vein on day 7, and the mice were given PFD gavage daily from day 7 onwards, and their survival was recorded. Lung tissues were collected on day 21 to detect pathological changes and the expression of collagen content and regulator of G protein signaling 2(RGS2). Pulmonary myofibroblasts were divided into MFB group and MFB + hUC-MSCs group, and different doses of PFD were added to each group, and the levels of RGS2 and fibrosis markers were detected in each group. Results Compared with other doses of PFD groups, the P 100 group significantly improved mouse survival and lung pathology, and significantly reduced collagen and fibrosis marker levels ( p < 0.05). The hUC-MSCs + P 30 group significantly improved mouse survival and lung pathology, significantly reduced collagen and fibrosis marker levels ( p < 0.05), and the efficacy was better than that of the P 100 and hUC-MSCs groups ( p < 0.05). RGS2 expression was significantly higher in the MSCs + P 30 group compared with the P 100 and hUC-MSCs groups ( p < 0.05). PFD increased RGS2 expression in MFB ( p < 0.05) in a dose-dependent manner. Fibrosis markers were more significantly reduced in the hUC-MSCs + PFD relative to the PFD alone group and the hUC-MSCs group. Conclusion The study suggested that hUC-MSCs combined with low-dose PFD showed a therapeutic effect better than that of the two treatments used separately. Its effect attenuating bleomycin-induced pulmonary fibrosis in mice is related with the increase of RGS2. Idiopathic pulmonary fibrosis pirfenidone umbilical cord-derived mesenchymal stem cells Regulator of G protein signaling 2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Idiopathic pulmonary fibrosis(IPF), a diffuse inflammatory disease of the lower respiratory tract with unknown etiology, is a chronic interstitial lung disease characterized by progressive dyspnea and progressive deterioration of lung function[ 1 ]. Its incidence is increasing year by year, the median survival is only 2.5–3.5 years, and the prognosis is worse than that of many types of cancer[ 2 , 3 ]. The 2015 edition of the clinical practice guidelines for the treatment of IPF states that the use of prednisone, azathioprine, N-acetylcysteine monotherapy, anticoagulants, platelet-derived growth factor receptor antagonists, endothelin receptor antagonists, and 5-phosphodiesterase inhibitors is strongly discouraged in patients with IPF, and that there are only four conditional recommended drugs, including pirfenidone(PFD), nidanib, N-acetylcysteine combination therapy, and antacid therapy[ 1 ]. The mechanism of PFD for IPF is still unclear, but the latest test confirmed that RGS2 is the basis of the anti-fibrotic effect of PFD which provides an a new direction to study the mechanism of PFD for IPF[ 4 ]. Although the emergence of PFD has brought some benefits to IPF patients, in the 2015 edition of the clinical practice guidelines for the treatment of IPF, PFD is defined as a conditional recommended drug for use due to its high effective dose, numerous adverse effects, and effectiveness only in mild to moderate IPF[ 1 , 5 , 6 ]. Therefore, the effect of monotherapy for PFD is not satisfactory, so it is important to find a reasonable treatment. Cell therapy based on stem cell technology has been a hot trend in recent years[ 7 ]. Human umbilical cord-derived mesenchymal stem cells(hUC-MSCs) are a type of adult stem cells with multi-directional differentiation potential derived from the mesoderm, and are widely used because of their simplicity of extraction, lack of ethical restrictions, strong immunomodulatory ability and low immunogenicity[ 8 , 9 ]. Clinical trials of hUC-MSCs for the treatment of various systemic diseases have been conducted worldwide, confirming the safety of hUC-MSCs in clinical applications[ 10 , 11 , 12 ]. Recent studies have shown that hUC-MSCs achieve immune modulation and inflammation control through paracrine secretion of multiple factors and microvesicles, which can reduce pro-fibrotic factors and collagen deposition, thus having therapeutic effects in animal models of bleomycin-induced early pulmonary fibrosis[ 13 , 14 ], but limited therapeutic effects in advanced pulmonary fibrosis[ 15 ]. Therefore, in this study, we established a mouse model of pulmonary fibrosis using bleomycin to investigate the effects of hUC-MSCs combined with low-dose PFD through observing the survival rate, lung pathological changes and detected the expression level of pulmonary fibrosis markers in mice. PFD and hUC-MSCs were combined to act on myofibroblasts to observe the effect of their combination on the expression of RGS2, which has antifibrotic effect, to preliminarily explore the possible mechanism of the combined treatment and provide a theoretical basis for the treatment of IPF with hUC-MSCs combined with PFD. Methods Reagents and cells PFD was purchased from Shanghai Adamas Reagents Co. Ltd.; hUC-MSCs were donated by Chongqing Stem Cell Therapy Engineering Technology Research Center; β-actin, type I collagen a1(Col1a1), type I collagen a2(Col1a2), α-smooth muscle actin(α-SMA), RGS2, calcium adhesion protein E(E-cad) and fibronectin(FN) primers were synthesized by Chengdu Kengke Zixi Biotechnology Co. Ltd.; ultrafiltration tubes were purchased from Millipore; other reagents were all domestic analytical purity. The mouse embryonic fibroblasts (NIH3T3) and human Embryonic Lung Fibroblasts(HLF-9) were purchased from Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Cell culture and drug treatments Cells were routinely cultured at 37 °C with 5 % CO2 in 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture (F12) supplemented with 10 % fetal bovine serum(FBS) and were used at < passage 6(P 6 ) for experiments. For dose–response experiments, human lung fibroblast cells were seeded into 12-well plates, starved in serum-free DMEM/F12 medium for 24 h, and then treated with the indicated concentrations of PFD for the indicated times. 6-well plate co-culture dishes were purchased from Thermo Fisher Scientific. Animal grouping and treatment SPF-grade 7-week-old C57BL/6 male rats were purchased from the Animal Experiment Center of Chongqing Medical University and housed in an SPF-grade breeding room at 22-26 ℃, 55-60% humidity, and 12 h/day light rotation, and modeling was started after 1 week of adaptation. Thirty C57BL/6 were divided into 6 groups using the random number method: control group (N), bleomycin model group (B), P 10 group (10 mg/kg PFD, P 10 ), P 30 group (30 mg/kg PFD, P 30 ), P 100 group (100 mg/kg PFD, P 100 ), and P 300 group (300 mg/kg PFD, P 300 ). Pulmonary fibrosis model was established by dripping bleomycin 3mg/kg into the lungs of mice at an equal rate through tracheal intubation, and the control group was given an equal amount of saline at an equal rate. After successful modeling, each PFD dose group was given 10 mg/kg, 30 mg/kg, 100 mg/kg, 300 mg/kg PFD suspension by gavage from day 7 after modeling, and the control group was given equal volume of saline once a day until day 21 after modeling. Another batch of 30 C57BL/6 was divided into 6 groups using the random number method: control group (N), hUC-MSCs control group (N+M), bleomycin model group (B), hUC-MSCs treatment group (B+M), P 100 group(100 mg/kg PFD, P 100 ) and hUC-MSCs+P 30 group (hUC-MSCs+30 mg/kg PFD, B+M+P 30 ). Modeling was performed by tracheal intubation with 3 mg/kg bleomycin drip, and on day 7 after modeling, hUC-MSCs control and hUC-MSCs treatment groups were injected with 5×10 5 /200 μL P4 generation hUC-MSCs via the tail vein of mice, and the non-MSCs group was given an equal amount of saline. Starting from day 7 after modeling, the P 100 groups and the hUC-MSCs+P 30 group (B+M+P 30 ) were given 100 mg/kg, and 30 mg/kg PFD suspension by gavage, respectively, and the control group was given an equal volume of saline once daily until day 21 after modeling. The survival of mice was observed and recorded, and lung tissues were collected on day 21. The mice survival curves, analysis of lung histopathology, determination of collagen content in the lungs, detection of mRNA, the markers of lung fibrosis, and detection of RGS2 expression will be briefly described in the following. Survival curves The survival of each group of mice was observed and recorded, and survival curves were plotted using Graphpad Prism 5.0. Lung histological analysis Paraformaldehyde-fixed mouse lung sections were analyzed with hematoxylin-eosin (HE) or Masson`s trichrome staining to assess fibrotic changes in the lungs. Three anterior, middle and posterior sections of each lung specimen of mice were taken for HE and Masson staining, respectively, and then five high magnification views of each section were selected for observation and scored separately using the modified Ashcroft method (scale range 0 to 8)[16]. The histopathological score of pulmonary fibrosis in each mouse was expressed as the mean score. Determination of intrapulmonary collagen content 80mg of lung tissue from the right lung was taken and determined according to the instructions of the Sircol method for measuring soluble collagen kit. A standard curve was made using collagen standards, and then the collagen concentration was calculated from the standard curve. Soluble collagen content was calculated according to the following formula: soluble collagen content = calculated collagen concentration × total volume of hydrolysate (1 mL)/80 mg × total wet weight of right lung tissue (mg). Fluorescence quantitative PCR Total RNA was extracted from each group of lung tissues using the TRIZOL method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. cDNA obtained was used for fluorescence quantitative PCR, and the expression of Col1a1, Col1a2 and α-SMA/ACTA2, RGS2, E-cad, and FN was detected using the β-actin gene as the internal reference gene levels. The genes, mRNA or protein are referred to using offcial gene symbols as provided by Te National Center for Biotechnology Information (NCBI;https:// www.ncbi.nlm.nih.gov/). Western Blot The expression of G protein signaling regulator 2 in lung tissues was detected in each group. Samples were electrophoresed and subjected to western blot using primary antibodies against RGS2 and β-actin. Acquisition and identification of hUC-MSCs P 2 generation hUC-MSCs were obtained from Chongqing Stem Cell Therapy Engineering Technology Research Center. hUC-MSCs were cultured and identified according to the methods reported in the literature[17]. hUC-MSCs were grown to P4 generation, and cell suspensions were collected after trypsin digestion, centrifuged and resuspended with PBS and counted to a final cell concentration of 2.5×10 6 /mL, placed on ice for use. Acquisition, grouping and treatment of myofibroblasts P 4 generation NIH3T3 and HLF-9 were spread in six-well plates at 1×10 6 , respectively, and when the cell fusion reached approximately 80%, they were replaced with fresh DMEM medium containing 1% fetal bovine serum and treated with 4ng/ml transforming growth factor-β1(TGF-β1) and cultured for 24 hours, i.e. myofibroblasts of both cell lines. Different concentrations of PFD were added in MFB: 0, 2, 4, 6, 8 and 10 mM, respectively, and the cells in each group were collected after 2h of incubation, and RGS2 mRNA expression was detected byRT- PCR to find out the effective concentration of RGS2 mRNA elevation caused by PFD.The effective concentration of PFD was added to MF separately, and the cells in each group were collected after 24h of culture separately, and the mRNA expression of fibrosis markers was detected by RT-PCR. The cells were divided into three groups according to different culture methods: NIH3T3 group (N) or HLF-9 group(H), hUC-MSCs group (M), and NIH3T3+hUC-MSCs group (N+M) or HLF-9+hUC-MSCs group (H+M). NIH3T3 or HLF-9 were cultured in the lower chamber of one well of the co-culture six-well plate alone (N or H). The P 4 generation hUC-MSCs were cultured in the upper chamber of the other well of the co-culture six-well plate alone (M). MFB were cultured in the lower chamber of the co-culture six-well plate while P4 generation hUC-MSCs were cultured in the upper chamber of the co-culture six-well plate (N+M or H+M). All were cultured using DMEM/F12 containing 5% fetal bovine serum. When the cell fusion of each group reached approximately 80%, the effective concentration of PFD or equal amount of PBS buffer was added, and each group of cells was collected after 24 h of culture respectively, and the mRNA expression of RGS2 and fibrosis markers were detected by PCR. The hUC-MSCs were divided into three groups: hUC-MSCs group (M), hUC-MSCs+TGF-β1 group (M+T), and hUC-MSCs+TGF-β1+PFD group (M+T+P). hUC-MSCs were cultured using DMEM/F12 containing 5% fetal bovine serum, and when the fusion of cells in each group reached approximately 80%, they were replaced with fresh DMEM containing 1% DMEM medium with fetal bovine serum, treated with 4ng/ml TGF-β1 and cultured for 24h. Cells in the M+T group were collected and detected by PCR for E-cad, FN,α-SMA mRNA expression; in the M+T+P group, PFD or equal amount of PBS buffer was added after 24h of TGF-β1 treatment and cells were collected after 24 h of culture and detected by PCR for E-cad, FN, α-SMA mRNA expression. Statistical analysis Data were expressed as mean±SEM. Comparisons between groups were made using Student's t test for unpaired observations or two-factor ANOVA and Bonferroni correction for multiple comparisons. p < 0.05 was considered statistically significant. Results Therapeutic effects of different doses of PFD in BLM induced pulmonary fibrosis mice Compared with the survival time in the model group(B), the low dose of PFD(10 mg/kg, P 10 ) alone could not prolong the survival time of mice with pulmonary fibrosis, and there was a trend to prolong the survival time in the low dose of PFD(30 mg/kg, P 30 ), the medium dose group (100 mg/kg, P 100 ) and the high dose PFD (300 mg/kg, P 300 ) alone, but the differences were not statistically significant ( P was 0.38, 0.08, 0.19, respectively) (Fig. 1 A). The Sircol method could detect the content of soluble collagen in the lungs of mice, and the collagen content of the right lung of mice in the model group was significantly higher compared with the control group ( p < 0.001). Compared with the model group, the collagen content of the P 10 group was not significantly changed; the collagen content of the P 30 group was reduced, and the difference was statistically significant ( p < 0.05); the right lung collagen of the P 100 group was significantly reduced ( p < 0.001); and the right lung collagen content of the P 300 group was reduced ( p < 0.001), but the reduction was not as great as that of the P 100 group ( p < 0.05) (Fig. 1 B) . The pathological changes in the mice of the model group on day 21 of modeling were: inflammatory cell exudation, widening of alveolar septa, formation of ground glass like, strip like, grid like structure at the base of both lungs, and formation of cystic changes of varying sizes in some areas, i.e., honeycomb lung. Compared with the lung pathology of the model group, there was no significant improvement in the P 10 group, a trend of improvement in the P 30 group ( p = 0.055), a decrease in the areas of lattice-like and honeycomb shape in the lungs of the P 100 group, and a statistical difference in the improvement of Ashcroft score ( p < 0.001). There was some improvement in the lung pathology of the P 300 group ( p < 0.05), but not as much as that of the P 100 group ( p < 0.05 ) (Fig. 1 C, 1 D). Masson staining stained the collagen in the lungs blue, which could reflect the severity of fibrosis in the lungs. A large amount of collagen deposition in the lung was seen microscopically in the model group. Compared with the model group, there was no significant change in intrapulmonary collagen deposition in the P 10 group; collagen deposition was significantly reduced in the P 30 , P 100 , and P 300 groups, but the collagen reduction in the P 30 and P 300 groups was not as great as that in the P 100 group ( p < 0.05) (Fig. 1 E, 1 F). Col1a1, Col1a2 and α-SMA are considered to be the main pulmonary fibrosis markers, and their levels reflect the degree of pulmonary fibrosis [ 18 ]. Col1a1 and Col1a2 mRNA levels were significantly higher in the model group compared with the control group. Compared with the model group, there was no significant change in Col1a1 mRNA in the P 10 group, and the expression of Col1a1 mRNA was significantly reduced in the P 30 , P 100 and P 300 groups, and the difference was statistically significant (Fig. 1 G). Col1a2 mRNA levels were reduced in all treatment groups except for the low-dose PFD P 10 group ( p < 0.05), and the most significant reduction in Col1a2 mRNA expression was observed in the P 100 group ( p < 0.001). Compared with the α-SMA mRNA level in the model group, there was no significant change in the P 10 group, a group decrease trend in P 30 and P 300 ( p = 0.07, p = 0.056), and a significant decrease in the P 100 group ( p < 0.05) (Fig. 1 I ). It can be seen that low-dose PFD (30 mg/kg) was the lowest effective dose for anti-fibrosis, medium-dose PFD (100 mg/kg) had the best efficacy among the groups with PFD alone, and high-dose PFD (300 mg/kg) was not as effective as medium-dose PFD for anti-fibrosis. Culture and characterization of hUC-MSCs hUC-MSCs cultured in DMEM/F12 medium at 37°C in a 5% CO2 incubator were assayed for surface-specific antigens of P 4 generation hUC-MSCs using flow cytometry. The results showed that the surface molecules CD34, CD45, and HLA-DR positivity of hUC-MSCs were less than 2%, and CD73, CD90, and CD105 positivity were higher than 95% (Fig. 2 A), and this result was in accordance with the standards published by the International Stem Cell Therapy Association in 2006. In addition, we also examined the multidirectional differentiation potential of hUC-MSCs, and our results showed that hUC-MSCs were differentiated to chondrogenic (Fig. 2 B), osteogenic (Fig. 2 C) and adipogenic cells (Fig. 2 D). Therapeutic effects of hUC-MSCs combined with P30 in BLM induced pulmonary fibrosis mice Studies have shown that hUC-MSCs can attenuate acute lung injury and early pulmonary fibrosis, but the therapeutic effect on established pulmonary fibrosis is controversial[ 10 , 11 ]. Preliminary experimental data from our group showed that hUC-MSCs alone by transcatheter tail vein injection were less effective in treating a mouse model of pulmonary fibrosis[ 19 , 20 ], therefore the therapeutic effect of hUC-MSCs in combination with PFD on middle and late-stage pulmonary fibrosis is unclear. In contrast, the efficacy of too high doses of PFD is also less satisfactory and has significant adverse effects. Therefore, we next explored the therapeutic effect of hUC-MSCs combined with the lowest effective dose of PFD (30 mg/kg, P 30 ) on pulmonary fibrosis in mice. The results showed that the hUC-MSCs combined with P 30 group significantly prolonged the survival time of mice (Fig. 3 A), and the difference was statistically significant ( p < 0.01), and the survival time of the hUC-MSCs combined with P30 group longer than the P 100 group. The combination group significantly reduced the collagen content in the lungs ( p < 0.001), and the collagen content was significantly lower than that in the P 100 group. The collagen content in the hUC-MSCs combined with P 30 group was significantly lower than that in the P 100 group (Fig. 3 B, p < 0.001)). Moreover, the combination group significantly improved bleomycin-induced pulmonary lesions with significantly better Ashcroft scores (P < 0.001) and significantly reduced intrapulmonary collagen deposition, all with better improvement than P 100 (Fig. 3 C- 3 F, p < 0.01 ). For the expression of pulmonary fibrosis markers, Col1a1, Col1a2, and α-SMA mRNA levels were significantly decreased in the hUC-MSCs combined with P 30 group relative to the model group (Fig. 3 G- 3 I, p < 0.01), where the reduced levels of Col1a1 and Col1a2 were statistically different compared to the P 100 group alone, while the reduced levels of α-SMA compared to the P 100 group tended to be statistically different ( p = 0.09). hUC-MSCs combined with low-dose P 30 elevated RGS2 mRNA and protein expression levels in mouse lung tissue Studies have shown that RGS2 is a novel mechanism for the antifibrotic effect of PFD[ 4 ]. Quantitative RT-PCR analysis in this study confirmed that, as expected, the treatment of pulmonary fibrosis in mice with the optimal effective dose of PFD (100 mg/kg) increased RGS2 mRNA levels in mouse lung tissues (Fig. 4 A, p < 0.05), and RGS2 mRNA levels in the hUC-MSCs combined with P 30 group were more elevated than those in the P 100 group ( p < 0.05). Western blot confirmed that RGS2 protein levels in lung tissue were also significantly increased after PFD treatment in mice (Fig. 4 B), and RGS2 protein levels in the hUC-MSCs combined with P 30 group were also more than those in the P 100 group. hUC-MSCs can affect the expression of RGS2 and the markers of pulmonary fibrosis of myofibroblasts by PFD treatment Studies have shown that MFB are the main cells of tissues after fibrosis and that TGF-β1 induces the conversion of fibroblasts to myofibroblasts[ 21 ]. In this experiment, two fibroblast model cells, MFB, were obtained after stimulation of two fibroblasts, NIH3T3 and HLF-9, for 24h using 4 ng/mL TGF-β1. hUC-MSCs and PFD for fibroblasts in subsequent experiments were added after TGF-β1 stimulation. Quantitative RT-PCR analysis confirmed that RGS2 mRNA was elevated in a concentration-dependent manner after PFD treatment of both MFB (Fig. 5 B, 5 C), and the elevation of RGS2 mRNA in both MFB treated with ≥ 6 mM PFD was statistically significant ( p < 0.05). As shown in Fig. 5 D, 5 E, the mRNA of fibrosis markers in both MFB decreased significantly after 24 h of PFD treatment, and the difference was statistically significant. The effect of PFD (8 mM PFD) on the expression of RGS2 and lung fibrosis markers within MFB were explored after co-culture of hUC-MSCs with MFB. The results showed that the differences in RGS2 mRNA levels were statistically significant in the H + M + P group compared with the H + P group (Fig. 5 F, p < 0.01), as well as in the N + M + P group compared with the N + P group (Fig. 5 G, p < 0.05), indicating that hUC-MSCs further elevated RGS2 mRNA after co-action of MFB with PFD. As shown in Fig. 5 H and 5 I, hUC-MSCs co-treatment of MFB with PFD resulted in a statistically significant decrease in mRNA for fibrosis markers in both MFB. We also examined the changes in mRNA levels of endothelial mesenchymal transition markers in hUC-MSCs. The results showed that E-cad mRNA was significantly increased and both FN and ACTA2 mRNA expression were significantly decreased after 8 mM PFD treatment of hUC-MSCs (Fig. 5 J, p < 0.05). Discussion Although PFD has been approved by the Food and Drug Administration(FDA) for the treatment of IPF, its therapeutic effects are limited and its adverse effects are large. In the present study, we used hUC-MSCs combined with low-dose PFD for the treatment of pulmonary fibrosis in mice. We found combined with hUC-MSCs, the low-dose PFD could exert antifibrotic effects superior to its 3-fold or even 10-fold dose alone. Therefore, it can be speculated that the combination of hUC-MSCs and PFD can enhance the antifibrotic efficacy of PFD, reduce the dosage of PFD, and decrease the incidence of adverse effects, in order to enhance the tolerability and compliance of IPF patients. Hisashi O et al. used three concentrations of 10, 30, and 100 mg/kg PFD to treat a mouse model of bleomycin-induced pulmonary fibrosis, and the results of this study showed that the antifibrotic effect of PFD was positively correlated with the dose[ 22 ]. However, the efficacy of PFD containing higher dose gradients on pulmonary fibrosis in mice has not been reported in this literature. In the present study, four dose gradients of 10, 30, 100, and 300 mg/kg PFD were applied to explore the therapeutic effects on pulmonary fibrosis, and contrary to expectations, the highest dose (300 mg/kg) PFD did not achieve better antifibrotic effects than the medium dose PFD (100 mg/kg). In clinical trials of PFD for IPF conducted by Azuma A et al. and Wijsenbeek MS et al. adverse effects of PFD were predominantly gastrointestinal reaction and were positively correlated with dose, with an incidence of 40–60%, and some patients discontinued the drug due to intolerance[ 23 , 24 ]. Combined with the observation that the appetite of mice in the high-dose PFD group was poorer than that in the medium and low-dose PFD groups, it can be assumed that the PFD dose exceeded the appropriate range, resulting in gastrointestinal adverse reactions and reducing the therapeutic effect of PFD. Therefore, we cannot expect to achieve better anti-fibrotic effect by increasing the dose of PFD in clinical practice. For this reason, in order to avoid the adverse effects of higher doses of PFD, the combination of the low dose, 30 mg/kg PFD, with hUC-MSCs was used in this study instead of 100 mg/kg PFD with hUC-MSCs. RGS2 is a known negative regulator of G protein signaling that inhibits the amplitude and duration of signals mediated by GQ-coupled GPCRs[ 25 , 26 ]. Some GQ-coupled GPCRs and their ligands are important drivers of pulmonary fibrosis[ 27 , 28 ]. Jang HS et al. demonstrated in animal experiments that knockdown of RGS2 leads to renal fibrosis in mice after unilateral ureteral obstruction (UUO), suggesting that endogenous RGS2 has an anti-fibrotic function[ 29 ]. Similarly, Xie Y et al. showed that the gene that they screened for rapid upregulation in human lung fibroblasts in response to pirfenidone using gene microarrays was RGS2, and confirmed that endogenous RGS2 exhibited anti-pulmonary fibrosis function and that RGS2 underlies the anti-fibrotic effect of PFD, thus providing some ideas to investigate the mechanism of PFD for the treatment of pulmonary fibrosis[ 4 ]. In our in vitro experiments, we observed that hUC-MSCs increased the expression of endogenous RGS2 in MFB, allowing pirfenidone to exert a stronger anti-fibrotic effect, which may be one of the reasons why hUC-MSCs combined with low-dose PFD could achieve better efficacy. However, the mechanism of how hUC-MSCs increase the expression of RGS2 needs to be explored, and the mechanism of how PFD exerts its antifibrotic effect through RGS2 also needs to be further investigated. The pathogenesis of IPF is still unclear, but the main pathological changes are the proliferation and aggregation of large numbers of myofibroblasts and the deposition of extracellular matrix, of which the main component is collagen [ 30 ]. MFB, as the main effector cell of IPF, has a negative correlation with the severity and prognosis of IPF disease [ 31 ]. It was found that in addition to their anti-fibrotic effects, hUC-MSCs still have a certain degree in pro-fibrotic effects, especially in the fibrotic environment, which can be converted to MFB through endothelial mesenchymal conversion[ 32 , 33 ], which may be one of the reasons for the controversial effects of hUC-MSCs in the treatment of pulmonary fibrosis. And PFD, as a multi-cytokine inhibitor, can inhibit the expression of cytokines such as TGF-β1, basic fibroblast growth factor(bFGF), and connective tissue growth factor(CTGF) of target cells (myo)fibroblasts, thus suppressing the proliferation of (myo)fibroblasts and the synthesis of collagen[ 34 , 35 ]. Therefore, in this study we examined the changes in mRNA levels of endothelial mesenchymal transition markers in hUC-MSCs. The results showed that E-cad mRNA was significantly increased and FN and α-SMA mRNA expression were both significantly decreased after PFD treatment of hUC-MSCs. This result indicated that PFD inhibited the conversion of hUC-MSCs to mesenchymal cells, thereby reducing the conversion of hUC-MSCs to MFB in order to give hUC-MSCs the opportunity to exert maximum anti-fibrotic effects, which may also be one of the mechanisms why hUC-MSCs combined with PFD could achieve better efficacy compared with PFD alone. Conclusion In conclusion, the results of the present study provide direct evidence that hUC-MSCs combined with low-dose PFD had a therapeutic effect on a mouse model of pulmonary fibrosis and delayed the progression of IPF, providing a new idea for the clinical treatment of IPF. In addition, this study also found that the possible mechanism of the efficacy of hUC-MSCs in combination with low-dose PFD may be related to a significant increase in the expression of the anti-fibrotic protein RGS2, but the in-depth mechanism of the combined treatment of both needs to be further studied and explored. Abbreviations hUC-MSCs, umbilical cord-derived mesenchymal stem cells; PFD, pirfenidone; P 10, 10 mg/kg PFD; P 30 , 30 mg/kg PFD; P 100 , 100 mg/kg PFD; P 300 , 300 mg/kg PFD; HE, hematoxylin-eosin ; PCR, polymerase Chain Reaction; RGS2, regulator of G protein signaling 2; MFB, myofibroblasts; IPF, diopathic pulmonary fibrosis; Col1a1, type I collagen a1; Col1a2, type I collagen a2; α-SMA, α-smooth muscle actin; ACTA2, actin alpha 2; E-cad, calcium adhesion protein E; FN, fibronectin; NIH3T3, mouse embryonic fibroblasts; HLF-9, human Embryonic Lung Fibroblasts; DMEM, Dulbecco's Modified Eagle's Medium; F12, Ham's F-12 Nutrient; FBS, fetal bovine serum; P 6 , passage 6; h, hours; N, control group; B, bleomycin model group; N+M, hUC-MSCs control group; B+M, hUC-MSCs treatment group; B+M+P 30 , hUC-MSCs+30 mg/kg PFD group; TGF-β1, transforming growth factor-β; N, NIH3T3 group; H, HLF-9 group; M, hUC-MSCs group, N+M, NIH3T3+hUC-MSCs group; H+M, HLF-9+hUC-MSCs group; M+T, hUC-MSCs+TGF-β1 group; M+T+P, hUC-MSCs+TGF-β1+PFD group; FDA, Food and Drug Administration; bFGF, basic fibroblast growth factor; CTGF, connective tissue growth factor. Declarations Acknowledgments We gratefully acknowledge Dr. Huafeng Pan at Chongqing Stem Cell Therapy Engineering Technology Research Center for providing hUC-MSCs. Funding This work was supported by National Nature Science Foundation of China (NSFC 81670018), Key project from Chinese Ministry of Science and Technology (2016YFA0101300). Availability of data and material All data generated or analyzed during this study are included in this published article and its Additional file 1. Authors, contributions XW, and ZF participated in research design. XW, HG, HJQ and OZ conducted experiments. XW, HJQ and OZ contributed materials and reagents. XW performed data analysis. XW, HML, ZF and LNC wrote or contributed to the writing of the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate No human participants, human data or human tissue involved in studies reported in this manuscript. All animal studies were approved by the Animal Ethics Committee of Chongqing Medical University. Author details 1 Division of pediatric pulmonology and immunology, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan, People’s Republic of China. 2 Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan, People’s Republic of China. 3 NHC Key Laboratory of Chronobiology , Sichuan University, Chengdu 610065, Sichuan, People’s Republic of China. 4 Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu 610075, Sichuan, People’s Republic of China. 5 Pediatric Research Institute, Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing 400014, People’s Republic of China. 6 Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, People’s Republic of China. 7 Chongqing Engineering Research Center of Stem Cell Therapy, Chongqing 400014, People’s Republic of China. References Raghu G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, et al . An official ATS/ERS/JRS/ALAT clinical practice guideline: treatment of idiopathic pulmonary fibrosis. An update of the 2011 clinical practice guideline. Am J Respir Crit Care Med. 2015; 192(2): e3-e19. Raghu G, Chen SY, Yeh WS, Maroni B, Li Q, Lee YC, et al. Idiopathic pulmonary fibrosis in US medicare beneficiaries aged 65 years and older: incidence, prevalence and survival, 2001-11. Lancet Respir Med. 2014;2:566–72. Hutchinson J, Fogarty A, Hubbard R, McKeever T. Global incidence and mortality of idiopathic pulmonary fibrosis: a systematic review. Eur Respir J 2015; 46(3): 795–806. Xie Y, Jiang H, Zhang Q, Mehrotra S, Abel P, Toews M, et al . Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis. Respir Res, 2016; 22;17(1):103. King TE Jr, Bradford WZ, Castro-Bernardini S, Fagan EA, Glaspole I, Glassberg MK, et al . A Phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N Engl J Med 2014; 37: 2083–92. Noble PW, Albera C, Bradford WZ, Costabel U, Glassberg MK, Kardatzke D, et al . Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. Lancet. 2011; 377: 1760–9. Ding DC, Chang YH, Shyu WC, Lin SZ. Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy. Cell Transplant. 2015; 24(3): 339–47. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiplehuman organs. Cell Stem Cell. 2008; 3(3): 301–13. Bianco P. “Mesenchymal” stem cells. Annu Rev Cell Dev Biol. 2014;30:677–704. Chang YS, Ahn SY, Yoo HS, Sung SI, Choi SJ, Oh WI, et al. Mesenchymal stem cells for bronchopulmonary dysplasia: Phase 1 dose-escalation clinical trial. J Pediatr. 2014; 64(5): 966–72. Azuma A, Nukiwa T, Tsuboi E, Suga M, Abe S, Nakata K, et al. Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2005; 171: 1040–7. 28.Wilson JG, Liu KD, Zhuo H, Caballero L, McMillan M, Fang X, et al. Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial. Lancet Respir Med. 2015; 3(1): 24–32. Reddy M, Fonseca L, Gowda S, Chougule B, Hari A, Totey S. Human adipose-derived mesenchymal stem cells attenuate early stage of bleomycin induced pulmonary fibrosis: comparison with pirfenidone. Int J Stem Cells. 2016; 9: 192–206. Moodley Y, Atienza D, Manuelpillai U, Samuel CS, Tchongue J, Ilancheran S, et al . Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin induced lung injury. Am J Pathol. 2009; 175: 303–13. Yan X, Liu Y, Han Q, et al. Injured micro-environment directly guides the differentiation of engrafted flk-1 mesenchymal stem cell in lung. Exp Hematol. 2007, 35: 1466–75. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988; 41: 467–70. Hua Z, Yi X, Yunqiu X, Rong Z, Daiyin T, Ting W, et al. Therapeutic effects of human umbilical cord-derived mesenchymal stem cells in acute lung injury mice. Sci Rep. 2017; 7: 39889. Fernandez IE, Eickelberg O. New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis. Lancet. 2012;18;380(9842):680–8. Zhang Y. The investigation of human umbilical cord mesenchymal stem cells on pulmonary fibrosis mice models[D]. Chongqing, Chongqing Medical University. 2015. Peng DY. Combination of human umbilical cord mesenchymal stem cells and nintedanib ameliorates mice pulmonary fibrosis induced by bleomycin[D]. Chongqing, Chongqing Medical University. 2017. Martin MM, Buckenberger JA, Jiang J, Malana GE, Knoell DL, Feldman DS, et al. TGF-beta1 stimulates human AT1 receptor expression in lung fibroblasts by cross talk between the Smad, p38 MAPK, JNK, and PI3K signaling pathways. Am J Physiol Lung Cell Mol Physiol. 2007; 293: L790-9. Oku H, Shimizu T, Kawabata T, Nagira M, Hikita I, Ueyama A, et al . Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis. Eur J Pharmacol 2008; 590(12/3): 400–8. Azuma A, Nukiwa T, Tsuboi E, Suga M, Abe S, Nakata K, et al . Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2005; 171: 1040–7. Wijsenbeek MS, Grutters JC, Wuyts WA. Early experience of pirfenidone in daily clinical practice in belgium and the netherlands: a retrospective cohort analysis. Adv Ther 2015; 32: 691–704. Bernstein LS, Ramineni S, Hague C, Cladman W, Chidiac P, Levey AI, et al . RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling. J Biol Chem. 2004;279:21248–56. Heximer SP, Watson N, Linder ME, Blumer KJ, Hepler JR. RGS2/G0S8 is a selective inhibitor of Gαq function. Proc Natl Acad Sci USA. 2008;94:14389–93. Tager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, et al . The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat Med. 2008; 14:45–54. Ghavami A, Hunt RA, Olsen MA, Zhang J, Smith DL, Kalgaonkar S, et al . Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptor-mediated signaling and adenylyl cyclase activity. Cell Signal. 2004;16:711–21. Jang HS, Kim JI, Noh M, Rhee MH, Park KM. Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis. Biochim Biophys Acta. 2014; 1842(9):1733–41. Thomas AW, Thirumalai RR. Mechanisms of fbrosis: therapeutic translation for fbrotic disease. Nat Med. 2012; 18(7): 1028–40. Enomoto N, Suda T, Kato M, Kaida Y, Nakamura Y, Imokawa S, et al . Quantitative analysis of fbroblastic foci in usual interstitial pneumonia. Chest. 2006; 130(1): 22–9. Gazdhar A, Grad I, Tamò L, Gugger M, Feki A, Geiser T. The secretome of induced pluripotent stem cells reduces lung fbrosis in part by hepatocyte growth factor. Stem Cell Res Ther 2014; 5: 123. Mezey E. The therapeutic potential of bone-marrow-derived stromal cells. J Cell Biochem. 2011, 112(10): 2683–2687. Oku H, Shimizu T, Kawabata T, Nagira M, Hikita I, Ueyama A, et al . Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced pulmonary fibrosis. Eur J Pharmacol. 2008; 20: 400–8. Myllarniemi M, Kaarteenaho R. Pharmacological treatment of idiopathic pulmonary fibrosis preclinical and clinical studies of pirfenidone, nintedanib, and N-acetylcysteine. Eur Clin Respir J . 2015; 10;2. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 30 May, 2022 Reviews received at journal 17 May, 2022 Reviews received at journal 02 May, 2022 Reviewers agreed at journal 18 Apr, 2022 Reviewers invited by journal 18 Apr, 2022 Editor assigned by journal 12 Apr, 2022 Submission checks completed at journal 11 Apr, 2022 First submitted to journal 31 Mar, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1507756","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":97925916,"identity":"6de7ce16-9532-40fe-a341-f6a0fd7c8526","order_by":0,"name":"Xian Wu","email":"","orcid":"","institution":"Sichuan University, Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xian","middleName":"","lastName":"Wu","suffix":""},{"id":97925917,"identity":"7a26a91a-71d7-4171-929e-c03099eff637","order_by":1,"name":"Hao Gou","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Gou","suffix":""},{"id":97925918,"identity":"2cf68270-bb54-464e-b082-f14a6c2eb4aa","order_by":2,"name":"Huijun Qiu","email":"","orcid":"","institution":"Chengdu University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huijun","middleName":"","lastName":"Qiu","suffix":""},{"id":97925920,"identity":"5b7ea67d-f822-4574-a2e1-4484d0992354","order_by":3,"name":"Hanmin Liu","email":"","orcid":"","institution":"Sichuan University, Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanmin","middleName":"","lastName":"Liu","suffix":""},{"id":97925921,"identity":"032a3f16-8d8f-4691-b9d9-3ee6c400546c","order_by":4,"name":"Zhou Fu","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhou","middleName":"","lastName":"Fu","suffix":""},{"id":97925922,"identity":"1ffcaeaf-f72a-41c4-9864-f7c210349093","order_by":5,"name":"Lina Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYDACCSB+wGAjx8/MfPgB8VoSGNKMJdvZ0gxI0XIo0eA8j4IEUTr4Zzcfe5C440CC8WEeBgOGGptowpbcOZZukHjmTp7ZYd4DDxiOpeU2ENJiIJFjJpHY9qzY7DBfggFjw2FitOR/A2o5nLi5mcdAgkgtOWxgLRuYidUicSMN5LA0Y4nDwEBOIMYv/DOSn0l8bANGZf/hww8+1NgQ1oIKEkhTPgpGwSgYBaMAFwAA5i8+ngwED5IAAAAASUVORK5CYII=","orcid":"","institution":"Sichuan University, Children’s Hospital of Chongqing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2022-03-31 04:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1507756/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1507756/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20363889,"identity":"8b88f942-13f5-406f-9006-f6321608173e","added_by":"auto","created_at":"2022-04-14 20:25:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2701399,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic effects of different doses of PFD in BLM induced pulmonary fibrosis mice. \u003cstrong\u003eA\u003c/strong\u003e The KaplaneMeier survival curves of different doses of PFD groups. \u003cstrong\u003eB\u003c/strong\u003e The total content of soluble collagen in the right lungs of different groups in mice. \u003cstrong\u003eC,E\u003c/strong\u003e Representative pictures of HE and Masson staining of lung sections of different groups in pulmonary fibrosis mice. Scale bars: 200 μm. \u003cstrong\u003eD,F \u003c/strong\u003eThe Ashcroft score of HE and Masson staining of different groups in pulmonary fibrosis mice. \u003cstrong\u003eG,H,I\u003c/strong\u003e The mRNA levels of main pulmonary fibrosis markers(Col1a1, Col1a2 and α-SMA) of different groups in mice.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1507756/v1/04bc417d842756ecd39b47ec.jpg"},{"id":20363885,"identity":"fdb010b9-d4fa-48d7-afbc-6b894fd3e7ad","added_by":"auto","created_at":"2022-04-14 20:25:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105022,"visible":true,"origin":"","legend":"\u003cp\u003eThe pluripotency of hUC-MSCs. \u003cstrong\u003eA\u003c/strong\u003e The expression of the surface markers CD34, CD45, HLA-DR, CD73, CD90, and CD105 on hUC-MSCs detected by flow cytometry. \u003cstrong\u003eB, C, D\u003c/strong\u003e The results of hUC-MSCs differentiation into chrondrocytes, osteocytes, and adipocytes.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1507756/v1/b85a7dfb4f7445f78d930d04.jpg"},{"id":20363887,"identity":"6f08bb2d-ff68-497b-83cb-dce8f54b21fb","added_by":"auto","created_at":"2022-04-14 20:25:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2961754,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic effects of hUC-MSCs combined with P\u003csub\u003e30\u003c/sub\u003e in BLM induced pulmonary fibrosis mice. \u003cstrong\u003eA\u003c/strong\u003e The KaplaneMeier survival curves of different groups. \u003cstrong\u003eB\u003c/strong\u003e The total content of soluble collagen in the right lungs of different groups in mice. \u003cstrong\u003eC,E\u003c/strong\u003e Representative pictures of HE and Masson staining of lung sections of different groups in pulmonary fibrosis mice. Scale bars: 200 μm. \u003cstrong\u003eD,F \u003c/strong\u003eThe Ashcroft score of HE and Masson staining of different groups. \u003cstrong\u003eG,H,I\u003c/strong\u003e The mRNA levels of main pulmonary fibrosis markers(Col1a1, Col1a2 and α-SMA) of different groups in pulmonary fibrosis mice.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1507756/v1/193935335bb8b2c9b15187a2.jpg"},{"id":20363886,"identity":"3156bac0-952d-4d82-abb7-1bb91040cc34","added_by":"auto","created_at":"2022-04-14 20:25:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":597935,"visible":true,"origin":"","legend":"\u003cp\u003eThe RGS2 mRNA and protein expression levels in mouse lung tissue of different groups. \u003cstrong\u003eA\u003c/strong\u003e The RGS2 mRNA levels detected by PCR in mouse lung tissue of different groups. \u003cstrong\u003eB\u003c/strong\u003e The RGS2 protein expression levels detected by western blot in mouse lung tissue of different groups.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1507756/v1/f93768932591a72fc205994a.jpg"},{"id":20364307,"identity":"6f3f942a-52c5-4232-9488-f4f66acf11c2","added_by":"auto","created_at":"2022-04-14 20:30:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1108574,"visible":true,"origin":"","legend":"\u003cp\u003eRGS2 suppresses the profibrotic effects in HLF-9 and NIH3T3. \u003cstrong\u003eA,B\u003c/strong\u003e HLF-9(\u003cstrong\u003eA\u003c/strong\u003e) and NIH3T3(\u003cstrong\u003eB\u003c/strong\u003e) were treated with various concentrations of PFD (0-10 mM) for 2 h and then were harvested for RT-PCR analysis of RGS2 and β-actin mRNA levels. \u003cstrong\u003eC,D\u003c/strong\u003e HLF-9(\u003cstrong\u003eC\u003c/strong\u003e) and NIH3T3(D) were treated with 8 mM PFD over a time course of 24 h and then were harvested for RT-PCR analysis of fibrosis markers mRNA levels. \u003cstrong\u003eE\u003c/strong\u003e Schematic diagram of single culture and co-culture of hUC-MSCs and MFB. \u003cstrong\u003eF,G\u003c/strong\u003e HLF-9(\u003cstrong\u003eF\u003c/strong\u003e) and NIH3T3(\u003cstrong\u003eG\u003c/strong\u003e) were single culture or co-culture with hUC-MSCs, then were treated with 8 mM PFD or equal amount of PBS buffer for 2 h and then were harvested for RT-PCR analysis of RGS2 and β-actin mRNA levels. \u003cstrong\u003eH,I\u003c/strong\u003e HLF-9(\u003cstrong\u003eH\u003c/strong\u003e) and NIH3T3(\u003cstrong\u003eI\u003c/strong\u003e) were single culture or co-culture with hUC-MSCs, and were treated with 8 mM PFD for 24 h and then were harvested for RT-PCR analysis of fibrosis markers mRNA levels. \u003cstrong\u003eJ\u003c/strong\u003e hUC-MSCs were treated with 4ng/ml TGF-β1 and cultured for 24h, and treated with 8 mM PFD or equal amount of PBS buffer for 24 h and then were harvested for RT-PCR analysis of E-cad, FN, α-SMA mRNA levels.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1507756/v1/261e6f810db7dbfe039fb0b7.jpg"},{"id":20364312,"identity":"bfd57eca-88db-4459-9408-d4e6de5b54e5","added_by":"auto","created_at":"2022-04-14 20:30:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":772423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1507756/v1/67bb318e-9c06-46eb-b8bc-2d25e1d923bf.pdf"},{"id":20363890,"identity":"ff5dee3f-ad0b-4924-94d3-6a93c7c64e1a","added_by":"auto","created_at":"2022-04-14 20:25:30","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12751352,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1507756/v1/8b1fde6c998ffd396e11f457.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Human Umbilical Cord Mesenchymal Stem Cells Combined with Pirfenidone upregulates the Expression of RGS2 in the Pulmonary Fibrosis in Mice","fulltext":[{"header":"Background","content":"\u003cp\u003eIdiopathic pulmonary fibrosis(IPF), a diffuse inflammatory disease of the lower respiratory tract with unknown etiology, is a chronic interstitial lung disease characterized by progressive dyspnea and progressive deterioration of lung function[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Its incidence is increasing year by year, the median survival is only 2.5\u0026ndash;3.5 years, and the prognosis is worse than that of many types of cancer[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe 2015 edition of the clinical practice guidelines for the treatment of IPF states that the use of prednisone, azathioprine, N-acetylcysteine monotherapy, anticoagulants, platelet-derived growth factor receptor antagonists, endothelin receptor antagonists, and 5-phosphodiesterase inhibitors is strongly discouraged in patients with IPF, and that there are only four conditional recommended drugs, including pirfenidone(PFD), nidanib, N-acetylcysteine combination therapy, and antacid therapy[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The mechanism of PFD for IPF is still unclear, but the latest test confirmed that RGS2 is the basis of the anti-fibrotic effect of PFD which provides an a new direction to study the mechanism of PFD for IPF[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although the emergence of PFD has brought some benefits to IPF patients, in the 2015 edition of the clinical practice guidelines for the treatment of IPF, PFD is defined as a conditional recommended drug for use due to its high effective dose, numerous adverse effects, and effectiveness only in mild to moderate IPF[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, the effect of monotherapy for PFD is not satisfactory, so it is important to find a reasonable treatment.\u003c/p\u003e \u003cp\u003eCell therapy based on stem cell technology has been a hot trend in recent years[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Human umbilical cord-derived mesenchymal stem cells(hUC-MSCs) are a type of adult stem cells with multi-directional differentiation potential derived from the mesoderm, and are widely used because of their simplicity of extraction, lack of ethical restrictions, strong immunomodulatory ability and low immunogenicity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Clinical trials of hUC-MSCs for the treatment of various systemic diseases have been conducted worldwide, confirming the safety of hUC-MSCs in clinical applications[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recent studies have shown that hUC-MSCs achieve immune modulation and inflammation control through paracrine secretion of multiple factors and microvesicles, which can reduce pro-fibrotic factors and collagen deposition, thus having therapeutic effects in animal models of bleomycin-induced early pulmonary fibrosis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], but limited therapeutic effects in advanced pulmonary fibrosis[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, in this study, we established a mouse model of pulmonary fibrosis using bleomycin to investigate the effects of hUC-MSCs combined with low-dose PFD through observing the survival rate, lung pathological changes and detected the expression level of pulmonary fibrosis markers in mice. PFD and hUC-MSCs were combined to act on myofibroblasts to observe the effect of their combination on the expression of RGS2, which has antifibrotic effect, to preliminarily explore the possible mechanism of the combined treatment and provide a theoretical basis for the treatment of IPF with hUC-MSCs combined with PFD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eReagents and cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePFD was purchased from Shanghai Adamas Reagents Co. Ltd.; hUC-MSCs were donated by Chongqing Stem Cell Therapy Engineering Technology Research Center; \u0026beta;-actin, type I collagen a1(Col1a1), type I collagen a2(Col1a2), \u0026alpha;-smooth muscle actin(\u0026alpha;-SMA), RGS2, calcium adhesion protein E(E-cad) and fibronectin(FN) primers were synthesized by Chengdu Kengke Zixi Biotechnology Co. Ltd.; ultrafiltration tubes were purchased from Millipore; other reagents were all domestic analytical purity. The mouse embryonic fibroblasts (NIH3T3) and human Embryonic Lung Fibroblasts(HLF-9) were purchased from Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Cell culture and drug treatments Cells were routinely cultured at 37 \u0026deg;C with 5 % CO2 in 1:1 mixture of Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium (DMEM) and Ham\u0026apos;s F-12 Nutrient Mixture (F12) supplemented with 10 % fetal bovine serum(FBS) and were used at \u0026lt; passage 6(P\u003csub\u003e6\u003c/sub\u003e) for experiments. For dose\u0026ndash;response experiments, human lung fibroblast cells were seeded into 12-well plates, starved in serum-free DMEM/F12 medium for 24 h, and then treated with the indicated concentrations of PFD for the indicated times. 6-well plate co-culture dishes were purchased from Thermo Fisher Scientific.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal grouping and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPF-grade 7-week-old C57BL/6 male rats were purchased from the Animal Experiment Center of Chongqing Medical University and housed in an SPF-grade breeding room at 22-26\u0026nbsp;℃, 55-60% humidity, and 12 h/day light rotation, and modeling was started after 1 week of adaptation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThirty C57BL/6 were divided into 6 groups using the random number method: control group (N), bleomycin model group (B), P\u003csub\u003e10\u003c/sub\u003e group (10 mg/kg PFD, P\u003csub\u003e10\u003c/sub\u003e), P\u003csub\u003e30\u003c/sub\u003e group (30 mg/kg PFD, P\u003csub\u003e30\u003c/sub\u003e), P\u003csub\u003e100\u003c/sub\u003e group (100 mg/kg PFD, P\u003csub\u003e100\u003c/sub\u003e), and P\u003csub\u003e300\u003c/sub\u003e group (300 mg/kg PFD, P\u003csub\u003e300\u003c/sub\u003e). Pulmonary fibrosis model was established by dripping bleomycin 3mg/kg into the lungs of mice at an equal rate through tracheal intubation, and the control group was given an equal amount of saline at an equal rate. After successful modeling, each PFD dose group was given 10 mg/kg, 30 mg/kg, 100 mg/kg, 300 mg/kg PFD suspension by gavage from day 7 after modeling, and the control group was given equal volume of saline once a day until day 21 after modeling.\u003c/p\u003e\n\u003cp\u003eAnother batch of 30 C57BL/6 was divided into 6 groups using the random number method: control group (N), hUC-MSCs control group (N+M), bleomycin model group (B), hUC-MSCs treatment group (B+M), P\u003csub\u003e100\u003c/sub\u003e group(100 mg/kg PFD, P\u003csub\u003e100\u003c/sub\u003e) and hUC-MSCs+P\u003csub\u003e30\u003c/sub\u003e group (hUC-MSCs+30 mg/kg PFD, B+M+P\u003csub\u003e30\u003c/sub\u003e). Modeling was performed by tracheal intubation with 3 mg/kg bleomycin drip, and on day 7 after modeling, hUC-MSCs control and hUC-MSCs treatment groups were injected with 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e/200\u0026nbsp;\u0026mu;L P4 generation hUC-MSCs via the tail vein of mice, and the non-MSCs group was given an equal amount of saline. Starting from day 7 after modeling, the P\u003csub\u003e100\u003c/sub\u003e groups and the hUC-MSCs+P\u003csub\u003e30\u003c/sub\u003e group (B+M+P\u003csub\u003e30\u003c/sub\u003e) were given 100 mg/kg, and 30 mg/kg PFD suspension by gavage, respectively, and the control group was given an equal volume of saline once daily until day 21 after modeling. The survival of mice was observed and recorded, and lung tissues were collected on day 21. The mice survival curves, analysis of lung histopathology, determination of collagen content in the lungs, detection of mRNA, the markers of lung fibrosis, and detection of RGS2 expression will be briefly described in the following.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurvival curves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe survival of each group of mice was observed and recorded, and survival curves were plotted using Graphpad Prism 5.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLung histological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaformaldehyde-fixed mouse lung sections were analyzed with hematoxylin-eosin (HE) or Masson`s trichrome staining to assess fibrotic changes in the lungs. Three anterior, middle and posterior sections of each lung specimen of mice were taken for\u0026nbsp;HE and Masson staining, respectively, and then five high magnification views of each section were selected for observation and scored separately using the modified Ashcroft method (scale range 0 to 8)[16]. The histopathological score of pulmonary fibrosis in each mouse was expressed as the mean score.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of intrapulmonary collagen content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e80mg of lung tissue from the right lung was taken and determined according to the instructions of the Sircol method for measuring soluble collagen kit. A standard curve was made using collagen standards, and then the collagen concentration was calculated from the standard curve. Soluble collagen content was calculated according to the following formula: soluble collagen content = calculated collagen concentration\u0026nbsp;\u0026times;\u0026nbsp;total volume of hydrolysate (1 mL)/80 mg\u0026nbsp;\u0026times;\u0026nbsp;total wet weight of right lung tissue (mg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence quantitative PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from each group of lung tissues using the TRIZOL method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. cDNA obtained was used for fluorescence quantitative PCR, and the expression of Col1a1, Col1a2 and\u0026nbsp;\u0026alpha;-SMA/ACTA2, RGS2, E-cad, and FN was detected using the\u0026nbsp;\u0026beta;-actin gene as the internal reference gene levels. The\u0026nbsp;genes, mRNA or protein are referred to using offcial gene symbols as provided by Te National Center for Biotechnology Information (NCBI;https:// www.ncbi.nlm.nih.gov/).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of G protein signaling regulator 2 in lung tissues was detected in each group. Samples were electrophoresed and subjected to western blot using primary antibodies against RGS2 and\u0026nbsp;\u0026beta;-actin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcquisition and identification of hUC-MSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP\u003csub\u003e2\u003c/sub\u003e generation hUC-MSCs were obtained from Chongqing Stem Cell Therapy Engineering Technology Research Center. hUC-MSCs were cultured and identified according to the methods reported in the literature[17]. hUC-MSCs were grown to P4 generation, and cell suspensions were collected after trypsin digestion, centrifuged and resuspended with PBS and counted to a final cell concentration of 2.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e/mL, placed on ice for use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcquisition, grouping and treatment of myofibroblasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP\u003csub\u003e4\u003c/sub\u003e generation NIH3T3 and HLF-9 were spread in six-well plates at 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e, respectively, and when the cell fusion reached approximately 80%, they were replaced with fresh DMEM medium containing 1% fetal bovine serum and treated with 4ng/ml transforming growth factor-\u0026beta;1(TGF-\u0026beta;1) and cultured for 24 hours, i.e. myofibroblasts of both cell lines.\u003c/p\u003e\n\u003cp\u003eDifferent concentrations of PFD were added in MFB: 0, 2, 4, 6, 8 and 10 mM, respectively, and the cells in each group were collected after 2h of incubation, and RGS2 mRNA expression was detected byRT- PCR to find out the effective concentration of RGS2 mRNA elevation caused by PFD.The effective concentration of PFD was added to MF separately, and the cells in each group were collected after 24h of culture separately, and the mRNA expression of fibrosis markers was detected by RT-PCR.\u003c/p\u003e\n\u003cp\u003eThe cells were divided into three groups according to different culture methods: NIH3T3 group (N) or HLF-9 group(H), hUC-MSCs group (M), and NIH3T3+hUC-MSCs group (N+M) or HLF-9+hUC-MSCs group (H+M). NIH3T3 or HLF-9 were cultured in the lower chamber of one well of the co-culture six-well plate alone (N or H). The P\u003csub\u003e4\u003c/sub\u003e generation hUC-MSCs were cultured in the upper chamber of the other well of the co-culture six-well plate alone (M). MFB were cultured in the lower chamber of the co-culture six-well plate while P4 generation hUC-MSCs were cultured in the upper chamber of the co-culture six-well plate (N+M or H+M). All were cultured using DMEM/F12 containing 5% fetal bovine serum. When the cell fusion of each group reached approximately 80%, the effective concentration of PFD or equal amount of PBS buffer\u003c/p\u003e\n\u003cp\u003ewas added, and each group of cells was collected after 24 h of culture respectively, and the mRNA expression of RGS2 and fibrosis markers were detected by PCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe hUC-MSCs were divided into three groups: hUC-MSCs group (M), hUC-MSCs+TGF-\u0026beta;1 group (M+T), and hUC-MSCs+TGF-\u0026beta;1+PFD group (M+T+P). hUC-MSCs were cultured using DMEM/F12 containing 5% fetal bovine serum, and when the fusion of cells in each group reached approximately 80%, they were replaced with fresh DMEM containing 1% DMEM medium with fetal bovine serum, treated with 4ng/ml TGF-\u0026beta;1 and cultured for 24h. Cells in the M+T group were collected and detected by PCR for E-cad, FN,\u0026alpha;-SMA mRNA expression; in the M+T+P group, PFD or equal amount of PBS buffer was added after 24h of TGF-\u0026beta;1 treatment and cells were collected after 24 h of culture and detected by PCR for E-cad, FN,\u0026nbsp;\u0026alpha;-SMA\u0026nbsp;mRNA expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were expressed as mean\u0026plusmn;SEM. Comparisons between groups were made using Student\u0026apos;s t test for unpaired observations or two-factor ANOVA and Bonferroni correction for multiple comparisons. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003eTherapeutic effects of different doses of PFD in BLM induced pulmonary fibrosis mice\u003c/h2\u003e\n \u003cp\u003eCompared with the survival time in the model group(B), the low dose of PFD(10 mg/kg, P\u003csub\u003e10\u003c/sub\u003e) alone could not prolong the survival time of mice with pulmonary fibrosis, and there was a trend to prolong the survival time in the low dose of PFD(30 mg/kg, P\u003csub\u003e30\u003c/sub\u003e), the medium dose group (100 mg/kg, P\u003csub\u003e100\u003c/sub\u003e) and the high dose PFD (300 mg/kg, P\u003csub\u003e300\u003c/sub\u003e) alone, but the differences were not statistically significant ( P was 0.38, 0.08, 0.19, respectively) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eThe Sircol method could detect the content of soluble collagen in the lungs of mice, and the collagen content of the right lung of mice in the model group was significantly higher compared with the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with the model group, the collagen content of the P\u003csub\u003e10\u003c/sub\u003e group was not significantly changed; the collagen content of the P\u003csub\u003e30\u003c/sub\u003e group was reduced, and the difference was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); the right lung collagen of the P\u003csub\u003e100\u003c/sub\u003e group was significantly reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001); and the right lung collagen content of the P\u003csub\u003e300\u003c/sub\u003e group was reduced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but the reduction was not as great as that of the P\u003csub\u003e100\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB) .\u003c/p\u003e\n \u003cp\u003eThe pathological changes in the mice of the model group on day 21 of modeling were: inflammatory cell exudation, widening of alveolar septa, formation of ground glass like, strip like, grid like structure at the base of both lungs, and formation of cystic changes of varying sizes in some areas, i.e., honeycomb lung. Compared with the lung pathology of the model group, there was no significant improvement in the P\u003csub\u003e10\u003c/sub\u003e group, a trend of improvement in the P\u003csub\u003e30\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.055), a decrease in the areas of lattice-like and honeycomb shape in the lungs of the P\u003csub\u003e100\u003c/sub\u003e group, and a statistical difference in the improvement of Ashcroft score (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was some improvement in the lung pathology of the P\u003csub\u003e300\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not as much as that of the P\u003csub\u003e100\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 ) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). Masson staining stained the collagen in the lungs blue, which could reflect the severity of fibrosis in the lungs. A large amount of collagen deposition in the lung was seen microscopically in the model group. Compared with the model group, there was no significant change in intrapulmonary collagen deposition in the P\u003csub\u003e10\u003c/sub\u003e group; collagen deposition was significantly reduced in the P\u003csub\u003e30\u003c/sub\u003e, P\u003csub\u003e100\u003c/sub\u003e, and P\u003csub\u003e300\u003c/sub\u003e groups, but the collagen reduction in the P\u003csub\u003e30\u003c/sub\u003e and P\u003csub\u003e300\u003c/sub\u003e groups was not as great as that in the P\u003csub\u003e100\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n \u003cp\u003eCol1a1, Col1a2 and \u0026alpha;-SMA are considered to be the main pulmonary fibrosis markers, and their levels reflect the degree of pulmonary fibrosis [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. Col1a1 and Col1a2 mRNA levels were significantly higher in the model group compared with the control group. Compared with the model group, there was no significant change in Col1a1 mRNA in the P\u003csub\u003e10\u003c/sub\u003e group, and the expression of Col1a1 mRNA was significantly reduced in the P\u003csub\u003e30\u003c/sub\u003e, P\u003csub\u003e100\u003c/sub\u003e and P\u003csub\u003e300\u003c/sub\u003e groups, and the difference was statistically significant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG). Col1a2 mRNA levels were reduced in all treatment groups except for the low-dose PFD P\u003csub\u003e10\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the most significant reduction in Col1a2 mRNA expression was observed in the P\u003csub\u003e100\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with the \u0026alpha;-SMA mRNA level in the model group, there was no significant change in the P\u003csub\u003e10\u003c/sub\u003e group, a group decrease trend in P\u003csub\u003e30\u003c/sub\u003e and P\u003csub\u003e300\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056), and a significant decrease in the P\u003csub\u003e100\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI ). It can be seen that low-dose PFD (30 mg/kg) was the lowest effective dose for anti-fibrosis, medium-dose PFD (100 mg/kg) had the best efficacy among the groups with PFD alone, and high-dose PFD (300 mg/kg) was not as effective as medium-dose PFD for anti-fibrosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eCulture and characterization of hUC-MSCs\u003c/h2\u003e\n \u003cp\u003ehUC-MSCs cultured in DMEM/F12 medium at 37\u0026deg;C in a 5% CO2 incubator were assayed for surface-specific antigens of P\u003csub\u003e4\u003c/sub\u003e generation hUC-MSCs using flow cytometry. The results showed that the surface molecules CD34, CD45, and HLA-DR positivity of hUC-MSCs were less than 2%, and CD73, CD90, and CD105 positivity were higher than 95% (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), and this result was in accordance with the standards published by the International Stem Cell Therapy Association in 2006. In addition, we also examined the multidirectional differentiation potential of hUC-MSCs, and our results showed that hUC-MSCs were differentiated to chondrogenic (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), osteogenic (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC) and adipogenic cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003eTherapeutic effects of hUC-MSCs combined with P30 in BLM induced pulmonary fibrosis mice\u003c/h2\u003e\n \u003cp\u003eStudies have shown that hUC-MSCs can attenuate acute lung injury and early pulmonary fibrosis, but the therapeutic effect on established pulmonary fibrosis is controversial[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Preliminary experimental data from our group showed that hUC-MSCs alone by transcatheter tail vein injection were less effective in treating a mouse model of pulmonary fibrosis[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e], therefore the therapeutic effect of hUC-MSCs in combination with PFD on middle and late-stage pulmonary fibrosis is unclear. In contrast, the efficacy of too high doses of PFD is also less satisfactory and has significant adverse effects. Therefore, we next explored the therapeutic effect of hUC-MSCs combined with the lowest effective dose of PFD (30 mg/kg, P\u003csub\u003e30\u003c/sub\u003e) on pulmonary fibrosis in mice. The results showed that the hUC-MSCs combined with P\u003csub\u003e30\u003c/sub\u003e group significantly prolonged the survival time of mice (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), and the difference was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the survival time of the hUC-MSCs combined with P30 group longer than the P\u003csub\u003e100\u003c/sub\u003e group. The combination group significantly reduced the collagen content in the lungs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the collagen content was significantly lower than that in the P\u003csub\u003e100\u003c/sub\u003e group. The collagen content in the hUC-MSCs combined with P\u003csub\u003e30\u003c/sub\u003e group was significantly lower than that in the P\u003csub\u003e100\u003c/sub\u003e group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)). Moreover, the combination group significantly improved bleomycin-induced pulmonary lesions with significantly better Ashcroft scores (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and significantly reduced intrapulmonary collagen deposition, all with better improvement than P\u003csub\u003e100\u003c/sub\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 ). For the expression of pulmonary fibrosis markers, Col1a1, Col1a2, and \u0026alpha;-SMA mRNA levels were significantly decreased in the hUC-MSCs combined with P\u003csub\u003e30\u003c/sub\u003e group relative to the model group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG-\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), where the reduced levels of Col1a1 and Col1a2 were statistically different compared to the P\u003csub\u003e100\u003c/sub\u003e group alone, while the reduced levels of \u0026alpha;-SMA compared to the P\u003csub\u003e100\u003c/sub\u003e group tended to be statistically different (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehUC-MSCs combined with low-dose P\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003e30\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003eelevated RGS2 mRNA and protein expression levels in mouse lung tissue\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eStudies have shown that RGS2 is a novel mechanism for the antifibrotic effect of PFD[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Quantitative RT-PCR analysis in this study confirmed that, as expected, the treatment of pulmonary fibrosis in mice with the optimal effective dose of PFD (100 mg/kg) increased RGS2 mRNA levels in mouse lung tissues (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and RGS2 mRNA levels in the hUC-MSCs combined with P\u003csub\u003e30\u003c/sub\u003e group were more elevated than those in the P\u003csub\u003e100\u003c/sub\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Western blot confirmed that RGS2 protein levels in lung tissue were also significantly increased after PFD treatment in mice (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), and RGS2 protein levels in the hUC-MSCs combined with P\u003csub\u003e30\u003c/sub\u003e group were also more than those in the P\u003csub\u003e100\u003c/sub\u003e group.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ehUC-MSCs can affect the expression of RGS2 and the markers of pulmonary fibrosis of myofibroblasts by PFD treatment\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eStudies have shown that MFB are the main cells of tissues after fibrosis and that TGF-\u0026beta;1 induces the conversion of fibroblasts to myofibroblasts[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. In this experiment, two fibroblast model cells, MFB, were obtained after stimulation of two fibroblasts, NIH3T3 and HLF-9, for 24h using 4 ng/mL TGF-\u0026beta;1. hUC-MSCs and PFD for fibroblasts in subsequent experiments were added after TGF-\u0026beta;1 stimulation.\u003c/p\u003e\n \u003cp\u003eQuantitative RT-PCR analysis confirmed that RGS2 mRNA was elevated in a concentration-dependent manner after PFD treatment of both MFB (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), and the elevation of RGS2 mRNA in both MFB treated with \u0026ge;\u0026thinsp;6 mM PFD was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, the mRNA of fibrosis markers in both MFB decreased significantly after 24 h of PFD treatment, and the difference was statistically significant.\u003c/p\u003e\n \u003cp\u003eThe effect of PFD (8 mM PFD) on the expression of RGS2 and lung fibrosis markers within MFB were explored after co-culture of hUC-MSCs with MFB. The results showed that the differences in RGS2 mRNA levels were statistically significant in the H\u0026thinsp;+\u0026thinsp;M\u0026thinsp;+\u0026thinsp;P group compared with the H\u0026thinsp;+\u0026thinsp;P group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), as well as in the N\u0026thinsp;+\u0026thinsp;M\u0026thinsp;+\u0026thinsp;P group compared with the N\u0026thinsp;+\u0026thinsp;P group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that hUC-MSCs further elevated RGS2 mRNA after co-action of MFB with PFD. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI, hUC-MSCs co-treatment of MFB with PFD resulted in a statistically significant decrease in mRNA for fibrosis markers in both MFB.\u003c/p\u003e\n \u003cp\u003eWe also examined the changes in mRNA levels of endothelial mesenchymal transition markers in hUC-MSCs. The results showed that E-cad mRNA was significantly increased and both FN and ACTA2 mRNA expression were significantly decreased after 8 mM PFD treatment of hUC-MSCs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eJ, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough PFD has been approved by the Food and Drug Administration(FDA) for the treatment of IPF, its therapeutic effects are limited and its adverse effects are large. In the present study, we used hUC-MSCs combined with low-dose PFD for the treatment of pulmonary fibrosis in mice. We found combined with hUC-MSCs, the low-dose PFD could exert antifibrotic effects superior to its 3-fold or even 10-fold dose alone. Therefore, it can be speculated that the combination of hUC-MSCs and PFD can enhance the antifibrotic efficacy of PFD, reduce the dosage of PFD, and decrease the incidence of adverse effects, in order to enhance the tolerability and compliance of IPF patients.\u003c/p\u003e \u003cp\u003eHisashi O et al. used three concentrations of 10, 30, and 100 mg/kg PFD to treat a mouse model of bleomycin-induced pulmonary fibrosis, and the results of this study showed that the antifibrotic effect of PFD was positively correlated with the dose[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the efficacy of PFD containing higher dose gradients on pulmonary fibrosis in mice has not been reported in this literature. In the present study, four dose gradients of 10, 30, 100, and 300 mg/kg PFD were applied to explore the therapeutic effects on pulmonary fibrosis, and contrary to expectations, the highest dose (300 mg/kg) PFD did not achieve better antifibrotic effects than the medium dose PFD (100 mg/kg). In clinical trials of PFD for IPF conducted by Azuma A et al. and Wijsenbeek MS et al. adverse effects of PFD were predominantly gastrointestinal reaction and were positively correlated with dose, with an incidence of 40\u0026ndash;60%, and some patients discontinued the drug due to intolerance[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Combined with the observation that the appetite of mice in the high-dose PFD group was poorer than that in the medium and low-dose PFD groups, it can be assumed that the PFD dose exceeded the appropriate range, resulting in gastrointestinal adverse reactions and reducing the therapeutic effect of PFD. Therefore, we cannot expect to achieve better anti-fibrotic effect by increasing the dose of PFD in clinical practice. For this reason, in order to avoid the adverse effects of higher doses of PFD, the combination of the low dose, 30 mg/kg PFD, with hUC-MSCs was used in this study instead of 100 mg/kg PFD with hUC-MSCs.\u003c/p\u003e \u003cp\u003eRGS2 is a known negative regulator of G protein signaling that inhibits the amplitude and duration of signals mediated by GQ-coupled GPCRs[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Some GQ-coupled GPCRs and their ligands are important drivers of pulmonary fibrosis[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Jang HS et al. demonstrated in animal experiments that knockdown of RGS2 leads to renal fibrosis in mice after unilateral ureteral obstruction (UUO), suggesting that endogenous RGS2 has an anti-fibrotic function[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, Xie Y et al. showed that the gene that they screened for rapid upregulation in human lung fibroblasts in response to pirfenidone using gene microarrays was RGS2, and confirmed that endogenous RGS2 exhibited anti-pulmonary fibrosis function and that RGS2 underlies the anti-fibrotic effect of PFD, thus providing some ideas to investigate the mechanism of PFD for the treatment of pulmonary fibrosis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In our in vitro experiments, we observed that hUC-MSCs increased the expression of endogenous RGS2 in MFB, allowing pirfenidone to exert a stronger anti-fibrotic effect, which may be one of the reasons why hUC-MSCs combined with low-dose PFD could achieve better efficacy. However, the mechanism of how hUC-MSCs increase the expression of RGS2 needs to be explored, and the mechanism of how PFD exerts its antifibrotic effect through RGS2 also needs to be further investigated.\u003c/p\u003e \u003cp\u003eThe pathogenesis of IPF is still unclear, but the main pathological changes are the proliferation and aggregation of large numbers of myofibroblasts and the deposition of extracellular matrix, of which the main component is collagen [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. MFB, as the main effector cell of IPF, has a negative correlation with the severity and prognosis of IPF disease [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It was found that in addition to their anti-fibrotic effects, hUC-MSCs still have a certain degree in pro-fibrotic effects, especially in the fibrotic environment, which can be converted to MFB through endothelial mesenchymal conversion[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which may be one of the reasons for the controversial effects of hUC-MSCs in the treatment of pulmonary fibrosis. And PFD, as a multi-cytokine inhibitor, can inhibit the expression of cytokines such as TGF-β1, basic fibroblast growth factor(bFGF), and connective tissue growth factor(CTGF) of target cells (myo)fibroblasts, thus suppressing the proliferation of (myo)fibroblasts and the synthesis of collagen[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, in this study we examined the changes in mRNA levels of endothelial mesenchymal transition markers in hUC-MSCs. The results showed that E-cad mRNA was significantly increased and FN and α-SMA mRNA expression were both significantly decreased after PFD treatment of hUC-MSCs. This result indicated that PFD inhibited the conversion of hUC-MSCs to mesenchymal cells, thereby reducing the conversion of hUC-MSCs to MFB in order to give hUC-MSCs the opportunity to exert maximum anti-fibrotic effects, which may also be one of the mechanisms why hUC-MSCs combined with PFD could achieve better efficacy compared with PFD alone.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the results of the present study provide direct evidence that hUC-MSCs combined with low-dose PFD had a therapeutic effect on a mouse model of pulmonary fibrosis and delayed the progression of IPF, providing a new idea for the clinical treatment of IPF. In addition, this study also found that the possible mechanism of the efficacy of hUC-MSCs in combination with low-dose PFD may be related to a significant increase in the expression of the anti-fibrotic protein RGS2, but the in-depth mechanism of the combined treatment of both needs to be further studied and explored.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ehUC-MSCs, umbilical cord-derived mesenchymal stem cells; PFD, pirfenidone; P\u003csub\u003e10,\u0026nbsp;\u003c/sub\u003e10 mg/kg PFD; P\u003csub\u003e30\u003c/sub\u003e, 30 mg/kg PFD; P\u003csub\u003e100\u003c/sub\u003e, 100 mg/kg PFD; P\u003csub\u003e300\u003c/sub\u003e, 300 mg/kg PFD; HE, hematoxylin-eosin ; PCR, polymerase Chain Reaction; RGS2,\u0026nbsp;regulator of G protein signaling 2;\u0026nbsp;MFB, myofibroblasts; IPF, diopathic pulmonary fibrosis; Col1a1, type I collagen a1; Col1a2, type I collagen a2; \u0026alpha;-SMA, \u0026alpha;-smooth muscle actin; ACTA2, actin alpha 2; E-cad, calcium adhesion protein E; FN, fibronectin; NIH3T3, mouse embryonic fibroblasts; HLF-9, human Embryonic Lung Fibroblasts; DMEM, Dulbecco\u0026apos;s Modified Eagle\u0026apos;s Medium; F12, Ham\u0026apos;s F-12 Nutrient; FBS, fetal bovine serum; P\u003csub\u003e6\u003c/sub\u003e, passage 6; h, hours; N, control group; B, bleomycin model group; N+M, hUC-MSCs control group; B+M, hUC-MSCs treatment group; B+M+P\u003csub\u003e30\u003c/sub\u003e, hUC-MSCs+30 mg/kg PFD group;\u0026nbsp;TGF-\u0026beta;1, transforming growth factor-\u0026beta;; N, NIH3T3 group; H, HLF-9 group; M, hUC-MSCs group, N+M, NIH3T3+hUC-MSCs group; H+M, HLF-9+hUC-MSCs group; M+T, hUC-MSCs+TGF-\u0026beta;1 group; M+T+P, hUC-MSCs+TGF-\u0026beta;1+PFD group; FDA, Food and Drug Administration; bFGF, basic fibroblast growth factor; CTGF, connective tissue growth factor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge Dr. Huafeng Pan at Chongqing Stem Cell Therapy Engineering Technology Research Center for providing hUC-MSCs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Nature Science Foundation of China (NSFC 81670018), Key project from Chinese Ministry of Science and Technology (2016YFA0101300).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its Additional file 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors, contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXW, and ZF participated in research design. XW, HG, HJQ and OZ conducted experiments. XW, HJQ and OZ contributed materials and reagents. XW performed data analysis. XW, HML, ZF and LNC wrote or contributed to the writing of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human participants, human data or human tissue involved in studies reported in this manuscript. All animal studies were approved by the Animal Ethics Committee of Chongqing Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDivision of pediatric pulmonology and immunology, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan, People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eKey Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan, People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eNHC Key Laboratory of Chronobiology , Sichuan University, Chengdu 610065, Sichuan, People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eAffiliated Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu 610075, Sichuan, People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003ePediatric Research Institute, Children\u0026rsquo;s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing 400014, People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e6\u003c/sup\u003eDepartment of Respiratory Medicine, Children\u0026rsquo;s Hospital of Chongqing Medical University, Chongqing, 400014, People\u0026rsquo;s Republic of China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e7\u003c/sup\u003eChongqing Engineering Research Center of Stem Cell Therapy, Chongqing 400014, People\u0026rsquo;s Republic of China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eRaghu G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, \u003cem\u003eet al\u003c/em\u003e. An official ATS/ERS/JRS/ALAT clinical practice guideline: treatment of idiopathic pulmonary fibrosis. An update of the 2011 clinical practice guideline. Am J Respir Crit Care Med. 2015; 192(2): e3-e19.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRaghu G, Chen SY, Yeh WS, Maroni B, Li Q, Lee YC, \u003cem\u003eet al.\u003c/em\u003e Idiopathic pulmonary fibrosis in US medicare beneficiaries aged 65 years and older: incidence, prevalence and survival, 2001-11. Lancet Respir Med. 2014;2:566\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHutchinson J, Fogarty A, Hubbard R, McKeever T. Global incidence and mortality of idiopathic pulmonary fibrosis: a systematic review. Eur Respir J 2015; 46(3): 795\u0026ndash;806.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXie Y, Jiang H, Zhang Q, Mehrotra S, Abel P, Toews M, \u003cem\u003eet al\u003c/em\u003e. Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis. Respir Res, 2016; 22;17(1):103.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKing TE Jr, Bradford WZ, Castro-Bernardini S, Fagan EA, Glaspole I, Glassberg MK, \u003cem\u003eet al\u003c/em\u003e. A Phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N Engl J Med 2014; 37: 2083\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNoble PW, Albera C, Bradford WZ, Costabel U, Glassberg MK, Kardatzke D, \u003cem\u003eet al\u003c/em\u003e. Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. Lancet. 2011; 377: 1760\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDing DC, Chang YH, Shyu WC, Lin SZ. Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy. Cell Transplant. 2015; 24(3): 339\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCrisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, \u003cem\u003eet al.\u003c/em\u003e A perivascular origin for mesenchymal stem cells in multiplehuman organs. Cell Stem Cell. 2008; 3(3): 301\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBianco P. \u0026ldquo;Mesenchymal\u0026rdquo; stem cells. Annu Rev Cell Dev Biol. 2014;30:677\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChang YS, Ahn SY, Yoo HS, Sung SI, Choi SJ, Oh WI, et al. Mesenchymal stem cells for bronchopulmonary dysplasia: Phase 1 dose-escalation clinical trial. J Pediatr. 2014; 64(5): 966\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAzuma A, Nukiwa T, Tsuboi E, Suga M, Abe S, Nakata K, \u003cem\u003eet al.\u003c/em\u003e Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2005; 171: 1040\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003e28.Wilson JG, Liu KD, Zhuo H, Caballero L, McMillan M, Fang X, \u003cem\u003eet al.\u003c/em\u003e Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial. Lancet Respir Med. 2015; 3(1): 24\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eReddy M, Fonseca L, Gowda S, Chougule B, Hari A, Totey S. Human adipose-derived mesenchymal stem cells attenuate early stage of bleomycin induced pulmonary fibrosis: comparison with pirfenidone. Int J Stem Cells. 2016; 9: 192\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMoodley Y, Atienza D, Manuelpillai U, Samuel CS, Tchongue J, Ilancheran S, \u003cem\u003eet al\u003c/em\u003e. Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin induced lung injury. Am J Pathol. 2009; 175: 303\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYan X, Liu Y, Han Q, \u003cem\u003eet al.\u003c/em\u003e Injured micro-environment directly guides the differentiation of engrafted flk-1 mesenchymal stem cell in lung. Exp Hematol. 2007, 35: 1466\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAshcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988; 41: 467\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHua Z, Yi X, Yunqiu X, Rong Z, Daiyin T, Ting W, \u003cem\u003eet al.\u003c/em\u003e Therapeutic effects of human umbilical cord-derived mesenchymal stem cells in acute lung injury mice. Sci Rep. 2017; 7: 39889.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFernandez IE, Eickelberg O. New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis. Lancet. 2012;18;380(9842):680\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhang Y. The investigation of human umbilical cord mesenchymal stem cells on pulmonary fibrosis mice models[D]. Chongqing, Chongqing Medical University. 2015.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePeng DY. Combination of human umbilical cord mesenchymal stem cells and nintedanib ameliorates mice pulmonary fibrosis induced by bleomycin[D]. Chongqing, Chongqing Medical University. 2017.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMartin MM, Buckenberger JA, Jiang J, Malana GE, Knoell DL, Feldman DS, \u003cem\u003eet al.\u003c/em\u003e TGF-beta1 stimulates human AT1 receptor expression in lung fibroblasts by cross talk between the Smad, p38 MAPK, JNK, and PI3K signaling pathways. Am J Physiol Lung Cell Mol Physiol. 2007; 293: L790-9.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOku H, Shimizu T, Kawabata T, Nagira M, Hikita I, Ueyama A, \u003cem\u003eet al\u003c/em\u003e. Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis. Eur J Pharmacol 2008; 590(12/3): 400\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAzuma A, Nukiwa T, Tsuboi E, Suga M, Abe S, Nakata K, \u003cem\u003eet al\u003c/em\u003e. Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2005; 171: 1040\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWijsenbeek MS, Grutters JC, Wuyts WA. Early experience of pirfenidone in daily clinical practice in belgium and the netherlands: a retrospective cohort analysis. Adv Ther 2015; 32: 691\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBernstein LS, Ramineni S, Hague C, Cladman W, Chidiac P, Levey AI, \u003cem\u003eet al\u003c/em\u003e. RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling. J Biol Chem. 2004;279:21248\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHeximer SP, Watson N, Linder ME, Blumer KJ, Hepler JR. RGS2/G0S8 is a selective inhibitor of G\u0026alpha;q function. Proc Natl Acad Sci USA. 2008;94:14389\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, \u003cem\u003eet al\u003c/em\u003e. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat Med. 2008; 14:45\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGhavami A, Hunt RA, Olsen MA, Zhang J, Smith DL, Kalgaonkar S, \u003cem\u003eet al\u003c/em\u003e. Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5-HT1A, 5-HT2A, and dopamine D2 receptor-mediated signaling and adenylyl cyclase activity. Cell Signal. 2004;16:711\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJang HS, Kim JI, Noh M, Rhee MH, Park KM. Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis. Biochim Biophys Acta. 2014; 1842(9):1733\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eThomas AW, Thirumalai RR. Mechanisms of fbrosis: therapeutic translation for fbrotic disease. Nat Med. 2012; 18(7): 1028\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eEnomoto N, Suda T, Kato M, Kaida Y, Nakamura Y, Imokawa S, \u003cem\u003eet al\u003c/em\u003e. Quantitative analysis of fbroblastic foci in usual interstitial pneumonia. Chest. 2006; 130(1): 22\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGazdhar A, Grad I, Tam\u0026ograve; L, Gugger M, Feki A, Geiser T. The secretome of induced pluripotent stem cells reduces lung fbrosis in part by hepatocyte growth factor. Stem Cell Res Ther 2014; 5: 123.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMezey E. The therapeutic potential of bone-marrow-derived stromal cells. J Cell Biochem. 2011, 112(10): 2683\u0026ndash;2687.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOku H, Shimizu T, Kawabata T, Nagira M, Hikita I, Ueyama A, \u003cem\u003eet al\u003c/em\u003e. Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced pulmonary fibrosis. Eur J Pharmacol. 2008; 20: 400\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003eMyllarniemi M, Kaarteenaho R. Pharmacological treatment of idiopathic pulmonary fibrosis preclinical and clinical studies of pirfenidone, nintedanib, and N-acetylcysteine. Eur Clin Respir J . 2015; 10;2.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Idiopathic pulmonary fibrosis, pirfenidone, umbilical cord-derived mesenchymal stem cells, Regulator of G protein signaling 2","lastPublishedDoi":"10.21203/rs.3.rs-1507756/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1507756/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe therapeutic effect of umbilical cord-derived mesenchymal stem cells(hUC-MSCs) in combination with pirfenidone(PFD) on pulmonary fibrosis in mice and its possible mechanism were investigated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were randomly divided into six groups: control group, model group, P\u003csub\u003e10\u003c/sub\u003e group, P\u003csub\u003e30\u003c/sub\u003e group, P\u003csub\u003e100\u003c/sub\u003e group and P\u003csub\u003e300\u003c/sub\u003e group. Modeled by tracheal intubation with 3 mg/kg bleomycin drip, each dose of PFD was given daily by gavage from day 7 onwards. Mice were observed continuously for 21 days and survival was recorded. Lung tissues were collected on day 21, and HE and Masson staining were performed to assess morphological changes and collagen deposition in the lungs. Collagen content was measured by Sircol method, and fibrosis marker levels were detected by PCR and Western Blot. Another batch of C57BL/6 mice was then randomly divided into five groups: hUC-MSCs control group, model group, P\u003csub\u003e100\u003c/sub\u003e group, hUC-MSCs treatment group and hUC-MSCs\u0026thinsp;+\u0026thinsp;P\u003csub\u003e30\u003c/sub\u003e group. 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e hUC-MSCs were injected into the tail vein on day 7, and the mice were given PFD gavage daily from day 7 onwards, and their survival was recorded. Lung tissues were collected on day 21 to detect pathological changes and the expression of collagen content and regulator of G protein signaling 2(RGS2). Pulmonary myofibroblasts were divided into MFB group and MFB\u0026thinsp;+\u0026thinsp;hUC-MSCs group, and different doses of PFD were added to each group, and the levels of RGS2 and fibrosis markers were detected in each group.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompared with other doses of PFD groups, the P\u003csub\u003e100\u003c/sub\u003e group significantly improved mouse survival and lung pathology, and significantly reduced collagen and fibrosis marker levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The hUC-MSCs\u0026thinsp;+\u0026thinsp;P\u003csub\u003e30\u003c/sub\u003e group significantly improved mouse survival and lung pathology, significantly reduced collagen and fibrosis marker levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the efficacy was better than that of the P\u003csub\u003e100\u003c/sub\u003e and hUC-MSCs groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). RGS2 expression was significantly higher in the MSCs\u0026thinsp;+\u0026thinsp;P\u003csub\u003e30\u003c/sub\u003e group compared with the P\u003csub\u003e100\u003c/sub\u003e and hUC-MSCs groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PFD increased RGS2 expression in MFB (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in a dose-dependent manner. Fibrosis markers were more significantly reduced in the hUC-MSCs\u0026thinsp;+\u0026thinsp;PFD relative to the PFD alone group and the hUC-MSCs group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe study suggested that hUC-MSCs combined with low-dose PFD showed a therapeutic effect better than that of the two treatments used separately. Its effect attenuating bleomycin-induced pulmonary fibrosis in mice is related with the increase of RGS2.\u003c/p\u003e","manuscriptTitle":"Human Umbilical Cord Mesenchymal Stem Cells Combined with Pirfenidone upregulates the Expression of RGS2 in the Pulmonary Fibrosis in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-14 20:25:28","doi":"10.21203/rs.3.rs-1507756/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-05-30T20:33:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-18T02:00:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-02T17:11:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212efb39-8d09-4711-bd02-95968dcb9f10","date":"2022-04-18T20:46:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-04-18T20:44:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-04-12T19:18:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-04-12T01:08:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Respiratory Research","date":"2022-03-31T04:37:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"58de7c2d-f1f6-438a-bb69-62c03efc90e4","owner":[],"postedDate":"April 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-09-22T13:14:22+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-14 20:25:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1507756","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1507756","identity":"rs-1507756","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}