| Background:Lung injury-induced fibrosis is a severe sequela of acute lung disease,including COVID-19.However,there are still no effective drugs to treat pulmonary fibrosis.Mesenchymal stem cells(MSCs)have the functions of immune regulation and tissue repair,and have good clinical application prospects in the treatment of pulmonary fibrosis caused by lung injury.In this study,we used human embryonic stem cell(hESC)-derived MSCs with unique abilities to modulate immunity and extracellular matrix production compared with primary MSCs,which were named as immune-and matrix-regulatory cells(IMRCs).The mechanism of these cells in repairing lung injury and treating pulmonary fibrosis,the distribution of cells in the body,and whether multiple administration of cells can increase the efficacy?These questions remain to be answered.Objective:To explore a mouse model of pulmonary fibrosis with bleomycin nasal nebulization.To explore the appropriate dose of IMRCs for the treatment of bleomycin-induced early pulmonary fibrosis in mice.To explore the distribution characteristics of IMRCs in mice after intravenous administration.To explore the appropriate treatment time and frequency of IMRCs for pulmonary fibrosis induced by Bleomycin(BLM)-induced lung injury in mice.To explore and compare the efficacy of IMRCs,UCMSCs and PFD on pulmonary fibrosis induced by BLM-induced lung injury in mice.To explore the mechanism of IMRCs for bleomycin-induced lung injury-induced pulmonary fibrosis in mice.Methods:To create a mouse model of pulmonary fibrosis by nasal nebulization of bleomycin,12 C57BL/6 mice were randomly divided into the control group(CTL)according to the random number table method,and the samples were drawn 7 days after nebulization of bleomycin.Group(BLM-D7),14 days after nebulization group(BLM-D14)and 21 days after nebulization group(BLM-D21),with 3 animals in each group.Animal aerosols were continuously nebulized by nasal inhalation exposure system for 3 days,30 minutes per day,the control group was nebulized with the same amount of normal saline,and the model group was nebulized on the 7th,14th and 21st days after the nebulization.Collagen expression was detected by two-photon technique and hcmatoxylin-cosin staining(HE staining)was used to evaluate lung structure and fibrosis degree.At the same time,one CTL group was taken as blank control.To explore the appropriate dose of IMRCs to treat bleomycin-induced early pulmonary fibrosis in mice.110 healthy C57BL/6 mice were randomly divided into normal control group(CTL),10 mice,bleomycin group(BLM),20 mice,and low-dose IMRCs treatment group(IMRCs-Low).26 mice according to the random number table method.Twenty-seven rats in the middle-dose IMRCs treatment group(IMRCs-Medium)and 27 rats in the high-dose IMRCs treatment group(IMRCs-High).The bleomycin group and the treatment group were given an intratracheal injection of bleomycin to induce lung injury and pulmonary fibrosis models in mice,while the control group was given an equal volume of normal saline.The IMRCs group was injected with IMRCs three times by tail vein on the 7th,14th and 21st day after injection of bleomycin,while the control group and bleomycin group were given an equal volume of normal saline.During the experiment,the body weight and survival of the mice were monitored,and pulmonary function-related indicators,including pressure-volume curve(PV loop),inspiratory capacity(IC),respiratory resistance(Rrs),static compliance,were measured by pulmonary function 28 days after modeling.Compliance(Crs),elastic resistance(Ers),tissue damping(G),tissue elasticity(H)and small animal lung CT scan analysis.To explore the distribution characteristics of IMRCs in mice after intravenous administration,we first performed imaging detection of in vitro organs.C57BL/6 mice were used,and DiR-labeled IMRCs were injected into the tail vein.One hour later,the anesthetized animals were dissected,and the lungs,livers and spleens were taken to detect the distribution of DiR-labeled cells in the isolated organs of mice.In addition,in vivo imaging detection was performed.C57BL/6 mice were used and divided into a negative control group and a test group.The negative control group was injected with normal saline or the same concentration of DiR in the tail vein,and the test group was injected with DiRlabeled human embryonic stem cells in the tail vein.Source IMRCs,1 hour,1day,3day.5day,7day,9day,15day,21 day,26day and 32 days after injection at 10 time points.After anesthetizing the animals,the distribution of DiR-labeled cells in mice was detected.To explore the appropriate treatment time and frequency of IMRCs for pulmonary fibrosis induced by BLM-induced lung injury in mice,70 healthy C57BL/6 mice were randomly divided into the control group(CTL)according to the random number table method,bleomycin group(BLM),stem cell therapy group(IMRCs-D1)1 day after modeling,stem cell therapy group(IMRCs-D7)7 days after modeling,and stem cells were administered on the 1st and 7th days Two treatment groups(IMRCs-D1&7),15 animals in each group.The bleomycin group and stem cell treatment group were injected with bleomycin into the trachea to induce lung injury and pulmonary fibrosis models in mice,while the control group was given normal saline,10 mice in the CTL group and 15 mice in the BLM group.The IMRCs-Dl group and the IMRCs-D7 group were injected with IMRCs on the 1st day and the 7th day after the injection of bleomycin for modeling,respectively.On the seventh day,IMRCs were injected twice by tail vein,and the control group and bleomycin group were given equal volume of normal saline.Pulmonary function-related indicators,including pressure-volume curve(PV loop),inspiratory capacity(IC),respiratory resistance(Rrs),and static compliance(static compliance),were measured by pulmonary function 21 days after modeling.compliance(Crs),elastic resistance(Ers),newtonian resistance(Rn),tissue damping(G),tissue elasticity(H),and forced vital capacity(FVC);the lungs were collected and analyzed by HE and Masson staining to evaluate the degree of pulmonary fibrosis;immunohistochemical methods were used to determine collagen type I(collagen,COLI),fibronectin(FN)and alpha smooth muscle actin(alpha smooth muscle actin.α-SMA)content;immunofluorescence technique was used to determine the expression of HOPX protein(Homeodomain only protein X,HOPX),type 2 alveolar surfactant(surfactant protein C,SPC)and F4/80 protein in lung tissue.To explore and compare the efficacy of IMRCs,UCMSCs and PFD on pulmonary fibrosis induced by BLM-induced lung injury in mice,106 healthy C57BL/6 mice were randomly divided into nonmal control group(CTL),bleomycin group(BLM),IMRCs treatment group IMRCs,primary UCMSCs treatment group UCMSCs,pirfenidone treatment group PFD,6 in CTL group,40 in BLM group,20 in each treatment group.The bleomycin group and the treatment group were given an intratracheal injection of bleomycin to induce lung injury and pulmonary fibrosis models in mice,while the control group was given normal saline.In the IMRCs group,IMRCs were injected into the tail vein on the 1st and 7th day after injection of bleomycin,and the UCMSCs group was injected with UCMSCs in the tail vein on the 1st and 7th day after injection of bleomycin.The rats in the control group and the bleomycin group were given an equal volume of normal saline from the 1st day to the 21st day after the injection of bleomycin.Pulmonary function-related indicators,including pressure-volume curve(PV loop),inspiratory volume(IC).respiratory resistance(Rrs),static compliance(Crs),and elastic resistance(Ers),were measured by pulmonary function 21 days after modeling.Tissue damping(G),tissue elasticity(H)and forced vital capacity(FVC)and CT scan analysis of small animal lungs,and lung volume analysis;lungs were collected,and the degree of pulmonary fibrosis was evaluated by HE and Masson staining.To explore the possible mechanism of IMRCs in the treatment of bleomycin-induced lung injury-induced pulmonary fibrosis in mice,110 healthy C57BL/6 mice were randomly divided into normal control group(CTL)according to the random number table method.10 rats,27 rats in the bleomycin group(BLM),27 rats in the IMRCs treatment group(IMRCs),and 27 rats in the IMRCs cell treatment group(IMRCs-MMP1-/-)in which matrix metallopeptidase 1(MMP1)was knocked out.The bleomycin group and the treatment group were given an intratracheal injection of bleomycin to induce lung injury and pulmonary fibrosis models in mice,while the control group was given an equal volume of normal saline.In the IMRCs group,normal IMRCs were injected into the tail vein twice on the 1st and 7th day after injection of bleomycin,and the IMRCs-MMP1-/-group was injected twice on the 1st and 7th day after bleomycin injection.MMP1 knockout IMRCs were injected into the tail vein,and the control group and bleomycin group were given an equal volume of normal saline.During the experiment,the weight of the mice was monitored,and the degree of lung tissue edema was observed by the lung coefficient on the 21st day after the model was established.Vital capacity(FVC),respiratory resistance(Rrs),static compliance(Crs),elastic resistance(Ers),and small animal lung CT scan were analysised.Results:The exploratory experiment of bleomycin nasal nebulization mouse fibrosis model,HE results showed that the lung tissue of mice in the CTL group was observed under the light microscope,the tissue structure was clear,the alveolar wall was not thickened,and the alveolar structure was intact.Uniform and extensive inflammatory infiltration in lung tissue were observed under the light microscope at 7,14 and 21 days after the mice were treated,the alveolar septa were significantly widened,fused fibrous foci and a large amount of extracellular matrix were seen,and the inflammatory infiltration gradually decreased with the passage of time,collagen deposition increased,the lung structure was gradually deformed and distorted from the normal and ordered alveolar structure,and finally formed a uniform and extensive fibrotic foci,and BLM aerosol particles could reach the pleural edge,resulting in the formation of pleural thickening fibrous foci.The twophoton results showed that the collagen in the lung tissue gradually changed from a regular grid to a disordered and irregular distribution,and from a thin strip to a coarse bundle.To explore the appropriate dose of IMRCs for the treatment of bleomycin-induced early pulmonary fibrosis in mice.(1).The mice were treated with low,medium and high doses of IMRCs three times at 7 days,14 days and 21 days after BLM modeling,which could slow down the weight loss trend of mice;(2).It can improve the lung function of mice.The PV circle in the three doses of IMRCs group was significantly higher than that in the BLM group,and the IC in the IMRCs-Low group was significantly lower than that in the BLM group and the IMRCs-High group.Crs in IMRCs-Low group was significantly lower than BLM group,Ers,G and H in three dose groups were significantly lower than BLM group,Rrs in IMRCs-Low and IMRCs-Medium group was significantly lower than BLM group and IMRCs-High group,the differences were all Statistical significance(P<0.05);(3).Can improve lung CT in mice.(4)IMRCs treatment can reduce lung inflammation and collagen deposition in bleomycin model mice.Ashcroft score in IMRCsLow and IMRCs-Medium treatment groups was significantly lower than that in BLM group,and the difference was statistically significant(P<0.05).In conclusion,IMRCs in the treatment of early fibrosis in BLM can slow down the trend of weight loss,improve lung function,CT and lung histopathological changes.The distribution characteristics of IMRCs in mice after intravenous administration These experimental results showed that after IMRCs were injected into mice through the tail vein,the distribution level of the lungs was the highest,followed by the liver and spleen.lower.After IMRCs were injected into mice through the tail vein,they were mainly distributed in the lungs,liver,etc.,and their distribution level was at a high level within 3 days,and gradually decreased with time,and decreased to 5 days after administration.It was about 50%of the peak value,and decreased to less than 3%of the peak value on the 32nd day of administration.The appropriate treatment time and frequency of IMRCs for BLM-induced lung injury-induced pulmonary fibrosis in mice were explored.The experimental results showed that:(1).IMRCs and IMRCs were administered on the 1st or 7th day after bleomycin modeling.Administration of IMRCs twice on day 1 and day 7 could slow down the weight loss trend of mice,and the IMRCs group was significantly higher than the BLM group,and the difference was statistically significant(P<0.05).(2)Lung coefficient.IMRCs-D1 group and IMRCs-D1&7 group were significantly lower than BLM group,and the difference was statistically significant(P<0.05).(3)IC were significantly higher than those in the BLM group,the PV ring and FVC in the IMRCs-D1&7 group were significantly higher than those in the IMRCs-D7 group,the Crs in the IMRCs-D1&7 group were significantly higher than those in the BLM group,the Rrs,Ers and G in the three treatment groups were significantly lower than those in the BLM group,The H of IMRCsD1 group and IMRCs-D 1&7 group was significantly lower than that of BLM group,and the difference was statistically significant(P<0.05).(4)The Ashcroft scores of the three IMRCs treatment groups were significantly lower than those of the BLM group,and the Ashcroft scores of the IMRCs-D1&7 groups were significantly lower than those of the IMRCs-D1 and IMRCs-D7 groups(P<0.05),and the Szapiel scores of the IMRCs-D1&7 groups were significantly lower The difference was statistically significant in BLM group,IMRCs-D1 group and IMRCs-D7 group(P<0.05);(5).IMRCs cell therapy can reduce the expression of COLI,FN and α-SMA in lung tissue,among which IMRCs-D1 group and IMRCs-D1 group-D1&7 group COLI,FN and α-SMA and expression were significantly lower than BLM group,IMRCs-D 1&7 group COLI,FN and α-SMA expression was significantly lower than IMRCs-D7 group,IMRCs-D1 group COLI protein expression was significantly lower than IMRCs-D7 group,the differences were statistically significant(P<0.05);(6).IMRCs cell treatment can reduce the expression of F4/80 protein,the expression of F4/80 in the three IMRCs cell treatment groups was significantly lower than that in the BLM group,IMRCs-D 1&7 group was significantly lower than the IMRCs-D7 group,and the difference was statistically significant(P<0.05);(7).IMRCs cell therapy can increase the expression of HOPX and SPC proteins,IMRCs-D1 group and IMRCsD1&7 group HOPX and SPC proteins The expression of SPC protein in IMRCs-D1&7 group was significantly higher than that in BLM group,the IMRCs-D1&7 group was significantly higher than IMRCs-D1 and IMRCs-D7 group,and the SPC protein expression in IMRCs-D7 group was significantly higher than that in BLM group,the differences were all statistically significant(P<0.05).In conclusion,IMRCs can improve lung function,inflammation,collagen deposition and increase the number of lung parenchyma cells in mice with pulmonary fibrosis,and have a certain time and frequency dependence,that is,the earlier the treatment time,the better the effect and the effect of two treatments better.The experimental results comparing the efficacy of IMRCs,UCMSCs and PFD on pulmonary fibrosis induced by BLM-induced lung injury in mice showed that:(1).It can slow down the weight loss trend of mice,and the body weight of IMRCs group was significantly higher than that of BLM group,and the difference was statistically significant(P<0.05).(2)The survival rate of bleomycin model mice was improved and the median survival time was prolonged.The survival curves of the IMRCs group and the PFD group were significantly better than those of the BLM group,and the difference was statistically significant(P<0.05);(3).After the bleomycin model was established Treatment with IMRCs,UCMSCs and pirfenidone could improve the lung coefficient of mice.The lung coefficient of IMRCs group and PFD group was significantly lower than that of BLM group,and the difference was statistically significant(P<0.05);(4).Treatment with IMRCs,UCMSCs and pirfenidone after modeling can improve lung inflammation and collagen deposition in mice.Ashcroft scores in the three treatment groups were significantly lower than those in the BLM group,and the IMRCs group was significantly lower than the UCMSCs and PFD groups.The differences were statistically significant(P<0.05);(5).The IMRCs,UCMSCs and pirfenidone treatment after bleomycin modeling can improve the lung function of mice.PV ring,FVC,IC in IMRCs group and Crs were significantly higher than those in the BLM group,and the Rrs,Ers,G and H in the IMRCs group were significantly lower than those in the BLM group,and the differences were statistically significant(P<0.05).(6)Pirfenidone treatment can improve lung CT and lung volume in mice,and the lung volume in the IMRCs group was significantly higher than that in the BLM group,and the differences were statistically significant(P<0.05).In conclusion,treatment with IMRCs,UCMSCs and pirfenidone after bleomycin modeling can improve the survival rate of mice,slow down the weight loss rate of mice,reduce the degree of pulmonary fibrosis,and improve lung function and lung CT in mice,and the therapeutic effect of IMRCs on lung injury-induced fibrosis is better than that of UCMSCs and pirfenidone.To explore the mechanism of IMRCs in the treatment of bleomycin-induced lung injury-induced pulmonary fibrosis in mice These experimental results show that:(1).Normal IMRCs can slow down the weight loss trend and improve lung coefficient of pulmonary fibrosis mice,but improvement of IMRCs knockout of MMP1 gene was not obvious;(2).Normal IMRCs could improve lung function in mice with pulmonary fibrosis.The PV circle,FVC,IC and Crs of the IMRCs group were significantly higher than those of the BLM group,and the Rrs and Ers of the IMRCs group were significantly lower than those of the BLM group.The difference was statistically significant(P<0.05),while the IMRCs-MMP1-/-group and the BLM group had no significant difference in pulmonary function indexes.(3).IMRCs treatment can reduce lung inflammation and collagen deposition in bleomycin model mice.Ashcroft score of IMRCs and IMRCs-MMP1-/treatment group was significantly lower than that of BLM group,and Ashcroft score of IMRCs group was significantly lower than that of BLM group.IMRCs-MMP1-/-group,the difference was statistically significant(P<0.05).In conclusion,the treatment of fibrosis in mice by IMRCs knocking out the MMP1 gene was significantly reduced.Conclusion:The mouse pulmonary fibrosis model produced by bleomycin nasal aerosolization has more uniform and extensive fibrosis lesions,and the aerosolized particles can reach the edge of the lung tissue,resulting in the formation of subpleural fibrous foci,which is more in line with the clinical characteristics of pulmonary fibrosis.Multiple tail vein injections of IMRCs ameliorated bleomycin-induced early pulmonary fibrosis in mice.After intravenous injection of IMRCs into mice,they are mainly distributed in the lungs,followed by the liver,etc.,with a half-life of 5.26 days,and will not remain in the body for a long time.Intravenous injection of IMRCs has a certain therapeutic effect on bleomycininduced lung injury inflammation and pulmonary fibrosis in mice,and has a certain time and frequency dependence,that is,the earlier the treatment time,the better the effect and twice the treatment effect is better.IMRCs,UCMSCs and PFD have certain therapeutic effects on bleomycin-induced lung injury and pulmonary fibrosis in mice,and IMRCs have better therapeutic effects on lung injury and fibrosis than UCMSCs and PFD.The high expression of MMP1 in IMRCs may be the mechanism of its treatment of bleomycininduced lung injury-induced pulmonary fibrosis in mice. |
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