| Objective:To explore whether regulatory T cells(Treg)induce epithelial-mesenchymal transition(EMT)and myofibroblast(MFB)transformation by secreting transforming growth factor-β1(TGF-β1)to promote radiation pulmonary fibrosis(RIPF),and to further study whether the effect of glycyrrhetinic acid(GA)in reducing RIPF is related to this mechanism.Methods:1.A single dose of 18 Gy 6MV-X ray was used to irradiate the chest of C57BL/6mice to establish RIPF model.The samples were taken on Day 2,Day 17,Month 3 and Month 5 after irradiation.HE and Masson staining were performed to evaluate lung tissue pathology,and immunofluorescence was performed to observe the expression of E-cadherin and α-smooth muscle actin(α-SMA)in lungs.The ratio of Treg/CD4+T cells in spleens,peripheral blood,and lungs was analyzed by flow cytometry,and CD4 and Fox P3 expression in lungs were detected by immunofluorescence.2.Treg cells were sorted from mice spleen and lymph nodes,after which the Treg cell medium(Treg CM)was prepared.Two kinds of co-culture systems including Treg cell-cell and Treg CM-cell were established.For comparison,the culture of mouse typeⅡ alveolar epithelial cells(MLE-12)or mouse embryonic fibroblasts(MEF)was as control group.The morphological changes of MLE-12 cells were observed,and the expression levels of epithelial,mesenchymal and MFB markers in MLE-12 or MEF cells after co-culture were detected by immunofluorescence,RT-q PCR,and Western Blot.The expression of TGF-β1 in the supernatant of Treg cells was detected by ELISA.3.The effect of GA on the Treg-induced EMT and MFB transformation:(1)They were divided into four groups: MLE-12 or MEF,MLE-12 or MEF+Treg,MLE-12 or MEF+Treg+GA,MLE-12 or MEF+GA.The morphological changes of MLE-12 cells in each group were observed,and the expression levels of epithelial,mesenchymal and MFB markers in MLE-12 or MEF cells in each group were detected by immunofluorescence,RT-q PCR,and Western Blot.The expression of TGF-β1 in the supernatant of each group was detected by ELISA.(2)Treg cell medium(Treg CM)and the Treg cell medium in the presence of GA [(Treg+GA)CM] were prepared.They were divided into four groups: MLE-12 or MEF,MLE-12 or MEF+Treg CM,MLE-12 or MEF+Treg CM+SB 525334(TGF-β1 receptor inhibitor),MLE-12 or MEF+(Treg+GA)CM.The morphological changes of MLE-12 cells in each group were observed,and the expression levels of epithelial,mesenchymal and MFB markers in MLE-12 or MEF cells in each group were detected by immunofluorescence,RT-q PCR,and Western Blot.4.The effect of GA on the RIPF: They were divided into NS(normal saline)group,IR(irradiation)+ NS group,IR + GA group,and GA group.The samples were taken on Day 2,Day 17,Month 3 and Month 5 after irradiation.HE and Masson staining were performed to evaluate lung tissue pathology,and immunofluorescence was performed to observe the expression of E-cadherin,Vimentin and α-SMA in lungs.The ratio of Treg/CD4+T cells in spleens,peripheral blood,and lungs was analyzed by flow cytometry,while CD4 and Fox P3 expression in lungs were detected by immunofluorescence.The expression level of TGF-β1 in peripheral blood and lungs of each group was detected by ELISA.5.The role of Treg cells in GA-improved RIPF: They were divided into NS group,IR+NS group,IR+GA group,GA group and IR+GA+Treg group(Treg cell reinfusion was implemented into mice on Day 17 after irradiation).The samples were taken on Month 3 and Month 5 after irradiation.The ratio of Treg/CD4+T cells in spleens,peripheral blood,and lungs was analyzed by flow cytometry.HE and Masson staining were performed to evaluate lung tissue pathology,and immunofluorescence was performed to observe the expression of E-cadherin,Vimentin and α-SMA in lungs.The expression level of TGF-β1 in peripheral blood and lungs of each group was detected by ELISA.Results:1.The occurrence of Treg cells infiltration in RIPF mice: Compared with the control group,HE staining of the irradiated group showed that the lungs appeared inflammatory infiltration on Day 2,Day 17 after irradiation,and the alveolar interval was gradually widened,resulting in fibrotic changes on Month 3 and Month 5 after irradiation.Masson staining showed that collagen deposition in lungs gradually increased on Month 3 and Month 5 after irradiation.Immunofluorescence showed the gradual decreasing of E-cadherin and increasing of α-SMA expression in lungs on Month 3 and Month 5 after irradiation.All of these indicated that the RIPF mice model was successfully constructed.Compared with the control group,the ratio of Treg/CD4+T cells in the spleens,peripheral blood,and lungs of the irradiated group significantly increased on Day 2,Day 17 after irradiation.The expression of CD4 and Fox P3 in lungs also significantly increased on Day 2,Day 17 after irradiation.2.Treg cells inducing EMT and MFB transformation:(1)Treg cells and MLE-12 cells were co-cultured: The morphology of MLE-12 cells in the Treg group was changed from cobblestone to spindle shape,and the connections between cells were looser.Compared with the control group,the immunofluorescence and Western Blot results in the Treg group showed that the expression of E-cadherin in MLE-12 cells was down-regulated,and the expression of Vimentin in MLE-12 cells was up-regulated,and the RT-q PCR results showed that the m RNA expression levels of Vimentin,Fibronectin1,PAI-1,MMP9,Snail1,ZEB1,and Twist in MLE-12 cells were all up-regulated(P<0.05).(2)Treg cells and MEF cells were co-cultured: Compared with the control group,the immunofluorescence and Western Blot results in the Treg group showed that the expression of α-SMA in MEF cells were up-regulated,and the RT-q PCR results showed that the m RNA expression of MMP9 and α-SMA in MEF cells was also up-regulated(P<0.05).3.The induction of EMT and MFB transformation by Treg cells related to its TGF-β1 secretion:(1)Treg CM and MLE-12 cells were co-cultured: The morphology of MLE-12 cells in the Treg CM group was changed from cobblestone-like to long spindle-shaped and was connected loosely.Compared with the control group,the immunofluorescence and Western Blot results in the Treg CM group showed that the expression of E-cadherin in MLE-12 cells was down-regulated and the expression of Vimentin in MLE-12 cells was up-regulated,and the RT-q PCR results showed that the m RNA expression levels of Vimentin,Fibronectin1,PAI-1,MMP9,Snail1,ZEB1,and Twist in MLE-12 cells were all up-regulated(P<0.05).(2)Treg CM and MEF cells were co-cultured: Compared with the control group,the immunofluorescence and Western Blot results in the Treg CM group showed that the expression of α-SMA in MEF cell was up-regulated,and the RT-q PCR results showed that the m RNA expression levels of Fibronectin1,MMP9,α-SMA in MEF cells were all up-regulated(P<0.05).(3)The ELISA results showed that the expression of TGF-β1 in the supernatant of the Treg group was significantly higher than that of the control group(P<0.05).4.GA can inhibit the EMT and MFB transformation induced by Treg cells:(1)The co-culture of Treg cells and MLE-12 cells was intervened by GA: Compared with the Treg group,the degree of morphological transformation of MLE-12 cells from cobblestone to spindle in the Treg+GA group was reduced.Immunofluorescence and Western Blot results showed that the E-cadherin expression was up-regulated and Vimentin expression was down-regulated in the Treg+GA group,and the RT-q PCR results showed that the m RNA expression levels of Vimentin,Fibronectin1,MMP9,ZEB1,and Twist in the Treg+GA group were all down-regulated(P<0.05).(2)The co-culture of Treg cells and MEF cells was intervened by GA: Compared with the Treg group,the immunofluorescence and Western Blot results in the Treg+GA group showed that the expression of α-SMA in MEF cell was down-regulated,and the RT-q PCR results showed that the m RNA expression levels of MMP9 and α-SMA were down-regulated(P<0.05).5.GA can inhibit the EMT and MFB transformation induced by TGF-β1 secreted by Treg:(1)Treg CM and(Treg+GA)CM were co-cultured with MLE-12 cells:Compared with the Treg CM group,the Treg CM+SB 525334(TGF-β1 receptor inhibitor)Group and(Treg+GA)CM group showed that the degree of morphological transformation of MLE-12 cells from cobblestone to spindle was reduced,and immunofluorescence and Western Blot results showed that the E-cadherin expression was up-regulated and Vimentin expression was down-regulated and the RT-q PCR results showed that the m RNA expression levels of Vimentin,MMP9 and ZEB1 were all down-regulated(P<0.05).(2)Treg CM and(Treg+GA)CM were co-cultured with MEF cells: Compared with the Treg CM group,the Treg CM+SB 525334 group and(Treg+GA)CM group in immunofluorescence and Western Blot showed that the expression of α-SMA in MEF cell was down-regulated,and the RT-q PCR results showed that the m RNA expression levels of MMP9 and α-SMA were down-regulated(P<0.05).(3)ELISA showed that the expression of TGF-β1 in the supernatant of the Treg group was significantly higher than that in the control group(P<0.05),while the expression of TGF-β1 in the supernatant of the Treg+GA group was lower than that in the Treg group(P<0.05).6.GA can improve RIPF and reduce Treg cell infiltration by decreasing the expression of TGF-β1 in mice after irradiation: Compared with the IR+NS group,the inflammation,pulmonary fibrosis and collagen deposition in lungs of the IR+GA group at different times after irradiation were all reduced(P<0.05).On Month 3 and Month 5after irradiation,the expression of E-cadherin was up-regulated,the expression of Vimentin and α-SMA were down-regulated in the IR+GA group(P<0.05).On Day 2,Day 17 after irradiation,the ratio of Treg/CD4+T cells in the spleens,peripheral blood and lungs decreased in the IR+GA group(P<0.05),and the expression of CD4 and Fox P3 in lungs also decreased(P<0.05).Compared with the NS group,the expression of TGF-β1 in peripheral blood and lungs of the IR+NS group increased gradually at different times after irradiation(P<0.05),while the expression of TGF-β1 in the IR+GA group was lower than that in the IR+NS group during the same period(P<0.05).7.Treg cells reinfusion can up-regulate the TGF-β1 expression in irradiated mice with GA intervention and inhibit GA-improved RIPF: Compared with the IR+GA group,the ratio of Treg/CD4+T cells in the spleens,peripheral blood,and lungs of the IR+GA+Treg group increased significantly on Month 3 and Month 5 after irradiation(P<0.05).The degree of pulmonary fibrosis and collagen deposition in lungs of the IR+GA+Treg group was significantly worsened(P<0.05),and the expression of E-cadherin was down-regulated and the expression of Vimentin and α-SMA were up-regulated in lungs of the IR+GA+Treg group(P<0.05),and the TGF-β1 expression in peripheral blood and lung tissues were increased(P<0.05).Conclusions:Treg cells can induce EMT and MFB transformation by secreting TGF-β1 to promote the process of RIPF.GA can improve RIPF,which may be related to the inhibition of EMT and MFB transformation induced by TGF-β1 secreted by Treg cells. |
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