Effect Of NRP1 Gene-Specific Knockout On The Process Of Radiation-Induced Pulmonary Fibrosis And Its Mechanism

Radiation-induced pulmonary fibrosis(RIPF)is a common complication of clinical radiotherapy for chest malignant tumors,and it is also one of the important reasons that limit the effect of radiotherapy for chest tumors.The main pathological manifestations of RIPF are inflammatory reactions caused by early acute radiation injury,including thickening of lung texture,widening of alveolar septum,and inflammatory infiltration.In the later stage,symptoms of fibrosis include increased fibrous connective tissue,alveolar structure destruction,and parenchymal cells Reduce,etc.,continuous progress can cause lung failure and endanger human life and healthThe pathogenesis of pulmonary fibrosis is complex and its mechanism has not been fully elucidated.In recent years,research focuses on transduction regulation of effector cells,cytokine networks and related signaling pathways.The occurrence and development of pulmonary fibrosis are often related to the damage of alveolar epithelial type II cells(AEC II)and macrophages.When the injury occurs,fibroblasts are easily activated,which in turn causes a series of cytokines Release,which in turn activates fibrosis-related signaling pathways,cytokine interactions,and causes epithelial-mesenchymal transition(EMT)and extracellular matrix(ECM)in alveolar epithelial cells.Excessive deposition,etc.,eventually lead to the occurrence and development of pulmonary fibrosis.Similarly,the formation of RIPF involves multiple factors and the interaction of multiple cells,and the exact mechanism of its occurrence needs further study,which is also a hot and difficult point of research today and in the future.Most of the preventive drugs for RIPF have not yet been marketed,and they have only stagnated in animal experiments,and there are still many problems that have not been properly resolved from experiment to clinical.However,with the widespread application of modern molecular biology and genomic technologies in the field of radiation and the continuous deepening of research on the pathogenesis and pathophysiological changes of the disease,I believe that we will have a deeper and comprehensive understanding of the relevant mechanisms of RIPF.Many new,potentially therapeutic targets.Therefore,targeted therapies targeting these related molecules will become a new trend in RIPF therapy.Background:Neuropilin-1(NRP1)is a multifunctional transmembrane glycoprotein originally found in the nervous system.It can be used as transforming growth factor-β(TGF-β),vascular endothelial growth factor(VEGF),platelet derived growth factor(PDGF),and semaphoring 3(Sema 3)Co-receptors of factors or other ligands,which in turn play a variety of biological functions in nervous,tumor and immune systems.Currently,NRP1 has been shown to play an important role in enhancing TGF-β1 signaling and promoting the occurrence of EMT,and according to our previous research,NRP1 can be expressed on regulatory T cells(Tregs),and at the same time It plays an important role in promoting the EMT transformation of fibroblasts and the radiation resistance of lung cancer cells.Therefore,the current researches on NRP1 mostly focus on immunoregulation,tumorigenesis and development,and related roles in the treatment process,but there are few reports on radiation pulmonary fibrosisResearch purposes:To investigate the effect of NRP1 on the differentiation of immune cells and the secretion of related cytokines during the RIPF process,and the correlation between the Wnt/β-catenin pathway and the TGF-β/Smads pathway-mediated EMT and the promotion of extracellular matrix deposition.The mechanism of action provides new therapeutic targets for the genesis and clinical prevention of RIPF.Methods:Based on the above research status and experimental results obtained,this study first established a C57BL/6J mouse RIPF model,and verified the success of the mouse RIPF model by H&E and Masson staining.Immunohistochemistry(IHC)was used to detect fibrosis markers and the protein level of NRP1.Then,the Cre-LoxP recombinase system and drug induction were used to construct a transgene C57BL/6J mice that specifically deleted the NRP1 gene in AEC Ⅱ cells(NRP1-/-;Cre+/0)and mouse tail genotype identification.Differential expression profiles of genes in lung tissue of control group and NRP1-/-;Cre+/0 mice were detected by RNA sequencing(RNA-Seq)technology and analyzed using bioinformatics technology.Next,the obtained mice were randomly divided into 4 weeks,8 weeks,16 weeks,and 24 weeks according to the time point after irradiation,and in each group,the mice were divided into a wild type group(Con)and a wild type according to a random number table method.Type simple irradiation group(IR),NRP1-/-;Cre+/0 group(KO-Con)and NRP1-/-;Cre+/0+ irradiation group(KO+IR),10 mice in each group,160 mice in total The RIPF mouse model was established in the irradiation group using 20 Gy(dose rate 2.0 Gy/min)X-ray whole chest irradiation.H&E staining and Masson staining were used to verify whether the model was successfully constructed.At the same time,siRNA-NRP1 interference fragment was used to specifically inhibit the expression of NRP1 gene in mouse lung epithelial cell line(MLE-12).After the establishment of the in vivo and in vitro models,the IHC method was first used to detect the expression of collagen I and a-smooth muscle actin(a-SMA)protein in the lung tissue of each group of mice;Flow cytometry was used to detect the proportion of Treg cells;Enzyme linked immunosorbent assay(ELISA)was used to detect the secretion of related cytokines in mouse bronchoalveolar lavage fluid(BALF)the amount.Then,the protein expression levels of NRP1,β-catenin,TGF-β1,and Smad2 were detected by Western bloting;real-time PCR(qRT-PCR))Method to detect NRP1,Collagen Ⅰ,α-SMA,β-catenin,TGF-β1,Smad2,E-cadherin,N-cadherin and Vimentin waveform in lung tissue of mice Relative expression levels of mRNAs such as proteins,and further explore the role of NRP1 in the occurrence and development of radiation-induced pulmonary fibrosis.Results:1.To explore the changes of NRP1 expression in the formation of radiation-induced pulmonary fibrosis(1)This study first established a mouse model of RIPF and verified the success of the model.That is,a single single irradiation of the mouse thorax by a single 20 Gy X-ray was performed to establish a mouse lung fibrosis model,and the degree of damage to mouse lung tissue was detected by H&E staining.The results showed that in the normal control group,the alveolar structure was intact and the alveolar walls were thin.In the RIPF group,the lung tissue gradually showed acute radiation injury 4-8 weeks after irradiation,that is,the alveolar space widened and the lung texture became thicker;8-16 weeks after irradiation,significant alveolar fusion and alveolar integrity gradually appeared Decreased,increased interstitial cells and inflammatory cell infiltration;24 weeks after irradiation,significant fibrous tissue proliferation was observed,large areas of the lungs became consolidated,and eventually pulmonary fibrosis formed.Masson staining was used to verify the RIPF mouse model.The results showed that the alveolar structure of the normal control group was clear,no blue collagen was deposited,and fibrosis was not obvious.In the RIPF model group,pulmonary fibrosis began to appear in the lung tissue of mice at 8 weeks after irradiation(blue),and only existed around the bronchiole.At 16 and 24 weeks after irradiation,obvious localized fibrotic areas appeared,and large blue collagen was visible.Deposition,suggesting that the mouse model of radioactive pulmonary fibrosis was successfully constructed(2)The expressions of NRP1 and fibrosis markers in lung tissues of RIPF model were further detected by qRT-PCR and IHC methods.The results showed that the expression of pulmonary fibrosis markers α-SMA and Collagen Ⅰ increased gradually with the time after irradiation(p<0.05).Similarly,the expression of NRP1 in mRNA and protein was also significantly increased(p<0.05),and the highest expression at 24 weeks after irradiation(p<0.05)was positively correlated with the expression ofα-SMA and Collagen Ⅰ and the degree of fibrosis.It is suggested that the increase of NRP1 expression is related to the progress of radiation pulmonary fibrosis in mice2.AEC Ⅱ cell-specific knockout NRP1 mouse model construction and sequencing of lung tissue gene expression profiles(1)Based on the results that have been determined that the increased expression of NRP1 is related to radiation-induced pulmonary fibrosis in mice,in order to further explore the role of NRP1 in the occurrence and development of RIPF,construct AECⅡ cell-specific knockout NRP1 mice(NRP1-/-;Cre+/0)model.The NRP1flox/flox mice purchased from Jackson Laboratories in the United States were used to mate with AEC Ⅱ cells expressing Cre recombinase-specific mice,and the mouse tail genomic DNA was extracted for genotype analysis to determine the mouse genotype.This resulted in NRPlfl/fl;Cre+/0 mice.Subsequently,the genotyped mice were specifically activated by the intraperitoneal administration of tamoxifen(TMX)to Cre recombinase in AEC Ⅱ cells,thereby achieving specific knockout of the NRP1 gene(NRP1-/-;Cre+/0)purpose.After 7 days of observation,AEC Ⅱ cells in the lung tissue of the mice were extracted and detected by triple immunofluorescence staining.NRP1-/-;AEC Ⅱ cells in the lung tissue of the Cre+/0 group were negative for NRP1 staining.The results showed that the NRP1 gene could be effectively knocked out in AEC Ⅱ cells of NRP1-/-;Cre+/0 mice.(2)Using RNA-seq technology to detect the differential expression profiles of genes in lung tissues of NRP1-/-;Cre+/0 and control mice.The results showed that AEC Ⅱ cell specific knockout of NRP1 could cause changes in gene expression profiles of mouse lung tissues.Compared with the control group,a total of 356 genes were differentially expressed(p<0.05),of which 93 expressions were up-regulated and 263 gene expression is down-regulated.In terms of biological processes,differentially expressed genes caused by AEC Ⅱ cell-specific knockout of NRP1 are mainly involved in lipid metabolism,neutrophil chemotaxis,regulation of signaling receptor activity,inflammatory response,and negative regulation of inflammatory response.etc.Further KEGG analysis revealed that most of the signaling pathways involved in the regulation of these differentially expressed genes are related to fibrosis,immunity,inflammation,ECM,and transcriptional regulation.3.Effect of AEC Ⅱ cell-specific knockout NRP1 on the occurrence and development of RIPF(1)First verify the effect of AEC Ⅱ cell-specific knockout NRP1 on the signs of RIPF mice.The results showed that the control group and NRP1-/-;Cre+/0 groups had no significant differences in various indicators.After irradiation and over time,the weight of IR mice slowed down,lung coefficient increased,and typical fibrotic pathological changes occurred in lung tissues,but the relevant indicators were significantly reduced after specific knockout of NRP1(p<0.05).In addition,the expression of NRP1 protein and mRNA in lung tissue of IR group increased gradually with time after irradiation,and reached the highest level at 24 weeks(p<0.05).The expression of NRP1 in KO-IR group was significantly lower than that in IR group(p<0.05).(2)In the early stage of RIPF formation,the effect of AEC Ⅱ cell-specific knockout of NRP1 on the inflammatory response during RIPF was discovered.After irradiation,in the lung tissue of mice in the NRP1-/-;Cre+/0 group,when After AEC Ⅱcells specifically knocked out NRP1,it could reduce the recruitment of CD4+Foxp3+Treg cells in the injured lung tissue and the proportion of these cells decreased at each time point(p<0.05).The proportion of Treg cells has little effect,indicating that the specific knockout of the NRP1 gene did reduce the proportion of thymus-derived CD4+Foxp3+Treg cells in mouse lung tissue.The results of inflammatory factor detection showed that the levels of IFN-γ,IL-2,TGF-β1,IL-17A and IL-4 in BALF of IR mice were significantly higher than those in the control group(p<0.05).The content of TGF-β1,IL-17A and IL-4 in BALF of group mice was decreased(p<0.05),while the content of IFN-γ and IL-2 was significantly lower than that of IR group at 4 weeks(p<0.05).After that,the expression of IFN-γ and IL-2 was gradually higher than that of IR group with time.(3)The effect of AEC Ⅱ cell-specific knockout NRP1 on the fibrosis stage in the RIPF process.The protein and mRNA expressions of Collagen-1,α-SMA,N-cadherin,and Vimentin in the IR group gradually increased(p<0.05),and the expression in the KO-IR group was significantly lower than that in the IR group at all time points.(p<0.05),but still higher than Con group(p<0.05).In terms of pulmonary fibrosis-related signaling pathways,the expression of β-catenin at the protein and mRNA levels in the irradiated group gradually increased(p<0.05),and the expression was significantly lower than that in the irradiated group(p<0.05).It is known that the TGF-β/Smad signaling pathway mainly plays a "switch" role in the process of inducing and developing RIPE.By examining the changes in the expression of this pathway-related factor at various time points,we found that compared with the control group,the expression of TGF-β1 and Smad 2 was significantly increased in the irradiation group at 16 and 24 weeks(p<0.05).The expression of NRP1 was significantly decreased after specific knockout(p<0.05).It can be seen that NRP1 can affect the activation of Wnt/β-catenin and TGF-β/Smad signaling pathways and thus affect the process of radiation-induced pulmonary fibrosis.4.In vitro studies on the role of NRP1 in the radiation response of lung epithelial cells(1)Construction of a cell model of NRP1 interference.First,Western blot and qRT-PCR were used to verify the inhibitory efficiency of the 3 siRNA fragments that specifically inhibited the NRP1 gene.The results showed that siRNA-NRP1-1 and siRNA-NRP1-3 in the three sirna-nrpl had significant inhibitory effect on NRP1 in MLE-12 cells(p<0.05).Finally,the best segment of siRNA-NRP1-3 was selected for subsequent experiments.Then,after 24 h of ionizing radiation at different doses and dose rates,the mRNA and protein levels of NRP1 in MLE-12 cells were verified.It was found that NRP1 increased in mRNA levels to varying degrees and that over time,its expression It increased further(p<0.05).It can be seen that radiation can induce the increase of NRP1 expression in MLE-12 cells,and the results are similar to those in vivo.According to the results,the irradiation conditions of 1.0 Gy/min and the total dose of 6 Gy were selected as the irradiation conditions for in vitro experiments(2)To investigate the effects of targeted inhibition of NRP1 on radiation-related pulmonary fibrosis-related signaling pathways in vitro.Targeted inhibition of NRP1 can significantly inhibit the expression of TGF-β1 in the TGF-β/Smads signaling pathway and its downstream key effector element Smad2 and the marker factorβ-catenin of the Wnt/β-catenin signaling pathway(p<0.05).And after the action of ionizing radiation,the expression of the three caused by radiation can be significantly suppressed(p<0.05).To further clarify this mechanism,we used inhibitors of these two signaling pathways to explore whether NRP1 affects the occurrence and development of RIPF by regulating the TGF-β/Smads and Wnt/β-catenin signaling pathways.The results confirmed that compared with the irradiation group,the expression of related markers can be significantly inhibited when two signaling pathway inhibitors are added after irradiation,and the combined inhibition of targeted NRP1 can further inhibit the effect,which is similar to that of the irradiation group.In contrast,when inhibitors were added separately,β-catenin expression decreased by 68.12%and TGF-β1 decreased by 20.52%;when NRP1 was also inhibited,β-catenin expression decreased by 75.51%and TGF-β1 decreased by 50.53%(p<0.05).It can be seen that the effect of NRP1 on RIPF can indeed be achieved by regulating the Wnt/β-catenin and TGF-β/Smads signaling pathways(3)By examining the effect of targeted inhibition of NRP1 on the expression of radiation-related pulmonary fibrosis,it was found that E-cadherin decreased significantly at 24 and 48 h after irradiation(p<0.05),and Vimentin and N-cadherin after irradiation The expression gradually increased(p<0.05).After targeted inhibition of NRP1,E-cadherin increased significantly,and the expression of N-cadherin and Vimentin decreased(p<0.05),and the inhibition of NRP1 gene after ionizing radiation could cause the expression of N-cadherin and Vimentin to decrease.This indicates that the inhibition of NRP1 gene can reduce the radiation-induced EMT in MLE-12 cells.Conclusion:1.In the lung tissue of RIPF mice,the expression of NRP1 is significantly increased,and it is positively correlated with the expression of lung fibrosis markers and the degree of lung fibrosis;2.Alveolar type Ⅱ epithelial cell-specific knockout of NRP1 can cause differential expression of genes involved in regulating alveolar structure,immune inflammation response,and fibrosis-related signaling pathways in mouse lung tissues;3.Alveolar type Ⅱ epithelial cell specific knockout NRP1 can effectively improve the function and integrity of lung tissue and tissue structure of RIPF mice;and reduce the degree of tissue damage and fibrosis,which is conducive to the prognosis of RIPF;4.Alveolar type Ⅱ epithelial cell specific knockout NRP1 can affect the differentiation and recruitment of Treg cells,the imbalance of Th cells and the secretion of related cytokines in the lung tissue of RIPF mice during the inflammatory response stage;5.The NRP1 gene can regulate the Wnt/β-catenin and TGF-β/Smads signaling pathways to affect the EMT transformation and ECM secretion of epithelial cells,which ultimately affects the occurrence and development of RIPF;6.Specific knockout of the NRP1 gene in mouse alveolar type Ⅱ epithelial cells significantly reduced radiation pneumonitis and subsequent pulmonary fibrosis.Targeting NRP1 may be another effective way to treat RIPF.