The Study Of Chmp1b/1a Function During Pulmonary Fibrosis Induced By Bleomycin

Objective:Idiopathic pulmonary fibrosis（IPF）is a destructive,progressive,and lethal disease with average 3-5 years of survival,and its pathogenesis is still not fully understood^[1].IPF is characterized by repeated alveolar epithelial cell injury（AEC）and repair,epithelial mesenchymal transdifferentiation,abnormal proliferation and activation of fibroblasts,and changes in extracellular matrix deposition,ultimately leading to destruction of lung parenchyma^[2],Epithelial cell damage and proliferation are key events in promoting IPF.ESCRT system is a multifunctional protein transport and membrane cleavage machinery involved in the regulation of signaling pathways of a variety of membrane-bound receptors^[3],maintaining the polarity of epithelial cells^[4],fibroblasts^[5];The multivesicular body is a precursor of the hallmark organelle lamellar body of type II alveolar epithelial cells.The important function of the ESCRT system is to regulate the formation of multivesicular bodies.As an important member of the ESCRT system,the distribution of Chmp1b/1a in lung tissue and its role in the development of pulmonary fibrosis have not been reported.Currently,there is great controversy about whether EMT of alveolar epithelial cells is one of the origins of mesenchymal cells.Some researchers used SPC-rt TA/tet O₇-Cre/lac Z system marker from the embryonic phase to show the presence of EMT during fibrosis^[6],while other scholars used SPC-Cre ER^T2 knock-in to show no EMT^[7].In response to this fatal disease,it is crucial to identify various cell types and search suitable molecular markers through co-localization of biomacromolecules,and track the alveolar epithelial and mesenchymal cells during pulmonary fibrosis by using cell identification and tracking techniques,and determine the state of differentiation and the path of differentiation of cells and explore the relevant factors that play an important role in the development of pulmonary fibrosis and the development of the disease course,which is crucial for finding effective treatments.This study sought to find a method for repeated immunohistochemical staining for stripping antibodies of the same section.This method was used to clarify the distribution of Chmp1b/1a in mouse lung tissues,and its co-localization with other common cell markers in lungs,and the relationship with intraperitoneal injection of Bleomycin-induced fibrotic lesions.AECII-specific knockdown of Chmp1b/1a in SPC-rt TA/tet O₇-Cre system transgenic mouse peripheral lung tissue and simultaneously established a mouse model of pulmonary fibrosis,and simultaneously establish a mouse model of pulmonary fibrosis,to determine whether EMT existed during pulmonary fibrosis and whether Chmp1b participated in the EMT process.Through the distribution of ADRP of lipofibroblast（LIF）markers in the fibrosis process and its co-localization with fibroblasts and macrophages,etc.We understood its relationship with the fibrosis process,thus to verify the effectiveness of immunohistochemical antibody stripping technology.Methods:This study is divided into four parts.In the first part,wild-type mice were selected as the study subjects.Bleomycin was injected intraperitoneally,and immunohistochemical antibody stripping and repeated staining and Realtime PCR and other methods were used to explore the changes of Chmp1b,Chmp1a and Tsg101expression of multiple members of ESCRT system in normal lung tissues and fibrotic lung tissues from two aspects:expression distribution and expression quantity,to confirm the correlativity between the expression pattern of Chmp1b in lung tissues and the fibrosis.In the second part,SPC-rt TA/tet O₇-Cre system transgenic mice AECII specific knockdown Chmp1b/1a,agarose gel electrophoresis,immunohistochemistry and other methods were used to identify the establishment of transgenic mouse model,to determine whether Chmp1b/1a has an effect on epithelial cell homeostasis;intraperitoneal injection of Bleomycin modeling,HE staining,immunohistochemistry,Realtime PCR and other methods were used to clarify whether Chmp1b/1a has an effect on fibrosis.In the third part,the SPC-rt TA/tet O₇-Cre/m Tm G system autoinducer marking and tracking airway epithelial descendant cells,immunohistochemistry,immunofluorescence and other methods were used to analyze the contribution of epithelial cells to EMT during pulmonary fibrosis and whether Chmp1b participated in EMT process.The fourth part attempts to immunohistochemically repeat staining the same slice of exfoliated antibody with different concentrations of urea and other basic stripping solution to select the best solution.Wild-type mice were injected intraperitoneally with Bleomycin,and immunohistochemical antibody stripping repeat staining,oil red staining and Realtime PCR were used to elucidate the distribution of lipoblastic cell marker ADRP during fibrosis,and the co-localization with the labeled macrophages,fibroblasts,etc.to understand its relationship with the fibrosis process;the truncated process molding was used to confirm the correlation between ADRP⁺cells and macrophage group transition and fibrosis process（the order was changed）,thus verifying the effectiveness of immunohistochemical antibody stripping techniques.1.Chmp1b/1a is a negatively correlated alveolar epithelial cell marker molecule in the pathogenesis of pulmonary fibrosis1.1 Animal grouping and pulmonary fibrosis model preparation:Male C57 BL/6 mice of 8-12 weeks were randomly divided into normal group and experimental group（pulmonary fibrosis model group）,with 6 mice in each group.The mice were injected with bleomycin 40 USP/kg on days 0 and 2,20 USP/kg on days 4 and 6,and 10 USP/kg on days 9,12,15,18,21,24,and 27.The mice in the normal control group were injected with the same volume of normal saline.The experimental group was sampled on the 14th,21st,and 28th days.1.2 Preparation of pathological specimens and HE,Masson staining:After the right lung tissues of the mice were embedded in paraffin and sectioned,HE staining and Masson staining were performed.1.3 Immunohistochemical staining:Immunohistochemical staining of right lung tissue sections was performed by SP method.The expression of Chmp1b/1a in ESCRT and lung tissue and fibrotic lung tissues was detected.1.4 Antibody stripping repeat staining:Stripping buffer 65 m M Tris-HCl p H 6.8,1%SDS,0.113 M 2-mercaptoethanol,0.1 M Na Cl,2 M urea stripping antibody,the same section was subjected to immunohistochemical repeated staining,and the colocalization of Chmp1b/1a with SPC,PCNA,Smad2,andα-SMA in normal mouse lung tissues and fibrotic lung tissues was observed.1.5 Realtime PCR:RNA was extracted from the left lung tissues of mice,and the expression levels of each ESCRT member（Chmp1b,Chmp1a,Tsg101）and fibrosis-related important molecules（Collagen,IL-1β,IL-6）were detected.2.Chmp1b/1a affects the differentiation of AECII in peripheral lung tissues of transgenic mice and aggravates pulmonary fibrosis to a certain extent2.1 Animal grouping and pulmonary fibrosis model preparation:The transgenic mice with genotype SPC-rt TA⁺/^-/tet O₇Cre⁺/⁺were mated with the mice with shcmb⁺/^-genotype,and thet were induced with Doxycyclin after successful mating,and 0.5mg/ml Doxycyclin was added to drinking water to induce until 30 days after birth（E0.5-P30）,and the genotypes were routinely identified,then the transgenic mice with genotype SPC-rt TA⁺/^-/tet O₇Cre⁺/^-/shcmb⁺/^-were selected as experimental group,and the mice of the same litter and same sex lacking of one of the three loci of SPC-rt TA/tet O₇-Cre/shcmb were taken as the control group,and were raised to a 12-week-old and intraperitoneally injected with bleomycin for molding.The operators didn’t know the genotypes of the mice,and Bleomycin was injected intraperitoneally for molding.Bleomycin was injected 40 USP/kg on days 0 and 2,20 USP/kg on the 4th and 6th days,and 10 USP/kg on the 9th,12th,15th,18th,21st,24th and 27th days,and during modeling the body weight changes and death were monitored,and the surviving mice completing the entire modeling process were sampled on the 28th day after modeling.The same experimental procedure was used for the study of Chmp1a.2.2 Preparation of pathological specimens and HE staining:The right lung tissues of the mice were embedded in paraffin and sectioned,and stained with HE staining.2.3 Immunohistochemical staining:Immunohistochemical staining of right lung tissue sections was performed by SP method.The fibrosis-related indexα-SMA was detected.2.4 Antibody stripping repeat staining:Stripping buffer 65 m M Tris-HCl p H 6.8,1%SDS,0.113 M 2-mercaptoethanol,0.1 M Na Cl,2 M urea stripping antibody,repeat immunohistochemical staining of the same section of antibody stripping（Cre,Chmp1b/Chmp1a）was performed to verify Chmp1b/1a knockdown in transgenic mice.Repeated immunohistochemical staining（Cre,Chmp1b,SPC）was performed on the same section of antibody stripping,and the effect of Chmp1b knockdown on alveolar epithelial homeostasis was observed.2.5 Agarose gel electrophoresis:DNA was extracted from the left lung tissues of mice,and the Chmp1b/1a knockdown of the transgenic mice was confirmed by agarose gel electrophoresis.2.6 Realtime PCR:RNA was extracted from the left lung tissues of mice,and the expression levels of Collagen,α-SMA,S100A4,and IL-6 were detected.The severity of fibrosis in control mice and transgenic mice was compared.3.Cell lineage tracking revealed the transdifferentiation fate of airway epithelial cells during pulmonary fibrosis3.1 Animal grouping and pulmonary fibrosis model preparation:SPC-rt TA mice（B6.Cg-Tg（SFTPC-rt TA）5Jaw/J）,tet O₇-Cre mice（B6.Cg-Tg（tet O-cre）1Jaw/J）were purchased from Jacksonlab;m Tm G mice（B6.129（Cg）-Gt（ROSA）26Sortm4（ACTB-td Tomato,-EGFP）Luo/J）were purchsed from the Model Animal Research Center Of Naijing University;three kinds of mice were mated to obtain the transgenic mice with genotype SPC-rt TA⁺/^-/tet O-Cre⁺/^-/m Tm G⁺/^-,and the transgenic mice of 8-12 weeks old were induced with Doxycyclin for 15 days and divided into control group and experimental group（fibrosis model group）,with 3 mice in each group.The mice were injected with bleomycin intraperitoneally every other day,1 USP bleomycin/mouse on days 0 and 2,and20 USP/kg on the 4th and 6th days,and 10 USP/kg on the 9th,12th,15th,18th,21st,24th,27th days.The mice in the normal control group were injected with the same volume of normal saline,and were sampled on the 28th day.3.2 Immunohistochemical staining:Immunohistochemical staining of right lung tissue sections was performed by SP method to detect the expression of m EGFP in fibrotic lung tissues.3.3 Antibody stripping repeat staining:The stripping buffer 65 m M Tris-HCl p H 6.8,1%SDS,0.113 M 2-mercaptoethanol,0.1 M Na Cl,2 M urea stripping antibody,immunohistochemical repeated staining of the same section（SPC,m GFP,α-SMA,S100A4）was performed,and the differentiation state of fibrotic mouse epithelial cells was observed.3.4 Immunofluorescence:The immunofluorescence was performed for m EGFP and PCNA,and the proliferation state of epithelial cells in the lesioned area of fibrotic mice was observed by confocal fluorescence microscopy.4.Immunohistochemical antibody stripping repeated staining showed that ADRP⁺CD206⁺cells were associated with pulmonary fibrosis in peripheral lung tissue injury4.1 Animal grouping and pulmonary fibrosis model preparationModeling construction by normal procedure:the animals were randomly divided into5 groups,normal control group and experimental group（pulmonary fibrosis model group）,6 in each group.The animals in the experimental group were intraperitoneally injected with Bleomycin,with bleomycin 40 USP/kg on days 0 and 2,20 USP/kg on days 4 and 6,and 10 USP/kg on days 9,12,15,18,21,24,and 27,and were sampled randomly on days7,14,21,and 28,respectively.The animals in the normal control group were injected with the same volume of normal saline,were sampled on day 28.Truncated process modeling:the animals were randomly divided into 6 groups,normal control groups and experimental groups（pulmonary fibrosis model groups）,6 mice in each group,the mice in the experimental groups were injected intraperitoneally with Bleomycin 1 time（40 USP/kg on day 0）,2 times（40 USP/kg on days 0 and 2）,3 times（40USP/kg on days 0 and 2,20 USP/kg on days 4）,4 times（40 USP/kg on days 0 and 2,20USP/kg on days 4 and 6）and continuous molded（is the same as modeling by normal procedure）,and the animals in the normal control groups were injected with the same volume of normal saline,and all were sampled on the 28th day.4.2 Preparation of pathological specimens and HE,Masson staining:After the right lung tissues of the mice were embedded in paraffin and sectioned,HE staining and Masson staining were performed to observe the changes of lung tissues in different sampling periods and different injection times.4.3 Immunohistochemical staining:Immunohistochemical staining of right lung tissue sections was performed by SP specimen method to detect the expression distribution of ADRP in the lung tissues of normal mice and fibrosis mice.4.4 Antibody stripping repeated:The base stripping buffer contains 65 m M Tris-HCl p H 6.8,1%SDS,0.113 M 2-mercaptoethanol,0.1 M Na Cl,and different concentrations of urea（0 M,1 M,2 M,4 M,6 M）were used for antibody stripping.The second immunohistochemical staining was performed.The most suitable urea concentration was selected and repeated immunohistochemical staining was performed to observe the co-localization of ADRP and macrophage markers（CD68,CD86,CD206）,S100A4 and Caspase9 in different sampling periods and of different injection times.4.5 Realtime PCR:RNA was extracted from the left lung tissues of mice,and the levels of expression of lipid fibroblast markers（ADRP,PDGFRα）,macrophage markers（CD68,CD86,CD206）and fibrosis-related important molecules（Collagen,α-SMA）were detected.Results:1.Chmp1b/1a is a negatively correlated alveolar epithelial cell marker molecule in the pathogenesis of pulmonary fibrosis1.1 Chmp1b/1a is mainly distributed in the Type II alveolar epithelial cells of the peripheral lung tissues and in the proximal airway epithelial cells.In the peripheral lung tissues,the expression pattern of Chmp1b/1a is highly coincident with the marker molecule SPC of traditional Type II alveolar epithelial cells.1.2 During the model-induced pulmonary fibrosis,Realtime PCR detected a decrease in the expression level of Chmp1b/1a and Tsg101.There are many PCNA-positive cells in the fibrotic lesion area with high expression ofα-SMA,but no Chmp1b/1a is expressed,and the staining intensity of Chmp1b in Chmp1b⁺cells adjacent to fibrotic lesions is lower than that of Chmp1b⁺cells away from the lesion area,and the staining intensity of SPC in the adjacent area of lesions and away from the lesion area is similar.1.3 Smad2 is expressed in many nuclei in normal area of peripheral lung tissue structure of the mice in the molding group,and Chmp1b is expressed in most of them simultaneously.There is no obvious Smad2 expression in the lung tissues of the control group.2.Chmp1b/1a affects the differentiation of AECII in peripheral lung tissues of transgenic mice and aggravates pulmonary fibrosis to a certain extent2.1 AECII-specific knockdown of Chmp1b/1a transgenic mice in the peripheral lung tissues of SPC-rt TA/tet O₇-Cre system was successfully established.The mouse tail DNA gel electrophoresis showed the amplified 629 bp band after recombination and the repeated immunohistochemical staining showed that Chmp1b/1a positive expression cells decreased in the alveolar Type II epithelial cells with Cre positive expression.2.2 Chmp1b knockdown had an effect on the alveolar epithelial homeostasis,and the expression of SPC in Cre⁺expression in AECII cells with Chmp1b knockdown decreased.2.3 The mortality of the transgenic mice with Chmp1a knockdown increased after intraperitoneal injection of Bleomycin,and the fibrosis index of Chmp1b knockdown transgenic mice increased.3 Cell lineage tracking revealed the transdifferentiation fate of airway epithelial cells during pulmonary fibrosis3.1 SPC-rt TA mice（B6.Cg-Tg（SFTPC-rt TA）5Jaw/J）,tet O₇-Cre mice（B6.Cg-Tg（tet O-cre）1Jaw/J）,m Tm G mice（B6.129（Cg）-Gt（ROSA）26Sortm4（ACTB-td Tomato,-EGFP）Luo/J）,three kinds of mice were mated successfully and the transgenic mice of labeled epithelial cells with genotype SPC-rt TA⁺/^-/tet O-Cre⁺/^-/m Tm G⁺/^-were obtained;antibody stripping repeat staining techniques can be used for Cell lineage tracking and mapping studies.3.2 In the lung tissues of mice,m EGFP⁺cells were present in the fibrous lesions.The co-localization ofα-SMA,S100A4 with m EGFP was observed in a few cells,which were not the main body of fibrotic lesions.3.3 In the lung tissues of modeled mice,most of the m EGFP⁺cells present in the fibrous lesion area were not co-localized with PCNA.4.Immunohistochemical antibody stripping repeated staining showed that the distribution area of ADRP⁺CD206⁺cells predicted the occurrence of pulmonary fibrosis4.1 Immunohistochemical antibody stripping repeat staining,the basic stripping buffer containing 65 m M Tris-HCl p H 6.8,1%SDS,0.113 M 2-mercaptoethanol,0.1 M Na Cl,and adding different concentrations of urea（2 M,4 M,6 M）when used;the best antibody peeling effect can be obtained when the urea concentration is 2 M.4.2 In the process of intraperitoneal injection of Bleomycin inducing pulmonary fibrosis in mice,sampled in the different periods and of different dosing times,the expression of ADRP in lipid fibroblast markers continued to increase,but the expression level of PDGFRαdecreased.4.3 In the early stage of modeling,ADRP⁺cells include lipid fibroblasts and macrophages（mainly M1）;late ADRP⁺cells are basically macrophages（M2）.The conversion of M1/M2 group of this ADRP⁺cells occurred on the 14th day sampling in the normal process or the 28th day sampling of 3 times of dosing in the truncated process.4.4 During the period of ADRP⁺cell group conversion,ADRP⁺CD206⁺cells are mainly distributed in the vicinity ofα-SMA and N-cadherin positive expression regions.In ADRP⁺CD206⁺cell-rich region AEC expressing Caspase-9 is higher,suggesting that this is the area of alveolar epithelial injury.4.5 During the fibrosis process,the ratio of co-localization of ADRP to S100A4gradually increased.There was co-localization with Caspase9 in different periods,but no co-localization relationship with PCNA.Conclusion:1.Chmp1b/1a can be used as a marker molecule for Type II alveolar epithelial cells in normal mouse peripheral lung tissues.There is a negative correlation between the expression pattern of Chmp1b/1a and the pathogenesis of pulmonary fibrosis in the lung tissues of mice with pulmonary fibrosis induced by intraperitoneal injection of Bleomycin,suggesting that ESCRT system may play an important role in pulmonary fibrosis.2.AECII-specific knockdown of Chmp1b/1a transgenic mice in the peripheral lung tissues of SPC-rt TA/tet O₇-Cre system was successfully established.Chmp1b knockdown resulted in a decrease in SPC of AECII,affecting the homeostasis of alveolar epithelial cells.The mortality of the transgenic mice with Chmp1a knockdown increased after intraperitoneal injection of Bleomycin,and the index of fibrosis of the transgenic mice with Chmp1a knockdown showed an upward trend.3.The SPC-rt TA/tet O₇-Cre/m Tm G system autoinducer successfully marking mouse epithelial cells.The antibody stripping repeated staining techniques can be used for Cell lineage tracking and localization studies.m EGFP-positive cells were found in the fibrotic lesion area of the molded mice.EMT occurred in a small number of epithelial cells,but most did not proliferate,and EMT is not the main body of fibrotic lesions.4.Immunohistochemical antibody stripping repeated staining techniques can be used for colocalization studies of cells.During the process of pulmonary fibrosis in molded mice,the expression level of ADRP of lipid fibroblast markers continued to increase,but the expression level of PDGFRαdecreased.Through co-localization method,it was found that ADRP⁺cells in the early stage of modeling included lip fibroblasts and macrophages（mainly M1）;late ADRP⁺cells were basically macrophages（M2）.This ADRP⁺cell M1/M2 group conversion is associated with pulmonary fibrotic pathology process,and the accumulation of ADRP-positive M2-polarized macrophages indicates that fibrotic damage is about to occur or has occurred.