Study On The Effect And Mechanism Of Blueberry Juice To Prevent And Alleviate Idiopathic Pulmonary Fibrosis Via TGF-β1/Smad2/3 And Stat3 Signaling

[Background and Objective]IPF is an interstitial lung disease with unknown origin,and is characterized by repeat alveolar epithelial cell injury,subsequent release of pro-inflammatory and pro-fibrotic cytokines and other mediators,the accumulation of activated fibroblasts and myofibroblasts in fibrotic foci,and abnormal accumulation of ECM,leading to loss of normal lung structure,end-stage lung disease,and resulting in respiratory failure and death finally.The incidence and prevalence of IPF appear to be upward and similar with liver and stomach cancers.In the"Guidelines for the Diagnosis and Treatment of IPF（2019 Edition）",it is mentioned that the prevalence of IPF in China is about2～29/100,000,which is common in elderly men with heavy smoking.In terms of medication treatment,there are only two clinic drugs,nintedanib（NTB）and pirfenidone,that have received regulatory approval by American FDA to treat IPF so far.However,these two drugs can only relieve but not cure IPF and there are side effects such as hepatotoxicity and adverse gastrointestinal reactions in these two drugs’treatment.As for surgical treatment,due to the limited clinical technology and donor sources,lung transplantation cannot be widely used in the clinical treatment of IPF.Therefore,it’s an urgent need to search new therapy to prevent and treat IPF.During the pathogenesis and development of IPF,overexpressed TGF-β1 is one of critical pathological features.TGF-β1 promotes Smad2/3 bind to Smad4,forming Smad complexes,then the Smad complexes are transmitted into nucleus to regulate the expressions of target proteins related to EMT,profibrotic mediators,and proliferation and differentiation to myofibroblasts of fibroblasts.Accordingly,given the effects of TGF-β1/Smad signaling pathway in IPF,including inducing extracellular matrix and EMT,block this pathway has become a vital therapeutic strategy in the treatment of IPF.Moreover,plentiful active Stat3 was observed in fibrotic lung tissue of IPF patients.Active Stat3 promotes EMT induced by TGF-β1/Smad signaling.However,the role of Stat3 in proliferation and differentiation of fibroblasts induced by TGF-β1/Smad signaling have not been a unanimous conclusion yet.Furthermore,some studies also believe that IPF is an immune-related disease,and the imbalance of immune response plays a key role in the formation and development of pulmonary fibrosis.There are more MDSCs found in the blood and lung tissues of IPF patients than in healthy people.These cells are related to the expression of Stat3,reduce the infiltration of T lymphocytes and suppress cellular immunity.Hence,Stat3 is also a key mediator in the formation and development of IPF.The past thirty years have seen a proliferation of studies that blueberry（in the form of whole fruits,juices and extracts,etc.）has precious value in disease prevention and health care,such as anti-inflammatory,antioxidant,anti-tumor and cardiovascular protection.However,no research reported that whether blueberry could exert a role in IPF and which mechanism is involved yet.Recently,a few literatures have grown up and it was discovered that BBJ could alleviate hepatic fibrosis.It is hopeful that BBJ could provide protection from IPF.Therefore,this study has conducted in-depth explorations on the effects and mechanisms of BBJ in preventing and relieving IPF.We detected the components and antioxidant capacity of BBJ,as well as its inhibitory effects on EMT and fibroblast proliferation and differentiation.We also verified the effect of BBJ in preventing and treating IPF in BLM-induced lung fibrosis mouse model.Meanwhile,the role of TGF-β1/Smad and Stat3 pathways and cellular immunity in BBJ’s anti-IPF effect was explored.Our work aims to bring the prospect of BBJ in IPF therapy as a preventive or adjuvant therapy to light,and prove the possibility of antioxidant-rich juice as one of safe,low toxicity and cheap therapies for disease.[Methods]The study used fresh blueberries to prepare BBJ.Firstly,the basic indicators of BBJ were measured.Secondly,the inhibitory effect of BBJ on the main pathological changes of IPF including EMT and fibroblasts’proliferation and differentiation was detected.Finally,bleomycin was applied to establish pulmonary fibrosis model to determine the preventive and alleviating effects of BBJ on IPF in vivo.1)BBJ preparation,composition determination,anthocyanidins,total flavonoids and phenols contents,soluble solid contents,and antioxidant activity detectionsBBJ was prepared by blender,centrifuge and filtration.BBJ was storage separately and protected from light at-80℃.LC/MS system was applied to confirm the possible compositions of BBJ,compared with available database.According to Agricultural Industry Standard of the People’s Republic of China,Determination of anthocyanidins in plant origin products-High performance liquid chromatography,the content of anthocyanidins in BBJ was analyzed.The total flavonoids content of BBJ was detected and rutin was applied for standard.The total phenols content of BBJ was determined by using chlorogenic acid as standard.Content of Soluble solid in BBJ was detected by Abbe refractometer.To determine the antioxidant ability of BBJ,DPPH assay,hydroxyl radical method and ABTS assay were used.Ascorbic acid was set as positive control and EC₅₀ of antioxidant ability was calculated.2)The inhibitory effect of BBJ on normal cell proliferation and EMTThe effect of BBJ and NTB on proliferations of LO2 and 293T cells were detected by MTT test.The IC₅₀ was calculated by Prism software.Human lung carcinoma epithelial cell,A549,was considered as the EMT model.MTT assay was applied to determine the inhibitory effect of BBJ on A549 proliferation and IC₅₀value was calculated by Prism.We used flow cytometry to analyze the influence of BBJ on cell apoptosis,ROS andΔΨm in A549.After EMT was induced by TGF-β1 and intervened by BBJ in A549,we observed the cell morphology.Cell migration of A549 was stimulated by TGF-β1 and BBJ was added.The anti-migrated activity of BBJ was determined by cell migration assay.Western blotting and IF tests were conducted to verify whether BBJ could reverse TGF-β1 induced upregulated or downregulated expressions of proteins involved in EMT（α-SMA,E-Cadherin,Vimentin,p-Smad2/3 and Smad2/3）and Stat3,p-Stat3.Thus,the effect of TGF-β1/Smad2/3 pathway and Stat3 on EMT blocked by BBJ was explored.Furthermore,the activities of BBJ on cell apoptosis,ΔΨm and ROS change in TGF-β1 stimulated A549 were also detected.3)Inhibition of BBJ on the proliferation and differentiation of fibroblastsThe Wister Rat was injected with BLM saline solution through tracheal.A week later,the rats were sacrificed and RPLF were extracted and cultured.The 3^rd-10^thgenerations of RPLF were used for following experiments.MTT assay was conducted to determine the anti-proliferation activity of BBJ on fibroblasts and IC₅₀ values were calculated,using NIH/3T3,HPF and RPLF for objects.Then cell apoptosis and ROS were detected by flowcytometry.The influence of BBJ onΔΨm,Stat3,p-Stat3 and cleaved Caspase-3 expressions was also disclosed in NIH/3T3.Differentiation of NIH/3T3 and HPF was induced by TGF-β1.After that,apoptosis and ROS change were tested by flowcytometry.The effect of BBJ on TGF-β1 inducedΔΨm change in NIH/3T3 was also determined.Western blotting and IF tests were applied to explore expressions of fibroblast differentiation related proteins,such asα-SMA,Collagen I,E-Cadherin,Vimentin,p-Smad2/3 and Smad2/3,as well as p-Stat3,Stat3,apoptotic proteins Bax and Bcl-2.As a result,it revealed that the change of TGF-β1/Smad2/3 signaling and Stat3 in the process of fibroblasts differentiation and proliferation blocked by BBJ.4)Explore the role of BBJ in preventing mice pulmonary fibrosis induced by BLM in vivoSPF male C57BL/6 mice were injected with saline contained BLM sulfate by intratracheal instillation,while normal saline with the same volume was injected into the sham group.Next day,these mice were randomly divided into four groups:sham group（Sham）,vehicle group（Veh）,BBJ-L（5 m L/kg body weight）group and BBJ-H（10 m L/kg body weight）group.The sham and vehicle group were treated with normal saline at the dose of 10 m L/kg body weight.Body weight was recorded every week.Mice were sacrificed after four weeks.Serum from mouse eyeball venous blood was collected at-80℃for Th1/Th2/Th17 related cytokines detection.The degree of lung fibrosis was evaluated by H&E stain.Masson stain and hydroxyproline contents detection were conducted to determine the accumulation of collagen in lung tissue.Furthermore,western blotting and IHC staining were applied to analyze fibrotic proteins（p-Smad3,α-SMA,Collagen I,and Vimentin）and Stat3,p-Stat3 in lungs,exploring preventive activity of BBJ on mice lung fibrosis and involved mechanism.5)Explore the role of BBJ in inhibiting mice pulmonary fibrosis induced by BLM in vivoMale C57BL/6 mice were bred in a SPF condition.To build lung fibrosis model,mice were anesthetized and injected with saline contained BLM sulfate by intratracheal instillation.Normal saline with the same volume was injected into the mice in sham group.A week later,mice injected with BLM were divided into three groups randomly,including vehicle,BBJ and NTB groups.After weight recorded,sham group and vehicle group were administrated by gavage at the dose of saline,BBJ and NTB group were administrated with BBJ and 3 mg/m L NTB by gavage separately,and the doses were all 10 m L/kg every day.Administration was conducted every day at the same time.Body weight,food and water consumption were recorded every three days.Twenty-one days later,mice were sacrificed at the end of study and serum from mouse eyeball venous blood was collected at-80℃for Th1/Th2/Th17 related cytokines detection.A few fresh lung tissues were cut into pieces and digested into single cells by collagenase.Then the different proportions of MDSCs,CD4⁺T cells and CD8⁺T cells in each group were analyzed by flowcytometry,to explore the differences of immune microenvironment among groups.The degree of lung fibrosis was evaluated by H&E stain.Masson stain and hydroxyproline contents detection were conducted to determine the accumulation of collagen in lung tissue.Furthermore,western blotting and IHC staining were applied to analyze fibrotic proteins（p-Smad3,p-Smad2/3,Smad2/3,E-Cadherin,α-SMA,Collagen I,and Vimentin）and Stat3,p-Stat3 in lungs,exploring inhibitive activity of BBJ on mice pulmonary fibrosis and involved mechanism and NTB was set as positive control.[Results]1)BBJ is rich in phytochemicals and has anti-oxidative activityThe yield rate of BBJ was 0.24 m L/g（fresh fruit）and concentration was 1.0146g/m L.Twenty-seven possible substances were determined in BBJ,including carbohydrates,vitamins,amino acids and peptides,lipids,plant hormones,phenols,and organic acids.Phenolic acids such as chlorogenic acid and ferulic acid,and flavonoids such as rutin,quercetin and quercetin 3β-D-glucoside were the detected phenols in BBJ.Total content of anthocyanidins in BBJ was 18.6 mg/kg.Cyanidin,petunidin chloride,peonidin,malvidin were detected in BBJ,and their contents were1.82,0.860,0.766 and 15.2 mg/kg,respectively.The total flavonoids content was603.89μg RE/g.The total phenols content was 1219.15μg CAE/g.There was 10.83%soluble solid in BBJ.And,in result of ABTS assay,soluble solid in BBJ required to eliminate 50%of free radicals were less than ascorbic acid.That is,the antioxidant activity of soluble solid in BBJ was higher than ascorbic acid.These results suggested that BBJ have better antioxidative activity.2)BBJ induced A549 apoptosis and blocked EMT stimulated by TGF-β1 through downregulating TGF-β1/Smad2/3 and Stat3 signaling.No obvious inhibitory effect was observed in LO2 and 293T when were treated with BBJ for 24 h.IC₅₀ values were all over 200μL/m L.And IC₅₀ values of NTB in LO2 and 293T were 18.21μM and>20μM for 24 h.These results suggested that BBJ do not have obvious proliferation inhibitory activity in normal cells.The inhibitory effect of BBJ on A549 proliferation was time-and dose-dependent manners.IC₅₀ value for 24 h was 107.63μL/m L.When A549 was treated with BBJ for 24 h,intracellular ROS andΔΨm were decreased apparently.However,no significant cell apoptosis was discovered when A549 was treated with less than 50μL/m L BBJ for 24 h.After harvest for 6 h,A549 was treated with 50μL/m L BBJ and 24 h later,cell apoptosis was detected via flowcytometry.ROS andΔΨm were decreased.After harvest for 6 h,A549 was stimulated with 5 ng/m L TGF-β1,no difference was observed in cell apoptosis from control group.But intracellular ROS was increased obviously,whileΔΨm were decreased.When treated with BBJ,increased ROS and decreasedΔΨm stimulated by TGF-β1 did not change.Cell morphology of A549 was changed from oval to fusiform induced by TGF-β1,which was one of interstitial phenotypes.Incubation with BBJ reversed this morphology change in A549.Meanwhile,TGF-β1 could give rise to A549 cell migration,and 50μL/m L BBJ arrested migration.The results of western blot and IF showed that BBJ could reverse upregulatedα-SMA,Vimentin,p-Smad2/3/Smad2/3 and p-Stat3/Stat3 as well as downregulated E-Cadherin induced by TGF-β1 in A549.These results suggested that BBJ inhibit EMT by downregulating TGF-β1/Smad2/3 and Stat3 signaling.3)BBJ stimulated fibroblasts apoptosis and reversed fibroblasts differentiation induced by TGF-β1 through TGF-β1/Smad2/3 and Stat3 signaling.The cell viabilities of NIH/3T3,HPF and RPLF were restrained by BBJ.IC₅₀values for 24 h were 95.66,195.37 and>200μL/m L,respectively.When these three types of fibroblasts were treated with BBJ for 24h,intracellular ROS was decreased significantly and cell apoptosis was induced.Furthermore,ΔΨm and p-Stat3expression was reduced by BBJ in NIH/3T3.BBJ upregulated cleaved Caspase 3 in NIH/3T3.Vimentin,α-SMA,Collagen I and the ration of p-Stat3/Stat3 was down-regulated and E-Cadherin was up-regulated in RPLF when dealt with 50μL/m L BBJ for 24 h.Thus,BBJ could cause fibroblasts apoptosis by Stat3 signaling inhibition.NIH/3T3 and HPF were treated with 50μL/m L BBJ after 6 h starvation and 1 h TGF-β1 treatment,cell apoptosis was discovered.After NIH/3T3 and HPF were treated with 5 ng/m L of TGF-β1,there was no significant divergence on cell apoptosis from control group.However,intracellular ROS was increased andΔΨm is decreased in NIH/3T3.NIH/3T3 and HPF received BBJ treatment for 24 h after TGF-β1stimulation,resulting in more cell apoptosis and reducedΔΨm in NIH/3T3 as well as more apoptotic cells in HPF.Results from western blotting and IF tests suggested that BBJ could reverse upregulatedα-SMA,Vimentin,Collagen I and ratio of p-Smad2/3/Smad2/3 as well as downregulated E-Cadherin,ratios of Bax/Bcl-2 and p-Stat3/Stat3 induced by TGF-β1 in NIH/3T3 and HPF.These results suggested that BBJ inhibit NIH/3T3 and HPF cell differentiation via TGF-β1/Smad2/3 and Stat3 signaling.What’s more,BBJ also induced fibroblasts apoptosis.4)BBJ could prevent BLM-induced mice pulmonary fibrosis formation viaTGF-β1/Smad2/3 and Stat3 pathway and mediating immunity.Pulmonary fibrosis was built by 2 mg/kg BLM and oral BBJ could restrain the formation of lung fibrosis.The results from Masson staining and hydroxyproline content detection suggested that BBJ could lead to less hydroxyproline contents and collagen formation in lung tissues.BBJ decreased the expressions of IL-10 and IL-17A.According to IHC and western blot experiments,oral BBJ could reverse upregulated expressions of p-Smad3,Collagen I,α-SMA,Vimentin and ratio of p-Stat3/Stat3 stimulated by BLM,to prevent from fibrotic pulmonary formation.5)BBJ released BLM-induced mice lung fibrosis by TGF-β1/Smad2/3 and Stat3 signaling and regulating immune response in lung.Pulmonary fibrosis was built by 1mg/kg BLM.After lung fibrosis was aggravated for 7 days,intragastric administration of BBJ,NTB and normal saline continued for21 days.In the period,body weights,food and water consumptions between groups were no difference.No significant difference was observed in histological structures of heart,liver,spleen and kidney.Obviously,oral BBJ and NTB could alleviate lung fibrosis.The results from Masson staining and hydroxyproline content detection suggested that BBJ could lead to less hydroxyproline contents and collagen formation in lung tissues.Lung fibrosis could cause increased proportion of MDSCs and decreased proportions of CD4⁺T and CD8⁺T cells,which were reversed by BBJ and NTB.BBJ induced the lower contents of IL-6,IL-10,IL-17A and IFN-γin serum,while NTB decreased IL-6,IL-10 and IFN-γsecretion.According to IHC and western blotting experiments,oral BBJ could reverse upregulated p-Smad2/3/Samd2/3,Collagen I,α-SMA,Vimentin and p-Stat3/Stat3 as well as downregulated E-Cadherin stimulated by BLM-induced lung fibrosis,to restrain progress of lung fibrosis.[Conclusion]1)BBJ had anti-oxidative effects with plentiful phytochemicals;2)EMT process could be blocked by BBJ through TGF-β1/Smad2/3 and Stat3 signal pathways;3)BBJ suppressed fibroblasts proliferation and differentiation induced by TGF-β1 via TGF-β1/Smad2/3 and Stat3 signal pathways;4)Animal studies proved that BBJ prevented from BLM-induced pulmonary fibrosis as well as relieved lung fibrosis caused by BLM through mediating immune response in lung and downregulating TGF-β1/Smad2/3 and Stat3 signaling.