Citrus Alkaline Extracts Mitigate Pulmonary Fibrosis Via Regulating Endoplasmic Reticulum Stress

Objective:Pulmonary fibrosis is a rare interstitial lung disease with a poor prognosis and limited treatment.Our previous researches have proved that Citrus alkaline extracts(CAE)can treat pulmonary fibrosis and inhibit lung fibroblasts,but the molecular mechanisms,especially in alveolar epithelial cell(AEC),are still unclear.First,this study completed the identification of CAE.Second,it verified the therapeutic effect of CAE on bleomycin-induced pulmonary fibrosis mice model.Then,it continued to explore the effects of CAE on endoplasmic reticulum stress(ERS),ATF3/PINK1 signaling pathway,and mitochondrial homeostasis in vivo and in vitro.At last,it explored the interaction and the possible cell factors between AEC ?s and lung fibroblasts.Methods:1.Preparation,identification,and quality control of CAE:Citrus pericarp was decocted by 95%ethanol.Then CAE,a kind of yellow powder,was extracted from the ethanol decoction through several steps,such as filtration,concentration,acid adjustment,alkali adjustment and extraction.The detection and the quality control work of CAE was performed through UPLC-ESI-MS/MS system.The total ion currents of CAE were obtained and used in extracting retention time,parent ion molecular weight,daughter ion molecular weight,peak intensity and etc.The plant secondary metabolism database,Metware,was used in the qualitative analysis of CAE basing on secondary spectrum.The multi-reaction monitoring mode was used in the relative quantitative analysis of CAE;2.Pulmonary fibrosis mice model induced by bleomycin:120 mice were divided into blank group,lumbar spinal needle group,indwelling needle group,and tracheotomy group.Pulmonary fibrosis mice model was created by intratracheal instillation of bleomycin using lumbar spinal needle,indwelling needle,or tracheotomy respectively.The general condition(changes in body weight and survival rate)was observed,the histopathological changes(H&E and Masson staining),and the hydroxyproline level were examined.3.Inhibitory effect of CAE on pulmonary fibrosis in mice:50 mice were divided into blank group,model group,CAE low-dose group(32 mg/kg/d),CAE medium-dose group(64mg/kg/d),and CAE high-dose group(96 mg/kg/d).Bleomycin-induced pulmonary fibrosis mice model was established by lumbar spinal needle.Then the intragastric administration with different concentrations of CAE was performed for 21 days.The histopathological changes(H&E,Masson,and Sirius Red staining)were observed,and the level of collagen deposition was detected in lung tissues.4.CAE regulates ATF3/PINK1 pathway by mitigating ERS in AEC ?s:Western Blot is used to detect the expression of ERS-related proteins,ATF3,and PINK1 in lung tissues;immunofluorescence is used to observe the overlap between BiP,a biomarker of ERS,and ATF3,ATF3 and PINK1,SPC,a biomarker of AEC ?s,and ATF3,SPC and PINK1,in lung tissues.Then A549s were divided into blank group,model group,CAE low-dose group(50 ?g/ml),CAE medium-dose group(100 ?g/ml),CAE high-dose group(200 ?g/ml),and control group(TUDCA,5 ?g/ml).A549s were treated by tunicamycin(TM)(2 ?g/ml),which is used to establish an ERS model in vitro,for 12 h,and complete medium,containing different concentrations of CAE,for 48 h.Western Blot was used to detect the expression of ERS-related proteins,ATF3 and PINK1 in A549s;immunofluorescence was used to observe the overlap between BiP and ATF3,ATF3 and PINK1,in A549s.5.CAE regulates mitochondrial homeostasis via ATF3/PINK1 pathway in AEC IIs:JC-10 was used to detect the level of mitochondrial membrane potential in A549s;ROS detection kit was used to detect the level of ROS in A549s.These two tests could be used to observe the mitochondrial function of A549s.A549s were silenced in gene ATF3 and PINK 1 through shRNA respectively,then the mitochondrial membrane potential was detected again to observe the mitochondrial function.6.CAE influences lung fibroblasts by regulating AEC ?s:After the intervention of TM and CAE,A549s were cultured with CAE-free low serum medium for 72 h,then the cell supernatant was collected to treat MRC5s.Western Blot was used to detect the collagen level in MRC5s;ELISA was used to detect the level of TGF-?1,IL-6,and IL-10 in the cell supernatant.Results:1.Preparation,identification,and quality control of CAE:About 1.56 mg CAE,a yellow powder,could be extracted from 1 kg Citrus pericarp on average.In the UPLC-ESI-MS/MS analysis,315 compounds were identified from CAE including 143 flavonoids and 32 alkaloids.80 of these 315 compounds were identified as being the active ingredients in CAE based on fragment ions,including 47 flavonoids,such as tangeretin,nobiletin,narirutin,neohesperidin,sinensetin,and hesperidin,and 5 alkaloids,such as synephrine,citrusin I,citpressine I,and ecgonine;there is no significant difference among the different samples of CAE.2.Pulmonary fibrosis mice model induced by bleomycin:Among lumbar spinal needle group,indwelling needle group,and tracheotomy group,the weight loss in tracheotomy group is most significant,the survival rate in indwelling needle group is highest(90%),meanwhile which in tracheotomy group is lowest(60%).The degree of alveolitis and pulmonary fibrosis in lumbar spinal needle group and tracheotomy group are higher than indwelling needle group,especially on day 21.The hydroxyproline level in lumbar spinal needle group and tracheotomy group are higher than blank group significantly(P<0.001).3.Inhibitory effect of CAE on pulmonary fibrosis in mice:The treatment of CAE repairs alveolar structure,reduces inflammation infiltration and collagen deposition,especially in CAE high-dose group.Compared with model group,the alveolitis score(P<0.05),pulmonary fibrosis score(P<0.001),and semi-quantitative analysis of collagen(P<0.001)in CAE high-dose group are reduced significantly,while the expression of COLlal and COL3 are also significantly reduced(P<0.05).4.CAE regulates ATF3/PINK1 pathway by mitigating ERS in AEC ?s:In lung tissues,the expressions of BiP,PERK,p-eIF2?,ATF4,and ATF3 in CAE groups are significantly reduced compared with model group,while PINK1 is significantly increased,and all the changes are dose-dependent.Among the three CAE groups,the CAE high-dose group has the most significant difference(P<0.05;P<0.001;P<0.01;P<0.001;P<0.01;P<0.01).The immunofluorescence changes of BiP and ATF3 have a large overlap,and the immunofluorescence changes of ATF3 or PINK1 also have a large overlap with SPC respectively.In A549s,the expressions of BiP,PERK,p-eIF2a,ATF4,and ATF3 are significantly reduced,while PINK1 is significantly increased,and all the changes are dose-dependent.Among the three CAE groups,CAE high-dose group has the most significant difference(P<0.05;P<0.001;P<0.01;P<0.001;P<0.01;P<0.01),and the immunofluorescence changes of BiP and ATF3 overlap greatly.5.CAE regulates mitochondrial homeostasis via ATF3/PINK1 pathway in AEC ?s:In A549s,the mitochondrial membrane potential in CAE groups are significantly reduced compared with model group,and the change is dose-dependent.Among the three CAE groups,CAE high-dose group has the most significant difference(P<0.001).The change in ROS is the same(P<0.01).After ATF3 is silenced in A549s,the expression of PINK1 in CAE groups and control group do not increase significantly compared with model group,although the mitochondrial membrane potential still increases slightly,but not significantly.After PINK1 is silenced in A549s,the mitochondrial membrane potential just increases slightly,but no significant difference.6.CAE influences lung fibroblasts by regulating AEC ?s:In MRC5s,the expressions of COLl?1 and COL3 are lower in CAE groups compared with model group,and the change is dose-dependent,but only CAE high-dose group has a significant difference(P<0.05;P<0.05).In the cell supernatant,the expressions of TGF-?1,IL-6,and IL-10 are significantly lower in CAE groups compared with model group,and the changes are dose-dependent.CAE high-dose group has significant difference(P<0.001;P<0.05;P<0.05).Conclusions:1.The active ingredients of CAE are mainly flavonoids and alkaloids.2.CAE can alleviate ERS in AEC ?s to regulate mitochondrial homeostasis via ATF3/PINK1 signaling pathway.3.CAE can reduce the secretion of collagen in lung fibroblasts indirectly by inhibiting the secretion of cell factors,such as TGF-?1,in AEC ?.