Protective Effects And Potential Mechanism Of Epicatechin On Sepsis-associated Acute Lung Injury In Mice

Objective:In order to explore new pharmacological drugs for the management of acute lung injury（ALI）,sepsis-associated ALI model was established to dynamically observe the characteristics of lung injury.On this basis,further works were carried out to investigate the effects of EC and its potential protective mechanism on inflammatory in LPS induced ALI in mice.Methods:In the first part of work,sepsis-associated ALI model was established by intraperitoneal injection of lipopolysaccharide（LPS）in mice.ALI model was evaluated by the general status,weights change,survival rate,histological changes of lung,alveolar capillary barrier ability,pulmonary inflammation and lung wet/dry weight ratio.The characteristics of pulmonary inflammatory injury were dynamically observed at different time points（6h,24h and 72h）in sepsis-associated ALI in mice.The degree of pulmonary inflammatory injury were evaluated by lung injury score,which were observed by hematoxylin and eosin（H&E）staining for histological analysis.In order to estimate the capability of alteration of the alveolar capillary barrier,total bronchoalveolar lavage fluid（BALF）protein concentration and the wet/dry（W/D）ratio were measured.We also tested the absolute number of neutrophils and TNF-αprotein concentration in BALF to acess the level of inflammatory response.In the second part of work,sepsis-associated ALI model as previously mentioned was established to evaluate the effects of EC on ALI.32 male C57BL6/N mice were randomly divided into 4 groups（n=8/each group）.Mice were given a dose of LPS（10mg/kg）by intraperitoneal injection（i.p.）route in a volume of 5mL/kg to establish ALI model.Control group:NS 5mL/kg ip,half an hour later nasal feeding NS 5mL/kg,12h later repeated nasal feeding NS at the same dose;EC group:NS 5mL/kg ip,half an hour later nasal feeding EC solution 15mg/kg（5mL/kg）,12h later repeated nasal feeding EC at the same dose;LPS group:LPS 10mg/kg ip,half an hour later nasal feeding NS5mL/kg,12h later nasal feeding NS 5mL/kg again;LPS+EC group:LPS 10mg/kg ip,half an hour later nasal feeding EC solution 15mg/kg（5mL/kg）,12h later repeated nasal feeding EC at the same dose.Briefly,the mice were killed and analyzed 24h after stimulation.Lung injury score were used for histological analysis observed by H&E staining.Total BALF protein concentration and the W/D ratio were measured to estimate the capability of alteration of the alveolar capillary barrier,the absolute number of neutrophils were tested to evaluate the level of inflammatory response.Inflammatory model were established in vitro in RAW264.7 cell.Appropriate concentration range of EC in RAW264.7 cell and EC together with 500ng/mL LPS in RAW264.7 cell were tested by CCK8 test.Concentration range of TNF-αand NO in supernatant were tested by ELISA to evaluate the level of inflammatory response in vitro in diferent groups.RAW264.7 cell were randomly divided into several groups:control group,LPS group,EC groups with different EC concentration.IL-6 in supernatant were tested in control group,LPS group and 50uM EC group to evaluate the effect of EC on inflammation.In the third part of work,the mechanism of EC on sepsis-associated ALI was observed.24 male C57BL6/N mice were randomly divided into 3 groups（n=8/each group）.Mice were given a dose of LPS（10mg/kg）by the i.p.route in a volume of5mL/kg to establish ALI model.Control group:NS 5mL/kg ip,half an hour later nasal feeding NS 5mL/kg,12h later repeated nasal feeding NS at the same dose;LPS group:LPS 10mg/kg ip,half an hour later nasal feeding NS 5mL/kg,12h later nasal feeding NS 5mL/kg again;LPS+EC group:LPS 10mg/kg ip,half an hour later nasal feeding EC solution 15mg/kg（5mL/kg）,12h later repeated nasal feeding EC at the same dose.Briefly,the mice were killed and analyzed 24 h after stimulation.4 samples were randomly selected from the LPS group and the LPS+EC group.The changes of gene expression in lung tissue between LPS group and LPS+EC group were detected by PCR-array assay to screen differentially expressed genes.MAPK signal pathway protein which included P38 and phosphorylated P38,ERK1/2 and phosphorylated ERK1/2,JNK and phosphorylated JNK were detected by Western blot.C-Jun as a subunit of AP1together with phosphorylated c-Jun expression were also detected to evaluate the effect of EC on P38MAPK-AP1 signal pathway.Though the method of computer aided drug design,the spatial structure of P38α（4L8M）protein and its interaction with Ligand were analysed.4L8M was prepared by Discovery Studio 3.0 and the models of the pharmacophore were constructed and the best one was screened.X-Score program was used to predict the possible relationship between EC and 4L8M by docking score.Furthermore,the inhibition effect of EC on P38 were proved by kinase inhibition test,and IC₅₀0 were calculated.Results:The sepsis-associated ALI model was successfully established by given a dose of LPS（10mg/kg）by the i.p.route in mice.At different time points of ALI,several different features were presented.In terms of the general status,there was no significant difference between control group and LPS group at 6h after LPS stimulation,while the general status were significantly worse at 24h and 72h after LPS stimulation.Body weight were significantly reduced at any time points after LPS stimulation.Some mice developed diarrhea and their eyelids were surrounded with secretions at 24h and 72h after LPS stimulation.Compared with control group,the histological changes of lung in LPS group were obvious at any time points,which included:increased neutrophils in the alveolar space and the interstitial space;appearance of hyaline membranes;proteinaceous debris filling the airspaces and increased alveolar septal thickening.The lung injury scores were significantly higher in LPS group than control group.The damage of alveolar structural integrity significantly happened in LPS group at 24h to 72h after LPS stimulation.Alteration of the alveolar capillary barrier in LPS group was also obvious.Compared with conrtrol group,total BALF protein concentration in LPS group were significantly increased at 24h to 72h after LPS stimulation and wet/dry weight ratio in lung were also significantly increased in LPS group at any time points after LPS stimulation.Compared with control group,the absolute number of neutrophils in BALF were significanly increased in LPS group at any time points and the concentrations of proinflammatory cytokine TNF-αin BALF were significanly increased in LPS group at6h after LPS stimulation,but no differences were observed between LPS group and control group at 24h to 72h after LPS stimulation.EC possessed the capacity of alleviating lung tissue injury,reducing the protein concentration in BALF and lung wet/dry weight ratio,inhibiting the release of inflammatory mediators and improving alteration of the alveolar capillary barrier in LPS-induced ALI in mice.EC also inhibited the expression of inflammatory mediators in vitro.After exposure to EC 24 hours,detection of IC₅₀0 was 1235.5 uM in RAW264.7cells,while exposure to EC together with LPS 24 hours,detection of IC₅₀ was 1041.4 uM in RAW264.7 cells.EC inhibited the expression of TNF-αand IL-6 in supernatant in LPS induced RAW264.7cell.EC also inhibited the expression of NO in LPS induced RAW264.7cell.EC significantly down-regulated the expression of Fos,Ksrl,Map3k1,Map2k1,Map2k3 mRNA in lung tissue in sepsis-associated ALI.EC also down-regulated the expression of phosphorylated P38,phosphorylated ERK1/2 and phosphorylated JNK,which were the key protein kinase of MAPK signaling pathway.Meanwhile,EC significantly down-regulated the expression of c-Jun and phosphorylated c-Jun protein.Molecular docking results indicated that EC presented the favorable binding interactions with P38α（4L8M）protein,with binding energy is-8.92kcal/mol.This studies also showed that Ala111,Glu71,Leu171 and Asp112 were the main amino acids contaced EC with 4L8M through forming a hydrogen bond.The p38αenzymatic reaction was carried out with transcription factor ATF2 as a kinase substrate,EC moderated p38αinhibitory activity with an IC₅₀ of 2.4uM on enzymatic level.Conclusion:Taken together,this study provides evidence of the protective activity of EC by alleviating lung tissue injury,inhibiting the release of inflammatory mediators and improving alteration of the alveolar capillary barrier in LPS-induced ALI in mice,which might be related to EC suppression of P38MAPK-AP1 signaling pathways via interaction with P38.