| Objective:To investigate the protective effect of chicoric acid（CA）preconditioning on acute lung injury（ALI）induced by sepsis in mice and its relationship with the TLR9/NF-κB signaling pathway.Methods:1 Animal experiment:To establish the model of acute lung injury in mice with sepsis by cecal ligation and puncture,and observe the effect of different concentrations of chicoric acid pretreatment on acute lung injury index,oxidative stress and inflammatory factors in mice.The rats were divided into four groups:control group（Con）,septic acute lung injury group（CLP）,10 mg·kg-1 CA preconditioning+CLP group(CLP+10 mg·kg-1CA),20 mg·kg-1 CA preconditioning+CLP group(CLP+20 mg·kg-1 CA),40 mg·kg-1CA preconditioning+CLP group(CLP+40 mg·kg-1 CA),and CLP+dexamethasone group(CLP+5 mg·kg-1 Dex).Methods:HE staining was used to detect the morphological changes of lung tissue,the ratio of dry and wet weight of lung tissue was used to evaluate the degree of pulmonary edema,BCA method was used to detect the total protein content in bronchoalveolar lavage fluid（BALF）,and ELISA was used to detect the content of TNF-α,IL-6 and IL-1β.2 Cell experiment:Lipopolysaccharide(LPS,5μg·ml-1)was used to treat Beas-2B cells,a normal human epithelial cell line,to establish an inflammatory model of lung injury and to observe the protective effect of chicoric acid pretreatment at different concentrations on cells.Experimental groups:control group（Con group）,model group(LPS group,5μg·ml-1LPS),LPS+10μg·ml-1 CA group(10μg·ml-1 group),LPS+25μg·ml-1 CA group(25μg·ml-1 group),LPS+50μg·ml-1 CA group(50μg·ml-1 group),LPS+5μg·ml-1Dex group（Dex group）.Methods:CCK-8 assay was used to detect the effect of chicoric acid on Beas-2B cells.Flow cytometry was used to detect the content of ROS and the apoptosis rate of Beas-2B cells.ELISA was used to detect the content of TNF-α,IL-6 and IL-1βin supernatant.3 Sequencing results:significant difference occur in that gene expression of mice in a blank group,a model group and a drug administration group,the GO enrichment result of a screened differential gene is mostly relate to immunity,the KEGG enrichment result has a crossed Toll-like receptor signal inflammatory pathway,molecular docking is performed on proteins correspond to genes in the cichoric acid and the Toll-like receptor signal pathway by using Dockduck software,and representative genes TLR9,IRF7,MYD88 and P65 are found to be well combined,he results of WB experiment showed that TLR9,IRF7,MYD88,P65 proteins were overexpressed in the model group compared with the blank group（P<0.01）.Compared with the model group,the protein expression in the treatment group decreased,and the difference was not significant in the low and medium concentration treatment groups,but significant in the high concentration treatment group and the positive drug group（P<0.01）.Results:1 Animal experiment:Compared with Con group,CLP group had more severe lung injury and pulmonary edema（P<0.05）,more total protein in BALF,more TNF-α,IL-6 and IL-1βin tissues（P<0.01）,and more gene expression in different groups.Compared with LPS group,the lung injury and pulmonary edema in mice pretreated with 10 mg·kg-1 cichoric acid were alleviated slightly,the total protein content in bronchoalveolar lavage fluid（BALF）,and the contents of TNF-α,IL-6 and IL-1βin tissues were reduced;Compared with CLP group,the lung injury and pulmonary edema in mice pretreated with 20 mg·kg-1 cichoric acid were alleviated,the total protein content in bronchoalveolar lavage fluid（BALF）and the contents of TNF-α,IL-6 and IL-1βin tissues were reduced（P<0.05）.Compared with LPS group,the lung injury was alleviated,the pulmonary edema was improved（P<0.05）,the total protein content in BALF was decreased（P<0.05）,and the contents of TNF-α,IL-6 and IL-1βin tissues were significantly decreased（P<0.05）in chicoric acid pretreatment group(40 mg·kg-1).Compared with CLP group,dexamethasone(5 mg·kg-1)significantly reduced the levels of TNF-α,IL-6 and IL-1βin BALF,lung edema and lung injury（P<0.01）.2 Cell experiment:Compared with Con group,LPS group increased the production of ROS（P<0.05）,cell apoptosis rate（P<0.05）,and the contents of TNF-α,IL-6 and IL-1βin cell supernatant（P<0.01）.Compared with LPS group,cichoric acid pretreatment(10μg·ml-1)decreased ROS release,cell apoptosis rate,and the contents of TNF-α,IL-6 and IL-1βin cell supernatant;Compared with LPS group,cichoric acid pretreatment(25μg·ml-1)decreased ROS release,cell apoptosis rate,and the contents of TNF-α,IL-6 and IL-1βin cell supernatant;Compared with LPS group,cichoric acid pretreatment(50μg·ml-1)decreased ROS release（P<0.05）,cell apoptosis rate（P<0.05）,and the contents of TNF-α,IL-6 and IL-1βin cell supernatant（P<0.05）;Compared with LPS group,dexamethasone treatment of5μg·ml-1 group decreased ROS release（P<0.05）,cell apoptosis rate（P<0.01）,and the contents of TNF-α,IL-6 and IL-1βin cell supernatant（P<0.01）.3 Sequencing results:significant difference occur in that gene expression of mice in a blank group,a model group and a drug administration group,the GO enrichment result of a screened differential gene is mostly relate to immunity,the KEGG enrichment result has a crossed Toll-like receptor signal inflammatory pathway,molecular docking is performed on proteins correspond to genes in the cichoric acid and the Toll-like receptor signal pathway by using Dockduck software,and representative genes TLR9,IRF7,MYD88 and P65 are found to be well combined,The results of WB experiment showed that TLR9,IRF7,MYD88,P65 proteins were overexpressed in the model group compared with the blank group（P<0.01）.Compared with the model group,the protein expression in the treatment group decreased,and the difference was not significant in the low and medium concentration treatment groups,but significant in the high concentration treatment group and the positive drug group（P<0.01）.Conclusion:In conclusion,the present study demonstrated that chicoric acid pretreatment has a certain protective effect on the sepsis acute lung injury mouse model,and can reduce the inflammation of Beas-2B cells induced by LPS.Through further exploring the mechanism of cichoric acid at the gene level by transcription sequencing technology,it is found that Toll-like receptor signal and TLR9,IRF7,MYD88,P65 and other proteins in NF-κB pathway are highly expressed in the model group with inflammation,and the expression is reduced in the pretreatment group.Therefore,it is speculated that the anti-inflammatory effect of cichoric acid may be related to the regulation of TLR9/NF-κB signal pathway. |
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