The Mechanism Of Classical Prescription Zhenwu Decoction On LPS-induced Acute Lung Injury:Redirection Of Cell Death Mediated By Endoplasmic Reticulum Stress-mitochondrial Autophagy-NLRP3 Inflammasome Pathway

Part Ⅰ: Network pharmacology study of Zhenwu Decoction on acute lung injury ObjectiveThe network pharmacology method was used to predict and analyze the key active substances,important targets and pathways of Zhenwu Decoction,and the molecular docking technology was used to clarify the possible molecular mechanism of Zhenwu Decoction in the intervention of acute lung injury.MethodsTCMSP library was used to search the main active ingredients and related targets of Zhenwu Decoction,and Uniprot library was used to search the gene names corresponding to target proteins.GeneCards and OMIM libraries were used to find targets related to acute lung injury.The active ingredient targets of Zhenwu Decoction were mapped to targets of acute lung injury,and the intersection targets were obtained by drawing Venn diagram.The active ingredients and intersection targets were visualized using Cytoscape.And import potential targets into STRING library to obtain PPI network;Metascape was used for GO analysis and KEGG analysis of intersection targets.Autodock Vina was used to conduct molecular docking between the top seven components of Zhenwu Tang and the 3D target structure in PDB,and Discovery Studio was used to conduct visual analysis of the docking conformation.Results:（1）94 target genes of the active components of Zhenwu Decoction;（2）7032 targets related to acute lung injury;（3）84 intersection targets of zhenwu Decoction active ingredient and acute lung injury targets were obtained;（4）The first seven components in zhenwu Decoction were 3β-acethoxylatractyloxone,saponin of ivy,β-sitosterol,kaempferol,stigmasterol,（+）-catechin and styrosnin;（5）Go analysis related to the activity of REDOX enzyme in acute lung injury.KEGG analysis involves energy metabolism,autophagy pathway,oxidative stress pathway,etc;（6）The top seven active ingredients showed good binding activities with the target ROS,CASP1,CAT,CHOP,GRP78,IL1 B,IL-18,LC3,NLRP3,NRF2,P62,SOD and TNF-α.Conclusions:Based on network pharmacology and molecular docking technology,this study took Zhenwu Decoction as the research object,systematically analyzed the important active ingredients contained in zhenwu Decoction,target target,biological process and related signal pathway,and discussed the affinity between the main active ingredients and target target.It was found that Zhenwu Decoction may pass through TRX1/TXNIP,NRF2/HO1 and PINK1/PARK2 signal pathways.It acts on ROS,ENaCα,CASP1,CAT,CHOP,GRP78,GSDMD,IL1 B,IL-18,LC3,NLRP3,NRF2,P62,SOD,TNF-α,VDAC1 and other targets to intervene in acute lung injury.This is the result of the interaction of multiple components,multiple key targets and multiple signal pathways,which provides a theoretical basis for zhenwu Decoction in the intervention of acute lung injury.In conclusion,this study lays a theoretical foundation for further experimental mechanism verification.Part Ⅱ: Pharmacodynamics of Zhenwu Decoction on LPS induced acute lung injury Objective:The mice model of Acute lung injury was constructed by tail vein injection of Lipopolysaccharide（LPS）to investigate the intervention effect and mechanism of Zhenwu Decoction on Acute lung injury（ALI）.Methods:A total of 56 male kunming mice were randomly divided into 4 groups（Control group,LPS group,LPS+DXM group,LPS+ZWT group）.Each group was given corresponding double steam water or therapeutic drugs by intragastric administration for 3 days.LPS was injected into tail vein on the fourth day to construct acute lung injury model.The survival rate and lung pathology of mice were observed.Lung W/D and blood gas were measured.Western blot and immunohistochemistry were used to detect ENaCα,ENaCβ and ENaCγ.Western blot analysis of Na⁺/K⁺-ATPase,AQP1,AQP5 levels.Results:（1）Compared with LPS group,the general condition of mice in LPS+ZWT group and LPS+DXM group was improved,and the survival rate was increased（P<0.05）.（2）The W/D value in LPS group was significantly higher than that in Control group（P<0.05）,and the W/D value in LPS+ZWT group and LPS+DXM group was significantly lower than that in LPS group（P<0.05）.（3）Compared with Control group,Pa O2,PaCO2 and pH in LPS group were decreased（P<0.05）;Compared with LPS group,Pa O2 values in LPS+DXM group and LPS+ZWT group were increased（P<0.05）,and PaCO2 and pH values in LPS+DXM group and LPS+ZWT group had no significant difference compared with LPS group（P>0.05）.（4）Lung lesion in LPS+ZWT group and LPS+DXM group was less than that in LPS group;Lung injury scores in LPS+ZWT and LPS+DXM groups were lower than those in LPS group（P<0.05）.（5）Compared with the Control group,the levels of ENaCα,ENaCβ and ENaCγ in LPS group were decreased by Western blot（P<0.05）.Compared with LPS group,ENaCα level was slightly decreased（P>0.05）,ENaCβ expression was increased（P<0.05）and ENaCγ expression was decreased（P<0.05）in LPS+DXM group.Compared with LPS group,the expressions of ENaCα,ENaCβ and ENaCγ in LPS+ZWT group were increased（P<0.05）.（6）Compared with the Control group,the levels of ENaCα,ENaCβ and ENaCγ in LPS group were significantly decreased（P<0.01）.Compared with LPS group,the levels of ENaCα,ENaCβ and ENaCγ in LPS+DXM group were increased（P<0.05）;Compared with LPS group,the levels of ENaCα,ENaCβ and ENaCγ in LPS+ZWT group were increased（P<0.05）.（7）Compared with Control group,the levels of Na⁺/K⁺-ATPase,AQP1 and AQP5 in LPS group were significantly decreased（P<0.01）;Compared with LPS group,the contents of Na⁺/K⁺-ATPase and AQP1 in LPS+DXM group were increased（P<0.05）,while the level of AQP5 was decreased（P<0.05）;Compared with LPS group,Na⁺/K⁺-ATPase,AQP1 and AQP5 levels were increased in LPS+ZWT group（P<0.05）.Conclusions:Zhenwu Decoction could reduce pulmonary edema induced by acute lung injury by up-regulating the expressions of ENaCα,ENaCβ,ENaCγ,Na⁺/K⁺-ATPase,AQP1 and AQP5 proteins,which could be one of the important mechanisms of Zhenwu Decoction in protecting ALI.Part Ⅲ: Mechanism of Zhenwu Decoction in intervention of LPS-induced acute lung injury Section Ⅰ: Endoplasmic reticulum stress-mitochondrial autophagy-NLRP3 inflammasome pathway mediated redirection of cell death patterns Objective:This part mainly observed whether Zhenwu Decoction could intervene in NLRP3 inflammasome mediated scortosis by regulating TRX-1 to inhibit er stress response and activating mitochondrial autophagy through PINK1/Parkin pathway.Methods:A total of 98 male mice were randomly divided into 7 groups（Control group,LPS group,LPS+DXM group,LPS+ZWT group,PX12 group,NAC group and H₂O₂group）.Each group was given double steam water or corresponding therapeutic drugs ingavage for 3 days.The PX12 treatment group,NAC treatment group and H₂O₂ treatment group were intraperitoneally injected with corresponding drugs 3h before modeling.Modeling,drawing materials as before.The levels of MPO,LDH,TNF-α,IL-18 and ATP were measured by ELISA.The mRNA expressions of IL-1β,TNF-α,GRP-78 and CHOP were detected by RT-PCR.Western Blot analysis of NLRP3,Caspase-1,IL-1β,GRP-78,pIRE1-α,pPERK,peIF2-α,CHOP,Calpain-1,TXNIP,TRX-1,VDAC-1,Pink-1,Parkin,P62,LC3-Ⅱ/Ⅰ levels.The levels of Caspase-1,TXNIP and NLRP3 were measured by immunofluorescence.MMP,mtROS and ROS levels were detected.Results:（1）Compared with the Control group,the contents of LDH,MPO,IL-18,TNF-α,TNF-αmRNA and ROS in LPS group were increased（P < 0.05）;Compared with LPS group,the levels of LDH,MPO,IL-18 and TNF-α in LPS+ZWT and LPS+DXM groups were decreased（P<0.05）.Zhenwu Decoction reduced LDH and IL-18 levels more significantly than dexamethasone（P<0.05）.TNF-αmRNA expression in LPS+ZWT group and LPS+DXM group was slightly lower than that in LPS group（P>0.05）.ROS level in LPS+ZWT group was significantly lower than that in LPS group（P< 0.05）.（2）Mechanism related to coke death: Compared with Control group,mRNA contents of NLRP3,Caspase-1,IL-1β and IL-1β in LPS group were significantly increased（P<0.01）;Compared with LPS group,the mRNA contents of NLRP3,Caspase-1,IL-1β and IL-1β in LPS+ZWT and LPS+DXM groups were decreased（P<0.05）.Moreover,Zhenwu Decoction reduced NLRP3 level more significantly than dexamethasone（P<0.05）.Compared with the Control group,the levels of NLRP3 and Caspase-1 and their MFI values in LPS group were significantly increased（P<0.01）.Compared with LPS group,the levels of NLRP3 and Caspase-1 and their MFI values were decreased in LPS+ZWT and LPS+DXM groups（P<0.05）,and the decrease was more obvious in LPS+ZWT group（P<0.05）.（4）Mechanisms related to er stress: Compared with the Control group,mRNA levels of GRP-78,pIRE1-α,pPERK,peIF2-α,CHOP,TXNIP,GRP-78 and CHOP were increased in LPS group（P<0.05）.Compared with LPS group,gr P-78 mRNA and CHOP mRNA contents in LPS+DXM group and LPS+ZWT group were decreased（P<0.05）,and the levels of GRP-78,pIRE1-α and TXNIP in LPS+DXM group were slightly decreased（P>0.05）.The levels of pPERK,peIF2-α,CHOP,and Calpain-1decreased in LPS+DXM group（P<0.05）,and the levels of GRP-78,pIRE1-α,pPERK,peIF2-α,CHOP,Calpain-1,and TXNIP decreased in LPS+ZWT group（P<0.05）.Compared with LPS+ZWT group,the levels of GRP-78,pIRE1-α,pPERK,peIF2-α,CHOP,Calpain-1 and TXNIP were increased in PX12 group（P<0.05）.Compared with Control group,TXNIP and MFI levels in LPS group were significantly increased（P<0.01）.Compared with LPS group,TXNIP and MFI levels were decreased in LPS+DXM group and LPS+ZWT group（P<0.05）.Compared with LPS+ZWT group,TXNIP and MFI levels in PX12 group were significantly increased（P < 0.01）.（5）Mechanisms related to mitochondrial injury and mitochondrial autophagy:Compared with the Control group,the levels of MMP and ATP in LPS group were decreased（P<0.05）,while the levels of VDAC-1 and mtROS were increased（P<0.05）;Compared with LPS group,MMP level was increased（P<0.05）,ATP content was slightly increased（P>0.05）,VDAC-1 and mtROS levels were slightly decreased（P>0.05）in LPS+DXM group.Compared with LPS group,MMP and ATP contents were increased（P<0.05）,VDAC-1 and mtROS levels were significantly decreased（P< 0.01）in LPS+ZWT group.Compared with the Control group,the expressions of LC3-Ⅱ/Ⅰ,Pink-1 and Parkin in LPS group were increased（P<0.05）,and the expression of P62 was decreased（P<0.05）.Compared with LPS group,the levels of LC3-Ⅱ/Ⅰ and PINK1 in LPS+DXM group were increased（P<0.05）,while the expressions of P62 and Parkin were slightly increased（P>0.05）.Compared with LPS group,the expressions of LC3-Ⅱ/Ⅰ,PINK-1 and Parkin were increased in LPS+ZWT group（P<0.05）,while the expression of P62 was decreased（P<0.05）.（6）Compared with Control group,ROS,TXNIP,mtROS and NLRP3 levels in LPS group were increased（P<0.05）;Compared with LPS group,the contents of ROS,TXNIP,mtROS and NLRP3 in LPS+ZWT group were decreased（P<0.05）,while the levels of ROS,TXNIP,mtROS and NLRP3 in LPS+DXM group were slightly decreased（P>0.05）.Compared with LPS+ZWT group,ROS,TXNIP,mtROS and NLRP3 levels were decreased in NAC group（P<0.05）,and ROS,TXNIP,mtROS and NLRP3 levels were increased in H₂O₂ group（P<0.05）.Section Ⅱ: Effects of Zhenwu Decoction on oxidative stress of lung cells induced by LPS in acute lung injury Objective:This part mainly observed the effects of Zhenwu Decoction on NRF-2,HO-1,TRX-1,GSH,SOD,CAT,ROS and MDA in lung tissues,and explored the regulatory effect of Zhenwu Decoction on oxidative stress of lung cells induced by LPS in acute lung injury.Methods:Seventy male kunming mice were randomly divided into 5 groups（Control group,LPS group,LPS+DXM group,LPS+ZWT group and ML385 treatment group）.Each group was given double steam water or corresponding drugs for 3 days,while the ML385 treatment group was given corresponding drugs intraperitoneally 3 hours before modeling,drawing materials as before.ROS levels were detected,NRF-2,HO-1 and TRX-1 levels were measured by Western blot,and GSH,SOD,CAT and MDA levels were measured by ELISA.Results:（1）Compared with the Control group,the levels of NRF-2,HO-1 and TRX-1 in LPS group were significantly decreased（P<0.05）,and the levels of NRF-2,HO-1 and TRX-1 in LPS+DXM group and LPS+ZWT group were increased（P < 0.05）;Compared with LPS+ZWT group,the levels of NRF-2,HO-1 and TRX-1 in ML385 treatment group were significantly decreased（P<0.01）.（2）Compared with Control group,the levels of GSH,SOD and CAT in LPS group were decreased（P<0.05）;Compared with LPS group,the levels of GSH,SOD and CAT in LPS+DXM and LPS+ZWT groups were increased（P<0.05）;Compared with LPS+ZWT group,the levels of GSH,SOD and CAT in ML385 treatment group were decreased（P<0.05）.（3）Compared with Control group,MDA and ROS in LPS group were increased（P<0.05）;Compared with LPS group,MDA content and ROS levels in LPS+DXM group were decreased（P<0.05）and slightly decreased（P>0.05）,while MDA and ROS levels in LPS+ZWT group were decreased（P<0.05）.Compared with LPS+ZWT group,MDA and ROS levels in ML385 treatment group were increased（P<0.05）.Conclusions:（1）Zhenwu Decoction inhibited endoplasmic reticulum stress and activated mitochondrial autophagy to intervene NLRP3 inflammasome mediated scortosis in acute lung injury.Zhenwu Decoction alleviates mitochondrial dysfunction,increases ATP content and reduces the generation of mtROS by enhancing mitochondrial autophagy,and ultimately redirects the mode of lung cell death by activating autophagy and inhibiting pyroapoptosis,so as to play a protective role against acute lung injury.（2）Zhenwu Decoction can up-regulate the expression of NRF-2 to increase the activities of downstream antioxidant factors and antioxidant enzymes,so as to eliminate excessive ROS in acute lung injury and fight against oxidative stress injury caused by ROS.Conclusions of this article:1.Using network pharmacology and molecular docking technology,zhenwu Decoction was found to pass through TRX1/TXNIP,NRF2/HO1 and PINK1/PARK2 signaling pathways.It acts on ROS,ENaCα,CASP1,CAT,CHOP,GRP78,GSDMD,IL1 B,IL-18,LC3,NLRP3,NRF2,P62,SOD,TNF-α,VDAC1 and other targets to intervene in acute lung injury.2.The mice model of acute lung injury was established by tail vein injection of LPS.The conclusions are as follows:（1）Zhenwu Decoction could reduce pulmonary edema induced by acute lung injury by up-regulating the expressions of ENaCα,ENaCβ,ENaCγ,Na⁺/K⁺-ATPase,AQP1 and AQP5 proteins,which may be one of the important mechanisms of zhenwu Decoction in protecting acute lung injury.（2）Zhenwu Decoction can inhibit endoplasmic reticulum stress and activate mitochondrial autophagy to intervene NLRP3 inflammasome mediated pyroapoptosis in acute lung injury.Zhenwu Decoction alleviates mitochondrial dysfunction,increases ATP content and reduces the generation of mtROS by enhancing mitochondrial autophagy,and ultimately redirects the mode of lung cell death by activating autophagy and inhibiting pyroapoptosis,so as to play a protective role against acute lung injury.（3）Zhenwu Decoction can up-regulate the expression of NRF-2 and increase the activities of downstream antioxidant factors and antioxidant enzymes,so as to eliminate excessive ROS in acute lung injury and fight against oxidative stress injury caused by ROS.