| Chapter Ⅰ: Network pharmacological study Objective:Based on the network pharmacological method,the pharmacological mechanism of Sini Decoction Plus Ginseng(SNRS)on acute liver failure(ALF)was studied.Methods:The potential active ingredients of SNRS were screened by TCMSP database,and their targets were predicted by Swiss Target Prediction database.ALF-related disease targets were screened by OMIM,Disgenet and Genecards databases,and common targets were obtained by intersecting with drug-related targets for PPI analysis.The drug-component-target-disease network was constructed by Cytoscape software,and topology analysis was performed.GO function and KEGG pathway enrichment analysis of common targets were performed by Metascape database,and molecular docking verification were performed with Auto Dock Vina software.Results:(1)139 active components and 1044 action targets of Sinijiashen Decoction,1222 related targets of ALF,and 167 common targets were screened.(2)The core components of SRNS in the intervention of ALF included Gomisin B 、Phaseolinisoflavan、3’-Methoxyglabridin、Panaxadiol、glyasperin B、Kaempferol,etc.And the core targets were AKT1,IL-6,TNF,MAPK1,MTOR,PIK3 CA,etc.(3)SNRS was involved in the positive regulation of protein phosphorylation,response to extracellular stimuli and cell response to chemical stress,and plays an intervention role in ALF through PIK3CA-AKT and necroptosis pathways.(4)Molecular docking verification showed that the core components of SNRS had certain binding activities with AKT1,TNF,MAPK1,PIK3 CG,BCL2,CASP3,CASP8,IL6,IGF1 R,MDM2,STAT3 and other target moleculars in PIK3CA-AKT and necroptosis pathway.Conclusion:Network pharmacological results showed that PIK3CA-AKT and necroptosis signaling pathway might be key targets for SNRS intervention in ALF.Chapter Ⅱ: In vivo experimental study Objective:This study aimed to investigate the protective effect of SNRS on D-Galactosamine(D-Ga IN)/Lipopolysaccharide(LPS)-induced ALF mice and its possible mechanism.Methods:84 ICR mice were random Ly divided into 4 groups of 21 in each group.Each group randomly took 15 animals for 24 h survival rate analysis,and the remaining 6were sterilized 8 hours after molding to collect specimens for testing.A single intraperitoneal injection of D-Ga IN(800 mg/kg)combined with LPS(20 μg/kg)was used to establish an ALF mouse model,which was filled with gastric lavage once at48 h,24 h and immediately after molding,respectively.The doses are as follows:SNRS group(12.31 g/kg/d),Silymarin group(200 mg/kg/d),blank group and model group(equal amount of distilled water).Liver pathology and ultrastructure were observed by optical microscopy and transmission electron microscopy.The hepatocyte apoptosis rate,the JC-1 polymer/monomer ratio and reactive oxygen species levels were detected by flow cytometry.The Caspase-3 expression level was detected by immunohistochemistry assay.The m RNA levels of PIK3 CA,AKT1 and UCP2 were detected by RT-PCR assay.The protein levels of(p-)MLKL,RIP3,PIK3 CA,AKT1 and UCP2 were detected by western blot assay.The activities of SOD,MDA,GSH,CAT and ATP synthase were detected by kit method.The levels of MPTP,HMGB1,NLRP3,IL-1β,IL-6,IL-10,TNF-α,Cyt-C,ATP,ADP and AMP were detected by ELISA.Results:Part 1:(1)SNRS improved the general condition and liver function of the mice with ALF,and increased the survival rate.(2)The liver of the model group showed hemorrhagic necrosis in pieces,with more erythrocyte exudation and inflammatory cell infiltration,some hepatocyte apoptosis,nucleus enlargement or fat vacuoles,etc.Compared with the model group,the liver pathology of the SNRS group was significantly improved,while the Silymarin group did not change much.(3)The levels of HMGB1,NLRP3,IL-1β,IL-6 and TNF-α in liver of the model group were significantly increased,and the IL-10 level was significantly reduced.Compared with the model group,the levels of HMGB1,NLRP3,IL-1β,IL-6 and TNF-α in liver of the SNRS group were significantly reduced,and the IL-10 level was significantly increased.Part 2:(1)The hepatocyte necrosis was very significant in the model group and significantly reduced in scope and degree of hepatocyte necrosis in the SNRS group.(2)The hepatocytes apoptosis rate and the Cyt-C level in liver of the model group were significantly increased.Compared with the model group,the hepatocytes apoptosis rate and the Cyt-C level in liver of the SNRS group were significantly reduced.(3)The protein levels of p-MLKL and RIP3 in liver of the model group were significantly increased.Compared with the model group,the protein levels of p-MLKL and RIP3 in liver of the SNRS group were significantly reduced.Part 3:(1)There were at least two forms of hepatocyte death in the model group,namely apoptosis and necrosis.The former included nuclear shrinkage,chromatin aggregation,increased cytoplasmic electron density,and mild shrinkage of few mitochondrial.The latter included discontinuous cell membrane,obvious loss of cytoplasmic content,dissolved cavitation of chromatin in nucleus,and serious damage of some mitochondrial structures.Compared with the model group,the SNRS group had milder hepatocyte damage,included mild swelling of mitochondria,and a small amount of autophagy in the cytoplasm.(2)The MPTP level in liver of the model group was significantly increased.Compared with the model group,the MPTP level in liver of the SNRS group was significantly reduced.(3)The JC-1 polymer/monomer ratio in hepatocytes of the model group was significantly reduced.Compared with the model group,the JC-1 polymer/monomer ratio in hepatocytes of the SNRS group was significantly increased.(4)The AMP level in liver of the model group was significantly increased,while the ADP and ATP levels along with ATP synthase activity were significantly reduced.Compared with the model group,the AMP level in liver of the SNRS group was significantly reduced,while the ADP and ATP levels along with ATP synthase activity were significantly increased.(5)The activities of SOD、GSH、CAT in liver of the model group were significantly reduced,while the MDA activity and the ROS level were significantly increased.Compared with the model group,the activities of SOD、GSH、CAT in liver of the SNRS group were significantly increased,while the MDA activity and the ROS level were significantly reduced.Part 4:(1)The m RNA levels of PIK3 CA and AKT1 in liver of the model group were significantly reduced,while the m RNA level of UCP2 was significantly increased.Compared with the model group,the m RNA levels of PIK3 CA and AKT1 in liver of the SNRS group was significantly increased,while the m RNA level of UCP2 was significantly reduced.(2)The protein levels of PIK3 CA and AKT1 in liver of the model group were significantly reduced,while the protein level of UCP2 was significantly increased.Compared with the model group,the protein levels of PIK3 CA and AKT1 in liver of the SNRS group were significantly increased,while the protein level of UCP2 was significantly reduced.Conclusion:(1)From the overall level,SNRS could inhibit liver NLRP3inflammasome-IL-1β pathway,reduce inflammatory response,and then improve liver function,tissue pathology and survival rates in mice with ALF.(2)From the cellular level,SNRS could inhibit a variety of hepatocyte death mode in mice with ALF,including apoptosis,necrosis and necroptosis.(3)From the organelle level,SNRS could improve liver energy metabolism and oxidative stress in mice with ALF,reduce the opening of mitochondrial permeability transition pore,thereby relieving mitochondrial damage and increasing mitochondrial transmembrane potential.(4)From the molecular level,SNRS could up-regulate the expression of PIK3CA-AKT1 pathway and down-regulate the expression of UCP2 pathway in liver of the mice with ALF.Chapter Ⅲ: In vitvo experimental study Objective:This study aimed to investigate the protective effect of SNRS on D-Ga IN/LPS-induced mouse AML12 hepatocyte and its possible mechanism.Methods:Logarithmic AML12 cells were routinely cultured in DMEM/F12 medium for 12 h.The cell after adhesion was randomized,that is,the blank group and the model group added 10% of the normal control serum;however,each SNRS group added the corresponding SNRS-containing serum(6%,8%,10%),and the insufficient serum was supplemented with normal control serum,which were cultured for 12 hours respectively.Except for the blank group,D-Ga IN(44 μg/m L)combined with LPS(100 ng/m L)was added to induce 24 h and the cell were collected later.The mitochondrial transmembrane potential level was detected by laser confocal microscope,the cell survival rate was detected by CCK-8 assay,and the apoptosis rate was detected by flow cytometry.The expression levels of IL-6,AKT1 and UCP2 in cell were detected by immunofluorescence staining.The activity of SOD,MDA,GSH,CAT and ATP level were detected by kit method.The m RNA levels of PIK3 CA,AKT1,UCP2,Caspase-3,Caspase-9,Bax and Bcl-2 in cell were detected by RT-PCR assay.The protein levels of(p-)PIK3CA,(p-)AKT1,UCP2,c-Caspase-3,c-Caspase-9,Bax,Bcl-2,RIP1,RIP3,(p-)MLKL,IFN-γ,IL-6 and TNF-α in cell were detected by western blot assay.Results:Part 1:(1)The cell survival rate of the model group was significantly reduced.Compared with the model group,the cell survival rates of the SNRS groups were significantly increased.(2)The protein levels of IFN-γ,IL-6 and TNF-α in the model group were significantly increased.Compared with the model group,the protein levels of IFN-γ,IL-6 and TNF-α in the SNRS groups were significantly reduced.(3)The IL-6 fluorescence intensity in the model group was significantly increased.Compared with the model group,the IL-6 fluorescence intensities in the SNRS groups were significantly reduced.Part 2:(1)The fluorescence intensity,protein and m RNA levels of UCP2 in the model group were significantly increased.Compared with the model group,the fluorescence intensities,protein and m RNA levels of UCP2 in the SNRS groups were significantly reduced.(2)The ATP content in the model group was significantly reduced.Compared with the model group,the ATP contents in the SNRS groups were significantly increased.(3)The mitochondrial membrane potential in the model group was significantly induced.Compared with the model group,the mitochondrial membrane potential levels in the SNRS groups were significantly increased.(4)The UCP2 fluorescence intensity in the model group was significantly increased.Compared with the model group,the UCP2 fluorescence intensity in the SNRS group was significantly reduced.(5)The cell survival rate in the model group was significantly reduced,and the protein levels of UCP2,RIP1,RIP3,and(p-)MLKL were significantly increased.Compared with the model group,the cell survival rates in the SNRS group and genipine group were significantly increased,while the protein levels of UCP2,RIP1,RIP3,and(p-)MLKL were significantly reduced.Part 3:(1)The apoptosis rate in the model group was significantly increased.Compared with the model group,the apoptosis rtes in the 10% SNRS group was significantly reduced.(2)The m RNA levels of Caspase-9,Caspase-3 and Bax along with the protein levels of c-Caspase-9,c-Caspase-3 and Bax in the model group were significantly increased,while the m RNA and protein levels of Bcl-2 were significantly reduced.Compared with the model group,the m RNA levels of Caspase-9,Caspase-3 and Bax along with the protein levels of c-Caspase-9,c-Caspase-3 and Bax in the SNRS groups were significantly reduced,and the m RNA and protein levels of Bcl-2 were significantly increased.(3)The activities of SOD,GSH and CAT in the model group were significantly reduced,while the MDA activity was significantly increased.Compared with the model group,the activities of SOD,GSH and CAT in the SNRS groups were significantly increased,while the MDA activities were significantly reduced.(4)The m RNA levels of PIK3 CA and AKT1 in the model group were significantly reduced.Compared with the model group,the m RNA levels of PIK3 CA and AKT1 in the SNRS groups were significantly increased.(5)The cell survival rate and the protein levels of(p-)PIK3CA,(p-)AKT1 and Bcl-2 in the model group were significantly reduced,while the protein level of Bax was significantly increased.Compared with the model group,the cell survival rate and the protein levels of(p-)PIK3CA,(p-)AKT1 and Bcl-2 in the SNRS group were significantly increased.Whether it was the model group or the SNRS group,after the addition of PIK3CA-AKT1 pathway inhibitor LY294002,the cell survival rate and the protein levels of(p-)PIK3CA,(p-)AKT1 and Bcl-2 were significantly reduced,while the protein level of Bax was significantly increased.Conclusion:(1)SNRS could reduce the inflammatory factor levels of AML12 cell induced by D-Ga IN/LPS,and improve cell survival.(2)SNRS could improve mitochondrial transmembrane potential and ATP generation by down-regulating the UCP2 pathway,thereby reducing necroptosis of AML12 cells induced by D-Ga IN/LPS,and improving cell survival.(3)SNRS could upregulate the PIK3CA-AKT1 pathway,reduce reactive oxygen species generation and apoptosis of AML12 cell induced by D-Ga IN/LPS,and improve cell survival.(4)SNRS could protect AML12 cells induced by D-Ga IN/LPS in a dose-dependent manner. |
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