| Objective We constructed an acute liver failure(ALF)animal model by using D-Gal N/LPS,observed the treatment of ALF mice with Sini Decoction plus Ginseng Soup(SNRS)through "Huiyang Gutuo" and explore the molecular mechanism of SNRS inhibiting hepatocyte necroptosis and exerting anti-inflammatory effect to protect the liver of ALF mice from D-Gal N/LPS attack,providing scientific evidence for SNRS in the treatment of ALF.Methods Part Ⅰ Identification of potential targets and pathways of SNRS to improve liver cell necroptosis in the treatment of ALF The raw data of ALF patients were collected from the Gene Expression Omnibus(GEO)under the National Center for Biotechnology Information(NCBI)platform.Among them,GSE14668,GSE38941,GSE62029,and GSE96851 are ALFs caused by HBV infection.GSE120652 and GSE74000 are ALFs caused by acetaminophen overdose.Through a series of differential expression analyses,necroptosis-related gene co-expression analysis,CIBERSORT analysis,and functional pathway annotation,the key targets and core pathways related to necroptosis and immunophenotype among the many targets of SNRS in the treatment of ALF were screened.Part II Construction of D-Gal N/LPS induced acute liver failure model We randomly divided 36 mice into 3 groups with 12 mice in each group using the random number table method.High dose group: intraperitoneal injection of D-Gal N(900mg/kg)and LPS(30μg/kg);medium-dose group: intraperitoneal injection of DGal N(800mg/kg)and LPS(20μg/kg);low dose group: intraperitoneal injection D-Gal N(700 mg/kg)and LPS(20 μg/kg).We determined the optimal modeling dose by comparing the 24-hour survival rates of mice in different groups.After determining the optimal modeling dose,we randomly divided 56 mice into blank control group(T0 group),2h exposure(T2 group),4h exposure(T4 group),6h exposure(T6 group),8h exposure(T8 group),10 h exposure(T10 group),and 12 h exposure(T12 group).By observing the changes in liver function and histopathology of mice injected with the optimal modeling dose over time,the optimal sampling node was finally determined.Part III Effects of PPARα on necroptosis in mice with acute liver failure To explore the molecular mechanism of peroxisome proliferator-activated receptor(PPAR)α in ALF.The 40 mice were randomly divided into 4 groups,namely the blank group(Control),model group(D-Gal N/LPS+DMSO),and PPARα inhibitor group(DGal N/LPS+GW6471),and PPARα agonist group(D-Gal N/LPS+WY14643).GW6471 and WY14643 are potent inhibitors and agonists of PPARα molecules,respectively.The drug was administered by intraperitoneal injection at a concentration of 10 mg/kg,2 hours before injection for modeling.The samples were collected at the 8th hour after the injection of the molding agent.Detection of mouse liver function and H&E staining of liver tissue.The expression of PPARα m RNA was detected by RT-PCR,the protein expressions of PPARα,TLR4,and p-MLKL in liver tissue were detected by Western Blot(WB),the protein expressions of RIP3,JNK,ASC,and NF-κB were detected by immunohistochemistry,and the HMGB-1 and TNF-α were detected by ELISA,ROS in living cells was detected by flow cytometry,and the activity of CAT in liver tissue was detected by colorimetric method.Part IV SNRS reduces necroptosis of hepatocytes in ALF mice by regulating PPARα This part of the experiment is divided into two levels.First,to clarify the efficacy and molecular mechanism of SNRS intervention in ALF mice,we randomly divided 100 SPF-grade male ICR mice into 4 groups: blank control group(Control),model group(D-Ga IN/LPS),traditional Chinese medicine group(D-Ga IN/LPS+SNRS),and positive control group(D-Gal N/LPS+Silymarin).There were 25 mice in each group.Among the 25 mice,15 mice were randomly selected for the observation of 24 h survival rate.Secondly,to confirm the relevant molecular mechanism of SNRS in the treatment of ALF is related to the promoting effect of SNRS on PPARα,we divided 60 mice into 6 groups according to the random number table.They were blank group(Control+DMSO),model group(D-Gal N/LPS+DMSO),traditional Chinese medicine group(D-Gal N/LPS+SNRS+DMSO),PPARα inhibitor group(DGal N/LPS+GW6471),PPARα agonist group(D-Gal N/LPS+WY14643)and traditional Chinese medicine+PPARα inhibitor group(D-Gal N/LPS+GW6471+SNRS).Each Chinese medicine group was treated with SNRS by gavage 48 h,24h before modeling,and 10 minutes after injection of a modeling agent.Finally,the mouse liver function and H&E staining of liver tissue were detected,and the 24-hour ALF mouse survival curve was drawn.RT-PCR was used to detect the m RNA expressions of PPARα,TLR4,JNK2,and CPT1A;The electron microscope was used to detect the liver tissue sections of the mice in each group;WB was used to detect the expression of MLKL,P-MLKL,RIP3,PPARα,ASC,CASP1,TLR4,p-JNK2 and JNK2 of the liver in each group;NF-κB expression in liver tissue was detected by immunohistochemistry.MDA,SOD,and GSH in liver tissue were detected by the colorimetric method.HMGB-1,NLRP3,IL-1β,IL-6,IL-10,TNF-a,CPT1 A,ADP,and ATP were detected by ELISA.Results Part Ⅰ Identification of potential targets and pathways of SNRS to improve liver cell necroptosis in the treatment of ALF Through bioinformatics analysis,five target genes were screened.Among them,CXCL8 and SPP1 were up-regulated in ALF liver tissue,and SPLI,SOD1,and NAMPT were down-regulated in ALF liver tissue.These five genes are both involved in the necroptosis phenotype of ALF.In addition to CXCL8 and NAMPT,several other genes showed correlations with infiltrating cells in the liver,including macrophages,plasma cells,and natural killer cells.The results of pathway analysis showed that peroxisome is a pathway that is related to necroptosis and is mainly enriched in ALF.Therefore,PPARα,which has an important regulatory effect on peroxisomes,may be an important way for SNRS to treat ALF and regulate necroptosis.Part II Construction of D-Gal N/LPS induced acute liver failure model We determined that at the dose of 800 mg/20 μg of Gal N/LPS,the mortality rate of ALF mice was close to our expected level(50%-80%).The samples were taken at the 8th hour after the injection of the modeling agent.At this time,the histopathological manifestations of the mouse liver were consistent with the pathological characteristics of ALF.At the same time,the serum ALT and AST of the mice both peaked at this time point.Therefore,after the injection of the modeling agent The 8th hour will be used as the best sampling time in subsequent experiments.Part III Effects of PPARα on necroptosis in mice with acute liver failure In the D-Gal N/LPS-induced ALF animal model,activation of PPARα can reduce the serum ALT level in mice,improve the liver pathology in mice,and alleviate hepatocyte damage.In contrast,inhibiting the expression of PPARα promoted the development of ALF.We then confirmed the modulation of PPARα by selected PPARα agonists(WY14643)and inhibitors(GW6471).Further mechanistic analysis showed that necroptosis-related molecules RIP3 and p-MLKL were significantly up-regulated in ALF.Inhibition of PPARα can significantly increase the expression of RIP3 and pMLKL,while activation of PPARα has the opposite effect.After activation of PPARα,the cell damage marker HMGB-1 was significantly decreased,and the proinflammatory mediator TNF-α was also decreased.TLR4 was also significantly upregulated in the ALF model,and activation of PPARα could significantly reduce the expression of TLR4,as well as the JNK and NF-κB pathways mediated by TLR4.At the same time,activation of PPARα could significantly reduce the levels of NLRP3 and ASC in liver tissue.The detection of oxidative stress-related indicators found that ROS in ALF was greatly increased,and the antioxidant CAT was significantly reduced.PPARα agonists can significantly reduce the release of ROS in liver tissue,and promote the activity of CAT to produce anti-oxidative stress,while PPARα inhibitors produce the opposite regulatory effect.Part IV SNRS reduces necroptosis of hepatocytes in ALF mice by regulating PPARα The SNRS can significantly improve the survival rate of ALF mice(80%),which is significantly different from the model group(20%)and the positive control group(33.3%)(P<0.05).SNRS could significantly reduce serum ALT levels in ALF mice(P<0.05),and improve the pathological conditions of liver tissue in ALF mice.Further mechanism analysis showed that the SNRS could promote the transcription and translation of PPARα.Then we confirmed that the use of SNRS could inhibit the expression of necroptosis-related proteins RIP3 and MLKL(P<0.05),and also partially inhibit the expression of phosphorylated MLKL(P=0.107).The use of SNRS can reduce the release of HMGB-1(P<0.05),and the expression of TLR4(P<0.05).At the same time,JNK pathway phosphorylation and NF-κB expression were also decreased(P<0.05).We then confirmed that NLRP3,ASC,and CASP1 were significantly upregulated in ALF liver tissue(P<0.05).SNRS could significantly reduce the levels of NLRP3 and ASC in liver tissue(P<0.05),but no inhibition of CASP1 protein expression was observed.The SNRS can significantly reduce the pro-inflammatory cytokines TNF-α,IL-6,and IL-1β in liver tissue(P<0.05),and at the same time promote the release of IL-10 anti-inflammatory cytokines in liver tissue(P<0.05).Regarding the upstream pathway of necroptosis,we believe that the β-oxidation pathway of liver tissue may be involved in the relevant mechanism.Transmission electron microscopy confirmed the mitochondrial morphological disorder in ALF liver tissue.After using SNRS,the ultrastructure of hepatocytes was significantly improved,the morphology of mitochondria was normal,CPT1 A in liver tissue showed an obvious upward trend(P<0.05),and the level of liver ATP also increased(P<0.05).Further experiments need to confirm whether SNRS plays a relevant therapeutic role by regulating PPARα.We confirmed that SNRS could reverse the inhibitory effect of GW6471 on PPARα(P<0.001),which indicated the exact regulation ability of SNRS on PPARα.Based on treatment with GW6471,adding SNRS could reverse the promoting effect of GW6471 on p-MLKL(P<0.05).The use of PPARα agonists could increase the levels of SOD and GSH(P<0.01).Inhibition of PPARα led to further reduction of SOD and GSH in ALF mice(P<0.05).Based on the inhibition of PPARα,increasing the use of SNRS could significantly increase the decreased SOD and GSH levels(P<0.05).As an important product of lipid peroxidation,MDA was significantly up-regulated in the ALF model group.PPARα inhibitor would further increase the level of MDA.On this basis,SNRS treatment could inhibit the promoting effect of GW6471 on MDA,with significant differences(P<0.05).The expression of CPT1 A,a key enzyme involved in fatty acid β-oxidation,was significantly up-regulated in the SNRStreated group,accompanied by increased ATP levels,which may be the relevant mechanism by which SNRS reduces necroptosis.Conclusion Activating the expression of PPARα can effectively improve liver function and pathological changes in liver tissue in mice.The relevant mechanisms may be related to reducing the occurrence of necroptosis,reducing the release of damage-associated molecular patterns(DAMPs),and reducing the stimulation of liver tissue inflammation.In terms of pathway regulation,promoting the expression of PPARα may inhibit TLR4-mediated activation of NF-κB and MAPK pathways and NLRP3/ASC inflammasome by reducing necroptosis.In addition,activation of PPARα can also reduce the level of oxidative stress in liver tissue.Traditional Chinese medicine SNRS can inhibit the necroptosis of hepatocytes and reduce liver cell damage and improve liver function by promoting the expression of PPARα,reducing oxidative stress in liver tissue,reducing liver inflammation,increasing the expression of key enzymes of β-oxidation in liver tissue,and increase the supply of ATP in hepatocytes. |
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