| Objectives To virtually identify the key target genes and pathways related to glycolysis in the treatment of acute liver failure(ALF)with Sini decoction plus ginseng(SNRS)through bioinformatics and network pharmacology,and to perform immune infiltration analysis of key target genes to provide a theoretical basis for the therapeutic effect of SNRS on affecting the reprogramming of glucose metabolism to regulate the polarization state of macrophages,and to provide reference ideas for subsequent experimental design.This study aims to investigate the pharmacodynamics of SNRS in the treatment of ALF by intraperitoneal injection of LPS/D-Gal N into ALF mice,to observe the effect of SNRS on the infiltration of hepatic macrophage subtypes,and to verify the effectiveness of SNRS in the treatment of ALF and its effect on the regulation of the polarization state of hepatic macrophages.Finally,we prepared and selected the optimal concentration of SNRS-containing serum,together with lipopolysaccharide(LPS),which was used to treat RAW264.7 macrophages of mice.Besides,we explored the effect of SNRScontaining serum on the energy metabolism mode and polarization status of LPSinduced activated macrophages based on the m TOR/HIF-1a pathway.It aims to provide a scientific basis for SNRS to reshape the polarization status of macrophages by interfering with immune metabolism for the treatment of ALF.MethodsPart I Immuno-infiltration analysis of potential target genes for ALF treatment by regulation of glycolysis with SNRSHuman liver tissue microarray data for HBV-associated acute liver failure(HBVALF)and acetaminophen-induced acute liver failure(APAP-ALF)were collected through the Gene Expression Omnibus(GEO)database under the National Center for Biotechnology Information(NCBI)platform;the differential genes expressed by each of the two were screened.Subsequently,the genes were intersected with glycolysis-related genes and SNRS effector target genes to obtain potential target genes for SNRS to intervene in ALF,and their gene functions were annotated;finally,immune cell infiltration in ALF and normal liver tissues were compared and analyzed,and the expression levels of the final obtained target genes were correlated with the degree of immune cell infiltration in ALF liver tissues.Part II Pharmacodynamic study of SNRS on LPS/D-Gal N-induced ALF modelA random number table method was utilized to randomly divide 63 ICR mice into blank group,model group,and Chinese medicine group,with 21 mice in each group.Both the model group and the Chinese medicine group were given a single intraperitoneal injection of D-Gal N(800mg/Kg)+ LPS(20μg/Kg),while the blank group was given a single intraperitoneal injection of the same amount of saline.Mice in the Chinese medicine group were treated with gastric administration of SNRS 48 h,24h before,and 10 min after modeling,while the blank group and the model group were treated with saline gastric administration at the same time points.At 8 h after injection of the molding agent,6 mice in each group were randomly selected for sample collection and specimen testing,and the remaining 15 mice in each group were analyzed for survival rate.During the experiment,the mental status,hair condition,diet,urine,and feces of the mice in each group were observed and recorded.Serum ALT and AST of mice were measured by automatic biochemical analyzer;TNF-α,HMGB-1,IL-1β,IL-6,and IL-10 were measured by enzyme-linked immunosorbent assay(ELISA);hematoxylin-eosin staining(HE)was performed on liver tissue sections to observe the pathological changes;immunofluorescence staining was used to observe the infiltration of M1 and M2 macrophages in liver tissues.Part III In vitro experimental study of SNRS on the remodeling of macrophage polarization stateExperiment 1 Preparation and concentration selection of SNRS-containing serumTwenty SD rats were divided into the SNRS group and the blank group by random number table method,with 10 rats in each group.SNRS group was administered with Chinese medicine and the blank group was administered with an equal volume of saline.The drug was administered twice daily for 3 days.Blood was collected 1 h after the last administration.The collected whole blood of rats was centrifuged,inactivated,and debacterized to produce SNRS-containing serum.RAW264.7 cells at logarithmic phase were inoculated in 6-well plates and added with LPS 100ng/ml to polarize RAW264.7 cells to M1 type.The CCK8 method was employed to detect the effects of different concentrations of blank serum and SNRS-containing serum on the proliferation of normally cultured RAW264.7 cells and LPS-induced activated RAW264.7 cells.And three serum concentrations(high,medium,and low)were set for the subsequent selection of optimal concentrations.After LPS-induced activation of RAW264.7 cells treated with high,medium,and low concentrations of drug-containing serum for 24 h,the concentrations of the IL-1β and the TNF-α were detected by ELISA,LD release by colorimetric assay,and the expression levels of m TOR,p-m TOR,and HIF-1α by Western Blot.Experiment 2 SNRS-regulated m TOR/HIF-1a signaling pathway affecting glycolytic reprogramming to reshape macrophage polarization stateRAW264.7 cells at the logarithmic phase were inoculated in 96-well plates.The effects of MHY1485 and Rapamycin on the proliferation of RAW264.7 cells without LPS-induced activation were examined using the CCK8 method;the effects of 10% drug-containing serum,10% drug-containing serum with MHY1485 and Rapamycin on the proliferation of RAW264.7 cells activated by LPS were also examined.RAW264.7 cells at logarithmic phase were inoculated in 6-well plates,and five experimental groups were set up:(1)blank group;(2)model group;(3)LPS+drugcontaining serum group;(4)LPS+drug-containing serum+m TOR agonist group;(5)LPS+blank serum+m TOR inhibitor group.After drug treatment for 24 h,cell morphology was observed by light microscopy;the ratio of M1(CD86+CD206)and M2(CD86-CD206+)cell subpopulations was analyzed by flow cytometry;the expression of m TOR,p-m TOR,and HIF-1a was detected by WB;the levels of IL-1β,TNF-α,and IL-10 in cell supernatants were detected by ELISA,and the concentrations of HK2,PFK-1,PKM2 and LDHA in cell extracts were detected by ELISA.Colorimetry was used to detect the LD content of cell supernatant,Ac-Co A,and ATP levels in cell extracts.In addition,transmission electron microscopy was used to observe the ultrastructure of cells in the blank group,model group and LPS+ drugcontaining serum group.ResultsPart I Immune infiltration analysis of potential target genes of SNRS-regulated glycolysis for the treatment of ALFIn this study,two expression profile microarray datasets of HBV-ALF(GSE38941,GSE14668)and one expression profile microarray dataset of APAP-ALF(GSE120652)were collected.The APAP-ALF differentially expressed gene set,the combined HBVALF differentially expressed gene set,the glycolytic gene set,and the SNRS target gene set were intersected using the Venn diagram online tool.Consequently,a total of five SNRS target genes regulating glycolysis for the treatment of HBV-ALF were identified: Hexokinase-2,Cyclin-dependent kinase 1,Superoxide dismutase [Cu-Zn],Vascular endothelial growth factor A,Glutamate oxaloacetate transaminase 1.(APAP-ALF did not acquire similar genes)The expression of HK2 and CDK1 was upregulated in the HBV-ALF group compared to the normal group,while the expression of SOD1,VEGFA,and GOT1 was downregulated.According to GO functional annotation and KEGG pathway enrichment analysis of the five target genes,the biological processes(BP)were mainly involved in "response to copper ions","cellular senescence","response to cadmium ions","response to axonal damage","follicular development",etc.;the cellular components(CC)were mainly enriched in mitochondria.KEGG enrichment pathways were mainly concentrated in hypoxia-inducible factor-1(HIF-1),carbon metabolism,etc.Differential analysis of immune infiltration suggested that the level of immune cell infiltration in HBV-ALF liver tissue was significantly increased compared with normal liver tissue.Immune cells infiltrating in HBV-ALF liver tissue mainly included dendritic cells,neutrophils,M2 macrophages,M1 macrophages,M0 macrophages,CD8+ T cells,CD4+ T cells and B cells.Correlation analysis of five action genes with the major immune cells infiltrating in HBV-ALF liver tissue suggested that SNRS could affect immune cell infiltration through action target genes.Part II Pharmacodynamic study of SNRS on LPS/D-Gal N-induced ALF modelThe ICR mice with ALF modeled by intraperitoneal injection of D-Gal N(800 mg/Kg)+ LPS(20 μg/Kg)had a poor general condition and an 80% mortality rate within 24 h.Compared with the blank group,the levels of ALT and AST were significantly increased;the release of TNF-α,HMGB-1,IL-1β and IL-6 was significantly increased;the liver histopathological damage was severe;the expression of M1 macrophage marker CD86 was significantly increased and the expression of M2 macrophage marker CD163 was relatively increased in liver tissue.The general condition of ALF mice treated with SNRS improved,and the 24-h mortality rate was 26.7%.Compared with the model group,ALT and AST levels were significantly reduced;TNF-α,HMGB-1 and IL-1β release was significantly reduced,IL-6 levels showed a decreasing trend,while IL-10 expression levels were significantly increased;liver histopathological damage was reduced;the expression of M1 macrophage marker CD86 was significantly reduced and the expression of M2 macrophage marker CD163 was significantly increased in liver tissue.Part III In vitro experimental study of SNRS on remodeling the polarization state of macrophagesExperiment 1 Preparation and concentration selection of SNRS-containing serumThere was no statistically significant difference in the cell vitality of the two groups when normal cultured RAW264.7 cells were treated with blank serum and drugcontaining serum at the same dilution concentration.Treatment of SNRS-containing serum at various dilution concentrations(2.5%,5%,10%,15%,and 20%)with LPSinduced activation of RAW264.7 cells reduced cell proliferation.Blank serum at 15% and 20% concentrations can reduce the proliferation of activated RAW264.7 cells induced by LPS.Gradient concentrations of 2.5%,5%,and 10% were selected for subsequent selection of optimal drug-containing serum concentrations.SNRS-containing serum inhibited the release of IL-1β and TNF-α from LPSinduced RAW264.7 cells in a dose-dependent manner.It also reduced the production of the glycolytic metabolite LD.The expression of m TOR/HIF-1a pathway-related proteins was significantly increased in RAW264.7 macrophages under an LPS-induced state,while SNRS-containing serum showed a dose-dependent decrease in pm TOR/m TOR ratio and relative expression of HIF-1a protein.Experiment 2 SNRS-regulated m TOR/HIF-1a signaling pathway affecting the reprogramming of glucose metabolism to remodel the polarization state of macrophagesMHY1485 and Rapamycin had no significant effect on the proliferation of RAW264.7 cells.The electron microscopic results revealed that the model group had severe swelling of cell mitochondria compared with the blank group,and the SNRScontaining serum could effectively mitigate the degree of mitochondrial swelling.Light microscopy and flow cytometry results suggested that M1-type and M2-type macrophages increased after LPS-induced activation of RAW264.7 cells;compared with the model group,treatment of LPS-induced RAW264.7 cells with drug-containing serum and Rapamycin decreased M1-polarized cells and increased M2-polarized cells;and the addition of MHY1485 to drug-containing serum reversed the effect of serum on M1 and M2 polarization distribution.The expression levels of IL-1β and TNF-α were significantly increased in the model group,and IL-10 tended to increase;LPS-induced RAW264.7 cells treated with drug-containing serum and Rapamycin could inhibit IL-1β and TNF-α expression and significantly increase IL-10 levels;while MHY1485 could reverse the anti-inflammatory effect of drug-containing serum.The p-m TOR/m TOR ratio and the relative expression of HIF-1a were significantly increased in the model group;after the intervention of LPS-induced activation of RAW264.7 cells with drug-containing serum and Rapamycin,the p-m TOR/m TOR ratio and the relative expression of HIF-1a were significantly decreased;and the addition of MHY1485 to the drug-containing serum reversed the effect of drug-containing serum reduced p-m TOR/m TOR ratio and HIF-1a relative expression.The expression levels of HK2,PFK1 and LDHA were significantly increased in the model group,and the LD content in the cell supernatant was significantly increased.Treatment of LPS-induced RAW264.7 cells with drug-containing serum and Rapamycin resulted in a significant decrease in the expression levels of HK2,PFK1,and LDHA and a significant decrease in LD content in the cell supernatant.MHY1485 reversed the regulatory effect of drugcontaining serum.Compared with the blank group,the Ac-Co A and ATP in RAW264.7 cells of the model group were significantly reduced;compared with the model group,the Ac-Co A and ATP contents in the LPS-induced RAW264.7 cells treated with the drug-containing serum and Rapamycin were significantly increased;however,when MHY1485 was added to the drug-containing serum,the Ac-Co A and ATP content was significantly lower than that of the drug-containing serum alone.ConclusionSNRS improved the general condition,increased survival,and decreased transaminase in ALF mice,regulated inflammatory mediator release,alleviated pathological tissue damage,and affected the distribution of hepatic macrophage subtype infiltration.Hepatic macrophages,a vital component of the hepatic intrinsic immune system,are involved in the immune damage associated with the pathogenesis of ALF.SNRS could improve immune metabolism by regulating the m TOR/HIF-1α signaling pathway,inhibiting HK2,PFK1,and LDHA expression,reducing LD secretion,increasing Ac-Co A production and promoting ATP synthesis.It suppressed M1-type macrophage activation,promoted M2-type macrophage activation,and reshaped macrophage polarization status.It also decreased the expression of proinflammatory factors IL-1β and TNF-α,increased the secretion of anti-inflammatory factor IL-10,and actively exerted anti-inflammatory and pro-repair effects.In addition,SNRS protected mitochondrial structure and maintained mitochondrial function of RAW264.7 cells. |
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