Study On The Mechanism Of Endotoxemia-induced Liver Injury Based On Single-cell Transcriptome Sequencing

Background:Endotoxemia is a kind of sepsis,usually defined as a clinical syndrome caused by the release of large amounts of endotoxin into circulation by bacteria in the blood or lesions.The progress of the disease expands rapidly and leads to septic shock and multiple organ failures,which further cause death.During the development of endotoxemia,mechanisms involving intrahepatic inflammatory activation and the related liver injury are key for the early development of endotoxemia and the subsequent activation and amplification of systemic proinflammatory responses.The cellular and molecular mechanisms which regulate the spatial distribution of intrahepatic immune cells at the early stage of endotoxemia are still key questions to be addressed.Aim:To understand the transformation of liver cells in different states during liver injury and find new targets for prevention and treatment of endotoxemia liver dysfunction,the sequencing of liver parenchymal cells and liver parenchymal cells in the early stage of endotoxemia and liver dysfunction was carried out by single-cell transcriptome sequencing technology.Methods:(1)Establishment and validation of the endotoxemia model in mice:6-8 weeks-old male C57BL/6J mice were used for the control group,the Poly(I:C)group,the LPS-3h group,and the LPS-12h group.Construction and intervention of each groups are described as follows:(1)Control group:intraperitoneal injection of PBS(11ul/g body weight)for 10 hours;(2)Poly(I:C)group:intraperitoneal injection of poly(I:C)(1ug/g body weight)for 7 hours,followed by intraperitoneal injection of PBS(10ul/g body weight)for 3 hours;(3)LPS-3h group:intraperitoneal injection of poly(I:C)(1ug/g body weight)for 7 hours and then intraperitoneal injection of LPS(10ug/g body weight)for 3hours;(4)LPS-12h group:intraperitoneal injection of poly(I:C)(1ug/g body weight)for 7 hours and then intraperitoneal injection of LPS(10ug/g body weight)for 12 hours.Model verification:After the intervention of each group,the mice were observed the response to the stimulus.The mice in the control group and the Poly(I:C)group were energetic and in good mental state,while the mice in the LPS-3h group and the LPS-12h group responsed obviously slowly but were not in shock,on this condition the model was considered to be established successful.(2)Detection of liver function test,morphology,and infiltration and distribution of macrophages in mice at the early stage of endotoxemia:(1)Using serum enzymology to detect hepatic insufficiency by testing the concentrations of alanine aminotransferase and aspartate aminotransferase of control group,Poly(I:C)group,LPS-3h group and LPS-12h group respectively.(2)The liver tissues of the mice in the control group,LPS-3h group and LPS-12h group were separated,prepared into paraffin sections,and then stained with hematoxylin-eosin to observe the nucleopyknosis and nucleolysis of hepatocytes to explore the liver morphological changes of mice during the early stage of endotoxemia.(3)The liver tissues of mice in the control group,Poly(I:C)group,LPS-3h group and LPS-12h group were separated and prepared for paraffin sections.The number and distribution of macrophages(F4/80~+)were observed by immunohistochemical staining to explore the changes in the number and distribution of macrophages in the liver of mice in the early stage of endotoxemia.(3)Single-cell RNA sequencing and bioinformatics analysis:(1)Cell suspension samples preparation for single-cell RNA sequencing:tissue digestion and centrifugation were used to prepare the liver parenchyma cell suspensions and non-parenchymal cell suspensions of mice in the control group,LPS-3h group and LPS-12h group,respectively.Single-cell suspensions are used for single-cell sequencing.(2)Identification of cell subtypes in single-cell transcriptome data:the first 40 principal component dimensions were used for data dimension reduction by principal component analysis,and cell clustering with a discrimination degree of 0.3 was performed using the function"Find All Marker"(min.pct=0.25,logfc.threshold=0.25,only.pos=TRUE)to screen the characteristic genes of each cell subset to identify intrahepatic cell heterogeneity in the early stage of endotoxemia.(3)Functional enrichment:characteristic genes of each cell subset were used to carry out functional enrichment using GO and KEGG databases to explore the functional differences between different cell subsets of liver parenchymal cells and non-parenchymal cells in the early stage of endotoxemia.(4)Intercellular interaction analysis:The R package cellchat(version 1.1.0)was used to analyze the cellular interaction between different subpopulations of liver parenchymal cells and non-parenchymal cells.Results:1.Impaired liver function and histomorphological changes in the early stage of endotoxemia mice.(1)The results of serum enzymatic test showed that the concentrations of serum.alanine aminotransferase(LPS-3h,LPS-12h VS control,Poly(I:C),57.0±10.7U/L,75.7±18.2U/L VS 31.0±2.7U/L,27.8±2.1U/L)and aspartate aminotransferase(LPS-3h,LPS-12h VS control,Poly(I:C),188.2±58.4U/L,240.8±60.5U/L VS 126.2±40.7U/L,144.8±41.8U/L)in the LPS-3h group and the LPS-12h group were significantly increased than those in the control group and the Poly(I:C)group(P<0.05);(2)The results of hematoxylin-eosin staining of paraffin sections showed that the ratio of nucleopyknosis and nucleolysis(LPS-3h,LPS-12h VS control,17.38±5.28%,24.98±6.28%VS 1.56±0.27%)was significantly increased in LPS-3h group and LPS-12h group compared with the control group(P<0.001).2.Changes of macrophage infiltration in early liver tissue of mice with endotoxemia.(1)Single-cell transcriptomic sequencing results showed that compared with the control group,the hepatic non-parenchymal cells in the LPS-3h group and the LPS-12h group had a significantly larger constituent ratio of macrophages;GO/KEGG functional enrichment results showed that compared with the control group,the intrahepatic macrophages in the LPS-3h and LPS-12h group had significantly up-regulated the pro-inflammatory,phagocytosis and myeloid cell recruitment functions(P<0.005).(2)The results of immunohistochemical staining showed that the number of macrophages(F4/80~+)(LPS-3h,LPS-12h VS control,Poly(I:C),156.23±54.85,245.96±38.74 VS 82.34±3.92,89.41±8.47)in the LPS-3h group and the LPS-12h group was significantly increased compared with the control group and the Poly(I:C)group(P<0.001).3.Hepatocyte phenotypic switching mediates macrophage recruitment and liver injury via CCL2.(1)pro-inflammatory macrophages in the LPS-3h group and LPS-12h group were the cell subtype with the highest interaction score with hepatocytes,and CCL2-CCR2 was the ligand-receptor pair with the strongest cellular interaction score.(2)hepatocytes were the main cell type expressing CCL2,and pro-inflammatory macrophages were the main cell type expressing CCR2 in the LPS-3h group.Conclusions:(1)Endotoxemia induces hepatic insufficiency and massive recruitment of macrophages in the liver during the early stage of endotoxemia.(2)CCL2 of hepatocytes is one of the mechanisms to promote the infiltration of macrophages in the mice liver during the early stage of endotoxemia.