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Objective: To establish a method for the induction of peripheral blood mononuclear cells to hepatocyte-like cells, and preliminarily investigate cell response to injury under the effect of acetaminophen (APAP). Methods: The surface marker CD45 of peripheral blood mononuclear cells wase detected cells by using flow cytometry and immunofluorescence methods. The cellular morphology of induced hepatocyte-like cells was observed under an inverted microscope. Real-time fluorescent quantitative PCR (RT-PCR) was used to detect the expression level of hepatocyte-specific genes, such as cytochrome (CY) P1A2, CYP3A4, CYP2C9, albumin (ALB), alpha-fetoprotein (AFP), and hepatocyte nuclear factor (HNF)4α mRNA. Immunofluorescence method was used to detect intracellular hepatocyte markers AFP, HNF4α, and ALB expression at the protein level. Biochemical analyzer was used to detect hepatocyte-specific secretory functions of AFP, ALB, and urea. Luciferase chemiluminescence method was used to detect the activity of key drug metabolizing enzyme CYP3A4. Colorimetric assay was used to detect the effect of the drug acetaminophen on hepatocyte-like cells, and alanine aminotransferase (ALT) was used as an indicator of liver cell injury. The statistical differences between the data were compared with t-test and rank-sum test. Results: The positive expression rate of CD45 cell surface markers isolated from peripheral blood mononuclear cells was about 98%, and hepatocyte-like cell morphology changes appeared on 15th day of induction. Compared with isolated mononuclear cells, CYP1A2, CYP3A4, CYP2C9, ALB, AFP and HNF4α mRNA was markedly elevated. The expression level of AFP, ALB and HNF4α protein were equally increased, and the secretory function of AFP, ALB and urea were enhanced. Compared with primary hepatocytes, CYP1A2, CYP2C9, AFP, HNF4α mRNA, and CYP3A4 mRNA did not decrease. The expression levels of AFP, ALB, and HNF4α proteins in the cells did not decrease, and the secretory function of AFP, ALB, and urea did not decrease. In addition, the CYP3A4 enzyme activity produced by hepatocyte-like cells was similar to that of primary hepatocytes. Compared with hepatocyte-like cells incubated without APAP, hepatocyte-like cells incubated with APAP had higher ALT level. Under the effect of APAP, the ALT level of hepatocyte-like cells was higher than isolated mononuclear cells. Conclusion: Peripheral blood mononuclear cells can be induced into hepatocyte-like cells with partial characteristics of hepatocytes, including the activity of CYP3A4, a key enzyme of hepatocyte drug metabolism. Additionally, preliminarily ALT secretory features reflect the hepatocytes injury under the effect of acetaminophen.
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Acetaminophen/pharmacology , Cell Differentiation , Cells, Cultured , Hepatocytes , Leukocytes, Mononuclear , RNA, MessengerABSTRACT
ObjectiveTo investigate the clinical features of patients with different types of acute drug-induced liver injury (DILI) through a retrospective analysis. MethodsClinical data were collected from 790 patients who were diagnosed with acute DILI in Beijing YouAn Hospital and Beijing Tongren Hospital affiliated to Capital Medical University from December 2010 to March 2019, and according to the type of damaged target cell, the patients were divided into hepatocellular injury type group with 554 patients, cholestasis type group with 99 patients, and mixed type group with 137 patients. The patients were evaluated based on severity grade and score, clinical outcome, and Hy′s rule. An analysis of variance was used for comparison of normally distributed continuous data between three groups, and the least significant difference t-test was used for further comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between groups. The chi-square test was used for comparison of categorical data between three groups. The Kruskal-Wallis H test was used for comparison of ranked data between three groups, and the Mann-Whitney U test was used for comparison between two groups. ResultsMost of the patients were female in all three groups, and the hepatocellular injury type group had a significantly higher proportion of female patients than the cholestasis type group (70.8% vs 54.5%, P＜0.05), and the Cholestasis type group had a significantly lower proportion of female patients than the mixed type group(54.5% vs 54.7%, P＜0.05). There were 244 patients with grade 3 hepatocellular injury type DILI (244/554, 44.4%), 56 patients with grade 3 cholestasis type DILI (56/99, 56.6%), and 46 patients with grade 3 mixed type DILI (46/137, 33.6%), and there was a significant difference between the three groups (χ2=36.589, P＜0.05). Drugs inducing liver injury included traditional Chinese medicine, Western medicine, combination of traditional Chinese medicine and Western medicine, and other drugs, among which traditional Chinese medicine was the most common cause of liver injury. There was a significant difference in the outcome at discharge between the patients with different types (H=14.390, P=0.001). Compared with the cholestasis type group, the hepatocellular injury type group had a significantly higher cure rate and significantly lower uncured rate and mortality rate (all P＜0.05). Among the 554 patients with hepatocellular injury type DILI, 388 (70.0%) met Hy′s rule and 166 (300%) did not meet Hy′s rule, and there was a significant difference in clinical outcome between these two groups (U=38 372.0, P=0.033). ConclusionDILI is more common in women, and most patients have hepatocellular injury type DILI. Traditional Chinese medicine is the main cause of liver injury. There is a high proportion of patients with severe DILI among the patients with hepatocellular injury type or cholestasis type. DILI often has good prognosis with a relatively low mortality rate. Hy′s rule cannot predict the death of patients with acute DILI.
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The mitochondria in liver tissue not only provides energy for substance metabolism in hepatocytes, but also participates in hepatocellular injury and even apoptosis. It also plays an important role in several pathological processes closely associated with hepatocellular injury and apoptosis, such as hepatitis, hepatic fibrosis, precancerous lesion, and liver cancer. This article elaborates on the association between mitochondria and hepatocellular injury from the following aspects: the important role of the change in mitochondrial membrane permeability in hepatocellular injury, hepatocellular injury accelerated by ATP synthesis disorder and consumption, and the association of abnormal Ca2+ homeostasis in hepatocellular mitochondria with hepatocellular injury, in order to provide a theoretical basis for mitochondria-targeted prevention and treatment of chronic liver diseases due to hepatocellular injury.
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Objcetive To investigate protective effect of pinocembrin ( PIN)on hepatocytes induced by hypoxia/reoxygenation ( H/R) as well as its relationship to the TLR 4/NF-κB signaling pathway .Methods The cells were randomly divided into 4 groups: control group, PIN group, hypoxia/reoxygenation injury group and PIN pretreatment group .The cell viability was measured with CCK-8.The apoptosis rate was determined by Annexin V-FITC/PI staining.The activity of ALT was detected .ELISA was used to evaluate the contents of TNF-αand IL-β.The mRNA and protein expression level of TLR 4, IκB-αand NF-κB P65 in cells was observed by quantita-tive real-time PCR or Western blot .Results The H/R stimulation decreased cell survival rate and enhanced the apoptosis .The activity of ALT was increased .The contents of TNF-αand IL-1βwere significantly enhanced , and the expression level of TLR4 and NF-κB P65 was markedly increased while IκB-αdecreased.After pretreatment with PIN, the cell survival rate increased while the apoptosis rate decreased .The activity of ALT was decreased. TNF-αand IL-1βwere reduced significantly and the expression level of TLR 4 and NF-κB P65 were decreased while IκB-αincreased.Conclusions PIN has protective effects on hypoxia/reoxygenation injury, which might be mediated in part by TLR4/NF-κB signaling pathway .
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Objective To investigate the protective effect of IL-37 on hepatocyte injury against hypoxia/reoxygenation (H/R) by promoting polarization of M2-type macrophages and its molecular mechanisms.Methods Real-time fluorescent quantitative PCR (qRT-PCR) and Western blot were used to detect the levels of IL-37 mRNA and protein in human monocyte-macrophage THP-1 cells with different polarizations.The lentivirus with IL-37 gene was infected into THP-1 cells.The levels of CD206,CD86,ARG1 and iNOS mRNA was detected by qRT-PCR.The levels of CD163 and CD86 protein was detected by flow cytometric analysis.THP-1 cells and L02 cells were co-cultured by Transwell and treated with H/R.The survival rate and apoptotic rate of L02 cells were detected.The levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in culture medium were measured.The levels of STAT6 and its phosphorylation in THP-1 cells were detected by Western blot.Results The levels of IL-37 mRNA and protein were up-regulated in M2-type macrophages.IL-37 promoted the polarization of M2-type macropahges.M2-type macrophages induced by IL-37 were cocultured with L02 cells,the survival rate was significantly increased by H/R treatment (P =0.015),while the apoptotic rate,ALT level and AST level were significantly decreased (P＜0.001).The level of phosphorylated STAT6 in THP-1 cells overexpressing IL-37 was up-regulated (P ＜ 0.01).Conclusions IL-37 can induce polarization of M2-type macrophages and protect hepatocyte injury against H/R.Its mechanism may be related to STAT6 signal pathways.
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Objective To study the effect of high copper on Wnt /β-catenin signaling pathway and oxidative stress. Methods BRL-3A cells were incubated with different concentrations of CuSO4.The cell growth and proliferation wre assessed using MRR method.The production of reactive oxygen species(ROS)and mitochondrial membrane potential (JC-1) were examined usingf flow cytometry. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were detected by microplate reader. The expression of protein was detected by Western Blot. Results①The results of MTT showed that CuSO4inhibited the growth and proliferation of BRL-3A cells in a time- and concentration-dependent manner(P<0.01).②Flow cytometry results showed that CuSO4induced a large amount of ROS and significantly decreased the fluorescence intensity of JC-1 in BRL-3A cells (P<0.01). ③ Microplate reader results showed that CuSO4increased the content of MDA and decreased the activity of SOD (P<0.05).④Western blotting assay showed that CuSO4significantly decreased the total expression levels of β-catenin and p-Ser 9-GSK-3β protein as well as nuclear levels of p-(S33+S37)-β-catenin and c-Myc (P<0.01) and increased expression levels of GSK-3β、DKK1、Dishevelled3 protein in BRL-3A cells. Conclusion High copper can induce oxidative stress and induce Wnt /β-catenin signaling pathway to deactivate liver cells,leading to hepatocellular injury.
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Objective:To evaluate the protective effect of Diclipterachinensis polysaccharide P2B on liver cell line L-02 injury in-duced by carbon tetrachloride ( CCl4 ) . Methods:The human liver L-02 cells were cultured, and the injury model was built by CCl4 . The L-02 cells were divided into the normal control group, the CCl4-damaged group, and the P2B sample groups (0. 125, 0. 250 and 0. 500 mg· ml-1 ). The contents of alanine aminotransferase ( ALT), aspartate aminotransferase ( AST), superoxide dismutase (SOD) and malondialdehyde (MDA) were determined by MTT assay. Results:Compared with the CCl4-damaged group, P2B could improve the activity of L-02 cells, and the activity of AST and ALT in the supernatant was significantly reduced, and the content of SOD in the cells was increased and that of MDA was decreased. Conclusion:P2B can significantly prevent L-02 cells from the damage induced by CCl4 in a dose-dependent manner, and the mechanism may be related with the anti-oxidative activity of P2B.
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OBJECTIVE:To study the improving effect of total flavone from Litchi chinensis (TFL) on the hepatocyte injury in rats with liver fibrosis. METHODS:The rats were given dimethylnitrosamine(DMN),ip,once a day in the first 3 d of every week,which lasted for 30 consecutive days to establish hepatocyte injury model. 60 rats were equally randomized into a normal control(isometric normal saline)group,a model(isometric normal saline)group and the groups of high and low-dose TFL(200 and 100 mg/kg). When the model was being established,drugs were administered,ig,once a day for 45 consecutive days except for normal control group. HE staining was performed,and then the rats’hepatocytes were observed under the microscope and path-ological stage (S1-S4) of liver tissue was analyzed. Masson staining and immunohistochemical staining were conducted,and then the rats’hepatocytes were observed under the microscope and calculation was made for the degree of liver fibrosis and the expres-sion of Bcl-2 and Bax. The activities of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in rats’serum were determined. RESULTS:The pathological stages of liver cell of rats in the model group were mainly stages S3 and S4 and the groups of high and low doses TFL were dominated by stages S1 and S2. Compared to the rats in the normal control group,those in the mod-el group had higher degree of liver fibrosis,expression of Bcl-2 and Bax and activities of AST and ALT in serum. Compared to the rats in the model group,those in the groups of high and low doses TFL had lower degree of liver fibrosis,higher expression of Bcl-2,lower expression of Bax,and lower activities of AST and ALT in serum. There were statistically significance (P<0.05). CONCLUSIONS:TFL can alleviate the hepatocyte injury in rats with liver fibrosis to some degree by a mechanism which may be related to the up-regulation the expression of Bcl-2 and the down-regulation of the expression of Bax.
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Objective To explore the regulating mechanism of bcl 2 gene in hepatocyte injury caused by obstructive jaundice in rats. Methods Normal rats' and bile duct ligated 7d,14d,21d rats' hepatocytes were isolated by in situ collagenase perfusion and primary culture. (1) bcl 2 mRNA was detected by RT PCR in all cells; (2) After normal rat and bile duct ligated 14d rat hepatocytes were added 100?M GCDC and kept for 24hrs, cells were evaluated by FCM and TUNEL. Results (1) Normal rat hepatocytes did not express bcl 2 by RT PCR technique. bcl 2 was expressed in 7-,14-,21-day BDL rats. (2) After adding 100?M GCDC and keeping for 24hrs, the apoptosis of bile duct ligated rat hepatocytes significantly decreased compared with that of normal rat hepatocytes. Conclusions (1) Bile duct ligated rat hepatocytes expressed bcl 2. (2) Hepatocellular expression of bcl 2 during obstructive jaundice is an adaptive phenomenon to resist apoptosis by bile salts.
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The mitochondria in liver tissue not only provides energy for substance metabolism in hepatocytes, but also participates in hepatocellular injury and even apoptosis. It also plays an important role in several pathological processes closely associated with hepatocellular injury and apoptosis, such as hepatitis, hepatic fibrosis, precancerous lesion, and liver cancer. This article elaborates on the association between mitochondria and hepatocellular injury from the following aspects: the important role of the change in mitochondrial membrane permeability in hepatocellular injury, hepatocellular injury accelerated by ATP synthesis disorder and consumption, and the association of abnormal Ca2+ homeostasis in hepatocellular mitochondria with hepatocellular injury, in order to provide a theoretical basis for mitochondria-targeted prevention and treatment of chronic liver diseases due to hepatocellular injury.