OBJECTIVES: To observe the evolution of intrahepatic macrophage polarization in mice with liver fibrosis induced by iron overload. METHODS: Thirty-two C57BL/6 mice (6-8 weeks) were randomized into control group (n=8) and liver fibrosis model group (n=24) induced by aidly intraperitoneal injection of iron dextran. At the 3rd, 5th, and 7th weeks of modeling, 8 mice in the model group were sacrificed for observing liver fibrosis using Masson, Sirius Red and immunohistochemical staining and detecting serum levels of ALT, AST and the levels of serum iron, ferritin, liver total Fe and ferrous Fe. iNOS⁺/F4/80⁺ cells and CD206⁺/F4/80⁺ cells were detected by double immunofluorescence assay to observe the proportion and distribution of M1 and M2 macrophages. The hepatic expressions of Arg-1, iNOS, IL-6, IL-10, and TNF‑α proteins were detected using Western blotting or ELISA, and the expression of CD206 mRNA was detected using RT-PCR. RESULTS: The mice in the model group showed gradual increase of fibrous tissue hyperplasia in the portal area over time, structural destruction of the hepatic lobules and formation of pseudolobules. With the passage of time during modeling, the rat models showed significantly increased hepatic expressions of α-SMA and COL-1, elevated serum levels of ALT, AST, Fe, ferritin, and increased liver total Fe and ferrous Fe levels. The expressions of M1 polarization markers IL-6, TNF‑α, and iNOS all increased with time and reached their peak levels at the 3rd week; The expressions of M2 polarization markers (IL-10 and Arg-1 proteins and CD206 mRNA) significantly increased in the 3rd week and but decreased in the 5th and 7th weeks. CONCLUSIONS Iron overload promotes M1 polarization of macrophages in mice. Liver fibrosis in the early stage promotes M2 polarization of macrophages but negatively regulate M2 polarization at later stages.
Animals ; Mice ; Mice, Inbred C57BL ; Iron Overload/pathology* ; Macrophages/metabolism* ; Male ; Liver Cirrhosis/etiology* ; Nitric Oxide Synthase Type II/metabolism* ; Interleukin-10/metabolism* ; Liver/pathology* ; Interleukin-6/metabolism* ; Mannose Receptor ; Tumor Necrosis Factor-alpha/metabolism* ; Mannose-Binding Lectins/metabolism* ; Arginase

Liver fibrosis is a dynamic wound-healing response characterized by the agglutination of the extracellular matrix (ECM). Si-Wu-Tang (SWT), a traditional Chinese medicine (TCM) formula, is known for treating gynecological diseases and liver fibrosis. Our previous studies demonstrated that long non-coding RNA H19 (H19) was markedly upregulated in fibrotic livers while its deficiency markedly reversed fibrogenesis. However, the mechanisms by which SWT influences H19 remain unclear. Thus, we established a bile duct ligation (BDL)-induced liver fibrosis model to evaluate the hepatoprotective effects of SWT on various cells in the liver. Our results showed that SWT markedly improved ECM deposition and bile duct reactions in the liver. Notably, SWT relieved liver fibrosis by regulating the transcription of genes involved in the cytoskeleton remodeling, primarily in hepatic stellate cells (HSCs), and influencing cytoskeleton-related angiogenesis and hepatocellular injury. This modulation collectively led to reduced ECM deposition. Through extensive bioinformatics analyses, we determined that H19 acted as a miRNA sponge and mainly inhibited miR-200, miR-211, and let7b, thereby regulating the above cellular regulatory pathways. Meanwhile, SWT reversed H19-related miRNAs and signaling pathways, diminishing ECM deposition and liver fibrosis. However, these protective effects of SWT were diminished with the overexpression of H19 in vivo. In conclusion, our study elucidates the underlying mechanisms of SWT from the perspective of H19-related signal networks and proposes a potential SWT-based therapeutic strategy for the treatment of liver fibrosis.
Humans ; RNA, Long Noncoding/genetics* ; Liver Cirrhosis/genetics* ; Liver/metabolism* ; Hepatic Stellate Cells/pathology* ; MicroRNAs/metabolism* ; Extracellular Matrix/metabolism* ; Drugs, Chinese Herbal

As the central organ of metabolism, the liver plays a pivotal role in the regulation of the synthesis and metabolism of various nutrients within the body. Ferroptosis, as a newly discovered type of programmed cell death caused by the accumulation of iron-dependent lipid peroxides, is involved in the physiological and pathological processes of a variety of acute and chronic liver diseases. Ferroptosis can accelerate the pathogenetic process of acute liver injury, metabolic associated fatty liver disease, alcoholic liver disease, viral hepatitis, and autoimmune hepatitis; while it can slower disease progression in advanced liver fibrosis and hepatocellular carcinoma. This suggests that targeted regulation of ferroptosis may impact the occurrence and development of various liver diseases. This article reviews the latest research progress of ferroptosis in various liver diseases, including acute liver injury, metabolic associated fatty liver disease, alcoholic liver disease, viral hepatitis, autoimmune hepatitis, liver fibrosis and hepatocellular carcinoma. It aims to provide insights for the prevention and treatment of acute and chronic liver diseases through targeting ferroptosis.
Humans ; Liver Diseases/etiology* ; Ferroptosis/physiology* ; Liver Neoplasms/pathology* ; Carcinoma, Hepatocellular/pathology* ; Liver Cirrhosis/etiology* ; Liver/pathology* ; Hepatitis, Autoimmune/metabolism* ; Liver Diseases, Alcoholic/metabolism*

OBJECTIVE: To explore the effect and mechanism of Dahuang Zhechong Pill (DHZCP) on liver fibrosis. METHODS: Liver fibrosis cell model was induced by transforming growth factor-β (TGF-β) in hepatic stellate cells (HSC-T6). DHZCP medicated serum (DMS) was prepared in rats. HSC-T6 cells were divided into the control (15% normal blank serum culture), TGF-β (15% normal blank serum + 5 ng/mL TGF-β), DHZCP (15% DMS + 5 ng/mL TGF-β), DHZCP+PDTC [15% DMS + 4 mmol/L ammonium pyrrolidine dithiocarbamate (PDTC)+ 5 ng/mL TGF-β], and PDTC groups (4 mmol/L PDTC + 5 ng/mL TGF-β). Cell activity was detected by cell counting kit 8 and levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the cell supernatant were determined by enzyme-linked immunosorbnent assay. Western blot was used to measure the expressions of p38 mitogen-activated protein kinase/nuclear factor kappa B/transforming growth factor-β1 (p38 MAPK/NF-κ B/TGF-β1) pathway related proteins, and the localization and expressions of these proteins were observed by immunofluorescence staining. RESULTS: DHZCP improves the viability of cells damaged by TGF-β and reduces inflammatory cytokines and ALT and AST levels in the supernatant of HSC-T6 cells induced with TGF-β (P<0.05 or P<0.01). Compared with the TGF-β group, NF-κ B p65 levels in the DHZCP group were decreased (P<0.05). p38 MAPK and NF-κ B p65 levels in the DHZCP+PDTC were also reduced (P<0.01). Compared with the TGF-β group, the protein expression of Smad2 showed a downward trend in the DHZCP, DHZCP+PDTC, and PDTC groups (all P<0.01), and the decreasing trend of Samd3 was statistically significant only in DHZCP+PDTC group (P<0.01), whereas Smad7 was increased (P<0.05 or P<0.01). CONCLUSION DHZCP can inhibit the process of HSC-T6 cell fibrosis by down-regulating the expression of p38 MAPK/NF-κ B/TGF-β1 pathway.
Animals ; Liver Cirrhosis/pathology* ; Drugs, Chinese Herbal/therapeutic use* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Male ; Signal Transduction/drug effects* ; Rats ; Disease Progression ; Cell Line ; Hepatic Stellate Cells/pathology* ; Rats, Sprague-Dawley

Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.
Animals ; Rats ; Cell Proliferation ; Collagen Type I/metabolism* ; Fibrosis ; Hepatic Stellate Cells ; HMGB1 Protein/genetics* ; Liver Cirrhosis/pathology* ; MicroRNAs/metabolism*

An effective therapeutic regimen for hepatic fibrosis requires a deep understanding of the pathogenesis mechanism. Hepatic fibrosis is characterized by activated hepatic stellate cells (aHSCs) with an excessive production of extracellular matrix. Although promoted activation of HSCs by M2 macrophages has been demonstrated, the molecular mechanism involved remains ambiguous. Herein, we propose that the vitamin D receptor (VDR) involved in macrophage polarization may regulate the communication between macrophages and HSCs by changing the functions of exosomes. We confirm that activating the VDR can inhibit the effect of M2 macrophages on HSC activation. The exosomes derived from M2 macrophages can promote HSC activation, while stimulating VDR alters the protein profiles and reverses their roles in M2 macrophage exosomes. Smooth muscle cell-associated protein 5 (SMAP-5) was found to be the key effector protein in promoting HSC activation by regulating autophagy flux. Building on these results, we show that a combined treatment of a VDR agonist and a macrophage-targeted exosomal secretion inhibitor achieves an excellent anti-hepatic fibrosis effect. In this study, we aim to elucidate the association between VDR and macrophages in HSC activation. The results contribute to our understanding of the pathogenesis mechanism of hepatic fibrosis, and provide potential therapeutic targets for its treatment.
Humans ; Hepatic Stellate Cells/pathology* ; Receptors, Calcitriol ; Liver Cirrhosis/pathology* ; Macrophages/metabolism*

Objective To study the effect and mechanism of pearl hydrolysate on hepatic sinusoidal capillarization in liver fibrosis. Methods Hepatic sinusoidal endothelial cells (HSEC) and hepatic stellate cells (HSC-LX2) were incubated with Hepu pearl hydrolysate.The proliferation of HSEC and HSC-LX2 was examined by MTT colorimetry.The cell cycle and apoptosis of HSC-LX2 were measured by flow cytometry.The changes of the microstructures such as fenestra and basement membrane of HSEC were observed by transmission electron microscopy. Results The intervention with leptin increased the viability of HSC-LX2 (P=0.041),decreased the viability of HSEC (P=0.004),and caused capillarization signs such as decreased number and diameter of fenestrae and formation of continuous basement membrane.The treatment with pearl hydrolysate at different doses increased and expanded the fenestrae of HSEC (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032),disintegrated the extracellular basement membrane of HSEC (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032),decreased the viability of HSC-LX2 (low dose:P=0.018;medium dose:P=0.013;high dose:P=0.009),and induced the apoptosis of HSC-LX2 (low dose:P=0.012;medium dose:P=0.006;high dose:P=0.005).Pearl hydrolysate exerted therapeutic effect on capillarization in a dose-dependent manner (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032).Moreover,high-dose pearl hydrolysate showed stronger effect on capillarization of hepatic sinuses than colchicine (P=0.034) and salvianolic acid B (P=0.038). Conclusion Hepu pearl hydrolysate can increase the viability of HSEC,restore the area of fenestrae,disintegrate the basement membrane,and decrease the viability and induce the apoptosis of HSC-LX2,demonstrating significant pharmacological effects on the capillarization of HSEC and HSC-LX2.
Humans ; Endothelial Cells/metabolism* ; Liver Cirrhosis ; Liver/pathology*

Objective: To investigate the effects of combined blockade of interleukin-33 (IL-33) and inducible co-stimulatory molecule (ICOS) on carbon tetrachloride-induced chronic liver fibrosis and imbalance of T helper lymphocyte subsets in mice. Methods: There were 40 BALB/c mice in each model and control group. Flow cytometry was used to determine the proportion of Th1/Th2/Th17 cells in the splenic lymphocyte suspension of mice, the expression levels of interferon γ, IL-4, and IL-17 in the splenic lymphocyte suspension of liver fibrosis mice after combined blockade of IL-33 and ICOS, and the pathological changes of liver histopathology in mice with liver fibrosis. Two independent sample t-test was used to compare data between groups. Results: Compared with the non-blocking group, the proportion of Th2 and Th17 cells in the IL-33/ICOS blocking group was significantly down-regulated (Th2: 65.96% ± 6.04% vs. 49.09% ± 7.03%; Th17: 19.17% ± 4.03% vs. 9.56% ± 2.03%), while the proportion of Th1 cells and Th1/Th2 ratio were up-regulated (Th1: 17.14% ± 3.02% vs. 31.93% ± 5.02%; Th1/Th2: 0.28 ± 0.06 vs. 0.62 ± 0.23), and the difference was statistically significant (t = 5.15, 6.03, 7.14, 4.28, respectively, with P < 0.05). After entering the chronic inflammation stage of liver fibrosis in mice (10 weeks), compared with the non-blocking group, the expression levels of IL-4 and IL-17 in the blockade group were significantly down-regulated [IL-4: (84.75 ± 14.35) pg/ ml vs. (77.88 ± 19.61) pg/ml; IL-17: (72.38 ± 15.13) pg/ml vs. (36.38 ± 8.65) pg/ml], while the expression of interferon γ was up-regulated [(37.25 ± 11.51) pg/ml vs. (77.88 ± 19.61) pg/ml], and the difference was statistically significant (t: IL-4: 4.71; IL-17: 5.84; interferon γ: 5.05, respectively, with P < 0.05). Liver histopathological results showed that hepatic necrosis, hepatic lobular structural disorder, and fibrous tissue hyperplasia were significantly lower in the blockade group than those in the non-blocking group at 13 weeks of liver fibrosis. Conclusion: Combined blockade of the ICOS signaling pathway and IL-33 can regulate Th2 and Th17 polarization, down-regulate the inflammatory response, and inhibit or prevent the occurrence and progression of fibrosis.
Mice ; Animals ; Interferon-gamma/metabolism* ; Interleukin-17/metabolism* ; Interleukin-33/metabolism* ; Cytokines/metabolism* ; Carbon Tetrachloride ; Th2 Cells ; Interleukin-4/metabolism* ; Liver Cirrhosis/pathology* ; Th1 Cells ; Th17 Cells/pathology* ; Immunity

Objective: To investigate the clinical value of plasma scaffold protein SEC16A level and related models in the diagnosis of hepatitis B virus-related liver cirrhosis (HBV-LC) and hepatocellular carcinoma (HBV-HCC). Methods: Patients with HBV-LC and HBV-HCC and a healthy control group diagnosed by clinical, laboratory examination, imaging, and liver histopathology at the Third Hospital of Hebei Medical University between June 2017 and October 2021 were selected. Plasma SEC16A level was detected using an enzyme-linked immunosorbent assay (ELISA). Serum alpha-fetoprotein (AFP) was detected using an electrochemiluminescence instrument. SPSS 26.0 and MedCalc 15.0 statistical software were used to analyze the relationship between plasma SEC16A levels and the occurrence and development of liver cirrhosis and liver cancer. A sequential logistic regression model was used to analyze relevant factors. SEC16A was established through a joint diagnostic model. Receiver operating characteristic curve was used to evaluate the clinical efficacy of the model for liver cirrhosis and hepatocellular carcinoma diagnosis. Pearson correlation analysis was used to identify the influencing factors of novel diagnostic biomarkers. Results: A total of 60 cases of healthy controls, 60 cases of HBV-LC, and 52 cases of HBV-HCC were included. The average levels of plasma SEC16A were (7.41 ± 1.66) ng/ml, (10.26 ± 1.86) ng/ml, (12.79 ± 1.49) ng /ml, respectively, with P < 0.001. The sensitivity and specificity of SEC16A in the diagnosis of liver cirrhosis and hepatocellular carcinoma were 69.44% and 71.05%, and 89.36% and 88.89%, respectively. SEC16A, age, and AFP were independent risk factors for the occurrence of HBV-LC and HCC. SAA diagnostic cut-off values, sensitivity, and specificity were 26.21 and 31.46, 77.78% and 81.58%, and 87.23% and 97.22%, respectively. The sensitivity and specificity for HBV-HCC early diagnosis were 80.95% and 97.22%, respectively. Pearson correlation analysis showed that AFP level was positively correlated with alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and γ-glutamyltransferase (GGT) with P < 0.01, while the serum SEC16A level was only slightly positively correlated with ALT and AST in the liver cirrhosis group (r = 0.268 and 0.260, respectively, P < 0.05). Conclusion: Plasma SEC16A can be used as a diagnostic marker for hepatitis B-related liver cirrhosis and hepatocellular carcinoma. SEC16A, combined with age and the AFP diagnostic model with SAA, can significantly improve the rate of HBV-LC and HBV-HCC early diagnosis. Additionally, its application is helpful for the diagnosis and differential diagnosis of the progression of HBV-related diseases.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; alpha-Fetoproteins/metabolism* ; Endoplasmic Reticulum/metabolism* ; Golgi Apparatus/metabolism* ; Vesicular Transport Proteins ; Liver Cirrhosis/complications* ; Hepatitis B/complications* ; ROC Curve ; Hepatitis B virus/metabolism* ; Biomarkers, Tumor

Objective: To explore carnosine dipeptidase 1 (CNDP1) potential value as a diagnostic and prognostic evaluator of hepatocellular carcinoma (HCC). Methods: A gene chip and GO analysis were used to screen the candidate marker molecule CNDP1 for HCC diagnosis. 125 cases of HCC cancer tissues, 85 cases of paracancerous tissues, 125 cases of liver cirrhosis tissues, 32 cases of relatively normal liver tissue at the extreme end of hepatic hemangioma, 66 cases from serum samples of HCC, and 82 cases of non-HCC were collected. Real-time fluorescent quantitative PCR, immunohistochemistry, western blot, and enzyme-linked immunosorbent assay were used to detect the differences in mRNA and protein expression levels of CNDP1 in HCC tissue and serum. Receiver operating characteristic (ROC) curves and Kaplan-Meier survival were used to analyze and evaluate the value of CNDP1 in the diagnosis and prognosis of HCC patients. Results: The expression level of CNDP1 was significantly reduced in HCC cancer tissues. The levels of CNDP1 were significantly lower in the cancer tissues and serum of HCC patients than those in liver cirrhosis patients and normal controls. ROC curve analysis showed that the area under the curve of serum CNDP1 in the diagnosis of HCC patients was 0.753 2 (95% CI 0.676-0.830 5), and the sensitivity and specificity were 78.79% and 62.5%, respectively. The combined detection of serum CNDP1 and serum alpha-fetoprotein (AFP) significantly improved the diagnostic accuracy (AUC = 0.820 6, 95% CI 0.753 5-0.887 8). The diagnostic sensitivity and specificity of serum CNDP1 for AFP-negative HCC patients were 73.68% and 68.75% (AUC = 0.793 1, 95% CI 0.708 8-0.877 4), respectively. In addition, the level of serum CNDP1 distinguished small liver cancer (tumor diameter < 3 cm) (AUC = 0.757 1, 95% CI 0.637 4-0.876 8). Kaplan-Meier survival analysis showed that CNDP1 was associated with a poor prognosis in HCC patients. Conclusion: CNDP1 may be a potential biomarker for the diagnostic and prognostic evaluation of HCC, and it has certain complementarity with serum AFP.
Humans ; Carcinoma, Hepatocellular/genetics* ; Liver Neoplasms/pathology* ; Prognosis ; Carnosine ; alpha-Fetoproteins/metabolism* ; Biomarkers, Tumor/genetics* ; Liver Cirrhosis/diagnosis* ; ROC Curve