Malignant proliferating liver cancer cells possess the ability to detect and respond to various body signals, thereby facilitating tumor growth, invasion, and metastasis. One crucial mechanism through which hepatocellular carcinoma (HCC) cells interpret these signals is crosstalk. Within liver cancer tissues, cancer cells engage in communication with hepatic stellate cells (HSCs), tumor-associated macrophages (TAMs), and immune cells. This interaction plays a pivotal role in regulating the proliferation, invasion, and metastasis of HCC cells. Crosstalk occurs in multiple ways, each characterized by distinct functions. Its molecular mechanisms primarily involve regulating immune cell functions through the expression of specific receptors, such as CD24 and CD47, modulating cell functions by secreting cytokines like transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF), and mediating cell growth and proliferation by activating pathways such as Wnt/β-catenin and Hedgehog. A comprehensive understanding of the mechanisms and interactions within crosstalk is essential for unraveling the pathogenesis of HCC. It also opens up new avenues for the development of innovative therapeutic strategies. This article reviews the relationship between crosstalk and the progression of HCC, offering insights and inspiration for future research.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Hepatic Stellate Cells/physiology* ; Disease Progression ; Signal Transduction/physiology* ; Transforming Growth Factor beta/metabolism* ; Cell Proliferation ; Hedgehog Proteins/metabolism* ; Tumor-Associated Macrophages ; Platelet-Derived Growth Factor/metabolism* ; Cell Communication/physiology*

OBJECTIVE: To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. METHODS: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. RESULTS: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL (P<0.05). CONCLUSION The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Hepatic Stellate Cells ; drug effects ; physiology ; Humans ; Injections ; Liver Cirrhosis ; drug therapy ; Tumor Suppressor Proteins ; genetics

OBJECTIVE: To investigate the inhibitory effect of genistein on activation of hepatic stellate cells (HSCs) and the role of the autophagy pathway regulated by PPAR-γ in mediating this effect. METHODS: Cultured HSC-T6 cells were exposed to different concentrations of genistein for 48 h, and HSC activation was verified by detecting the expressions of -SMA and 1(I) collagen; autophagy activation in the cells was determined by detecting the expressions of LC3-II and p62 using Western blotting. The autophagy inhibitor 3-MA was used to confirm the role of autophagy in genistein-induced inhibition of HSC activation. A PPAR-γ inhibitor was used to explore the role of PPAR-γ in activating autophagy in the HSCs. RESULTS: Genistein at concentrations of 5 and 50 μmol/L significantly inhibited the expressions of -SMA and 1(I) collagen ( < 0.05), markedly upregulated the expressions of PPAR-γ and the autophagy-related protein LC3-II ( < 0.05) and significantly down-regulated the expression of the ubiqutin-binding protein p62 ( < 0.05) in HSC-T6 cells. The cells pretreated with 3-MA prior to genistein treatment showed significantly increased protein expressions of -SMA and 1(I) collagen compared with the cells treated with genistein only ( < 0.05). Treatment with the PPAR-γ inhibitor obviously lowered the expression of LC3-II and enhanced the expression p62 in genistein-treated HSC-T6 cells, suggesting the activation of the autophagy pathway. CONCLUSIONS PPAR-γ- regulated autophagy plays an important role in mediating genistein-induced inhibition of HSC activation .
Anticarcinogenic Agents ; pharmacology ; Autophagy ; Collagen Type I ; Genistein ; pharmacology ; Hepatic Stellate Cells ; Humans ; PPAR gamma ; physiology

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
Epithelial Cells/*physiology ; Epithelium/metabolism ; Hepatic Stellate Cells/*physiology ; Humans ; Liver/*cytology/injuries/*physiology ; Liver Cirrhosis/etiology/prevention & control ; Liver Regeneration/*physiology ; Mesenchymal Stromal Cells/physiology ; Myofibroblasts/physiology

To observe the effect of Ligusticum wallichii-containing serum on the expressions of Toll-like receptor 4 and myeloid differentiation factor 88 in hepatic stellate cells. Clean-grade SD rats were randomly divided into 5 groups and orally given L. wallichii decoction, colchicine and normal saline for 7 d to prepare L. wallichii-containing serums. Except for the blank group, all of the remaining groups were stimulated with LPS 1 mg x L(-1) for 24 h. After being intervened, the L. wallichii-containing serums were cultured in 5% CO2 incubator at 37 degrees C for 24 hours. The expression of TLR4 and MyD88 were detected by RT-PCR and Western blot. After HSC was stimulated with LPS, TLR4 and MyD88 mRNA and protein expressions were significantly higher than the blank control group (P < 0.01). After being intervened with L. wallichii-containing serum, TLR4 and MyD88 mRNA and protein expressions were notably lower than the model group (P < 0.05 or P < 0.01). In conclusion, L. wallichii-containing serum could regulate the TLR4 signaling pathway and show the anti-fibrosis effect by inhibiting the expression of TLR4 and MyD88 in LPS-induced HSCs.
Animals ; Female ; Hepatic Stellate Cells ; drug effects ; metabolism ; Ligusticum ; Lipopolysaccharides ; pharmacology ; Liver Cirrhosis, Experimental ; drug therapy ; Myeloid Differentiation Factor 88 ; genetics ; physiology ; Phytotherapy ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4 ; genetics ; physiology

BACKGROUND/AIMS: The Wnt/beta-catenin signaling pathway has been reported to play an important role in liver fibrosis. This study was designed to investigate whether mesoderm-specific transcript homologue (Mest), a strong negative regulator of Wnt/beta-catenin signaling, could inhibit liver fibrosis. METHODS: pcDNA-Mest was transfected into hepatic stellate cells (HSCs) and rats. Rats were randomly divided into four groups: normal group (normal saline), treatment group (pcDNA-Mest+CCl4), control group (pcDNA-neo+CCl4), and model group (normal saline+CCl4). Changes in liver pathology were evaluated by hematoxylin and eosin and Masson's trichrome staining. The levels of alanine transaminase, aspartate transaminase, lactic dehygrogenase, hyaluronic acid, and laminin in the serum and hydroxyproline in the liver were detected by biochemical examination and radioimmunoassay, respectively. The expression and distribution of beta-catenin, alpha-smooth muscle actin (alpha-SMA), Smad3, and tissue inhibitor of metalloproteinase type I were determined, and the viability of the HSCs was tested. RESULTS: Our data demonstrate that Mest alleviated CCl4-induced collagen deposition in liver tissue and improved the condition of the liver in rats. Mest also significantly reduced the expression and distribution of beta-catenin, alpha-SMA and Smad3 both in vivo and in vitro, in addition to the viability of HSCs in vitro. CONCLUSIONS: We found that Mest attenuates liver fibrosis by repressing beta-catenin expression, which provides a new therapeutic approach for treating liver fibrosis.
Animals ; Carbon Tetrachloride/toxicity ; Cells, Cultured ; Hepatic Stellate Cells/physiology ; Liver Cirrhosis, Experimental/*physiopathology ; Male ; Proteins/*physiology ; Random Allocation ; Rats, Wistar ; Transfection ; Wnt Signaling Pathway/*physiology ; beta Catenin/metabolism

The lipids present in hepatic stellate cells (HSCs) lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Activation of HSCs is crucial to the development of fibrosis in liver disease. During activation, HSCs transform into myofibroblasts with concomitant loss of their lipid droplets and production of excessive extracellular matrix. Release of lipid droplets containing retinyl esters and triglyceride is a defining feature of activated HSCs. Accumulating evidence supports the proposal that recovering the accumulation of lipids would inhibit the activation of HSCs. In healthy liver, quiescent HSCs store 80% of total liver retinols and release them depending on the extracellular retinol status. However, in injured liver activated HSCs lose their retinols and produce a considerable amount of extracellular matrix, subsequently leading to liver fibrosis. Further findings prove that lipid metabolism of HSCs is closely associated with its activation, yet relationship between activated HSCs and the lipid metabolism has remained mysterious.
Animals ; Cholesterol ; metabolism ; Hepatic Stellate Cells ; physiology ; Humans ; Lipid Metabolism ; Triglycerides ; metabolism ; Vitamin A ; metabolism

This study examined the effect of copper ions on the proliferation of hepatic stellate cells (HSCs) and the role of oxidative stress in this process in order to gain insight into the mechanism of hepatic fibrosis in Wilson's disease. LX-2 cells, a cell line of human HSCs, were cultured in vitro and treated with different agents including copper sulfate, N-acetyl cysteine (NAC) and buthionine sulfoximine (BSO) for different time. The proliferation of LX-2 cells was measured by non-radioactive cell proliferation assay. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of platelet-derived growth factor receptor β subunit (PDGFβR), ELISA to determine the level of glutathione (GSH) and oxidized glutathione (GSSG), dichlorofluorescein assay to measure the level of reactive oxygen species (ROS), and lipid hydroperoxide assay to quantify the level of lipid peroxide (LPO). The results showed that copper sulfate over a certain concentration range could promote the proliferation of LX-2 cells in a time- and dose-dependent manner. The effect was most manifest when LX-2 cells were treated with copper sulfate at a concentration of 100 μmol/L for 24 h. Additionally, copper sulfate could dose-dependently increase the levels of ROS and LPO, and decrease the ratio of GSH/GSSG in LX-2 cells. The copper-induced increase in mRNA and protein expression of PDGFβR was significantly inhibited in LX-2 cells pre-treated with NAC, a precursor of GSH, and this phenomenon could be reversed by the intervention of BSO, an inhibitor of NAC. It was concluded that copper ions may directly stimulate the proliferation of HSCs via oxidative stress. Anti-oxidative stress therapies may help suppress the copper-induced activation and proliferation of HSCs.
Cell Line ; Cell Proliferation ; drug effects ; Copper ; administration & dosage ; Dose-Response Relationship, Drug ; Hepatic Stellate Cells ; cytology ; drug effects ; physiology ; Humans ; Ions ; Liver Cirrhosis ; metabolism ; Oxidative Stress ; drug effects ; physiology ; Oxygen ; metabolism

BACKGROUNDIn addition to hematopoietic effect, the erythropoietin is known as a multifunctional cytokine with anti-fibrosis and organ-protective activities. The purpose of this study was to evaluate the effect of recombinant human erythropoietin (rhEPO) on hepatic fibrosis and hepatic stellate cells (HSCs).

METHODSCarbon tetrachloride (CCl(4)) induced hepatic fibrosis mice models were used for in vivo study and HSCs line for in vitro study. CCl(4) and rhEPO (0, 200 or 1000 U/kg) was injected intraperitoneally in BALB/c mice three times a week for 4 weeks. Immunohistochemistry and immunoblotting were performed to evaluate expressions of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), and fibronectin in explanted liver. Immunoblotting of α-SMA, phophorylated Smad-2 and Smad-2/3 was performed in HSCs treated with TGF-β1 and/or rhEPO.

RESULTSExpressions of TGF-β1, α-SMA, and fibronectin were increased in CCl(4) injected mice livers, but significantly attenuated by co-treatment with CCl(4) and rhEPO. Co-treatment of rhEPO markedly suppressed fibrosis in Masson's trichrome compared with treatment of only CCl(4). TGF-β1 increased phosphorylated α-SMA, Smad-2 expressions in HSCs, which were decreased by rhEPO co-treatment.

CONCLUSIONSTreatment of rhEPO effectively suppressed fibrosis in CCl(4)-induced liver fibrosis mice models. Anti-fibrosis effect of rhEPO could be related to inhibition of TGF-β1 induced activation of HSCs.

Animals ; Carbon Tetrachloride ; toxicity ; Cells, Cultured ; Erythropoietin ; pharmacology ; therapeutic use ; Fibronectins ; analysis ; Hepatic Stellate Cells ; drug effects ; Liver Cirrhosis, Experimental ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta ; antagonists & inhibitors ; physiology

OBJECTIVETo investigate the mechanisms of Aldosterone stimulating hepatic stellate cells(HSCs) contraction via Ca2+-independent pathways.

METHODSHSC-T6 cell line was pre-disposed with Aldo 10mumol/L. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The concentration variation of intracellular free calcium in rat HSC was observed by laser confocal microscopy. Besides, HSC-T6 cell line was under pre-disposal treatment with the blocking agents of Aldo receptor -antisterone, protein kinase C (PKC) special blocking agent-Stauro, Rho kinase blocking agent-Y27632 and MLCK special blocking agent-ML-7 respectively prior to stimulation with aldosterone. RT-PCR was used to detect the expression of Rock2, RhoAGTP and RhoGEF in Ca2+- independent pathways mediated by Rho-kinase.

RESULTSAldo could induce HSCs contraction. The concentration of intracellular free calcium in rat HSCs had no change after pre-disposal treatment with Aldo. The mRNA expressions of Rock2, RhoAGTP and RhoGEF increased significantly after treatment with Aldo (0.770+/-0.049, 0.960+/-0.096, 0.180+/-0.006, P is less than 0.01).When inhibited with antisterone, the mRNA expressions of the three elements were (0.440+/-0.166, 0.370+/-0.180 and 0.050+/-0.001, P is less than 0.01), lower than that of Aldo group, but higher in ML-7+Stauro + Aldo groups (0.940+/-0.066, 1.330+/-0.192 and 0.160+/-0.007, P is less than 0.05) as compared to the control group (0.140+/-0.023, 0.540+/-0.111 and 0.110+/-0.012). In the Y27632 + ML-7 + Stauro+Aldo group, the mRNA expression of RhoGEF (0.290+/-0.004, P is less than 0.01)was higher than that of the ML-7 + Stauro + Aldo group (0.160+/-0.007).

CONCLUSIONAldo could induce HSCs contraction via Ca2+-independent pathways and Rho-Rock pathway involved in the process.

Aldosterone ; pharmacology ; Animals ; Cell Line ; Hepatic Stellate Cells ; drug effects ; metabolism ; physiology ; Rats ; Signal Transduction ; drug effects ; rho-Associated Kinases ; metabolism