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1.
Chinese Journal of Hepatology ; (12): 1132-1136, 2021.
Article in Chinese | WPRIM | ID: wpr-922702

ABSTRACT

Hepatic fibrogenesis (HF) is the common consequence of various chronic liver diseases (CLD) induced by a variety of pathogenic factors. The mechanism of HF involves the interactions within different types of liver cells, cytokines, chemokines, cell mediators and multiple signaling pathways in a way of networks. As a result, excessive production and deposition of extracellular matrix (ECM) mainly composed of type I and type III fibril forming collagen destroys the original morphology, structure and function of the liver. The activation of hepatic stellate cells (HSCs), the major scar forming cells in liver, plays a crucial role in hepatic fibrogenesis. MicroRNAs are a group of short, single stranded, non-coding RNAs that can inhibit mRNA expression at the transcriptional and post transcriptional levels. They can be loaded and transferred as cargos by exosomes, to regulate the function of nearby and distant receptive cells. The expressions of many microRNAs such as miR-21, miR-29, miR-708, miR-101, miR-455, miR-146, miR-193 change significantly in activated HSCs, which regulate the activation, fibrogenic function, proliferation, apoptosis and autophagy of HSCs via affecting target genes expression and signaling pathway molecules. They are important substances and regulatory mechanism that mediate the initiation and progression of HF.


Subject(s)
Cell Proliferation , Gene Expression Regulation , Hepatic Stellate Cells , Humans , Liver Cirrhosis/pathology , MicroRNAs/genetics
2.
Article in Chinese | WPRIM | ID: wpr-887961

ABSTRACT

In order to investigate the effect of salidroside on inhibiting liver fibrosis and its relationship with CXC chemokine ligand 16(CXCL16) in vivo and in vitro, totally 45 C57 BL/6 J male mice were randomly divided into normal group, model group and salidroside group, with 15 mice in each group. The mice in model group and salidroside group were injected intraperitoneally with 15% carbontetrachloride(CCl_4) olive oil solution to establish liver fibrosis model, and the mice in normal group were injected intraperitoneally with the same dose of olive oil. Salidroside group was given with 100 mg·kg~(-1 )salidroside by gavage, while the normal group and model group received the same amount of double distilled water by gavage. All mice were sacrificed after 5 weeks of intragastric administration. The pathological changes of mouse liver were observed by hematoxylin-eosin(HE) staining, and the degree of liver fibrosis was observed by sirius red staining. The protein expressions of collagen Ⅰ(ColⅠ), α-smooth muscle actin(α-SMA), fibronectin(FN), CXCL16, phosphorylated Akt(p-Akt), Akt in liver tissues were detected by Western blot. Hepatic stellate cell line JS 1 was cultured in vitro and divided into control group, model group(100 μg·L~(-1) CXCL16) and salidroside group(100 μg·L~(-1) CXCL16+1×10~(-5) mol·L~(-1) salidroside). Cell migration was detected by cell scratch, the mRNA expressions of ColⅠ and α-SMA were detected by RT-PCR, and the protein expressions of p-Akt and Akt were detected by Western blot. As compared with the normal group, the protein expressions of ColⅠ, α-SMA, FN, CXCL16, and p-Akt in the model group were significantly increased, and salidroside could reduce the expression of these indicators(P<0.05 or P<0.01). In vitro, CXCL16 could promote the migration of JS 1, increase the mRNA expressions of ColⅠ and α-SMA in JS 1, and enhance Akt phosphorylation in JS 1(P<0.05 or P<0.01). As compared with the model group, salidroside could inhibit the migration of JS 1 induced by CXCL16(P<0.05), and reduce the high expression of ColⅠ and α-SMA mRNA and the phosphorylation of Akt in JS 1 induced by CXCL16(P<0.05). In conclusion, salidroside might attenuate CCl_4-induced liver fibrosis in mice by inhibiting the migration, activation and Akt phosphorylation of hepatic stellate cells induced by CXCL16.


Subject(s)
Animals , Carbon Tetrachloride , Chemokine CXCL16 , Glucosides , Hepatic Stellate Cells , Liver/pathology , Liver Cirrhosis/genetics , Male , Mice , Phenols
3.
Article in Chinese | WPRIM | ID: wpr-878948

ABSTRACT

Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen Ⅰ were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-β1(TGF-β1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen Ⅰ, TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.


Subject(s)
Animals , Caco-2 Cells , Hepatic Stellate Cells/metabolism , Humans , Liver Cirrhosis/genetics , NF-kappa B/metabolism , Rats , Signal Transduction
4.
Int. j. morphol ; 38(6): 1767-1778, Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1134510

ABSTRACT

SUMMARY: Acrylamide (ACR) is a cytotoxic and carcinogenic material. It is a product of a Maillard reaction during the cooking of many types of fried fast food, e.g. potato chip fries, and chicken nuggets. ACR has a severe toxic effect on different body organs. This study investigates the hepatotoxic effect of ACR, and the protective effect of ascorbic acid and silymarin. For this purpose, forty adult, male, albino rats were divided into four groups and received the following treatments for fourteen days: Group I: (the control) normal saline; Group II: ACR only; Group III: ACR and ascorbic acid; and Group IV: ACR and silymarin. Under a light microscope, the liver from rats treated with ACR only presented disturbed liver architecture, degenerated hepatocytes, reduced glycogen contents, congested central vein, and increased collagen fibres with areas of fibrosis. Immunohistochemical examination revealed an increased mean number of CD68-, and α-SMA-positive cells. This indicates the presence of large numbers of stellate macrophages (Kupffer cells) and Hepatic stellate cells (HSCs). The combination of ACR with either ascorbic acid or silymarin resulted in less hepatic degeneration, less fibrosis and fewer CD68 and α-SMA positive cells compared to the ACR only group. In conclusion, treatment with silymarin or ascorbic acid along with ACR appears to alleviate ACR-induced hepatotoxicity with more protection in silymarin treated rats.


RESUMEN: La acrilamida (ACR) es un material citotóxico y cancerígeno. Es producto de la reacción de Maillard durante la cocción de muchos tipos de comida rápida y frita, por ejemplo: papas fritas y nuggets de pollo. ACR tiene un efecto tóxico severo en diferentes órganos del cuerpo. Este estudio investigó el efecto hepatotóxico del ACR y el efecto protector del ácido ascórbico y la silimarina. Con este fin, cuarenta ratas albinas machos adultas se dividieron en cuatro grupos y recibieron los siguientes tratamientos durante catorce días: Grupo I (control), solución salina normal; Grupo II, solo ACR; Grupo III, ACR y ácido ascórbico; y Grupo IV, ACR y silimarina. Bajo microscopio óptico, el hígado de ratas tratadas con ACR solo presentó alteración de su arquitectura, entre ellos hepatocitos degenerados, contenido reducido de glucógeno, vena central congestionada y aumento de fibras de colágeno con áreas de fibrosis. El examen inmunohistoquímico reveló un aumento del número medio de células CD68 y α-SMA positivas. Esto indica la presencia de un gran número de macrófagos estrellados (células de Kupffer) y células estrelladas hepáticas (HSC). La combinación de ACR con ácido ascórbico o silimarina resultó en menos degeneración hepática, menos fibrosis y menos células positivas para CD68 y α-SMA en comparación con el grupo de ACR solo. En conclusión, el tratamiento con silimarina o ácido ascórbico junto con ACR parece aliviar la hepatotoxicidad inducida por ACR.


Subject(s)
Animals , Male , Rats , Ascorbic Acid/pharmacology , Silymarin/pharmacology , Acrylamide/toxicity , Liver/drug effects , Immunohistochemistry , Antigens, CD/analysis , Actins/analysis , Hepatocytes , Hepatic Stellate Cells , Liver/metabolism , Liver/pathology
5.
Article in Chinese | WPRIM | ID: wpr-828870

ABSTRACT

OBJECTIVE@#To investigate the role of endoplasmic reticulum (ER)-stress of Kupffer cells (KCs) and KCs-derived tumor necrosis factor- (TNF-) in medicating apoptosis of hepatic stellate cell (HSC).@*METHODS@#Sixty male SD rats were randomized into control group, model group, ER- stress group, depletion group and KCs block group (=15). The 4 groups of rats were given intraperitoneal injections (twice a week for 8 weeks) of normal saline (2 mg/kg); 40% CCl4 solution (in peanut oil, 2 mg/kg); 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg); and 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg) followed by clodronate liposomes (50 mg/kg), respectively. After the treatments, samples of the liver tissue and serum were collected from the rats from the 4 groups to isolate KC cells, which were co-cultured with LX2 cells. In the depletion group, the rats were injected with anti-rat TNFR mAb (0.35 mg/kg) via the portal vein before isolating the KCs. Liver function examination, Eirius red staining, ELISA, immuno- histochemical staining, and RT-PCR were performed to assess the liver function, liver fibrosis, KC phenotypes, expression of the in fl ammatory factors, and the number of active HSC was detected. The isolated KCs were treated with tunicamycin before co-culture with LX2 cells, and ELISA, RT-PCR and Western blot were performed to examine KC phenotypes, in fl ammatory factors, LX2 cell apoptosis and TNFR/caspase8 pathway activity.@*RESULTS@#Compared with the rats in the control group, the rats in the model group had significantly increased ALT and AST levels, Sirius red staining-positive area, and Desmin-positive cells (activated HSCs) ( < 0.05) with significantly lowered number of CD16-positive KCs (M1), and TNF- protein and mRNA expression ( < 0.05). Compared with those in the model group, the rats in ER-stress group showed significantly decreased ALT and AST levels, Sirius red staining positivity and Desmin-positive cells ( < 0.05) and increased number of CD16-positive KCs and TNF- expressions ( < 0.05). In the depletion group, compared with the ER-stress group, the rats had significantly increased ALT and AST levels of, Sirius red staining positivity and Desmin-positive cells ( < 0.05) and reduced CD16- positive KCs and TNF-expressions ( < 0.05). In the cell co-culture experiment, the model group showed significantly reduced TUNEL-positive LX2 cells, CD16-positive cells, and expressions of TNFR1, cleaved caspase- 8 and cleaved caspase- 3 in the KCs ( < 0.05) with increased Desmin-positive LX2 cells ( < 0.05). Compared with the model group, the ER- stress group exhibited significantly increased TUNEL-positive LX2 cells, CD16-positive cells and expressions of TNFR, cleaved caspase-8 and cleaved caspase-3 in the KCs ( < 0.05) and decreased Desmin-positive LX2 cells ( < 0.05). In the depletion group, blocking TNFR resulted in significantly decreased expressions of cleaved caspase-8 and cleaved caspase-3 compared with those in ER- stress group ( < 0.05) although there was no significant changed in TNFR expression.@*CONCLUSIONS@#ER stress of KCs promotes the transformation of KCs towards M1 phenotype and increases the expression of TNF-, which triggers the apoptosis of HSCs through the TNFR/caspase-8 pathway.


Subject(s)
Animals , Apoptosis , Caspase 8 , Endoplasmic Reticulum Stress , Hepatic Stellate Cells , Kupffer Cells , Male , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha
6.
Article in Chinese | WPRIM | ID: wpr-772043

ABSTRACT

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 .


Subject(s)
Anticarcinogenic Agents , Pharmacology , Autophagy , Collagen Type I , Genistein , Pharmacology , Hepatic Stellate Cells , Humans , PPAR gamma , Physiology
7.
Article in Chinese | WPRIM | ID: wpr-774575

ABSTRACT

Herpetone( HPT) is a bioactive lignan extracted from Herpetospermum pedunculosum,which can protect liver,lower aminotransferase and inhibit hepatitis B virus. However,HPT has a poor oral bioavailability due to its poor water solubility. And there is no report about whether HPT has an anti-hepatic fibrosis activity. To improve the dissolution of HPT and study its anti-hepatic fibrosis activity and mechanism,the study group prepared herpetone nanosuspensions( HPT-NS) by the miniaturized media milling method. The formulation and process of HPT-NS were optimized by the single factor experiment. Scanning electron microscopy was used to observe morphology of HPT-NS. Dialysis method was used to study dissolution of HPT-NS in vitro. CCK8 method was used to assess the effect of HPT-NS on proliferation of the rat hepatic stellate cells( HSC-T6). Flow cytometry was used to assess the effect of HPT-NS on apoptosis and cell cycle of HSC-T6. The mean particle size of optimized HPT-NS was( 196±7) nm with a polydispersity index of 0.279±0.009.SEM showed that HPT-NS was in a regular rod shape. The cumulative dissolution rate of HPT-NS reached 93% in 18 h,and was higher than that of herpetone coarse suspensions( HPT-CS,28%). CCK8 experiment showed that the inhibition rate of HPT-NS on HSC-T6 was higher than that of HPT-CS. Flow cytometry showed that HPT-NS could block HSC-T6 cells in G2/M phase and induce apoptosis of HSC-T6 cells,with a significantly stronger effect than HPT-CS. The results revealed that HPT-NS significantly increased the in vitro dissolution of HPT,and enhanced the inhibitive effect on HSC-T6 cell proliferation by blocking cells in the G2/M phase and inducing late apoptosis.


Subject(s)
Animals , Cell Line , Hepatic Stellate Cells , Lignans , Liver Cirrhosis , Rats
8.
Article in Chinese | WPRIM | ID: wpr-774134

ABSTRACT

The contractile force of hepatic stellate cells plays a very important role in liver damage, hepatitis and fibrosis. In this paper, a method based on polydimethylsiloxane (PDMS) thin micropillar arrays is proposed to measure the contractile force of human hepatic stellate cell line LX-2, which enables dynamic measurement of the subcellular distribution of force magnitude and direction. First, thin micropillar arrays on glass bottom dish were fabricated using two-step casting process in order to meet the working distance requirement of 100× objective lens. After hydrophilic treatment and protein imprint, cells were seeded on the micropillar arrays. LX-2 cells, which were quiesced by growth in serum-free medium, were activated by adding fetal bovine serum (FBS). The deflections of the micropillars were achieved by image processing technique, and then the contractile force of cells exerted on the micropillars was calculated according to mechanical simulation results, and was analyzed under both quiescent and activated conditions. The experimental results show that the average traction force of quiescent cells is about 20 nN, while the contractile force of activated cells increased to 110 nN upon adding FBS. This method can quantify the contractile force of LX-2 cell on subcellular scale in both quiescent and activated states, which may benefit pathology study and drug screen for chronic liver diseases resulted from liver fibrosis.


Subject(s)
Cell Line , Hepatic Stellate Cells , Cell Biology , Humans , Image Processing, Computer-Assisted , Mechanical Phenomena
9.
Article in English | WPRIM | ID: wpr-773987

ABSTRACT

OBJECTIVE@#To investigate the potential antifibrotic mechanisms of Chinese medicine Ganshuang Granules (, GSG) and to provide clinical therapeutic evidence of its effects.@*METHODS@#A cirrhotic mouse model was established by intraperitoneally injecting a mixture of CCl (40%) and oil (60%) at 0.2 mL per 100 g of body weight twice a week for 12 weeks. After 12-week modeling, GSG was intragastric administrated to the mice for 2 weeks, and the mice were divided into low-, medium- and high-dose groups at doses of 1, 2 and 4 g/(kg·day), respectively. Liver morphology changes were observed using Masson's trichrome staining and B-ultrasound. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA) in serum were detected using an automatic biochemistry analyzer. The expressions of desmin, smooth muscle actin (SMA) and Foxp3 in liver were detected by immunoflfluorescence. The regulatory T cell (Treg) frequency was determined through flflow cytometry analysis. Collagen-I, SMA, IL-6, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and transforming growth factor β1 (TGF-β1) expression levels were measured using quantitative polymerase chain reaction (qPCR).@*RESULTS@#Masson's staining result showed fewer pseudolobule structures and fibrous connective tissue in the GSG-treatment groups than in the spontaneous recovery group. Ultrasonography showed that GSG treatment reduced the number of punctate hyperechoic lesions in mice cirrhotic livers. The serum ALT, AST, HA levels were significantly ameliorated by GSG treatment (ALT: F=8.104, P=0.000; AST: F=7.078, P=0.002; and HA: F=7.621, P=0.001). The expression levels of collagen-I and SMA in the cirrhotic livers were also attenuated by GSG treatment (collagen-I: F=3.938, P=0.011; SMA: F=4.115, P=0.009). Tregs, which were elevated in the fibrotic livers, were suppressed by GSG treatment (F=8.268, P=0.001). The expressions of IL-6, TNF-α and IL-1β increased, and TGF-β levels decreased in the cirrhotic livers after GSG treatment (IL-6: F=5.457, P=0.004; TNF-α: F=6.023, P=0.002; IL-1β: F=6.658, P=0.001; and TGF-β1: F=11.239, P=0.000).@*CONCLUSIONS@#GSG promoted the resolution/regression of cirrhosis and restored liver functions in part by suppressing Treg cell differentiation, which may be mediated by hepatic stellate cells.


Subject(s)
Animals , Disease Models, Animal , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Hepatic Stellate Cells , Liver Cirrhosis, Experimental , Drug Therapy , Allergy and Immunology , Male , Mice , Mice, Inbred BALB C , T-Lymphocytes, Regulatory
10.
Yonsei Medical Journal ; : 561-569, 2019.
Article in English | WPRIM | ID: wpr-762078

ABSTRACT

PURPOSE: Liver fibrosis is a major cause of morbidity and mortality and the outcome of various chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the key event in liver fibrosis. Studies have confirmed that miR-140-3p plays a potential regulatory effect on HSC activation. However, whether miR-140-3p mediates the liver fibrosis remains unknown. MATERIALS AND METHODS: Expression of miR-140-3p was detected by real-time quantitative PCR (qPCR). Cell proliferation was measured by MTT, while cell apoptosis rate was determined via flow cytometry. Western blot assay was used to detect the expression of cleaved PARP. The fibrogenic effect was evaluated by expression of α-smooth muscle actin and desmin. Functional experiments were performed in transforming growth factor β1 (TGF-β1)-induced HSC-T6 cells with transfection of anti-miR-140-3p and/or siPTEN. Target binding between miR-140-3p and PTEN was predicted by the TargetScan database and identified using luciferase reporter assay and RNA immunoprecipitation. RESULTS: TGF-β1 induced the activation of HSC-T6 cells, and miR-140-3p expression varied according to HSC-T6 cell activation status. Knockdown of miR-140-3p reduced cell proliferation and the expressions of α-SMA and desmin, as well as increased apoptosis, in TGF-β1-induced HSC-T6 cells, which could be blocked by PTEN silencing. Additionally, inactivation of the AKT/mTOR signaling pathway stimulated by miR-140-3p knockdown was abolished when silencing PTEN expression. PTEN was negatively regulated by miR-140-3p via direct binding in HSC-T6 cells. CONCLUSION: miR-140-3p is an important mediator in HSC-T6 cell activation, and miR-140-3p knockdown suppresses cell proliferation and fibrogenesis in TGF-β1-induced HSC-T6 cells, indicating that miR-140-3p may be a potential novel molecular target for liver fibrosis.


Subject(s)
Actins , Apoptosis , Blotting, Western , Cell Proliferation , Desmin , Flow Cytometry , Hepatic Stellate Cells , Immunoprecipitation , Liver Cirrhosis , Liver Diseases , Luciferases , Mortality , Polymerase Chain Reaction , RNA , Transfection , Transforming Growth Factors
11.
Article in English | WPRIM | ID: wpr-776615

ABSTRACT

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.


Subject(s)
Apoptosis , Cell Proliferation , Cells, Cultured , Drugs, Chinese Herbal , Pharmacology , Hepatic Stellate Cells , Physiology , Humans , Injections , Liver Cirrhosis , Drug Therapy , Tumor Suppressor Proteins , Genetics
12.
São Paulo; s.n; s.n; 2019. 98 p. tab, ilus, graf.
Thesis in Portuguese | LILACS | ID: biblio-1024842

ABSTRACT

O carcinoma hepatocelular (HCC) é a neoplasia primária mais frequente que acomete o fígado, a quarta principal causa de morte relacionada ao câncer e apresenta mau prognóstico. A fibrose hepática está presente em grande parte dos casos de HCC e é um dos principais fatores de risco para esta afecção. Segundo estudos prévios do grupo, a ß-ionona (BI), presente em uvas e aromatizantes de vinho, apresenta potencial quimiopreventivo na hepatocarcinogênese principalmente por reduzir o número e tamanho de lesão pré neoplásica (LPN) e inibir a proliferação celular. No entanto, até o presente não foram identificados na literatura estudos que investigaram o efeito deste isoprenóide no processo fibrótico e na hepatocarcinogênese a ele associada. Desta forma, este estudo pretendeu investigar o potencial efeito quimiopreventivo da BI na hepatocarcinogênese associada à fibrose hepática. Para tanto, ratos machos Wistar foram tratados com óleo de milho (OM) [0,25 ml / 100 g de peso corporal (p.c.); Grupo de OM] ou BI (16mg / 100g p.c.; Grupo BI) durante 18 semanas. A partir da 2ª semana, todos os animais receberam uma dose intraperitoneal de dietilnitrosamina (DEN - 50 mg / Kg p.c.) uma vez por semana até a 16ª semana. Os animais foram eutanasiados em diferentes períodos do protocolo experimental: na 10a semana (grupos OMP1 e BIP1), na 14a semana (grupos OMP2 e BIP2) e na 18ª semana (grupos OMP3 e BIP3). O isoprenóide demonstrou, de maneira inédita na literatura, inibir o desenvolvimento da fibrose hepática em diferentes estágios da hepatocarcinogênese (pontos 1, 2 e 3) por reduzir (p < 0,05) a porcentagem de área marcada para picrosirius. Além disso, BI reduziu a porcentagem de área positiva para α- SMA (p < 0,05) e as concentrações de hidroxiprolina (p < 0,05) no ponto 2. Foi observada ação quimiopreventiva da BI nas fases iniciais da hepatocarcinogênese (pontos 1 e 2) mesmo em modelo associada a fibrose por reduzir (p < 0,05) o número e porcentagem de área do corte ocupada por LPN GSTP positivas. Este efeito não foi observado em fase mais avançada da hepatocarcinogênese (ponto 3). Corroborando este dado não foram observadas diferenças em relação ao número de tumores (p>=0,05) avaliados por imageamento e por análise histopatológica. No entanto, quando comparados ao seu controle (OMP3), os animais do grupo BIP3 apresentaram menor mortalidade e menor incidência (p < 0,05) de HCC high, considerado um tipo mais agressivo de HCC, sugerindo que este composto possa atuar na agressividade das células tumorais. O grupo BIP2 demonstrou ainda menor proliferação celular (p < 0,05) quando comparado ao grupo OMP2. Assim foram avaliadas as vias de proliferação celular PI3K/AKT e MAPK/ERK, bem como as proteínas p21 e p53, relacionadas a progressão do ciclo celular. Não foram observadas(p≥0,05) alterações nestas vias por parte do isoprenóide. O presente estudo demonstrou ação protetora da BI no desenvolvimento de fibrose, bem como na hepatocarcinogênese a ela associada. Contudo, são necessárias análises complementares para elucidar mecanismos pelos quais a BI atua na carcinigênese hepática associada à fibrose


Hepatocellular carcinoma (HCC) is the primary liver cancer, the fourth leading cause of death related to cancer and presents a poor prognosis. Hepatic fibrosis is present in most cases of HCC and represents one of the main risk factors for this condition. According to previous studies of the group, ß-ionone (BI), present in grapes and wine flavorings, has a potential chemopreventive in hepatocarcinogenesis mainly by reducing the number and size of preneoplastic lesions (LPN) and inhibiting cell proliferation. However, to date, no studies have been identified in the literature that investigated the effect of this isoprenoid on the fibrotic process and in its association with hepatocarcinogenesis. Thus, this study aimed to investigate the potential chemopreventive effect of BI in hepatocarcinogenesis associated with hepatic fibrosis. Male Wistar rats were treated with corn oil (OM) [0.25 ml / 100 g body weight (b.w.); OM] or BI group (16mg / 100g b.w; BI group) for 18 weeks. From week 2, all animals received an intraperitoneal dose of diethylnitrosamine (DEN - 50 mg / kg b.w.) once in a week until week 16. The animals were euthanized at different periods of the experimental protocol: at week 10 (groups OMP1 and BIP1), at week 14 (groups OMP2 and BIP2) and week 18 (groups OMP3 and BIP3). The isoprenoid, for the first time in the literature, shown to inhibit the development of liver fibrosis at different stages of hepatocarcinogenesis (points 1, 2 and 3) by reducing (p <0.05) the percentage of the area labeled for picrosirius. Also, BI reduced the percentage of α-SMA positive area (p <0.05) and hydroxyproline concentrations (p <0.05) at point 2. BI chemopreventive action was observed in the early stages of hepatocarcinogenesis (point 1 and 2) even in a model associated with fibrosis for reducing (p <0.05) the number and percentage of the liver section area occupied by GSTP positive LPN. This effect was not observed at a later stage of hepatocarcinogenesis (point 3). Corroborating this data, no differences were observed regarding the number of tumors (p>=0.05) evaluated by imaging and histopathological analysis. However, when compared to its control (OMP3), animals from the BIP3 group had lower mortality and lower incidence (p<0.05) of HCC high, considered a more aggressive type of HCC, suggesting that this compound may act in aggressiveness of tumor cells. The BIP2 group also showed lower cell proliferation (p <0.05) when compared to the OMP2 group. Thus, PI3K / AKT and MAPK / ERK cell proliferation pathways were evaluated, as well as p21 and p53 proteins, related to cell cycle progression. No changes were observed in these pathways by the isoprenoid (p≥0.05). The present study demonstrated the protective action of BI in the development of fibrosis, as well as its association with hepatocarcinogenesis. However, further analysis is needed to elucidate mechanisms by which BI acts on fibrosis-associated liver carcinogenesis


Subject(s)
Animals , Male , Rats , Norisoprenoids/adverse effects , Liver Cirrhosis/complications , Neoplasms/prevention & control , Terpenes/therapeutic use , Fibrosis/drug therapy , Carcinoma, Hepatocellular/classification , Hepatic Stellate Cells , Carcinogenesis/pathology
13.
Article in Chinese | WPRIM | ID: wpr-773772

ABSTRACT

OBJECTIVE@#To investigate the effects of endothelial progenitor cells (EPCs) under shear stress on the biological function such as proliferation, adhesion, migration, apoptosis and expression of α-smooth muscle actin (α-SMA), collagen-I and collagen-Ⅲ of hepatic stellate cells (HSCs).@*METHODS@#HSCs and EPCs were inoculated into the upper and lower layers of the co-culture chamber respectively and co-incubated for 24 hours. Then, 12 dyne/cm shear stress was applied to EPCs cells for another 24 hours. After that, proliferation, adhesion, migration and apoptosis of HSCs were detected by cell counting kit-8 (CCK-8) kit, cell adherent assay, Boyden cell migration assay and flow cytometry respectively. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expression of alpha -SMA, collagen I and collagen-Ⅲ in HSCs.@*RESULTS@#Under shear stress, EPCs ecological niche could obviously inhibit the proliferation, adhesion and migration of HSCs, promote the apoptosis of HSCs, and down-regulate the mRNA and protein expression of collagen-I, collagen-Ⅲ in HSC cells.@*CONCLUSIONS@#Under shear stress, EPCs ecological niche could inhibit the fibrosis development of HSCs to a certain extent.


Subject(s)
Actins , Apoptosis , Cell Proliferation , Cells, Cultured , Collagen Type I , Endothelial Progenitor Cells , Hepatic Stellate Cells
14.
Article in English | WPRIM | ID: wpr-713310

ABSTRACT

BACKGROUND/AIMS: Nonalcoholic steatohepatitis (NASH) is prevalent in both economically developed and developing countries. Twenty percent of NASH progresses to cirrhosis with/without hepatocellular carcinoma, and there is an urgent need to find biomarkers for early diagnosis and monitoring progression of the disease. Using immunohistochemical and immunoelectron microscopic examination we previously reported that expression of matrix metalloproteinase-1 (MMP-1) increased in monocytes, Kupffer cells and hepatic stellate cells in early stage NASH. The present study investigated whether serum MMP-1 levels reflect disease activity and pharmaceutical effects in NASH patients. METHODS: We measured the serum levels of MMPs, tissue inhibitors of metalloproteinases (TIMPs), and several cytokines/chemokines in patients with histologically proven early and advanced stages of NASH and compared them with those in healthy controls. RESULTS: Serum MMP-1 levels in stage 1 fibrosis, but not in the more advanced fibrosis stages, were significantly higher than in healthy controls (P=0.019). There was no correlation between serum MMP-1 level and fibrosis stage. Serum MMP- 1 levels in NASH patients represented disease activity estimated by serum aminotransferase values during the follow-up period. In contrast, MMP-2, MMP-9 and TIMPs did not change with disease activity. Consistent with the finding that MMP-1 is expressed predominantly in monocytes and Kupffer cells, serum levels of monocyte chemotactic protein-1 and granulocyte-colony stimulating factor were significantly increased in NASH with stage 1 fibrosis. CONCLUSIONS: These results suggest that serum MMP-1 levels represent disease activity and may serve as a potential biomarker for monitoring the progression of NASH.


Subject(s)
Biomarkers , Carcinoma, Hepatocellular , Chemokine CCL2 , Cytokines , Developing Countries , Early Diagnosis , Fibrosis , Follow-Up Studies , Hepatic Stellate Cells , Humans , Kupffer Cells , Liver Cirrhosis , Matrix Metalloproteinase 1 , Matrix Metalloproteinases , Metalloproteases , Monocytes , Non-alcoholic Fatty Liver Disease
15.
Int. j. morphol ; 35(2): 676-683, June 2017.
Article in Spanish | LILACS | ID: biblio-893039

ABSTRACT

Uno de los endulzantes más comúnmente utilizado es la fructosa. La fructosa es directamente metabolizada en el hígado y se puede transformar en glucosa, posteriormente es almacenada como glicógeno constituyéndose en una fuente de energía para los hepatocitos. Todo el exceso de fructosa se convierte en lípidos ejerciendo un efecto tóxico sobre el hígado, similar al producido por el exceso de alcohol, pudiendo provocar hígado graso no alcohólico (NAFLD). El objetivo de esta revisión es reunir hallazgos recientes en relación al efecto de la ingesta de fructosa en altas concentraciones y su relación con el NAFLD.


One of the most commonly used sweeteners is fructose. Fructose is directly metabolized in the liver and can be converted into glucose, later stored as glycogen constituting a source of energy for the hepatocytes. All excess fructose is converted into lipids by exerting a toxic effect on the liver, similar to that produced by excess of alcohol, and can cause nonalcoholic fatty liver (NAFLD). The aim of this review is to gather recent findings regarding the effect of fructose intake at high concentrations and its relationship with NAFLD.


Subject(s)
Humans , Non-alcoholic Fatty Liver Disease/chemically induced , Non-alcoholic Fatty Liver Disease/pathology , Fructose/adverse effects , Hepatic Stellate Cells/drug effects , Fructose/metabolism , Fructose/pharmacology , Liver Cirrhosis/chemically induced
16.
Ann. hepatol ; 16(1): 48-56, Jan.-Feb. 2017. graf
Article in English | LILACS | ID: biblio-838085

ABSTRACT

Abstract: Liver fibrosis resulting from chronic liver injury are major causes of morbidity and mortality worldwide. Among causes of hepatic fibrosis, viral infection is most common (hepatitis B and C). In addition, obesity rates worldwide have accelerated the risk of liver injury due to nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Also liver fibrosis is associated with the consumption of alcohol, or autoimmune hepatitis and chronic cholangiophaties. The response of hepatocytes to inflammation plays a decisive role in the physiopathology of hepatic fibrosis, which involves the recruitment of both pro- and anti-inflammatory cells such as monocytes and macrophages. As well as the production of other cytokines and chemokines, which increase the stimulus of hepatic stellate cells by activating proinflammatory cells. The aim of this review is to identify the therapeutic options available for the treatment of the liver fibrosis, enabling the prevention of progression when is detected in time.


Subject(s)
Humans , Animals , Liver Cirrhosis/drug therapy , Anti-Inflammatory Agents/therapeutic use , Time Factors , Signal Transduction/drug effects , Cell Communication/drug effects , Cytokines/metabolism , Treatment Outcome , Inflammation Mediators/metabolism , Disease Progression , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Liver Cirrhosis/mortality , Anti-Inflammatory Agents/adverse effects
17.
Article in English | WPRIM | ID: wpr-158428

ABSTRACT

Tissue fibrosis, characterized by excessive accumulation of aberrant extracellular matrix (ECM) produced by myofibroblasts, is a growing cause of mortality worldwide. Understanding the factors that induce myofibroblastic differentiation is paramount to prevent or reverse the fibrogenic process. Integrin-mediated interaction between the ECM and cytoskeleton promotes myofibroblast differentiation. In the present study, we explored the significance of integrin alpha 11 (ITGA11), the integrin alpha subunit that selectively binds to type I collagen during tissue fibrosis in the liver, lungs and kidneys. We showed that ITGA11 was co-localized with α-smooth muscle actin-positive myofibroblasts and was correlatively induced with increasing fibrogenesis in mouse models and human fibrotic organs. Furthermore, transcriptome and protein expression analysis revealed that ITGA11 knockdown in hepatic stellate cells (liver-specific myofibroblasts) markedly reduced transforming growth factor β-induced differentiation and fibrotic parameters. Moreover, ITGA11 knockdown dramatically altered the myofibroblast phenotype, as indicated by the loss of protrusions, attenuated adhesion and migration, and impaired contractility of collagen I matrices. Furthermore, we demonstrated that ITGA11 was regulated by the hedgehog signaling pathway, and inhibition of the hedgehog pathway reduced ITGA11 expression and fibrotic parameters in human hepatic stellate cells in vitro, in liver fibrosis mouse model in vivo and in human liver slices ex vivo. Therefore, we speculated that ITGA11 might be involved in fibrogenic signaling and might act downstream of the hedgehog signaling pathway. These findings highlight the significance of the ITGA11 receptor as a highly promising therapeutic target in organ fibrosis.


Subject(s)
Animals , Collagen , Collagen Type I , Cytoskeleton , Extracellular Matrix , Fibrosis , Hedgehogs , Hepatic Stellate Cells , Humans , In Vitro Techniques , Kidney , Liver , Liver Cirrhosis , Lung , Mice , Mortality , Myofibroblasts , Phenotype , Transcriptome , Transforming Growth Factors
18.
Article in English | WPRIM | ID: wpr-27728

ABSTRACT

BACKGROUND/OBJECTIVE: Orostachys japonicus A. Berger (Crassulaceae) has been used in traditional herbal medicines in Korea and other Asian countries to treat various diseases, including liver disorders. In the present study, the anti-fibrotic effects of O. japonicus extract (OJE) in cellular and experimental hepatofibrotic rat models were investigated. MATERIALS/METHODS: An in vitro hepatic stellate cells (HSCs) system was used to estimate cell viability, cell cycle and apoptosis by MTT assay, flow cytometry, and Annexin V-FITC/PI staining techniques, respectively. In addition, thioacetamide (TAA)-induced liver fibrosis was established in Sprague Dawley rats. Briefly, animals were divided into five groups (n = 8): Control, TAA, OJE 10 (TAA with OJE 10 mg/kg), OJE 100 (TAA with OJE 100 mg/kg) and silymarin (TAA with Silymarin 50 mg/kg). Fibrosis was induced by treatment with TAA (200 mg/kg, i.p.) twice per week for 13 weeks, while OJE and silymarin were administered orally two times per week from week 7 to 13. The fibrotic related gene expression serum biomarkers glutathione and hydroxyproline were estimated by RT-PCR and spectrophotometry, respectively, using commercial kits. RESULTS: OJE (0.5 and 0.1 mg/mL) and silymarin (0.05 mg/mL) treatment significantly (P < 0.01 and P < 0.001) induced apoptosis (16.95% and 27.48% for OJE and 25.87% for silymarin, respectively) in HSC-T6 cells when compared with the control group (9.09%). Further, rat primary HSCs showed changes in morphology in response to OJE 0.1 mg/mL treatment. In in vivo studies, OJE (10 and 100 mg/kg) treatment significantly ameliorated TAA-induced alterations in levels of serum biomarkers, fibrotic related gene expression, glutathione, and hydroxyproline (P < 0.05-P < 0.001) and rescued the histopathological changes. CONCLUSIONS: OJE can be developed as a potential agent for the treatment of hepatofibrosis.


Subject(s)
Animals , Apoptosis , Asian Continental Ancestry Group , Biomarkers , Cell Cycle , Cell Survival , Fibrosis , Flow Cytometry , Gene Expression , Glutathione , Hepatic Stellate Cells , Humans , Hydroxyproline , In Vitro Techniques , Korea , Liver , Liver Cirrhosis , Models, Animal , Rats , Rats, Sprague-Dawley , Silymarin , Spectrophotometry , Thioacetamide
19.
Article in English | WPRIM | ID: wpr-29539

ABSTRACT

Transplantation of bone marrow derived stem cells (BMSCs) has been reported inhibits liver fibrosis. Several in vitro studies by co-culturing BMSCs and hepatic stellate cells (HSCs) indirectly or directly in 2D models showed inhibition of HSC as the key player in liver fibrosis. In this study, we investigated direct effect of BMSCs on HSCs by co-culturing BMSCs and HSCs in 3D model as it represents the liver microenvironment with intricate cell-cell and cell-matrix interactions. Primary isolated rat HSCs and BMSCs were directly co-cultured at 1:1 ratio with hanging drop method. The monoculture of rat HSCs served as positive control. Mono-culture and co-culture samples were harvested on day 3, 5 and 7 for histological analysis. The samples were analyzed for extracellular matrix deposition by Masson's Trichrome staining, tenascin-C immunocytochemistry, resting HSC's state as shown by positive Oil Red O stained cells. Our results indicated CD90+CD34− BMSCs anti-liver fibrosis potency as evidenced by higher proportion of Oil Red O-positive cells in the co-culture group compared to the monoculture group and the significant decrease in extracellular matrix deposition as well as the decrease in tenascin-C expression in the co-culture group (p<0.05) compared to the monoculture group. These findings demonstrate that BMSCs have a potential therapeutic effect against liver fibrotic process through their capacity to inhibit HSCs activation and their effect in minimizing extracellular matrix deposition.


Subject(s)
Animals , Bone Marrow , Coculture Techniques , Extracellular Matrix , Fibrosis , Hepatic Stellate Cells , Immunohistochemistry , In Vitro Techniques , Liver , Liver Cirrhosis , Methods , Rats , Stem Cells , Tenascin
20.
Article in English | WPRIM | ID: wpr-82852

ABSTRACT

In the past, liver cirrhosis was considered an irreversible phenomenon. However, many experimental data have provided evidence of the reversibility of liver fibrosis. Moreover, multiple clinical studies have also shown regression of fibrosis and reversal of cirrhosis on repeated biopsy samples. As various etiologies are associated with liver fibrosis via integrated signaling pathways, a comprehensive understanding of the pathobiology of hepatic fibrogenesis is critical for improving clinical outcomes. Hepatic stellate cells play a central role in hepatic fibrogenesis upon their activation from a quiescent state. Collagen and other extracellular material components from activated hepatic stellate cells are deposited on, and damage, the liver parenchyma and vascular structures. Hence, inactivation of hepatic stellate cells can lead to enhancement of fibrolytic activity and could be a potential target of antifibrotic therapy. In this regard, continued efforts have been made to develop better treatments for underlying liver diseases and antifibrotic agents in multiple clinical and therapeutic trials; the best results may be expected with the integration of such evidence. In this article, we present the underlying mechanisms of fibrosis, current experimental and clinical evidence of the reversibility of liver fibrosis/cirrhosis, and new agents with therapeutic potential for liver fibrosis.


Subject(s)
Biopsy , Collagen , Fibrosis , Hepatic Stellate Cells , Liver Cirrhosis , Liver Diseases , Liver
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