Establishment of a novel co-cultured liver cancer model based on tumor microenvironment / 中华肿瘤杂志

Objective: To construct a new co-cultured liver cancer research model composed of activated hepatic stellate cells (aHSC) and liver cancer cells, explore the efficacy difference between it and traditional model, so as to establish a liver cancer research model in vitro and in vivo that can reflect the real clinical efficacy. Methods: A new co-culture model of liver cancer consisting of aHSC and liver cancer cells was constructed. The differences in efficacy between the new co-culture model and the traditional single cell model were compared by cytotoxicity test, cell migration test, drug retention test and in vivo tumor inhibition test. Western blot was used to detect the drug-resistant protein P-gp and epithelial-mesenchymal transition-related proteins. Masson staining was used to observe the deposition of collagen fibers in tumor tissues of tumor-bearing mice. CD31 immunohistochemical staining was used to observe the microvessel density in tumor tissues of tumor-bearing mice. Results: The cytotoxicity of single cell model and co-culture model was dose-dependent. With the increase of curcumin (CUR) concentration, the cell viability decreased, but the cell viability of single cell model decreased faster than that of co-culture model. When the concentration of CUR was 10 μg/ml, the cell viability of the co-culture model was 62.3% and the migration rate was (28.05±3.68)%, which were higher than those of the single cell model [38.5% and (14.91±5.92)%, both P<0.05]. Western blot analysis showed that the expressions of P-gp and vimentin were up-regulated in the co-culture model, which were 1.55 and 2.04 fold changes of the single cell model, respectively. The expression of E-cadherin was down-regulated, and the expression level of E-cadherin in the single cell model was 1.17 fold changes of the co-culture model. Drug retention experiment showed that the co-culture model could promote drug efflux and reduce drug retention. In vivo tumor inhibition experiment showed that the m-HSC+ H22 co-transplantation model had faster tumor growth and larger tumor volume than those of the H22 single cell transplantation model. After CUR treatment, the tumor growths of m-HSC+ H22 co-transplantation model and H22 single cell transplantation model were inhibited. Masson staining showed that the deposition of collagen fibers in tumor tissues of m-HSC+ H22 co-transplantation model mice was more than that of H22 single cell transplantation model. CD31 immunohistochemical staining showed that the microvessel density in tumor tissue of m-HSC+ H22 co-transplantation model was higher than that of H22 single cell transplantation model. Conclusions: The aHSC+ liver cancer cell co-culture model has strong proliferation and metastasis ability and is easy to be resistant to drugs. It is a new type of liver cancer treatment research model superior to the traditional single cell model.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles enhance the regenerative capability of fibrotic liver / 器官移植

Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle (hUC-MSC-EV) in the regeneration of fibrotic liver. Methods C57BL/6 mice were randomly divided into the 70% normal liver resection group (Oil+PHx group), 70% liver fibrosis resection group (CCl4+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl4+PHx+MSC-EV group), with 8 mice in each group. LX-2 cell lines were assigned into the phosphate buffer solution (PBS) group, transforming growth factor (TGF)-β group and TGF-β+MSC-EV group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice after partial liver resection were detected in each group. The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group. The messenger RNA (mRNA) expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in LX-2 cells were detected in each group, and their effects on HGF expression in mouse liver were observed. Results Compared with the Oil+PHx group, the serum levels of AST, ALT and LDH were up-regulated, and the degree of fibrosis was more severe, the positive area of Sirius red and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl4+PHx group. Compared with the CCl4+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the Oil+PHx group, the protein expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) were lower in the CCl4+PHx group. Compared with the CCl4+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-SMA protein was decreased in the TGF-β+MSC-EV group, the differences were statistically significant (all P < 0.05). The expression level of HGF protein in the CCl4+PHx group was lower than that in the Oil+PHx group, whereas the difference was not statistically significant (P > 0.05). The expression level of HGF protein in the CCl4+PHx+MSC-EV group was higher than that in the CCl4+PHx group, and the difference was statistically significant (P < 0.05). Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver. hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.

Vitamin D receptor (VDR) mediates the quiescence of activated hepatic stellate cells (aHSCs) by regulating M2 macrophage exosomal smooth muscle cell-associated protein 5 (SMAP-5) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Non-coding RNA-mediated TGF-β1/Smads Signaling Pathway and Liver Fibrosis， and Intervention by Active Components of Chinese Medicinal： A Review / 中国实验方剂学杂志

Liver fibrosis is a wound healing response that occurs in the setting of chronic liver injury and is caused by imbalance in the synthesis and degradation of extracellular matrix （ECM）. If left untreated， it can progress to liver cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cell （HSC） is now well established as a central driver of liver fibrosis. The activated HSC will transform into myofibroblasts that produce ECM protein. Transforming growth factor-β1 （TGF-β1） can induce the activation of hepatic stellate cell （HSC）， and TGF-β1/Smads signaling pathway is one of the important pathways to promote liver fibrosis. Non-coding RNA （ncRNA） does not encode proteins during the transcription but plays an important regulatory role in the post-transcriptional process of genes. Accumulating evidence shows that the occurrence of liver fibrosis is closely related to the abnormal expression of ncRNA which participates in the activation of HSC by regulating TGF-β1 signal transduction and then affects the process of liver fibrosis. MiRNA-mediated TGF-β1/Smads signaling pathway can not only promote liver fibrosis but also play a role in anti-fibrosis. Long non-coding RNA （lncRNA） not only promotes the development of liver fibrosis by binding to target genes but also enhances TGF-β1 signal transduction by acting as competitive endogenous RNA. circular RNA （circRNA） acts as a ''sponge'' to regulate TGF-β1/Smads pathway， thereby inhibiting HSC activation and exerting the anti-liver fibrosis effect. Chinese medicinal plays an essential part in the prevention and treatment of liver fibrosis， and the active components can inhibit TGF-β1/Smads pathway by regulating the expression of miRNA， thus alleviating liver fibrosis. This article reviews the role and mechanism of miRNA-， lncRNA- and circRNA-mediated TGF-β1/Smads signaling pathway in liver fibrosis and summarizes the anti-liver fibrosis effect of active components of Chinese medicinals by regulating miRNA-mediated TGF-β1/Smads signaling pathway， which can serve as a reference for clinical treatment of liver fibrosis and the development of new drugs.

Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro / 中国血吸虫病防治杂志

Objective To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. Methods The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. Results The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1. Conclusion rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

Molecular mechanism of Cigu Xiaozhi formula interfering with HSC-T6 cell activation by regulating HIF-1α signaling pathway based on computer aided drug design / 药学学报

In this study, we investigated the effect of Cigu Xiaozhi formula on HSC-T6 activity in hypoxic microenvironment based on network pharmacology and computer-aided drug design, and predicted and verified its possible targets and related signaling pathways. The potential active components and targets of Cigu Xiaozhi formula were screened by searching Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Encyclopaedia of Traditional Chinese Medicine (ETCM) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases, and the liver fibrosis related targets retrieved from Gene Cards and Pharm GK database were integrated to obtain the potential targets of Cigu Xiaozhi formula in the treatment of liver fibrosis. GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on Omic Share platform, and Cytoscape software was used to construct the "potential active ingredient-key target-pathway" network. The active components and target proteins were subjected to molecular docking analysis by Auto Dock software. According to the results of molecular dynamics simulation and binding free energy calculation, the top 5 active components with degree were scored. The active components stigmasterol and β-sitosterol were subjected to molecular docking. CoCl2 was used to induce HSC-T6 cells to construct hypoxia model in vitro. The cell viability was detected by CCK-8 assay, and the optimal time and concentration of hypoxia model of HSC-T6 cells was determined to be 100 µmol·L-1 CoCl2 for 24 h. Under hypoxia condition, HSC-T6 cells were activated, the wound healing rate was significantly increased, and the fluorescence signal of activation marker protein α-smooth muscle actin (α-SMA) was significantly enhanced. However, 6% drug-containing serum could inhibit the activation of HSC-T6 cells, and the wound healing rate was significantly decreased, and the fluorescence signal of α-SMA was significantly weakened. Further studies showed that the expressions of hypoxia-inducible factor-1α (HIF-1α), α-SMA and key proteins of Hedgehog (Hh) signaling pathway in HSC-T6 cells were up-regulated under hypoxia, while the expressions of HIF-1α, α-SMA, Patched-1 (Ptch-1) and glioma related oncogene homology-1 (Gli-1) were down-regulated in 6% drug-containing serum group, the YC-1 group and the cyclopamine group. These results indicated that HIF-1α and Hh signaling pathways were involved in the activation of HSC-T6 cells, and the traditional Chinese medicine Cigu Xiaozhi formula could inhibit the activation of HSC-T6 cells, and the mechanism may be related to the inhibition of HIF-1α expression and the blocking of Hh signaling pathway. In conclusion, Cigu Xiaozhi formula can inhibit the activation of HSC-T6 cells by directly acting on HIF-1α and Hh signaling pathway, and exert an anti-hepatic fibrosis effect. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Gansu University of Chinese Medicine, in compliance with the Institutional Animal Care Guidelines.

Effects of praziquantel isomers on the proliferation and activation of the LX-2 human hepatic stellate cell line / 中国血吸虫病防治杂志

Objective To compare the effects of levo-praziquantel (L-PZQ) and dextro-praziquantel (D-PZQ) on the proliferation and activation of the human hepatic stellate cell line LX-2 in vitro. Methods LX-2 cells were stimulated with transforming growth factor-β (TGF-β). LX-2 cell proliferation was measured using the CCK-8 assay after 24 h stimulation with 0 to 50 μg/mL concentrations of praziquantel, and the gene and protein expression of type Ⅰ collagen (collagen Ⅰ), type Ⅲ collagen (collagen Ⅲ) and α-smooth muscle actin (α-SMA) was quantified in LX-2 cells using quantitative real-time PCR (qPCR) and Western blotting assays 24 h and 48 h following stimulation with 15 μg/mL praziquantel to detect LX-2 cell activation. Results There were significant differences in the survival rate of LX-2 cells between L-PZQ and D-PZQ treatments at all concentrations (F = 6.119 and 79.180, both P values < 0.05). Either L-PZQ or D-PZQ at a concentration of < 30 μg/mL showed no remarkableeffectsonthe LX-2 cell proliferation (both P values > 0.05), and L-PZQ at a concentration of > 50 μg/mL and D-PZQ at a concentration of > 40 μg/mL inhibited the LX-2 cell proliferation (both P values < 0.05), while D-PZQ at concentrations of 40 μg/mL and 50 μg/mL showed greater inhibition on LX-2 cell proliferation than L-PZQ (t = 3.419 and 8.776, both P values < 0.05). There were significant differences in the collagen Ⅰ, collagen Ⅲ and α-SMA expression in LX-2 cells at both transcriptional (F = 21.55, 79.99 and 46.70, all P values < 0.05) and translational levels (F = 20.12, 30.29 and 32.93, all P values < 0.05) among the blank control group, TGF-β stimulation group, L-PZQ treatment group and D-PZQ treatment group. L-PZQ treatment resulted in remarkable inhibition on collagen Ⅲ and α-SMA gene expression in LX-2 cells (both P values < 0.05); however, the treatment showed no remarkable inhibition collagen Ⅰ gene expression or collagen Ⅰ, collagen Ⅲ or α-SMA protein expression in LX-2 cells (all P values > 0.05). In addition, D-PZQ treatment resulted in significant inhibition on collagen Ⅰ, collagen Ⅲ and α-SMA expression in LX-2 cells at both translational and transcriptional levels (all P values < 0.05), and D-PZQ showed higher inhibition on collagen Ⅰ, collagen Ⅲ and α-SMA gene expression in LX-2 cells than L-PZQ (all P values < 0.05). Conclusions Both L-PZQ and D-PZQ inhibit the proliferation and activation of LX-2 cells, and D-PZQ shows a higher inhibitory activity than L-PZQ.

Role of mitogen-inducible gene 6 in the activation of human hepatic stellate cells and deposition of extracellular matrix induced by sodium arsenite / 环境与职业医学

Background Arsenic is a well-known environmental toxicant. Hepatic fibrosis could occur dueto excessive or long-term exposure to arsenic, while associated molecular mechanisms remain undefined. Mitogen-inducible gene 6 (Mig-6) exhibits a protective effect on numerous diseases or cancers. However, the specific role of Mig-6 in the mechanisms of arsenite-induced hepatic fibrosis remains indistinct. Objective To investigate the specific role of Mig-6 in the activation of hepatic stellate cells (HSC) and the deposition of extracellular matrix (ECM) induced by sodium arsenite (NaAsO2). Methods Human hepatic stellate cells (Lx-2) were treated with 0, 1.875, 3.75, 7.5, and 15 μmol·L−1 of NaAsO2 for 24 h, or with 7.5 μmol·L−1 NaAsO2 for 0, 12, 24, 48, and 72 h. Additionally, Lx-2 cells were transfected by pcDNA3.1(+)/Mig-6, then treated with 7.5 μmol·L−1 NaAsO2 for 24 h; a blank control group, a pcDNA3.1(+)-control group, a pcDNA3.1(+)/Mig-6 group, and an arsenic (7.5 μmol·L−1 NaAsO2) group were also set up. After transfection, the cells and culture supernatants were collected, and the protein levels of Mig-6, α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) in Lx-2 cells were identified by Western blotting analysis; moreover, the secretion levels of main ECM components in supernatants such as hyaluronic acid (HA), laminin (LN), collagens IV (COL-IV), and procollagen-III (PIIINP) were tested by ELISA. Results The Mig-6 expression decreased in the 3.75, 7.5, and 15 μmol·L−1 NaAsO2 groups (0.561±0.095, 0.695±0.048, and 0.401±0.030) compared to the control group (1.000±0.000) in Lx-2 cells (P<0.05). After administration with 7.5 μmol·L−1 of NaAsO2 for 24, 48, and 72 h, the Mig-6 expression (0.856±0.036, 0.515±0.077, 0.491±0.060) decreased compared with the 0 h group (1.000±0.000) (P<0.05). After over-expression of Mig-6, the results of Lx-2 activation related protein levels showed that compared to the control group, the α-SMA and TGF-β1 expression were up-regulated in the arsenic group (P<0.05); meanwhile, the α-SMA and TGF-β1 in the Mig-6 over-expression combined arsenic exposure group reduced compared to the arsenic (7.5 μmol·L−1) group (P<0.05). The results of ELISA showed that compared with the control group, the HA, LN, PIIINP, COL-IV in the arsenic group were up-regulated (P<0.05); while compared to the arsenic group, the HA, LN, PIIINP, and COL-IV in the Mig-6 over-expression combined with arsenic exposure group were decreased (P<0.05). Conclusion Arsenic down-regulates Mig-6 expression in HSC, and over-expression of Mig-6 can reverse the activation of HSC and ECM deposition induced by arsenic exposure. It suggests that Mig-6 plays a protective role in arsenic-induced HSC activation and ECM deposition.

Mechanism study of the protective effects of selective cyclooxygenase-2 enzyme inhibitors on the liver of rats with type 2 diabetes mellitus combined with nonalcoholic steatohepatitis via Rho/ROCK pathway / 中华肝脏病杂志

Objective: To investigate whether the selective cyclooxygenase-2 enzyme inhibitors celecoxib has protective effect on the liver of rats with type 2 diabetes mellitus (T2DM) combined with nonalcoholic steatohepatitis (NASH) via inhibiting the expression of Rho/ROCK pathway. Methods: Forty male SD rats were randomly divided into four groups: type 2 diabetes mellitus combined with nonalcoholic steatohepatitis (T2DM-NASH) group, T2DM-NASH + celecoxib group, control group, and control+celecoxib group. The T2DM-NASH and T2DM-NASH + celecoxib groups were fed with high-sugar and fat diet, and the control group and control + celecoxib group were fed with basal diet (25 kJ/kg). Four weeks later, streptozotocin (STZ, 30 mg/kg) was intraperitoneally injected into the NASH group and T2DM-NASH + celecoxib group to induce T2DM model, and the control group and control + celecoxib group were intraperitoneally injected with isovolumic citric acid-sodium citrate buffer. Four weeks after STZ injection, the T2DM-NASH + celecoxib group and the control + celecoxib group were gavaged with celecoxib (10 mg·kg·d) dissolved in normal saline for 4 weeks, and the remaining two groups of rats were gavaged with isovolumic normal saline for 4 weeks. Animals were sacrificed at the end of the 12- weeks, and the liver tissue was collected. Liver pathological changes were observed by HE staining. The expressions of RhoA, RhoA, ROCK1 and ROCK2 proteins in liver were detected by immunohistochemistry and western blot. The expressional condition of RhoA, ROCK1 and ROCK2 mRNA in liver were detected by real-time quantitative PCR. The differences were compared between protein and mRNA expression among the groups by analysis of variance and t-test. Results: Compared with the control group and the control + celecoxib group, the liver tissue of the T2DM-NASH group and the T2DM-NASH + celecoxib group had severe steatosis, and there was partial inflammatory cell infiltration under the light microscope. The expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were significantly increased (P < 0.05) in each liver tissue, while liver steatosis was reduced to certain extent in T2DM-NASH + celecoxib group than T2DM-NASH group, and the expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were decreased in each liver tissue of T2DM-NASH group (P < 0.05). Conclusion: The selective cyclooxygenase-2 enzyme inhibitors celecoxib has a protective effect on the liver of rats with T2DM-NASH, and its effect may be achieved by inhibiting the expression of Rho/ROCK pathway.

Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin, filamin A and cortactin in activated hepatic stellate cells / 中华肝脏病杂志

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.

Research advances in hepatic fibrosis related signal pathways and anti-hepatic fibrosis drugs / 临床肝胆病杂志

Hepatic fibrosis is a pathological process in which the liver is subjected to various acute and chronic injuries for a long time, resulting in activation of hepatic stellate cells, the imbalance between the production and degradation of extracellular matrix, and the deposition of extracellular matrix in the liver, and it is jointly controlled by multiple cellular signal transduction pathways and a series of cellular information molecular networks. If there is no effective treatment, with the progression of the disease, liver fibrous nodules will form, destroy normal liver structure and function, and finally develop into liver cirrhosis, the decline of liver function, and even liver cancer. This article summarizes the research advances in the signaling pathways, receptors, and non-coding RNAs involved in liver fibrosis and the corresponding anti-hepatic fibrosis drugs/molecules.

Effects of sodium arsenite on liver fibrosis and expression of epithelial-mesenchymal transformation-related proteins in SD rats / 环境与职业医学

Background Long-term exposure to sodium arsenite leads to its accumulation in the liver and liver injury as a result. Previous studies showed that mesenchymal cells play an important role in hepatic fibrosis, and epithelial-mesenchymal transformation (EMT) is considered to be a main source of mesenchymal cells. Objective To investigate the effects of sodium arsenite at different doses on liver fibrosis and EMT-related protein expressions in SD rats. Methods Twenty-four healthy weaned SD rats, half male and half female, were randomly divided into four groups according to body weight, with 6 rats in each group. The four groups were control group (gavage with 10.0 mL·kg−1 physiological saline), 2.5 mg·kg−1 sodium arsenite group, 5.0 mg·kg−1 sodium arsenite group, and 10.0 mg·kg−1 sodium arsenite group. All rats were gavaged 6 d per week for 36 weeks and weighed once a week, the serum and liver tissues of rats were collected and weighed, then the organ coefficient was calculated. Hematoxylin-eosin staining and Masson's trichrome staining were used to determine the pathological changes of hepatic fibrosis in rats. The serum secretion levels of hyaluronic acid (HA), laminin (LN), procollagen Ⅲ N-terminal propeptide (PⅢNP), and collagen Ⅳ (COL-Ⅳ) in rats were detected by enzyme-linked immunosorbent assay (ELISA). The protein expressions of HSCs activation-related proteins, such as α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1), as well as EMT-related markers, such as E-cadherin, N-cadherin, Vimentin, and Snail, were detected by Western blotting. Results Compared with the control group, the 10.0 mg·kg−1 sodium arsenite group showed decreased body weight (P<0.05) and increased liver coefficient (P<0.05) of female and male rats. The pathological staining showed that, compared with the control group, a large number of inflammatory cells were observed in liver tissue of rats exposed to sodium arsenite, liver parenchymal cells were also liquefied, necrotic, and denatured, and the collagen positive staining area of liver tissue showed an upward trend along with the increase of arsenic exposure dose (P<0.05). The results of ELISA and Western blotting showed that the serum secretion levels of HA, LN, PⅢNP, and COL-Ⅳ in the 5.0 and 10.0 mg·kg−1 sodium arsenite groups were higher than those in the control group and the 2.5 mg·kg−1 sodium arsenite group (P<0.05). Compared with the control group, the expressions of α-SMA and TGF-β1 proteins in liver tissue were increased in each sodium arsenite exposure group (P<0.05), the expression levels of E-cadherin protein were decreased (P<0.05), and the expression levels of N-cadherin, Vimentin, and Snail were increased (P<0.05). Conclusion Sodium arsenite exposure can induce HSCs activation and liver fibrosis injury in SD rats, resulting in increased extracellular matrix secretion levels, accompanied by EMT in liver tissue, suggesting that EMT is closely related to the process of liver fibrosis caused by arsenic.

Liver structural injury in leptin-deficient (ob/ob) mice: lipogenesis, fibrogenesis, inflammation, and apoptosis / Lesión estructural del hígado en ratones con deficiencia de leptina (ob/ob): lipogénesis, fibrogénesis, inflamación y apoptosis

SUMMARY: Nonalcoholic fatty liver disease (NAFLD) might progress the steatosis to nonalcoholic steatohepatitis (NASH), reaching a cirrhosis state and possibly hepatocellular carcinoma. The liver of three-month-old C57BL/6J mice (wild-type, WT group, n=10) and leptin- deficient obese mice (ob/ob group, n=10) were studied, focusing on the mechanisms associated with the activation of the hepatic stellate cells (HSCs) and pro-fibrogenesis. The obese ob/ob animals' liver showed steatosis, increased lipogenesis gene expressions, inflammation, increased pro-inflammatory gene expressions, inflammatory infiltrate, and potential apoptosis linked to a high Caspase 3 expression. In ob/ob mice, liver sections were labeled in the fibrotic zones by anti-alpha-smooth muscle actin (alpha-SMA) and anti-Reelin, but not in the WT mice. Moreover, the alpha-SMA gene expression was higher in the ob/ob group's liver than the WT group. The pro-fibrogenic gene expressions were parallel to anti- alpha-SMA and anti-Reelin immunofluorescence, suggesting HSCs activation. In the ob/ob animals, there were increased gene expressions involved with lipogenesis (Peroxisome proliferator-activated receptor-gamma, Cell death-inducing DFFA-like effector-c, Sterol regulatory element-binding protein-1c, and Fatty acid synthase), pro-fibrogenesis (Transforming growth factor beta1, Smad proteins- 3, Yes-associated protein-1, Protein platelet-derived growth factor receptor beta), pro-inflammation (Tumor necrosis factor-alpha, and Interleukin-6), and apoptosis (Caspase 3). In conclusion, the results in obese ob/ob animals provide a clue to the events in humans. In a translational view, controlling these targets can help mitigate the hepatic effects of human obesity and NAFLD progression to NASH.

RESUMEN: La enfermedad del hígado graso no alcohólico (HGNA) puede progresar de la esteatosis a esteatohepatitis no alcohólica (ENA), alcanzando un estado de cirrosis y posiblemente carcinoma hepatocelular. Se estudió el hígado de ratones C57BL / 6J de tres meses de edad (tipo salvaje, grupo WT, n = 10) y ratones obesos con deficiencia de leptina (grupo ob/ob, n = 10), centrándose en los mecanismos asociados con la activación de las células estrelladas hepáticas (HSC) y profibrogénesis. El hígado de los animales obesos ob/ob mostró esteatosis, aumento de la expresión génica de la lipogénesis, inflamación, aumento de la expresión génica proinflamatoria, infiltrado inflamatorio y posible apoptosis ligada a una alta expresión de Caspasa 3. En ratones ob/ob, las sec- ciones de hígado se marcaron en las zonas fibróticas con anti-alfa- actina de músculo liso (alfa-SMA) y anti-Reelin, pero no en los ratones WT. Además, la expresión del gen alfa-SMA fue mayor en el hígado del grupo ob/ob que en el grupo WT. Las expresiones génicas profibrogénicas fueron paralelas a la inmunofluorescencia anti-alfa-SMA y anti-Reelin, lo que sugiere la activación de las HSC. En los animales ob/ob, hubo un aumento de las expresiones génicas involucradas con la lipogénesis (receptor activado por proliferador de peroxisoma gamma, efector c similar a DFFA inductor de muerte celular, proteína de unión al elemento regulador de esterol-1c y sintasa de ácidos grasos), pro-fibrogénesis (factor de crecimiento transformante beta 1, proteínas Smad-3, proteína-1 asociada a Yes, receptor beta del factor de crecimiento derivado de plaquetas de proteínas), proinflamación (factor de necrosis tumoral alfa e interleucina-6) y apoptosis (caspasa 3). ). En conclusión, los resultados en animales obesos ob/ob proporcionan una pista de los eventos en humanos. Desde un punto de vista traslacional, el control de estos objetivos puede ayudar a mitigar los efectos hepáticos de la obesidad humana y la progresión de HGNA a ENA.

Role of autophagy in liver fibrosis / 临床肝胆病杂志

Autophagy refers to the process in which organelles and proteins in eukaryocytes are degraded in lysosomes and their degradation products are reused, and it plays an important role in cell proliferation, differentiation, and homeostasis. In recent years, the role of autophagy in liver fibrosis has attracted more and more attention, and intervention of autophagy may become a new method for the treatment of liver fibrosis. This article summarizes the process and function of autophagy and its role in liver fibrosis. These data reveal the complex mechanism of action of autophagy in liver fibrosis and point out the need to find more reliable and definite mechanisms and targets for autophagy intervention in the future, so as to provide new ways for the treatment of liver fibrosis.

Effects of persistent Echinococcus multilocularis infections on hepatic fibrosis in mice / 中国血吸虫病防治杂志

Objective To investigate the effects of persistent Echinococcus multilocularis infections on hepatic fibrosis in mice, so as to provide insights into the understanding of liver fibrogenesis induced by E. multilocularis infections and the treatment of alveolar echinococcosis. Methods Hepatic stellate HSC-T6 and LX-2 cells were exposed to the sera (25, 50 and 100 μL) from Meriones unguiculatus infected with E. multilocularis, and E. multilocularis, germinal layer cells (GCs) and protoscoleces (PSCs) for 48 hours, respectively. The cell proliferation was measured using a CCK-8 assay, and the levels of collagen 1 (Col1) and α-smooth muscle actin (α-SMA) were measured in the culture supernatant of HSC-T6 cells using ELISA. In addition, the serum and liver samples were collected 1, 2, 4, 6, 8 months post-infection with E. multilocularis, respectively. The serum Col1 and α-SMA concentrations were measured using enzyme-linked immunosorbent assay (ELISA), and the deposition of collagen fibers was examined in mice livers using Sirius red staining. Results The sera of E. multilocularis-infected gerbils promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences seen in the proliferative rate of HSC-T6 (FHSC-T6 = 126.50, P < 0.05) and LX-2 cells (FLX-2 = 201.50, P < 0.05) among different serum groups, with the highest proliferative rate of HSC-T6 (573.36% ± 206.34%) and LX-2 cells (940.38% ± 61.65%) found following exposure to 100 μL mouse sera. Exposure to serum from E. multilocularis-infected gerbils resulted in an increase in the Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, with the greatest Col1 (20.99 ng/mL ± 2.01 ng/mL) and α-SMA levels (305.52 pg/mL ± 16.67 pg/mL) measured following exposure to 100 μL sera. The metacestodes (142.65% ± 9.17% and 189.99% ± 7.75%), GCs (118.55% ± 8.96% and 122.54% ± 0.21%) and PSCs of E. multilocularis (156.34% ± 17.45% and 160.59% ± 31.41%) all promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences in the proliferative rates of HSC-T6 (FHSC-T6 = 11.24, P < 0.05) and LX-2 cells among groups (FLX-2 = 47.72, P < 0.05). Exposure to E. multilocularis resulted in an increase in Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, and the highest Col1 (4.43 ng/mL ± 2.23 ng/mL) and α-SMA levels (285.20 pg/mL ± 90.67 pg/mL) were detected following treatment with E. multilocularis metacestodes. In addition, a persistent increase was seen in the deposition of collagen fibers in mice livers 1 to 8 months post-infection with E. multilocularis, with the greatest Col1 level (280.26 ng/mL ± 23.04 ng/mL) seen 6 months post-infection and the highest α-SMA level (33.68 ng/mL ± 4.45 ng/mL) detected 8 months post-infection, respectively. Conclusions Persistent E. multilocularis infections promote hepatic stellate cell proliferation, induce an increase in mouse serum Col1 and α-SMA levels, and cause elevated deposition of collagen fibers in mice livers. The infective stage of E. multilocularis is a critical period for inducing hepatic fibrosis of alveolar echinococcosis.

Regulatory effects of microRNAs on hepatic stellate cell activation and liver fibrogenesis / 中华肝脏病杂志

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.

Research advances in drug carrier systems targeting hepatic stellate cells / 临床肝胆病杂志

Hepatic stellate cells (HSCs) are an important target for the treatment of liver fibrosis. Safe and effective targeted delivery of therapeutic agents to HSCs, improvement of drug therapeutic effect, and reduction of toxic and side effects of off-target drugs are important measures for the development of anti-liver fibrosis drugs. A number of protein markers are elevated in activated HSCs, and thus their ligands are used for the specific delivery of anti-fibrotic drugs. This article summarizes the research advances in the treatment of liver fibrosis by targeting HSCs from the aspects of the type of drug carriers and target receptors.

miR-214 promotes the progression of liver fibrosis by activating hepatic stellate cells / 上海交通大学学报（医学版）

Objective • To explore the biological role of miR-214 in the activation of hepatic stellate cells (HSCs) and the procession of liver fibrosis and its possible mechanism. Methods • Quantitative real-time PCR (qPCR) was used to detect the expression of miR-214 in the activation of HSCs and the progression of liver fibrosis in rats induced by CCl4 (liver fibrosis model). HSC-T6 cells were treated with lentivirus infection and divided into miR-214 overexpression group, miR-214 knockout group and negative control lentivirus group (mock group). qPCR and Western blotting were used to detect the gene and protein expression levels of collagen type 1 (COL1) and α-smooth muscle actin (α-SMA) in the three groups, respectively. Transwell assay and flow cytometry were used to detect the migration and apoptosis of HSCs in the three groups, respectively. Double luciferase reporter gene assay was used to detect weather Hif1an was the target gene of miR-214, and then qPCR and Western blotting were used to detect the expression levels of HIF1AN in the three groups, the activation of HSCs and the liver fibrosis model. Results • miR-214 was upregulated during HSCs activation and the progression of liver fibrosis (both P<0.05). Compared with the mock group, the gene and protein expressions of COL1 and α-SMA in the miR-214 overexpression group were increased, and HSCs migration ability was increased and apoptosis rate was decreased (all P<0.05); the expressions of COL1 and α-SMA in the miR- 214 knockdown group were decreased, and HSCs migration ability was decreased (all P<0.05). Double luciferase reporter gene assay showed that Hif1an was the target gene of miR-214. Compared with the mock group, the gene and protein expressions of HIF1AN in the miR-214 overexpression group were decreased (both P<0.05), and those in the miR-214 knockdown group were increased (both P<0.05). The expression of HIF1AN was decreased during HSCs activation and the progression of liver fibrosis (all P<0.05). Conclusion • miR-214 may promote the migration and activation of HSCs by targeting Hif1an, and then promote the progression of liver fibrosis, suggesting that miR-214 may be a new marker and potential target for the treatment of liver fibrosis.

Endoplasmic reticulum stress-induced apoptosis up-regulation of DR5 via a CHOP-dependent mechanism in HSC-T6 cells / 中国临床药理学与治疗学

AIM: To investigate the inhibitory effects of endoplasmic reticulum stress(ERS) and TRAIL on hepatic stellate cells in vitro and how their interaction affect the apoptosis of hepatic stellate cells. METHODS: Take thapsigargin (TG) as the endoplasmic reticulum stress-inducing agents, ursodeoxycholic acid (UDCA) for the endoplasmic reticulum stress inhibitors, SP600125 as a c-Jun N-terminal kinase(JNK) inhibitor, HSC-T6 cells were divided into normal control group, DMSO group, TRAIL group, TG group, UDCA group, siCHOP group and SP600125 group. The apoptosis rate of HSC-T6 cell was detected by flow cytometry. Small interference RNA was applied to silence C/EBP homologous protein(CHOP) gene. The protein expression levels of Caspase-8 were detected by immunohistochemistry method. The ERS marker protein CHOP and TRAIL receptor DR5 expression levels were determined by RT-PCR and Western blot. RESULTS:TG (1 μmol/L, 2 μmol/L, 4 μmol/L, 8 μmol/L, 16 μmol/L) increased cell apoptosis rate of HSC-T6. RT-PCR and Western blot showed that the endoplasmic reticulum stress protein marker CHOP could induce the upregulation of TRAIL receptor DR5 and Caspase-8. Moreover, siCHOP and the JNK inhibitor SP600125 could reduce the expression of DR5 and Caspase-8 in HSC cells. CONCLUSION: These results indicated that CHOP and JNK may be a potential factor regulating DR5 expression, and play an important role in the process of apoptosis of hepatic stellate cells.

Research progress in tumor microenvironment of pancreatic cancer / 中华消化外科杂志

Pancreatic cancer is an aggressive malignant tumor with poor prognosis. On the one hand, it has a narrow therapeutic window due to the lack of specific markers and obvious clinical symptoms. Once diagnosed, it has often developed to an advanced stage. On the other hand, located in a vital region of the body, pancreatic operation is difficult and the postoperative recurrence rate is high. Therefore, surgical treatment is only sui-table for a small number of early patients. Pancreatic cancer has a tumor microenvironment with the characteristic of dense stroma, hypoxia, paucity of blood vessels and highly immunosuppression. It is often insensitive to traditional radiation and chemotherapy. Therefore, strategies targeting on tumor microenvironment have a potential prospect. This article reviews the research progress in tumor microenvironment of pancreatic cancer, in order to provide the references in the further research and treatment of pancreatic cancer.