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OBJECTIVE@#To screen the active components from Fuzheng Huayu Recipe (FZHY) and redesign a new recipe composed of the active components, and validate the effect of active components formulation from FZHY against liver fibrosis.@*METHODS@#Thirty-two components from FZHY were evaluated for their activities against liver fibrosis respectively, with 6 kinds of cell models in vitro, including oxidative stressed hepatocyte in L-02, hypoxia injured/proliferative hepatic sinusoidal endothelial cells in SK-HEP-1 and human hepatic sinusoidal endothelial cells (HHSEC), and activated hepatic stellate cell in LX-2. The comprehensive activity of each component against liver fibrosis was scored according to the role of original herbs in FZHY and cell functions in fibrogenesis. Totally 7 active components were selected and combined with equal proportion to form a novel active components formulation (ACF). The efficacy of ACF on liver fibrosis were evaluated on activation of LX-2 and proliferation of HHSEC in vitro and in liver fibrosis model mice induced by dimethylnitrosamine (DMN). Totally 72 mice were divided into 6 groups using a random number table, including normal, high-dose ACF control (20 µ mol/L × 7 components/kg body weight), model, low-, medium-, high-dose ACF groups (5, 10, 20 µ mol/L × 7 components/kg body weight, respectively). Hematoxylin eosin and Sirius red stainings were used to observe inflammation and fibrosis change of liver tissue; scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were utilized to observe the effect of ACF on ultrastructure of hepatic sinusoids.@*RESULTS@#Fifteen components from FZHY showed higher scores for their activity on against liver fibrosis. Among them, 7 components including tanshinone II A, salvianolic acid B, cordycepin, amygdalin, quercetin, protopanaxatriol, and schizandrin B were recombined with equal proportions to form ACF. ACF at 1,2, 4 µ mol/L showed strong inhibitory effects on activation of LX-2 and proliferation of HHSEC in vitro (all P<0.01). Compared with the model group, ACF attenuated liver collagen deposition, improved sinusoidal capillarization in a dose-dependent manner (all P<0.05).@*CONCLUSION@#ACF exerts a satisfactory effect against experimental liver fibrosis and attenuates sinusoidal capillarization, which warrant a further research and development for herbal components formulation on liver fibrosis.
Subject(s)
Animals , Mice , Body Weight , Drugs, Chinese Herbal/adverse effects , Endothelial Cells , Liver , Liver Cirrhosis/drug therapyABSTRACT
Objective To investigate the effect of liver CD8 + T lymphocytes on co-cultured hepatic stellate cells (HSCs) after the application of Fuzheng Huayu prescription in a moues model of acute liver injury, as well as the mechanism of action of Fuzheng Huayu prescription in preventing liver fibrosis. Methods A total of 18 specific pathogen-free male C57BL/6NCrl Vr mice were randomly divided into normal group, model group, and Fuzheng Huayu prescription group, with 6 mice in each group. The mice in the Fuzheng Huayu prescription group were given Fuzheng Huayu prescription for 5 days in advance. At 12 hours before the experiment, 10% CCl 4 was injected intraperitoneally at a dose of 2 mL/kg body weight. Blood was collected from the main abdominal vein, and the serum was separated to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Part of the liver was used for pathological observation. After the mice were pretreated with medication in vivo, flow cytometry was used for the sorting of mouse liver CD8 + T lymphocytes, which were then co-cultured with the mouse HSC cell line (JS 1) in a 96-well plate at a ratio of 2∶ 1, and after co-culture for 24 and 48 hours, qPCR was used to measure the changes in the mRNA expression of Col.I and α-SMA. An analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the SNK- q test or the least significant difference t -test was used for further comparison between two groups. Results The model group had significantly higher activities of ALT and AST than the normal group (both P < 0.000 1), and compared with the model group, the Fuzheng Huayu prescription group had a significantly lower degree of increase in ALT activity ( P < 0.001). HE staining showed that the Fuzheng Huayu prescription group had a significantly lower degree of hepatocyte degeneration and necrosis compared with the model group. Compared with the normal group, the total lymphocytes, CD45, CD4 - CD8 + T and CD8 + CD28 - T in the model group increased significantly, while the proportion of the above lymphocytes in the Fuzheng Huayu formula group decreased significantly compared with the model group ( P < 0.001). CD8 + T lymphocytes isolated from the liver of mice in each group were co-cultured with JS 1 for 48 hours, and compared with the control group (JS 1 cultured alone) and the normal group, the model group had a significant increase in the mRNA expression of α-SMA (both P < 0.01) and significantly higher mRNA expression of Col.I than the control group and the normal group (normal mouse liver CD8 + T lymphocytes co-cultured with JS 1) (both P < 0.001). The Fuzheng Huayu prescription group had significantly lower mRNA expression levels of α-SMA and Col.I than the model group (both P < 0.01). Conclusion Fuzheng Huayu prescription can indirectly inhibit activated HSCs by altering the functional phenotype of CD8 + T lymphocytes in mouse liver.
ABSTRACT
OBJECTIVE@#MicroRNAs (miRNAs) may be viable targets for treating renal interstitial fibrosis (RIF). Fuzheng Huayu recipe (FZHY), a traditional Chinese compound herbal medicine, is often used in China to treat fibrosis. This study sought to assess the mechanisms through which FZHY influences miRNAs to treat RIF.@*METHODS@#RIF was induced in rats by mercury chloride and treated with FZHY. Hydroxyproline content, Masson's staining and type I collagen expression were used to evaluate renal collagen deposition. Renal miRNA profiles were evaluated using a miRNA microarray. Those miRNAs that were differentially expressed following FZHY treatment were identified and subjected to bioinformatic analyses. The miR-21 target gene phosphatase and tensin homolog (PTEN) expression and AKT phosphorylation in kidney tissues were assessed via Western blotting. In addition, HK-2 human proximal tubule epithelial cells were treated using angiotensin II (Ang-II) to induce epithelial-to-mesenchymal transition (EMT), followed by FZHY exposure. miR-21 and PTEN expressions were evaluated via quantitative reverse transcription-polymerase chain reaction (qRT-PCR), while E-cadherin and α-smooth muscle actin (α-SMA) expressions were assessed by immunofluorescent staining and qRT-PCR. Western blotting was used to assess PTEN and AKT phosphorylation.@*RESULTS@#FZHY significantly decreased kidney collagen deposition, hydroxyproline content and type I collagen level. The miRNA microarray identified 20 miRNAs that were differentially expressed in response to FZHY treatment. Subsequent bioinformatic analyses found that miR-21 was the key fibrosis-related miRNA regulated by FZHY. FZHY also decreased PTEN expression and AKT phosphorylation in fibrotic kidneys. Results from in vitro tests also suggested that FZHY promoted E-cadherin upregulation and inhibited α-SMA expression in Ang-II-treated HK-2 cells, effectively reversing Ang-II-mediated EMT. We also determined that FZHY reduced miR-21 expression, increased PTEN expression and decreased AKT phosphorylation in these cells.@*CONCLUSION@#miR-21 is the key fibrosis-related miRNA regulated by FZHY. The ability of FZHY to modulate miR-21/PTEN/AKT signaling may be a viable approach for treating RIF.