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Objective To study the effect and mechanism of pearl hydrolysate on hepatic sinusoidal capillarization in liver fibrosis. Methods Hepatic sinusoidal endothelial cells (HSEC) and hepatic stellate cells (HSC-LX2) were incubated with Hepu pearl hydrolysate.The proliferation of HSEC and HSC-LX2 was examined by MTT colorimetry.The cell cycle and apoptosis of HSC-LX2 were measured by flow cytometry.The changes of the microstructures such as fenestra and basement membrane of HSEC were observed by transmission electron microscopy. Results The intervention with leptin increased the viability of HSC-LX2 (P=0.041),decreased the viability of HSEC (P=0.004),and caused capillarization signs such as decreased number and diameter of fenestrae and formation of continuous basement membrane.The treatment with pearl hydrolysate at different doses increased and expanded the fenestrae of HSEC (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032),disintegrated the extracellular basement membrane of HSEC (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032),decreased the viability of HSC-LX2 (low dose:P=0.018;medium dose:P=0.013;high dose:P=0.009),and induced the apoptosis of HSC-LX2 (low dose:P=0.012;medium dose:P=0.006;high dose:P=0.005).Pearl hydrolysate exerted therapeutic effect on capillarization in a dose-dependent manner (low dose:P=0.020;medium dose:P=0.028;high dose:P=0.032).Moreover,high-dose pearl hydrolysate showed stronger effect on capillarization of hepatic sinuses than colchicine (P=0.034) and salvianolic acid B (P=0.038). Conclusion Hepu pearl hydrolysate can increase the viability of HSEC,restore the area of fenestrae,disintegrate the basement membrane,and decrease the viability and induce the apoptosis of HSC-LX2,demonstrating significant pharmacological effects on the capillarization of HSEC and HSC-LX2.
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Humanos , Células Endoteliales/metabolismo , Cirrosis Hepática , Hígado/patologíaRESUMEN
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.
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Animales , Ratones , Peso Corporal , Medicamentos Herbarios Chinos/efectos adversos , Células Endoteliales , Hígado , Cirrosis Hepática/tratamiento farmacológicoRESUMEN
[ABSTRACT]AIM:Toexplorethedevelopmentofhepaticsinusoidalcapillarizationintheearlystageofliverfi-brosis induced by carbon tetrachloride (CCl4) in rats.METHODS:Clean SD rats were randomly divided into normal con-trol group (group N, n=6) and liver fibrotic model group (group M, n=32).The rats in group N were intraperitoneal in-jected with saline and the rats in group M were intraperitoneal injected with CCl 4(2 mL/kg, twice a week for 4 weeks).At the end of the 3rd day and the 1st, 2nd and 4th weeks, all rats were killed and then the samples were collected .The patho-logical changes in the livers were observed by HE staining and Masson straining .The development of hepatic sinusoidal capillarization was observed by transmission electron microscopy (TEM) and immunohistochemical staining .The cell sur-face expression of vascular endothelium-associated marker CD31, collagen type Ⅳ(Col IV) and laminin (LN) was deter-mined.RESULTS:HE and Masson staining showed the formation of liver fibrosis after treatment with CCl 4 for 4 weeks. TEM showed that the fenestrate diameter of liver sinusoidal endothelial cells (LSECs) grew down, the fenestrate numbers of LSECs were decreased along with the development of liver fibrosis , and the consecutive basement membrane was formed at the end of the experiment .The expression of CD31 was significantly increased along with the development of defenestration , and the expression of Col IV and LN was significantly increased after the treatment with CCl 4 for 2 weeks and 4 weeks , re-spectively .CONCLUSION:The typical hepatic sinusoidal capillarization was detected in the early stage of liver fibrosis , and the deposition of LN in the liver sinusoidal walls was the mainly factor of formation of the consecutive basement mem -brane .
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Objective To investigate the role of cyclooxygenase-2 (COX-2) in sinusoidal capillarization in liver cirrhotic rats. Methods The SD rats were intraperitoneally injected with carbon tetrachloride (CCl4) twice a week for 8 weeks to induce liver cirrhosis. The rats were randomly divided into three groups: normal control group (n= 10), model control group (n= 15) and rofecoxib treated group (received 10 mg/kg of rofecoxib daily, n = 15). Liver histopathology was examined by light microscopy, and sinusoidal ultrastructure was observed by transmission electron microscopy. Furthermore, the level of basement membrane proteins (collagen type Ⅳ, laminin) and their localizations in liver were determined by Western blotting and immunohistochemistry, respectively, and the microvessel density was detected following vWF (yon Willebrand factor) immunolabeling on liver tissue sections. Results Fibrotic areas were reduced in rofecoxib treated group compared with that in model group (30.7±8.9 vs 23.5±6.5,P<0. 05). The light and electron microscopy showed that the pathologic changes including loss or reduction in number of sinusoidal endothelial fenestrate, the accumulation of extracellular matrix in the Disse's space and development of subendothelial basal lamina (basement membrane formation) were more severe in model group than those in rofecoxib treated group. Compared with model group, administration of rofecoxib resulted in significant decrease in microvessel density (11.3 ± 1.6 vs. 6.4 ±0. 7, P<0. 01). Rofecoxib could significantly decrease the expression of type Ⅳ collagen and laminin at protein levels (3.0±0.5 and 3.0±0.5, respectively) when compared with model group (3.8±0.4 and 3.7±0. 5, respectively). Conclusion The results indicate that early administration of rofecoxib may reduce sinusoidal capillarization.
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Objective To study the relationship between expression of cyclooxygenase-2 (COX-2) and vas-culax endothelial growth factor (VEGF) with liver sinusnidal capillarization (LSC) of chronic hepatitis B (CHB). Methods We studied liver biopsies from 200 patients with CHB COX-2 ,VEGF immunohistochemical stain were ob-served to accomplish relationship between expression of COX-2,VEGF and LSC. Results LSC occupy above 80% in the group. There were manifestation in mild-LSC (focal) , middle-LSC (sheet-shape) and severe-LSC (widespread). Electron microscope shown the laceration in the endothelium of sinuses and formation of basal lamina and budding for-mation lumen of blood vessel and fat-storing cell convert myofibroblast. Expression of COX-2, VEGF, Co-Ⅳ and retic-ulum, collagen and elastic fibers with mild or severe in LSC is manifest locking relate. Conclusion Increased ex-pression of COX-2 ,VEGF in liver tissue of CHB may facilitate LSC and hepatic fibrosis.
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Objective To study the relationship between the liver sinusoidal pathological changes and portal hypertension during the dimethylnitrosamine (DMN) induced liver fibrosis in rats. Methods The rat liver fibrosis model was established by peritoneal injection of DMN (at a dose of 10 mg/kg, 3 times a week, for 4 weeks) in 40 male Wister mice. In the control group of 24 healthy mice, saline solution was injected peritoneally. The dynamic changes of liver fibrosis were observed at different time points (1, 2, 3 day, 1, 2, 4, 6 and 8 week). The pressure of portal vein (Ppv) was directly measured by the intubation of mesentery anterior vein in 5 mice of the model and 3 of the control. The expressions of type Ⅳ collagen (Col Ⅳ), laminin (LM) and type Ⅰ collagen (Col Ⅰ) were detected by immunohistochemistry. Hepatic ultrastructure was observed by electron microscopy. Results Positive expression of Col Ⅳ was observed in the normal liver sinusoidal walls. The various intensities of positive staining of Col Ⅳ were observed in the liver sinusoidal walls of the fibrosis model. Positive expressions of LM and Col Ⅰ increased, and the strongest positive staining displayed in the 4 week model rats. Meanwhile, the fenestrae in the sinusoidal endothelial cells (SECs) were lost and the basal membrane was formed. There was a remarkable positive correlation between the Ppv and the expressions of LM in the DMN induced rat fibrosis( P