Curcumol Alleviates Liver Fibrosis With Blood-stasis Syndrome In Rats And LSEC Ultrastructure Changes In Vivo And In Vitro

Background & Aims: Previous studies have confirmed that liver fibrosis is reversible,but there is a lack of effective and safety drug for liver fibrosis.This study established the liver fibrosis with blood-stasis syndrome in rats model for investigating the therapeutic effect of curcumol on blood-stasis of hepatic fibrosis in rats,for addressing the effects of curcumol on ultrastructure of liver sinusoidal endothelial cells(LSEC)in vivo and in vitro,and for exploring the mechanism of action of curcumol anti liver fibrosis.Methods:(1)Established liver fibrosis with blood-stasis syndrome model in rats and isolated,cultured and identified rat primary LSEC.(1)Established liver fibrosis with blood-stasis syndrome model in rats.Rat model of liver fibrosis with blood-stasis syndrome was induced by norepinephrine plus bovine serum albumin plus ethanol plus carbon tetrachloride(CCl4).In the first 3 weeks,the rats were injected subcutaneously with 40%CCl4 peanut oil mixture at a dose of 0.3 mL/100 g,2times weekly.In the fourth week,the rats were treated with 50%CCl4 peanut oil mixture in the same way for a total of 5 weeks.From the second week,the rats were injected with 0.1 g/L of norepinephrine into the tail vein,once a day.The rats were administrated for a period of 3 weeks.At the beginning of the first day and the first 10 days,the rats were injected with 50 g/L calf serum albumin by cauda vein.10% ethanol was administered to rats.The blood-stasis syndrome score scale was used to observate the establishment ofblood-stasis syndrome in rats model.HE staining and Masson staining detection were used to investigate the liver tissue fibrosis lesions.Fully automatic biochemical analyzer was performed to detect serum liver function(ALT and AST)and liver fibrosis four indexes(HA,LN,P?NP,Col-?)levels in rats,all were for comprehensively observating whether the model replication was successful.(2)Isolated,cultured and identified rat primary LSEC.LSECs were isolated by collagenase perfusion,Percoll density gradient sedimentation method and cell selective adherence method.And identified by scanning electron microscopy and di-Ac-LDL immunofluorescence staining analysis.(2)Effects of curcumol on liver fibrosis with blood-stasis syndrome in vivo.The experiment was divided into 6 groups,including normal control group,liver fibrosis with blood-stasis syndrome model group,colchicine group,salvianolic acid B group,curcumol in low dose group and high dose group.Norepinephrine,bovine serum albumin,ethanol and carbon tetrachloride(CCl4)were used to induce rat model of liver fibrosis with blood-stasis syndrome.After establishing the model,we used drugs to intervene.The expression of ET-1 protein was determined by enzyme-linked immunosorbent assay(ELISA).Scanning electron microscopy and transmission electron microscopy detection were used to observe the ultrastructural changes of LSECs in liver tissue.And the liver index,blood stasis syndrome score,pathological examination(HE staining and Masson staining),serum liver function and liver fibrosis four indexes level were also determinated to illustrate the roles of curcumol on liver fibrosis with blood-stasis syndrome.(3)Curcumol alleviated LSEC ultrastructure changes in vitro.The activated LSECs induced by leptin,were treated with different concentrationof curcumol.The ultrastructural changes of LSECs were viewed by scanning electron microscope and transmission electron microscope.All were for understanding the effect of curcumol on activated LSECs induced by leptin.Results:(1)The results of establishment of liver fibrosis with blood-stasis syndrome model in rats and isolation,culture and identification of rat primary LSEC.(1)The results of establishment of liver fibrosis with blood-stasis syndrome model in rats.Rats' hair was sparse and dull.Dark red or purple tongue with ecchymosis,varices in tongue base and tail ecchymosis found in the rats demonstrated that blood-stasis syndrome formed markedly,blood-stasis syndrome score was significantly higher than the control group(P < 0.01).The rats represented hepatomegaly and their dark purple liver,which were surfaced with tiny particles,had internal nodules.The liver tissue were found obvious fibrosis lesions as shown by HE staining and Masson staining detection.Liver index,serum ALT,AST and liver fibrosis four indexes level were all higher than the control group(P < 0.05 or P < 0.01).(2)The results of isolation,culture and identification of rat primary LSEC.The isolated and cultured LSEC grew well and the had typical morphology.The cells grew regularly,from round,ovoid to fusiform growth.The surface of the cells has unique pore(fenestrae)structure and multiple fenestrae arranged in a sieve plate like structure as shown by scanning electron microscopy detection.All together showed that the isolated and cultured LSEC were high purity.(2)The results of effects of curcumol on liver fibrosis with blood-stasis syndrome in vivo.Curcumol reduced blood-stasis syndrome score,liver index,serum liver function ALT,AST and liver fibrosis four indexes level(P< 0.05 or P < 0.01).Pathology detection(HE and Masson staining)resultsshowed that after treatment with curcumol,the rats' liver fiber damage significantly ameliorated.Curcumol remarkably reduced degree of liver fibrosis compared with model group(P < 0.05)by the degree of liver fibrosis semi-quantitative analysis.According to the results of scanning electron microscopy(sem)and transmission electron microscopy(tem),curcumol increased the numbers of the LSEC fenestrae and made the continuity of basement membrane disappear.ET-1 protein expression in model group was increased significantly compared with normal control group(P < 0.01),curcumol down regulated the expression of ET-1(P < 0.01)by ELISA.The effects of curcumol,colchicine and salvianolic acid B on treating liver fibrosis were almost the same,except for the blood stasis syndrome and LSEC fenestrae in fibrotic liver tissue.Curcumol and salvianolic acid B had better effects on improving blood stasis syndrome than colchicine.In terms of increasing LSEC fenestrae,curcumol worked best compared with colchicine and salvianolic acid B.(3)Curcumol alleviated LSEC ultrastructure changes in vitro.The activated LSEC induced by leptin showed that the number of fenestrae significantly reduced or even disappeared,the diameter of fenestrae was smaller than control group,representing "defenestration" phenomenon analyzed by scanning electron microscopy and transmission electron microscopy detection.After treatment with curcumol,the number of pore increased markedly,the pore became bigger compared with leptin activation model group.Conclusions: Curcumol significantly ameliorates liver fibrosis with blood-stasis syndrome,its mechanism is related to the down-regulation of ET-1 protein expression and alleviation of hepatic sinusoidal capillarization.Curcumol plays a positive role on pathological changes of LSECultrastructure,including open fenestrae,in vivo and in vitro.These findings will provide an important way for the prevention and treatment of liver fibrosis.