| Liver Fibrosis(Hepatic fibrosis,HF) is the accumulation of extracellular matrix,or scar,in response to acute or chronic liver injury.The accumulation of ECM proteins distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of nodules of regenerating hepatocytes defines cirrhosis.The imbalance between synthesis and degradation of extracellular matrix can form HF,and the hepatic stellate cells(HSC) is the major source of extracellular matrix cell in this process,which plays central role in the development of HF.Silymarin(silymarin,SL),extracted from the seeds of silymarin,have been refined to extract the mixture of flavonoids,of which silybin has the richest content,the highest activity and its pharmacological effect is the strongest, but Silybin is insoluble in water,poor oral absorption,low bioavailability, all those will affect its clinical application.Therefore,the research to develop its new dosage form both home and abroad are very actively,this experiment was based on a new type of drug delivery systems(NDDS) to made a long-term targeting agents,use lactic acid - glycolic acid copolymer[poly(lactic-co -glycolic acid),PLGA]as a carrier and polymer as materials,to produce Silybin PLGA microspheres targeting to the liver with long-term freeze-dried preparations, not only targeting to the liver,but also can slow the release of drug,which may resolve the problem of patients' needs to take drug every day.Liver fibrosis is induced by carbon tetrachloride(CCl4) and alcohol.All rats are divided into seven groups including silybin microsphere 50mg/kg 25mg/kg and 12.5mg/kg group,silybin meglumine injection group(25mg/kg),and silybin capsule group(25mg/kg),with the angle of liver function, pathomorphological observation of liver tissue and ECM-related components to observe the effect with silybin microsphere。Manufacturing model:40%CCl4(dissolved in peanut oil,s.c.,3ml/kg,twice a week) were administrated,10%Kyoto Erguotou as drinking water for twelve weeks.All treatment began with the first day of manufacturing model.Body weight were measured every two weeks,and keeping all the animals with noraml feeding.Rats from all groups were sacrisfied respectively at eighth and twelfth week and serum criteria were detected,which relevant with liver function and the serum levels associated to the public hyaluronic acid(HA),laminin(LN), as well as the content of hydroxyproline(Hyp),the expression ofα-smooth muscle actin(α-SMA),transforming growth factorβ1(TGF-β1), platelet-derived growth factor-BB(PDGF-BB),connective tissue growth factor (CTGF) in liver tissue,and describe the liver tissue morphological characteristics of pathological.1 The effects of Silybin microsphere injection on biochemical indicators of liver function in liver fibrosis rats1.1 The effects of Silybin microsphere injection on ALT,AST in serum of liver fibrosis ratsResults showed that:the content of ALT,AST in serum of the model group were significantly higher(P＜0.001),After the 8w and 12w administration, compared to the model group,all the treatment group can lower the content of ALT,AST in serum significantly(P＜0.05 or P＜0.001).After 8w administration, compared with silybin meglumine injection and Silybin capsule group,silybin microspheres 50mg/kg and 25mg/kg group were significantly lower in ALT levels (P＜0.01 or P＜0.001)。After 12w treatment,there is no significant difference among the treatment group.1.2 The effects of Silybin microsphere injection on ALP in serum of liver fibrosis ratsResults showed that:the content of ALP in serum of the model group was significantly increased(P＜0.001).Compared with model group,after treatment of 8w,silybin microspheres 50mg/kg group can significantly reduce the content of ALP(P＜0.05).However,after 12w treatment,the content of ALP in serum in each group of silybin microsphere were significantly lower than silybin capsule group.1.3 The effects of Silybin microsphere injection onγ-GGT in serum of liver fibrosis ratsThe results showed that:the content ofγ-GGT in serum of the model group was significantly increased(P＜0.001).Compared with model group,after 8w administration,silybin microspheres 50mg/kg and 25mg/kg group were significantly decreased(P＜0.01 or P＜0.001).After 12w administration,compared to the Silybin capsule group,the decrease in each of the Silybin microspheres groups are more significant]y(P＜0.05 or P＜0.01).1.4 The effects of Silybin microsphere injection on thecontent of TP in serum of liver fibrosis ratsThe results showed:the content of TP in serum of model group is decreased significantly(P＜0.001).Compared with model group,after 8w administration, various doses group of silybin microspheres was significant increased the TP content in serum than model group and silybin meglumine injection group(P＜0.01 or P＜0.001).After 12w administration,all dose group of the Silybin microspheres content TP can be increased,but had no significant difference compared with the model group.Synthesis and liver function-related biochemical indicators of the five test results showed that silybin microspheres can protect the liver cells, improve liver function,thereby delaying the occurrence of liver fibrosis.2 the effects of Silybin microsphere injection on ECM-related components2.1 The effects of Silybin microsphere injection on the content of HA in serum of liver fibrosis ratsThe results showed that:the content of HA in the liver tissue of rats in model group was significantly increased(P＜0.001).ffter 8w treatment,silybin microsphere 50mg/kg group was lower in serum HA levels than model group.Compared with silybin methylamine injection,after administration of 12w,.all dose groups of silybin microsphere had a high level of HA(P＜0.001).2.2 The effects of Silybin microsphere injection on the content of LN in serum of liver fibrosis ratsThe results showed that:the content of LN in the liver tissue of rats in model group was significantly increased(P＜0.001).Compared with model group, after 8w administration,all groups had no significant difference;and after 12w,we can see the serum content LN in Silybin 50mg/kg microspheres group was significantly lower than that of model group,silybin methylamine injection and capsule groups(P＜0.05).2.3 The effects of Silybin microsphere injection on the content of Hyp in hepatic tissue of liver fibrosis ratsThe results showed:the content of Hyp in the liver tissue of rats in model group was significantly increased(P＜0.001).Compared with model group,after 8w administration,the Hyp content in liver tissue of rats in silybin microspheres 50mg/kg group was significant decreased(P＜0.01).After 12w administration,the Hyp content in liver tissue of rats in Silybin microspheres 50mg/kg group,25mg/kg group,12.5mg/kg group,compared with the model group, were significantly lower(P＜0.01),and inter-group comparison had no significant difference.Integrated part of this experiment the three indicators of test results, to speculate Silybin microsphere injection to liver fibrosis by inhibiting the process of extracellular matrix synthesis or promote its degradation,thus reducing the content of extracellular matrix,and ultimately can stow down the progress of liver fibrosis. 3 The study on the effect of Silybin microsphere injection on liver fibrosis -related indicators3.1 The effect of Silybin microsphere injection on the content ofα-SMA in liver fibrosis ratsResults showed that:the content ofα-SMA in liver tissue of liver fibrosis ratswas significantly increased in the model group(P＜0.001).Compared with the model group,after 8 weeks administration,the treatment groups were significantly lower inα-SMA content(P＜0.05或P＜0.001).And after 12 weeks treatment,each dose of the silybin microspheres had a lowerα-SMA lever than model group.3.2 The effect of Silybin microsphere injection on the content of TGF-β1 in liver fibrosis ratsThe results showed that:the content of TGF-β1 in liver tissue of model group was significantly increased(P＜0.001).Compared with model group,the treatment groups were significantly lower in liver tissue of TGF-β1 levels (P＜0.01 or P＜0.001);after 12w administration,the content of TGF-β1 in Silybin microsphere group was reduced significantly than model group and silybin capsules group(P＜0.01或P＜0.001).3.3 The effect of Silybin microsphere injection on the content of PDGF-BB in liver fibrosis ratsThe results showed that:the content of PDGF-BB in iver tissue of the model group were significantly higher(P＜0.001).After administration of 8w and 12w,Silybin microspheres 50mg/kg group,compared with model group and silybin capsules group,were lower in the content of PDGF-BB,(P＜0.05 or P＜0.001).After 12w administration,each treatment groups was more lower in the content of PDGF-BB than model group(P＜0.05).3.4 The effect of Silybin microsphere injection on the content of CTGF in liver fibrosis ratsThe results showed that:the content of CTGF in the liver tissue of the model group were significantly higher(P＜0.001).Compared with model group, after 8w administration,the treatment groups were significantly lower in liver tissue CTGF levels(P＜0.01 or P＜0.001).After 12w administration the CTGF levels of Silybin microspheres 50mg/kg and 25mg/kg group were lower than silybin meglumine injection(P＜0.01 or P＜0.001).Summing up the above four indicators of test results,speculate the anti-fibrotic mechanism of Silybin microspheres is possible by inhibiting TGF-β1,CTGF,then reduce the synthesis of extracellular matrix,and by inhibiting PDGF,then the prevent the proliferation of hepatic stellate cells.From not only one targets to influence liver fibrosis. 4 Pathomorphological observation of liver tissue4.1 Pathomorphological of visual observationThe morphology of liver in normal group showed that color is red,the edge is sharp,surface of liver is mooth and delicate;In model group,liver become swollen obviously and showing a yellow color,small nodules and irregular vascular protuberance on surface of liver,in treatment groups,there were small nodule or not obvious,slightly uneven liver surface.4.2 Pathomorphological of liver visual observationHE staining and trichrome(Masson) staining showed that histological features of hepatic tissue each group is obviously with optical microscope.HE staining showed that no obvious abnormalities features in liver tissue, hepatic lobule is integral and no degeneration and necrosis cell in liver in normal group.Trichrome staining showed that:the collagen fibers on vessel wall,no fiber interval formation.HE staining showed that structure of hepatic lobule is not integral,fatty degeneration,small duct hyperplasia in portal area,a little bile in hepatic cell.All this histological features is obvious in administration group at the end of 8 weeks.Trichrome staining showed that fibrous hyperplasia can be seen clearly in portal area,fibrous hyperplasia in hepatic lobule around the central vein more obvious,pseudolobule lobules have not yet proliferation.HE staining showed hepatic lobule damaged become more slighter,fatty degeneration become more slighter portal area slightly expanded.Trichrome staining showed that small amount of fibrous tissue hyperplasia in portal area,fibrous hyperplasia in hepatic lobule around the central vein not obvious(figure3) in each administration group.HE staining showed that cell of hepatic lobule disappearance,moderate hyperplasia of the fibrous tissue will be separated into different sizes pseudolobule,the majority of cell become fatty degeneration,focal necrosis visible,small duct hyperplasia in portal area and inflammation infiltration lymphocytes more obviously,portal area widely expanded,fiber bundle is appearance between portal area and central vein.All this histological features is the cirrhosis manifestations at early stages(figure2).Trichrome staining showed that a lot offibrous hyperplasia can be seen clearly in portal area,pseudolobule lobules have proliferation,fibrous hyperplasia will be separated into different sizes pseudolobule. HE staining showed hepatic lobule not disappearance,the moderate of cell become fatty degeneration,focal necrosis visible at some times,portal area slightly expanded,fibrous hyperplasia it is visable in hepatic lobule. Trichrome staining showed that fibrous hyperplasia is inhibited and it is not obvious fibrous hyperplasia in hepatic lobule around the central vein (figure4)Conclusion:Silybin microspheres powder injection has Protective Effect on keeping morphology and construct of liver cell,inhibit fibrous hyperplasia.After staining the hepatic,to a comparison between the normal group and the model group show us:Silybin microsphere injection can protect the liver cells,to maintain the structure of hepatic tissue,to prevent the proliferation of collagen fibers and so on.To sum up,Silybin microsphere injection has the effect close to silybin meglumine injection and capsules,such as in the reduction of the serum ALT, AST andγ-GGT level,increasing TP content and lower in serum HA,LN and liver tissue Hyp content in liver tissue,inhibiting TGF-β1,PDGF-BB,CTGF expression level in liver.So silybin microsphere attenuate liver injury,protect hepatocyte,decrease synthesis and increase degradation of ECM,which can inhibit the development of liver fibrosis,to provide experimental basis for Clinical applications and drug development.Moreover,silybin microspheres 50mg/kg showed stronger beneficial effect than silybin capsule and Silybin microsphere injection groups,which is probably based on the effect of liver targetting.All part of a comprehensive indicator of the experiment,in Silybin microspheres 50mg/kg,25mg/kg and 12.5mg/kg three doses at different time points of the contrast effect,it was proposed that the reference clinical dose is:Silybin microspheres 50mg/kg,medication once erery 15 days and consecutive 12w.Therefor,silybin microsphere,as a potent anti-liver fibrosis new drug, own a prospective future in the clinical application,further research is necessary to clearify the mechanism. |
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