| Objective: To study the effects of Shuganning on alcoholic hepatic fibrosis and to reveal its pharmacological mechanism,and to provide experimental basis for clinical application.Methods:?1?50 male Wistar rats were fed by garage with mixed liquid of alcohol-pyrazol-corn oil?once per day?to replicate the rat models of alcoholic liver fibrosis;meanwhile 10 other rats of the same batch were fed with normal saline?NS?,as the control group.After 16 weeks,survived model rats?n=40?were randomly divided into 5 groups with 8 rats in each group: a model group,a Shuganning high dose group,a Shuganning middle dose group,a Shuganning low dose group and an Anluo Huaxian Wan group.Rats of the control group and the model group were injected with 5 mL·kg-1 normal saline;rats of the Shuganning high,middle and low dose groups were injected with 4.8,2.4,and 1.2mL·kg-1 Shuganning injection,respectively;and rats of the Anluo Huaxian Wan group were given 0.75 g·kg-1 Anluo Huaxian Wan.Each group was administered once a day for 8 weeks.During the treatment,the model animals were continued with modeling.After 24 hours of the last treatment,heart blood samples were collected from the rats of each group and the blood serum was separated.The levels of alanine aminotransferase?ALT?and aspartate aminotransferase?AST?in serum were measured using the microplate method;four indexes of liver fibrosis were measured using the method of chemiluminescence detection: the serum hyaluronic acid?HA?,the laminin?LN?,the type III procollagen?PIIINP?and the type IV collagen?IV-C?.Rats were sacrificed after collecting blood,liver tissues were taken and weighed,and the liver indexes were documented.A small piece of liver tissue was taken and made into liver tissue homogenate.The malondialdehyde?MDA?and superoxide dismutase?SOD?levels were detected.Pieces of liver tissue were fixed in 10% neutral formalin,dehydrated,embedded in paraffin,sectioned,and stained withhematoxylin-eosin?HE?and Masson.After all,microscopic observations were conducted for the pathological changes of the liver and liver fibrosis conditions.?2?Active rat hepatic stellate cells?HSC-T6?modles were prepared.The MTT method is used to observe the effects of Shuganning on HSC-T6 cells' proliferation,and to select appropriate drug concentration and time of action.A Shuganning high,medium and low dose group was set up with a concentration of 2.5%,1.25% and 0.625%,respectively.Aterwards,the groups were incubated together with HSC T6 cells for 24 hours.A normal control group was also set up?medium with equal volume?.A flow cytometry machine?FCM?was used to ckeck the effects of different drug concentrations on HSC-T6 cells' apoptosis.qPCR was used to detect the effects of different drug concentrations on the collagen protein expression of HSC-T6 ? and? cells.Results:?1?Rats of the control group were observed healthy,activate,with clean and shiny fur,water eating normally,urine without exception.defecation Modeled rats were stimulated after been administrated with the mixed liquid and were afterwards got into a drowsiness state.Modeled rats became less active after three weeks modeling,and phenomena such as hair became darker,yellow,losing,standing up and so on were observed,and decrease water feeding,loose stools.After treatment,the conditions of rats of the Shuganning high,middle,and delow dose groups as well as the Anluo Huaxian Wan group were significantly improved.As compared to the control group,the ALT and AST levels in the serum of the model group increased greatly?P <0.01?;the HA,LN,PIIINP,and IV-C levels were also significantly higher?P <0.01?;the body weight decreased largely?P <0.05?;and the liver indexes were significantly higher?P <0.01?;the MDA levels were significantly higher?P <0.01?;the SOD levels decreased largely?P < 0.01?;As compared to the model group,the ALT and AST levels in the serum of the Shuganning high,middle,and low dose groups as well as the Anluo Huaxian Wan group decreased greatly?P <0.01?;the HA,LN,PIIINP,and IV-C levels were also significantly lower?P <0.05 or P<0.01?;the body weight increased largely?P <0.05?;and the liver indexes were significantly lower?P <0.05 or P <0.01?;the MDA levels were significantly lower?P< 0.05 orP < 0.01?;the SOD levels increased largely?P < 0.05 orP < 0.01?.It could be observed by naked eyes that livers of the control group rats were with moderate sizes,deep red in color,smooth,soft,and sharp.Livers of the control group rats were swollen,dark red or brown in color,rough,and with small nodules,brittle,and dull.Livers of the treated group rats were relatively swollen,dark red or brown in color,rough,and relatively brittle.The following results were revealed by the pathological detections: It was observed from the HE stained samples that liver cells of the control group arranged neatly and the liver lobular structure was complete.As for the model group,liver tissue steatosis appeared;a large number of liver cells showed vacuolization along with punctuate and focal necrosis with inflammatory cell infiltration;a large number of fibrous tissue were around the portal area deposition;and the liver lobular structure was partly destroyed.While as compared to the model group,liver tissue steatosis,hepatocellular necrosis,inflammatory cell infiltration and deposition of fibrous tissue in the portal area were obviously lower in the treated groups.It was observed from the Masson stained samples that liver cells were red,and collagen fibers green.A few collagen fibers were observed at the portal areas of the control group;a large number of collagen fibers were deposited at the green portal area of the model group,which partly destroyed the lobular structure;however,green collagen fibers in the treated groups were obviously less,especially in the Shuganning high dose group.?2?Obviously,Shuganning reduces HSC-T6 cells' proliferation,and the effect is increased with the increase of drug concentration and the extension of time,and showing a certain dose effect relationship and time effect relationship.The results of flow cytometry showed that the early apoptosis rate and the late apoptosis and mortality in the low,middle and high dose group of Shuganning injection were higher than those in the control group,and the difference of middle and high dose group are both significant vs control group?P<0.05?.qPCR results showed that the Shuganning drug groups can significantly inhibit the collagen protein expression of the HSC-T6 ? and III cells,and the difference was extremely significant?P < 0.01?as compared with the control group.Conclusion: Shuganning has definite effect in alcoholic liver fibrosis.its mechanism may be related to effects such as protectingliver cells,reducing ECM deposition,inhibiting lipid peroxidation,inhibiting HSCs activation,inducting HSCs apoptosis,and inhibiting collagen protein expression of the HS-T6 ? and III cells. |
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