The Protective Effect And Mechanism Of The Total Lignans Of Tibetan Melon Seeds On Liver Fibrosis

Objective:Hepatic fibrosis?HF?is an important link of further development of various chronic liver diseases to liver cirrhosis and even liver cancer.At present,there is no safe and effective drug for the treatment of HF in clinical practice.However,in recent yearsthe unique therapeutic effects of traditional Chinese medicine and natural products in the treatment of HF have drawn more and more attention from researchers.Herpetospermum seed?HS?,a common medication used in Chinese tibetan medicine,is the dried ripe seed of Herpetospermum caudigerum Wall.H.caudigerum grows in shrubs and forest margins and along roadsides,and it has a bitter taste.In Tibet,it is a well-known treatment and is used in traditional medicine for the treatment of liver diseases,cholic diseases,and dyspepsia.A preliminary study showed that its chemical constituents mainly include fatty acids,lignans,amino acids and polysaccharides.^?10?Extracts of HS have been reported to display a broad spectrum of biological activities,including hepatoprotective,anti-fatigue,anoxiaresistant,and antiviral activities.A large number of studies have shown that total lignans from Herpetospermum seed?TLHS?have a good curative effect for the treatment of various kinds of liver injure,such as viral liver injury,alcoholic liver injury and chemical liver injury.These liver injury are closely related to the development of HF.The group by a large number of studies have shown that the main effective parts of HS prevention and treatment of liver disease is the total lignans.However,the research on the treatment and prevention of hepatic fibrosis in the treatment of TLHS has not been reported,and the mechanism of action is still unclear.Therefore,this study intends to establish carbon tetrachloride-induced rat HF model to study whether it has a protective effect on rat HF.Then,we explored the mechanism of action of TLHS on HF by culturing rat hepatic KC and HSC in vitro.Method:1.Protective effect and mechanism of TLHS on carbon tetrachloride-induced HF in rats:?1?Carbon tetrachloride-induced HF model:SD rats abdominal subcutaneous injection An olive oil solution containing 40%CCl₄ was injected for the first time at a rate of 5 ml/kg followed by 3 ml/kg every two to three days for 9 weeks.The state and weight changes of the rats were recorded during the period.SD rats were randomly divided into blank group,model group,Glycyrrhizin group,TLHS(100/200/400 mg/kggroups?hereinafter referred to as TLHSL/TLHSM/TLHSH groups?a total of six groups.CCl₄-induced HF model was established except for the blank group.The rats were given gavage daily,while the blank group and model group were given the same amount of normal saline.?2?Detecting the serum levels of ALT,AST and ALP in rat serum by automatic biochemical analyzer;?3??4?ELISA method was used to detect the serum levels of hepatic fibrosis indicators such as transforming growth factor?TGF-?1?,hyaluronic acid?HA?,Hydroxyproline?HYP?levels and rat liver homogenate superoxide dismutase?SOD?levels.2.Effects of TLHS on proliferation and apoptosis of the HSC-T6:The experiment was divided into control group,colchicine?0.1?g/ml??positive drug?group,total lignan 20/30/40?g/ml?hereinafter referred to as TLHSL/TLHSM/TLHSH groups?a total of five groups.The logarithmic growth phase of HSC-T6 were added to each group of corresponding drugs for culture.CCK8 assay was used to determine the inhibitory rate of total lignan on proliferation of HSC-T6 at 24,48,72h.The effects of TLHS on apoptosis and cell cycle of HSC-T6 were using Flow cytometry.3.The mechanism of TLHS on HF:?1?The experiment was divided into control group,model group?LPS?,colchicine?0.1?g/ml?,total lignans?30/40/50?g/ml??hereinafter referred to as TLHSL/TLHSM/TLHSHgroups?.KC in logarithmic growth phase were taken,except for the control group,drugs were added for 24 hours before adding LPS for 6 hours.ELISA method was used to detect the effect of TLHS on the expression oftransforming growth factor-?1?TGF-?1?and tumor necrosis factor-??TNF-??in KC.?2?The experimental group is the same as the previous group?2?and theprotein expressions of B cell lymphoma-2?Bcl-2?,Bcl-2-associated X protein?Bax?and Transcription factors-kB?NF-?B?were detected using Western blotting.Result:1.Compared with the blank group,hepatic necrosis of hepatocytes was found by pathological and Masson trichrome staining in model liver of model group,A large number of fibrous connective tissue hyperplasia,pseudolobule formation,suggesting that carbon tetrachloride-induced HF model was successful.?2?Compared with the model group,the levels of ALT,AST and ALP in the serum of the TLHS groups were significantly decreased?P<0.05,P<0.01?;the concentration of SOD in the liver tissue homogenate was significantly?P<0.05,P<0.01?.The degree of hepatic injury was significantly reduced,the connective tissue was significantly reduced and the HF score was decreased.The levels of serum HA,HYP and TGF-?1 in serum were significantly decreased?P<0.05,P<0.01?.2.Compared with control group24,48,and 72 h proliferation inhibition rate of HSC-T6 was obviously elevated in TLHS groups?P<0.01?.Early-and late-stage apoptosis rates of HSC-T6 cells obviously increased in TLHS groups?P<0.01?.G0/G1 phase cell number obviously increased in total lignans groups?P<0.01?and G2/M phase cell number have no significant difference?P>0.05?,S phase cell numbers obviously decreased?P<0.01?.3.The results of this study show that compared with LPS group,TLHS can inhibit the secretion of TGF-?1 and TNF-?in KC?P<0.01?.Simultaneously,TLHS can inhibit the proliferation of HSC and induce its apoptosis through decrease the Bcl-2,NF-?B expression and increase Bax expression?P<0.05,P<0.01?.Conclusion:In summary,in vivo experiments in rats showed that TLHS had a significant protective effect on carbon tetrachloride-induced rat HF,and could repair the liver injury and reduce the proliferation of fibrous connective tissue have obvious effect.Cell experiments showed that the protective effect of the total lignan fraction of TLHS on HF was related to inhibiting the proliferation of HSC-T6 and inducing it's apoptosis.At the same time it may be related to its inhibition of KC secretion of liver fibrosis related cytokines TGF-?1 and TNF-?.In addition,the inhibition of hepatic stellate cells proliferation and induce its apoptosis may be related to lower the expression of Bcl-2,NF-?B.