Research Of The Therapeutic Material Basis Of Carapax Trionycis On Anti-hepatic Fibrosis

Trionycis Carapax is a kind of traditional Chinese medicine derived from the shell of Trionyx sinensis Wiegmann with a long application history.This medicine has a variety functions like nourishing“Yin”and suppressing“Yang”,reducing fever and removing steam,resolving hard lump,which has been recorded in China Pharmacopoeia.Some diseases such as fever due to“Yin”deficiency,hyperactivity of“Yang”due to“Yin”deficiency,dizziness,hand and foot spasm can be relieved by this medicine.Based on the major functions of Carapax Trionycis,there have been reported that the extracts of Carapax Trionycis have great effects of anti-hepatic fibrosis.Modern pharmacology have showed that the formation of liver fibrosis are due to the excessive deposition of liver fiber tissue during the repair process,which is a reversible pathological phenomenon after liver damage.Further development of liver fibrosis will lead to various chronic liver diseases,which is the pathological basis of liver cirrhosis and liver cancer.Under the guidance of the theory of traditional Chinese medicine,Carapax Trionycis have played an important role in the Chinese patent medicine,such as Biejia Kangxian Recipes,Biejia Ruangan Tablets,Biejiajian Pills,which have significant curative effects on anti-hepatic fibrosis.In order to explore the main compositions of the Therapeutic Material Basis of Carapax Trionycis and its mechanism of treating hepatic fibrosis,based on previous studies this paper used the interdisciplinary research methods,such as Traditional Chinese Medicine Chemistry,Analytical Chemistry,Pharmacology and Molecular Biology,and modern science and technology to make a comprehensive research of Carapax Trionycis.The polypeptide substances of Carapax Trionycis in vinegar were extracted by Ultrasound-Assisted Water Extraction Method,then separated into two different molecular weight fragments（molecular weight more than 6 KD and less than 6 KD）by membrane dialysis.In this study,an inhibitory rate on the proliferation of hepatic stellate cells（HSC-T6）were used as an indicator to select the active polypeptides.The results showed that the polypeptides,molecular weight less than 6 KD,inhibited hepatic fibrosis more effectively,which may be the effective parts of Carapax Trionycis polypeptides.Then the Carapax Trionycis polypeptides of molecular weight less than 6 KD were further separated into 0-3 KD and 3-6 KD by membrane dialysis.To compare these two polypeptides’activity,their proliferations in HSC-T6 cells were used as the detection index.The MTT results indicated that the expression of TGF-β1 induced by HSC-T6 were significantly decreased by two active sites and they can both limit the proliferation of HSC-T6.Thus,the effect of 0-3 KD were better than 3-6KD.An animal model of hepatic fibrosis have been built and the comparative study between 0-3 KD and 3-6 KD on the pharmacodynamics of the effective polypeptides have been carried out in this paper.The results showed that the degree of liver fibrosis in the drug experimental groups were significantly lower than CCl₄ group by appearance of the liver.The organ coefficient of the main organs showed that heart,liver and kindney of mice in the drug experimental groups were decreased significantly（P<0.05）.HE staining and Masson staining results also showed that the degree of liver fibrosis of mice in the experimental groups were more alleviated.In the comparison of the anti-hepatic fibrosis effect between experimental groups and the positive medicine groups（Yu Gan Long group and Colchicine group）,their effectiveness were similar.Therefore,it is suggested that Carapax Trionycis effective polypeptides have markedly apharmacological actions on anti-hepatic fibrosis.In this paper,the techniques and methods of molecular biology were used to study the mechanism of the Carapax Trionycis effective polypeptides.The expression of COLⅣ,HPCⅢ,LN,HA,TNF-α,TGF-β1,IL-1βand IL-6 in supernatants of liver tissue homogenates were measured by ELISA.The results showed their expression were significantly decreased by Carapax Trionycis effective polypeptides（P<0.05）.Immunohistochemical staining of CTGF,EGFR,MMP9,p-AKT,p-p38,α-SMA and TGF-β1 of liver tissue showed that the expression of CTGF,EGFR,MMP9,p-AKT,p-p38,α-SMA and TGF-β1were significantly decreased and the MMP9 expression were increased（P<0.05）.The expression of p-JNK、p-ERK1/2、Smad7、p-p38、TIMP-1、MMP-1、p-Smad2 of liver tissue were detected by Western Blot and the results showed the expression of p-JNK,p-ERK1/2,p-p38,MMP-1 and p-Smad2 were significantly decreased and Smad7 and TIMP-1 expression were increased（P<0.05）,which implied Carapax Trionycis effective polypeptides activating the MAPK,Smads and TIMPs/MMPs signaling pathway to inhibit fibrogenesis.Through HPLC and triple TOF 5600 MS,the chemical composition of 0-3 KD and 3-6 KD effective polypeptides were analyzed and identified.More than 800 amino acid sequences were identified respectively with score of 20 points.18 amino acid sequences were identified with score of 40 points in 0-3 KD,7 amino acid sequences were identified with score of 40 points in 3-6 KD.These research discussed the structure and composition of the anti-hepatic fibrosis effective polypeptides of Carapax Trionycis,which have important significance on the Therapeutic Material Basis of Carapax Trionycis on anti-hepatic fibrosishas,and lay the foundation of synthesising anti-hepatic fibrosis peptide drugs.This paper used the variety of interdisciplinary research methods to explore the effective constituents and action mechanism of Carapax Trionycis,illustrate its anti-hepatofibrosis pharmacodynamic material basis,prodiving scientific basis and laying the foundation of exploiting anti-fibrotic new drugs.