Study On The Synthesis And Modification Of Anti-hepatic Fibrosis Active Peptides Of Carapax Trionycis

Carapax Trionycis, as both traditional Chinese medicine and food, was included in the《Chinese Pharmacopoeia》over the years. It has such activities as replenishing "yin", restraining "yang", softening and resolving hard masses, reducing fever and removing steam. Carapax Trionycis is one of the most widespread medicines for the therapy of liver fibrosis clinically. Traditional Chinese medicine compounds with Carapax Trionycis, such as Compound "Biejia Ruangan Tablet、Turtle Kangxian Pill、Biejiajian Pills" and so on, show curative effect when used for the therapy of liver fibrosis. Previous studies had demonstrated that extracts of Carapax Trionycis were able to protect liver against fibrosis in CC14animal models. Moreover, three active ingredients were first isolated from Carapax Trionycis and proved to be significantly anti-hepatic fibrosis by our study group. By determination and analysis, they were peptides whose sequences were H-G-R-F-G (572.3)、 N-P-N-P-T (542.16) and N-D-D-Y (526.2) respectively.Based on the precious studies, above active peptides of Carapax Trionycis were took as lead compounds and synthesized by Fmoc solid-phase method in this treatise. By the analysis and purification of RP-HPLC, the purity of finished products was over98%, the molecular weight of which was identical with the theoretical value by mass-spectrum identification, all of which proved the correctness of peptides’sequence. In order to investigate the activity of synthetic peptides of Carapax Trionycis on treatment of hepatic fibrosis, three synthetic peptides were applied and examined the effect on hepatic stellate cells (HSCs) by the assay of MTS and enzyme-linked immunosorbent assay (ELISA), The results showed the synthetic peptides of Carapax Trionycis A (H-G-R-F-G) and B (N-P-N-P-T) had significantly anti-hepatic fibrotic effect.Protein expression of Col Ⅰ、Col Ⅲ、Smad3、p-Smad3、α-SMA、TIMP-1and MMP-land apoptosis of hepatic stellate cells (HSC) were analyzed by Western blotting and Annexin V-FITC/PI staining respectively. Synthetic peptides A and B brought into play to the effect of anti-hepatic fibrosis by the following three pathways.①The expression of Col Ⅰ、ColⅢ and α-SMA proteins in the TGF-β1+synthetic peptides of Carapax Trionycis groups decreased significantly relative to that in the TGF-β1groups, which inhibited the synthesis and secretion of Col I and ColⅢ;②The level of p-Smad3protein in the TGF-β1+synthetic peptides of Carapax Trionycis groups decreased significantly relative to that in the TGF-β1groups, which inhibited phosphorylation of Smad3and transformation of ECM;③The content and protein expression of TIMP-1in experimental groups decreased significantly, but the level of MMP-1protein increased compared with the TGF-β1groups. Our results indicated that synthetic peptides of Carapax Trionycis A and B showed anti-hepatic fibrosis activity.Because of existing immunogenic epitope、short half-lives and low stability for synthetic peptides of Carapax Trionycis in vivo, seven chemical modifications were synthesized by solid-phase method through replacing corresponding L-amino acid with non-natural D-amino acid in peptide A; Stability of seven modifications was evaluated in solution by reversed-phase high performance liquid chromatography (RP-HPLC) from several aspects as different temperature、buffers and pH value. The results indicated that the stability of seven modifications increased significantly compared to synthetic peptide A; The modifications’activity on treatment of hepatic stellate cells (HSCs) was detected by CCK-8assay. The results indicated that the synthetic peptide A and its modifications all showed anti-hepatic fibrosis effect. However, the modifications possessed better stability. Therefore, above information provided here may help offer scientific foundation in looking for new anti-hepatic fibrosis medicine.