Studies On The Chemical Structrue Of Velvet Antler Polypeptides (Cervus Nippon Temminck) And Its Anti-hepatic Fibrosis

The velvet antler has been used clinically for thousands of years in our country. Yet, as a traditional chinese traditional resource , the velvet antler has not been adequately made use of. It is for this reason that people have not found the particular active composition for a long period .So it is unable to set up the quality standard of the velvet antler.Velvet antler can grow up rapidly, so people consider that it has a particular physiological characteristic that can make the cartilage, bone, epidermis, vessel, nerve and other tissues grow rapidly in velvet antler. Scientists in New Zealand have proverd that there are a great deal of growth factors and its receptors in the tip of velvet antler and they also consider that the reason of growthing rapidly of velvet antler is that these gorwth factors working in phase. Are there any peculiar active composition in the velvet antler except that people have discovered? Does is it for these peculiar active composition that the velvet antler growing up so rapidly ( 1-2cm/d)?As we know that these growth factors are likely to be protein or polypeptides, and studies show that the the active composition are mostly protein and polypeptides which occupy over 50% in velvet antler. So it is the emphases of this study to search the unique physiological characteric in the polypeptides of velvet antler.Our pre-study on the pharmacology activation of velvet antler polypeptides showed that it has nice curative effect to acute or chronic hepatic injury and liver regeneration after partial hepatectomy; velvet antler polypeptides have the function of improving the immunity of body and good anti-inflammation effect. Forthermore, velvet antler polypeptides have good therapeutic effect in wound healing and cell regeneration. So we can consider that there are certain ingredient in velvet antler polypeptides which have anti-hepatic fibrosis effections.According to these studies, we studies on anti-hepatic fibrosis of velvet antler polypeptides and its active mechamism. Furthermore in order to discover a new peculiar active ingredient from velvet antler polypeptides, we carried on such as the chemical composition analysis of research, separation and purification of one kind of polypeptide and its chemical structure analysis. A novel plasmid of the antler velvet polypeptide was constructed and expressed in high level in E.coli. Fusion protien reproduced from recombinant polypeptides and its purification is simple.Result:1. The velvet antler polypeptides (VAPs) was obtained by steps of separatingwith buffer, sinking with the alcohol, and Sephades G-25, molecular weight of which is less than 10KDa through SDS-PAGE electrophoresis and MALDI-TOF MS analyses. Content of peptides is 64.7% in VAPs.2. Female Wistar rats were injected carbon tetrachloride subcutaneously to be as hepatic-fibrosis models which were treated by VAPs ( 5, 10, 20 mg/kg) injecting subcutaneously too. After the 45d treatment, animals were sacrificed and the various indicators were determined. The result showed that high-dose and medium-dose showed more obvious effect.⑴Changes of the serous various indicators: after treatment with VAPs, release of ALT and AST decreased significantly, reduction of serous HA and LN obviously, at the same time, the content of total protein and albumin increased significantly.⑵Changes of the various indicators in liver homogenates: after treatment with VAPs, activity of SOD and GSH was significantly raised and the content of MDA was increased significantly, the hepatic Hyp was decreased too.⑶Histomorphological changes of livers: after treatment with VAPs, the destructions were tightened, prohterations of collagen fibers were also lightened, fiber cords were loosened and narrowed. Swelling of liver cells and degeneration alleviated, infiltrating cells decreased. These improvements were more significant in high and medium-dose groups .⑷immunohistochemical results: after treatment with VAPs, expression of the typeⅠ,Ⅳcollagen and LN protein in liver tissues were decreased significantly.⑸Western-blot and RT-PCR: after treatment with VAPs, expression of TGF-β1 and MMP-2, TIMP-1mRNAs in liver were decreased significantly.3. we get a polypeptides named nVAP ( natural velvet antler polypeptides ) which showed a single peak in HPLC chromatography and single band in SDS-PAGE. Its structure was determined by combination of MALDI-TOF MS analyses,N-terminal Edman degradation and amino analyses. It shows that the nVAP consists of a single chain of 32 amino acid residues ,its molecular weight is 3245.7 Da and the amino acid sequence is VLSAT DKTNV LAAWG KVGGN APAFG AEALE RM. nVAP showed significantly increase the proliferation of human liver cell line ( L-02 ) and rat spleen cells in culture and protective effect on injury of L-02 cell line by carbon tetrachloride.4. A new-designed nVAP gene was constructed according to the code preference of the expression system and its amino acid sequence. Then, we constructed a novel plasmid, pET-28a/VAP, a high expression system in E.coli ( BL21 ). The recombinant polypeptides express a fusion protien and its purification is simple. We named rVAP and the ratio of rVAP is 6.4mg/L. The research of the rVAP activity prove that it can promet proliferation of L-02 cell line and rat spleen cells in culture. Furthermore, the difference beween biologic activity of rVAP and nVAP in the same dose is not significant.Conclusion :1. The research has confirmed that VAPs can treat the disease of hepatic fibrosis notably. Anti-hepatic-fibrosis of VAPs might be:①protecting liver cells, alleviating its damage, improving liver functions by hydroxyl radical scavening activity and great effect of antioxidation, thereby eliminating the start-up factor which might be related to the occurrence and development of hepatic fibrosis;②promoting regeneration and repairing of hepatic cells;③intervening the expression of TGF-β1 and its signal conduction and thus intervening the progress of fibrosis;④decreasing the expression of collagenⅠ,Ⅳand LN statistically, lightening capillarization of hepatic sinusoids, improving microcirculation of liver;⑤decreasing the gene expression of MMP-2, improving exist-environment of liver cells and decreasing the gene expression of TIMP-1, prompting degradation of ECM.2. nVAP is a monomer polypeptides and its consists of a single chain of 32 amino acid residues. Its molecular weight is 3245.7Da and its homology is less than 50% to compare the known proteins. So we supposed that it might be a new polypeptide from velvet antler. nVAP showed significantly increase the proliferation of human liver cell line ( L-02 ) and rat spleen cells in culture and protective effect on injury of L-02 cell line by carbon tetrachloride. Therefore, nVAP is one of fundamental compositions that anti-hepatic-fibrosis of VAPs.3. The prokaryotic expression engineering bacteria of nVAP was constructed successfully and it is a high expression system in E.coli. So we conquered the shortcoming of low yield and high cost to obtain nVAP from velvet antler and offered the material foundation for profound research and extensive utilization of velvet antler ploypeptides.