Study On The Isolation And Identification Of Active Peptides Of Carapax Trionycis And Its Anti-hepatofibrosis Mechanism

Carapax Trionycis derived from Trionyx sinensis Wiegmann,a traditional Chinese medicine and food dual purpose.Carapax Trionycis has a long history of clinical application as disease prevention and treatment,first recorded in the "Shen Nong’s herbal classic" of the Eastern Han Dynasty,contained in the "China Pharmacopoeia edition over the years.It possesses several properties,such as softing and resolving hard masses,replenishing “yin”,restraining “yang”,abating heat and removing steam,treatment for yin-deficiency fever,endogenous deficient wind and abdominal mass.Clinical evidences indicate that the traditional Chinese medicine compounds containing Carapax Trionycis such as,but not limited to,“Trionycis Kangxian Pill,Biejia Ruangan Tablet and Biejiajian Pill”,show curative effects when applied for the treatment of hepatic fibrosis.However,the molecular mechanisms involved in Carapax Trionycis anti-fibrotic effects have not been completely characterized.Our previous study shows that Carapax Trionycis extracts restrain the rat hepotafibrosis,equal or superior to accepted anti-hepatofibrosis drugs like Fuzhenghuayu capsule,we further find that Carapax Trionycis extracts with molecular weight less than 6 KDa are anti-hepatofibrosis active peptide site.On the basis of previous research,this paper explore the effective anti-hepatofibrosis foundation of Carapax Trionycis by using Chinese medicine chemistry,analytical chemistry and molecular biology,preliminary determing the antihepatofibrosis active component of Carapax Trionycis for laying the foundation of exploiting anti-fibrotic new drugs.Objectives: To interpreting the Carapax Trionycis anti-hepatofibrosis pharmacodynamic material basis and action mechanism for providing theoretical foundation of developing and applying anti-liver fibrosis new drugs.Methods: According to the early stage of laboratory study,we isolated active site P6(molecular weight less than 6 KDa)into six different molecular weight fragment via low temperature ultrasonic extraction and dialysis membrane separation technology,screening optimum active fragment by MTT and Western blot.19 different subfractions were isolated from extracts with molecular weight less than 1 KDa(N1)by Sephadex G-15 gel filtration chromatography.ELISA assay was used to screen optimum subfraction.Analysis and identification of active peptides were detected by Triple TOF 5600 LC-MS.Besides,we conduct the anti-liver fibrosis action mechanism of active site P6 and subfraction by modern molecular biological techniques.Results: Our results showed that the N1 did not clearly affect cell viability but inhibited TGF-β1-induced HSC-T6 proliferation,and exerted excellently anti-hepatofibrosis effect.MTT and ELISA results showed that F7 isolated from N1 was the optimum subfraction,and we analyzed and identified 110 different peptide compounds of F7.The anti-fibrotic action mechanism results of F7 showed that the protein expression of α-SMA,Collagen I and TIMP-1 were significantly decreased by F7,whereas MMP-1 expression was increased,corresponding with the level of HA,α-SMA,Collagen III and Collagen IV cytokines expressions decreased significantly after treatment with F7(P<0.05,P<0.01).The active site P6 did not clearly affect cell viability but suppressed TGF-β1-induced HSC-T6 proliferation(P<0.05),the mRNA expression of α-sma,Collagen I,Timp-1,Timp-2,Tgf-β1,Tnf-α,Il-1β and Il-6 were significantly decreased by P6 in TGF-β1-induced HSC-T6 cells(P<0.05,P<0.01).α-SMA,Collagen I,TIMP-1,TIMP-2,TGF-β1,TGF-βR1,pSmad2,pSmad3,Jun D and p-p65 protein expression were decreased obviously,TNF-α,IL-1β and IL-6 inflammatory cytokines were also decreased,while Smad7 protein expression was increased(P<0.05,P<0.01).Conclusion: As a whole,both of active site P6 and F7 exerted antihepatofibrosis effect by inhibiting HSC-T6 activation and proliferation induced by TGF-β1 and accelerating degradation of ECM.Besides,we found that P6 inhibited fibrogenesis of activated HSC-T6 cells via TGF-β1/Smad and NF-κB signaling pathway.We preliminarily explored the effective constituents and its action mechanism,further illustrated the Carapax Trionycis anti-hepatofibrosis pharmacodynamic material basis for prodiving scientific basis and laying the foundation of exploiting antifibrotic new drugs.