The Development Of A Novel AcSDKP Tetrapeptide Which Has ACE Resistant Capacity And Antifibrotic Activities

Objective:AcSDKP, an N-terminal acetylated endogenous tetrapeptide, which isreleased from its precursor thymosin β4after the hydrolysis ofprolyl oligopeptidase (POP), generally keeps nanomolar level in the blood. As akind of multifunctional physiological regulatory factors, AcSDKP has diversebiological activity，such as inhibiting the proliferation of cardiac fibroblast,restraining the growth of hematopoietic stem cells, etc. However, the digestion ofAcSDKP by angiotensin-converting enzyme (ACE) widely existed in vivo leadsto the structural and functional loss of AcSDKP, thereby handicapping the clinicalapplication of AcSDKP. Our study intended to synthesize AcSDKP isomers by Damino acids substitutions using the standard Fmoc-based solid-phase synthesistechnique, which were expected to resist ACE degradation and retain the activityof nature AcSDKP. This work is expected to provide theoretical and experimentalbasis for AcSKDP antifibrotic application.Methods:AcSDKP isomers are prepared using the standard Fmoc-based solid-phase synthesis technique. Then, the derived AcSDKP isomers were purified by HPLC.After that, the ACE-resistant ability of AcSDKP isomers was measured by HPLC;the impacts of AcSDKP isomers on the proliferation of L929cells and cardiacfibroblasts were detected by MTT and BrdU assay. Meanwhile, the influence ofAcSDKP isomers on differentiation of BMSC to macrophages was analyzed byflow cytometry using the F4/80marked macrophage maturation. The effect ofAcSDKP isomers on the expression of cardiac fibroblasts P-Smad2was analyzedby the western blot assay. And the impact of AcSDKP isomers on hepatic fibrosiswas also studied on the constructed SD rat liver fibrosis model. H&E stainingwas used to detect histopathological change in liver tissue. The total protein(TP),albumin(ALB), alanine aminotranferease (ALT) and aspartate aminotransferase(AST) in the serum were measured using the Automatic biological detector.Results:Finally, three kinds of AcSDKP isomers with good anti-ACE degradationability were successfully derived, i.e. AcSD(d)KP, AcSDK(d)P and AcSD(d)K(d)P,with a purity above98%. Furthermore, the AcSD(d)K(d)P isomer can inhibit theproliferation of L929cells and cardiac fibroblasts, the differentiation of BMSC tomacrophages and the expression of cardiac fibroblasts P-Smad2, as demonstratedby experiments. And the animal experiment indicates that AcSD(d)K(d)P hasantifibrotic activity and show some protective effect to liver.Conclusion:The AcSDKP isomers with ACE-resistant capacity, which can restrain theproliferation of fibroblasts, the differentiation of macrophages and the liverfibrosis of SD rats, are successfully built. The study accumulates information onthe application of AcSDKP isomers as anti-fibrosis drugs.