Abstract:

Objective To explore the protective effect of velvet antler peptide （VAP） pretreatment on the myocardial H9c2 cell injury of the rats induced by tert-butyl hydroperoxide（TBHP）， and to clarify the effect of VAP on miR-133a/transforming growth factor-β（TGF-β）/Smad axis and its mechanism. Methods The H9c2 cells were divided into blank control group， blank serum group， TBHP group， TBHP+low dose（100 mg·kg^－1） of VAP group， TBHP+ high dose（400 mg·kg^－1） of VAP group， and miR-133 inhibitor（miR-133 inhibitor） group. The cells in blank control group were given no treatment， and the cells in the other groups were treated with VAP for 24 h， then were treated with 200 μmol·L^－1 TBHP； the cells in miR-133 inhibitor group were transfected with miR-133 inhibitor for 24 h，treated with 100 mg·kg^－1 VAP for 24 h+200 μmol·L^－1 TBHP. MTT assay was used to detect the survival rates of the H9c2 cells in various groups；the levels of troponin T（cTnT）， and cardial troponin T（cTnI）in culture supernatant of the cells in various groups were detected by enzyme-linked immunosorbent assay（ELISA） method and creatine kinase-MB（CK-MB）； the expression levels of miR-133 in the H9c2 cells in various groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method；the expression levels of transforming growth factor-β1（TGF-β1），phosphorylated Smad2/3（p-Smad2/3）， and Smad4 proteins the in H9c2 cells in various groups were detected by Western blotting method. Results The MTT results showed that compared with TBHP group， the survival rates of H9c2 cells in TBHP+low dose of VAP group and TBHP+high dose of VAP group were increased （P<0.05）， but the survival rate of the H9c2 cells in miR-133 inhibitor group had no significant difference （P>0.05）. The ELISA assay results showed that compared with blank control group the levels of cTnT， cTnI，and CK-MB in the H9c2 cells in TBHP group were increased （P<0.05）； compared with TBHP group， the levels of cTnT， cTnI，CK-MB in the H9c2 cells in TBHP+low dose of VAP group and TBHP+high dose of VAP group were decreased （P<0.05）.The RT-qPCR results showed that compared with blank control group， the expression level of miR-133a in the H9c2 cells in TBHP group was decreased（P<0.05）；compared with TBHP group， the expression levels of miR-133a in the H9c2 cells in TBHP+ low dose of VAP group and TBHP+ high dose of VAP group were increased （P<0.05 or P<0.01），and the expression level of miR-133a in the cells in miR-133 inhibitor group was decreased（P<0.01）. The Western blotting results showed that compared with blank control group， the expression levels of TGF-β1， p-Smad2/3， and Smad4 proteins in the H9c2 cells in TBHP group were increased（P<0.05）； compared with TBHP group， the expression levels of TGF-β1， p-Smad2/3， and Smad4 proteins in the H9c2 cells in TBHP+low dose of VAP and TBHP+ high dose of VAP groups were decreased （P<0.05）. Conclusion VAP pretreatment can protect the myocardial H9c2 cell injury of the rats induced by TBHP through regulating the TGF-β/Smad signaling pathway with miR-133a.

Key words: Velvet antler polypeptide, Myocardial injury, MicroRNA-133a, Transforming growth factor-β, Serum pharmacology