Abstract:

Objective To investigate the protective effect of a composite hydrogel against hydrogen peroxide（H?O?）-induced oxidative stress injury in the cardiomyocytes， and to clarify its possible mechanism. Methods The mice were subcutaneously injected with 100 μL of hydrogel. After normal feeding for 1， 14， and 28 d， the mice were sacrificed. Tissue samples were collected and subjected to HE staining to observe the histocompatibity of the hydrogel. The primary cardiomyocytes isolated from 1-day-old SD rats were used to establish an oxidative stress injury model. The primary cardiomyocyties were divided into control， H₂O₂ and H₂O₂+Hydrogel groups. The primary cardiomyocytes in control group were cultured normally， the primary cardiomyocytes in H₂O₂ group were treated with 200 μmol·L^-1 H₂O₂ for 24 h， and the primary cardiomyocytes in H₂O₂+Hydrogel group were incubated with 1 g·L^-1 composite hydrogel and 200 μmol·L^-1 H₂O₂ for 24 h. The viabilities of cardiomyocytes in various groups were assessed by cell counting kit-8（CCK-8） method. Dihydroethidium （DHE） and 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） staining were used to assess the reactive oxygen species（ROS） levels in the cells. The expressions of filamentous actin（F-actin） in the cells in various groups were detected by phalloidin fluorescence staining； the expressions of connexin 43 （Cx43） and cardiac troponin T （cTnT） proteins in the cardiomyocytes in various groups were detected by immunofluorescence method. The apoptotic rates of cardiomyocytes in various groups were assessed with TUNEL staining method. The expression levels of apoptosis-related proteins B-cell lymphoma-2（Bcl-2）and Bcl-2-associated X protein （Bax） in the cardiomyocytes in various groups were assessed by Western blotting method. Results The HE staining results showed that the inflammatory cells around the implanted hydrogel were less infiltrated， and the inflammatory reaction of subcutaneous implantation was less. Compared with control group， the viability of cardiomyocytes in H₂O₂ group was significantly decreased （P<0.05）， the level of ROS in the cells was increased（P<0.05）， the expression levels of Cx43， cTnT and F-actin proteins in the cells were decreased （P<0.001）， the apoptosis rate of cardiomyocytes were decreased （P<0.01）， the expression level of Bcl-2 protein in the cells was increased （P<0.001）， and the expression level of Bax protein was decreased（P<0.01）. Compared with H₂O₂ group， the viability of cardiomyocytes was significantly increased （P<0.05）， the level of ROS in the cells was decreased （P<0.01）， the expression levels of cTnT， Cx43 and F-actin proteins were increased （P<0.01），the apoptotic rate of cardiomyocytes were significantly decreased （P<0.001）， the expression level of Bcl-2 protein in the cells were decreased （P<0.01），and the expression level of Bax protein was increased （P<0.01）. Conclusion Hydrogel may promote the expression of cardiomyocyte-related proteins by scavenging ROS in the environment and inhibit the cardiomyocyte apoptosis to achieve the protective effect on the cardiomyocytes under oxidative stress.

Key words: Hydrogel, Cardiomyocyte, Hydrogen peroxide, Oxidative stress injury, Apoptosis