ABSTRACT
Z-VAD-FMK was combined with hypoxia-reoxygenation (H/R) injury to establish a necroptosis model of H9c2 cells to mimic the pathological changes of myocardial ischemia reperfusion injury (MIRI) in vitro and to study the effect and mechanism of tilianin against myocardial ischemia-reperfusion injury. A cell counting kit-8 (CCK-8) was used to detect cell viability, and commercial kits were used to detect lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatant. Hoechst 33342/PI immunofluorescence staining was used to detect cell death. DCFH-DA, BBcellProbeTMM61, and JC-1 probes were used to detect reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP), and mitochondrial membrane potential (MMP), respectively. An enzyme-linked immunosorbent assay (ELISA) method was used to detect the release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). The results show that the cell viability, SOD activity, and MMP of the model group induced by H/R injury decreased, as compared with control group, but the necroptosis rate, LDH level, and ROS release increased significantly. Furthermore, mPTP of the model group cells opened, and TNF-α, IL-1β, and IL-6 levels were significantly higher. Molecular docking modeling showed that tilianin can bind to calmodulin-dependent protein kinase II (CaMKII), and Western blot results showed that compared with control group, the expression levels of p-CaMKII and phospho-mixed lineage kinase domain-like protein increased in the model group, and tilianin could decrease the expression level of these proteins. The above results indicate that tilianin can protect H9c2 cells by inhibiting the phosphorylation of CaMKⅡ at threonine 287, protecting mitochondrial function, and inhibiting the opening of mPTP to prevent necroptosis. This study has value for research on new methods to treat H/R injury.
ABSTRACT
Objective: To explore the protective effect and mechanism of total flavonoids of Dracocephalum moldevica (TFDM) on doxorubicin-induced cardiotoxicity. Methods: H9c2 cells were induced with 1 μmol/L doxorubicin for 24 h to establish a cardiotoxicity model. H9c2 cells were randomly divided into control group, model group, and drug intervention group (four subgroups of 5, 25, 50, and 100 μg/mL). After the intervention of TFDM, the doxorubicin cardiotoxicity model was established in the other groups except the control group. The cell counting Kit-8 method was used to determine the viability of H9c2 cells induced by doxorubicin injury after the intervention of TFDM. The effects of lactate dehydrogenase release, intracellular superoxide dismutase and malondialdehyde in each group were determined by kit method. The apoptosis rate of each group was detected by flow cytometry using Annexin-V FITC/PI double staining method. Reactive oxygen species (ROS) and mitochondrial membrane potential in each group were detected by DCFH-DA and JC-1 probes. The expressions of p38MAPK, ERK1/2, and PI3K/Akt pathway-related proteins were detected by Western blotting. Results: Compared with the control group, the cell viability of the model group induced by doxorubicin was decreased, the release of lactate dehydrogenase and the content of malondialdehyde were increased, the activity of superoxide dismutase was decreased, the apoptosis rate was increased, the release of reactive oxygen species was increased significantly, and the mitochondrial membrane potential was decreased significantly. However, TFDM increased H9c2 cell viability, decreased LDH and MDA levels, increased SOD activity, decreased apoptosis rate, significantly decreased ROS release, and significantly increased MMP in a dose-dependent manner. The difference was statistically significant. The results of Western blot showed that the expression levels of p-PI3K, p-Akt, p-ERK1/2, and Bcl-2 were decreased, and the expression levels of p-p38MAPK, Bax and Caspase-3 were significantly increased compared with the control group. However, in the TFDM-treated group, the expression of p-PI3K, p-Akt, p-ERK1/2, and Bcl-2 protein was increased, and the protein expression of p-p38MAPK, Bax, and Caspase-3 protein was decreased. Conclusion: TFDM can protect cardiomyocytes, and its protective mechanism may be related to the resistance to oxidative stress, protection of cardiomyocyte mitochondria, and regulating MAPK enzyme family proteins, and PI3K/Akt signaling pathway and subsequent release of apoptotic cytokines to inhibit apoptosis.