Molecular Mechanism Of Angelin - Induced Cardiomyocyte Hypoxia / Reoxygenation Injury Based On Autophagy

ObjectiveThe major pathological manifestations of myocardial ischemia reperfusion injury (MIRI) in coronary heart disease are ischemic cell damages caused by ischemia and hypoxia. Recent evidences indicated that autophagy plays a "double edge-sword" role in MIRI. Beclin 1 and mTOR mediated signaling pathways are two classical signaling pathways that activate autophagy, may be the important regulation mechanism that made the "double edge-sword" effects of autophagy turn to cell survival. Our previous studies have shown that Orientin could protect myocardium from MIRI though anti-apoptosis and induction of autophagy. On that basis, the present study intended to build cardimyocyte hypoxia reoxygenation (H/R) injury model on rat, to explore the main targets of Orientin for prevention and treatment of MIRI, to verify the hypothesis that "the core mechanism of Orientin (Ori) against hypoxia-reoxygenation injury of myocardial cells was induction of modest autophagy by regulating mTOR and Beclin 1 pathways, to encourage the ’double edge-sword’effects of autophagy turn to cell survival", and finally to provide pharmacological evidences for Miao medicine Ndut mongx youx against MIRI.Methods and ResultsThe H/R injury model of primary cultured neonatal rat cardiomyocytes was built firstly. The Experiments were divided into seven goups:Control group, H/R group, low concentration of Orientin treated group (3 μmol/L, Ori-3 group), middle concentration of Orientin treated group (10 μmol/L, Ori-10 group), high concentration of Orientin treated group (30 μmol/L, Ori-30 group), autophagy inhibitor group (30 μmol/L Orientin+100 nm Wortmannin, OW group), and Resveratrol treated group (5 μmol/L, Res group). Myocardial cells were incubated with drug for 12h, and then followed hypoxia-reoxygenation treatment. This research was divided into four parts to study the molecular mechanism of Orientin against myocardial cell hypoxia/reoxygenation injury.1. The pharmacodynamic research of Orientin against hypoxia reoxygenation injury of myocardial cellsMorphology changes of myocardial cells by light microscope, myocardial cell vitality detected by CCK-8, the activity of myocardial enzymes in the culture medium through Biochemical analyzer, the ultrastructure changes of myocardial cells by electron microscopy were measured in each group, to evaluate the damage degree of myocardial cells and do pharmacodynamics researches on Orientin against H/R injury of myocardial cells.The results showed that:Compared with Control group, the cell vitality of myocardial cells in HR group fell about 50%(P< 0.001), the activity of LDH and CK-MB in culture medium was significantly higher (P< 0.001), and pathological damage of myocardial cells was observed under light microscope and electron microscope, these results suggested myocardial cells treated by hypoxia-reoxygenation suffered a high degree of damage. After pretreated by Orientin for 12h, compared with the HR group, the myocardial cell vitality obviously increased (P< 0.001), the activity LDH and CK-MB significantly decreased (P< 0.001), and the pathological changes of myocardial cells were obviously improved by high concentration of Orientin. These results indicated that Orientin could significantly reduce the hypoxia-reoxygenation injury in myocardial cells. In the OW group, compared with Ori-30 group, myocardial cell vitality fell about 22% after hypoxia and reoxygenation (P< 0.001), the activity LDH and CK-MB increased obviously (P< 0.001), the pathological damage degree in myocardial cells was similar with HR group, indicating Wortmannin could impair the protection of Orientin against hypoxia reoxygenation injury of myocardial cells.2. The effects of Orientin on autophagy and apoptosis induced by hypoxia-reoxygenation injury in myocardial cellsMyocardial cell apoptosis was detected by flow cytometry and TUNEL method, and the protein expression level of Bcl-2 and the Bcl-XL was measured by Western Blot to judge the degree of cell apoptosis in each group. The autophagosomes and autolysosomes in myocardial cells detected by electron microscopy, the distribution of LC3 immunofluorescence in myocardial cells measured by laser confocal microscopy, and LC3-Ⅱ/LC3-I ratio and Beclin 1 protein expression level detected by Western Blot were used to assess the degree of cell autophagy.The results showed that: In the HR group, compared with Control group, myocardial cell apoptosis rate increased significantly (P< 0.001), and Bcl-2 expression level also increased but no statistical difference. In the HR group, LC3 immunofluorescence expression in myocardial cells significantly enhanced, LC3 positive cell rate rise obviously, autophagosomes were observed under the electron microscope, and the LC3-Ⅱ/LC3-Ⅰ ratio and the expression level of Beclin 1 had a rising trend. These results suggested apoptosis and autophagy inmyocardial cells could be induced by hypoxia-reoxygenation injury. Compared with the HR group, myocardial cell apoptosis caused by hypoxia-reoxygenation injury could inhibited by middle and high concentration of Orientin, and expression level of Bcl-2 increased with the increase of the dose of Orientin, suggesting that the Orientin could prevent myocardial cell apoptosis caused by hypoxia-reoxygenation injury through the increasing expression level of Bcl-2. But there was no defference on the expression level of Bcl-XL between each group. In the Ori-30 group, compared with HR group, the LC3 positive cell rate rise to 62%(P< 0.05), the numbers of autophagosomes obviously increased, and the LC3Ⅱ/LC3 Ⅰ ratio and the expression level of Beclin 1 also further strengthened, the experimental results indicated that Orientin could significantly induce autophagy. In OW group, the level of apoptosis increased significantly (P< 0.001), while the expression level of Bcl-2 decreased obviously, suggesting Wortmannin could weak the effects of Orientin against apoptosis. Compared with Ori-30 group, the LC3 punctate dots and the numbers of autophagosomes decreased, the LC3-Ⅱ/LC3-Ⅰ ratio and the expression level of Beclin 1 were dropped. All results suggested Wortmannin could inhibit induction of autophagy by Orientin.3. The molecular mechanism of Orientin against hypoxia-reoxygenation injury in myocardial cells through the mTOR related signaling pathwaysThe expression and phosphorylation level of Akt, AMPK, mTOR, p70S6K, etc., which are the important signaling molecules in mTOR signal pathway, were detected by Western Blot for studying the regulation of autophagy by Orientin through mTOR related signaling pathway.The results showed that:The phosphorylation level of Akt in Ori-30 group was higher than that in HR group and OW group (P< 0.05), indicating that PI3K-Akt signaling pathways played a role in high concentration Orientin against hypoxia-reoxygenation injury. Compared with HR group, in Ori-30 group, the mTOR phosphorylation was partially inhibited (P< 0.01 or P< 0.001), the Raptor expression level decreased obviously (P< 0.001), the phosphorylation level of p70S6K also decreased, while the phosphorylation level of AMPK rose, these results indicated Orientin could suppress the mTORC1 activity by AMPK activation. In OW group, the phosphorylation of mTOR and p70S6K was higher than Ori-30 group. With dose of Orientin increased, the Rictor protein expression levels significantly decreased (P< 0.001 vs. HR group), suggesting Orientin could inhibit the activity of mTORC2.4. The molecular mechanism of Orientin against hypoxia-reoxygenation injury in myocardial cells through the interaction between Beclin 1 and Bcl-2mRNA expression level of Beclin 1 was measured by Real Time-PCR to study the regulation of Orientin on gene transcription of beclin 1 in the induction of autophagy. The phosphorylation level of Beclin 1, Bcl-2 and the Bcl-XL detected by Western Blot and the interaction between Beclin 1 and Bcl-2 on endoplasmic reticulum detected by Co-IP to study the molecular mechanisms of Orientin on the Beclin 1/Bcl-2 interaction in H/R.The results showed that:Compared with HR group, the mRNA expression level of beclin 1 increased significantly in Ori-30 group (P< 0.05), while the mRNA expression level of beclin 1 in OW group decreased, indicating that the regulation of Orientin on mRNA expression level of beclin 1 involved in induction of autophagy. Compared with HR group, the phosphorylation level of Ser87 in Bcl-2 and Ser62 in Bcl-XL in Orientin-treated groups was reduced, suggesting that Orientin could inhibit phosphorylation of Bcl-2 and Bcl-XL. While the phosphorylation level of Ser234 and Thr119 in Beclin 1 in Orientin-treated groups was all increased, suggesting that Orientin could induce phosphorylation of Beclin 1 in Ser234 and Thr119. The interaction between Beclin 1 and Bcl-2 detected by Co-IP was significantly higher than that in HR group, indicating that Orientin could enhance interaction between Beclin 1 and Bcl-2.Conclusions1. Orientin could protect against hypoxia-reoxygenation injury of myocardial cells with a dose dependent way. Orientin could obviously enhance the cell vitality, reduce the leakage of myocardial LDH and CK-MB, and significantly improve morphology and ultrastructure changes in myocardial cells damaged by hypoxia-reoxygenation injury.2. The protection of the myocardial cells by Orientin against hypoxia reoxygenation injury may be associated with activated PI3K-Akt signaling pathway, which could phosphorylate Beclin on Ser234 to prevent Beclin 1 mediated myocardial cell excessive autophagy in H/R.3. Orientin could suppress the myocardial cell apoptosis induced by hypoxia reoxygenation injury though the rise of the expression level of Bcl-2.4. Orientin could induce moderate myocardial cell autophagy by AMPK-mTORC1 signaling pathway, which play a role in resistance to hypoxia-reoxygenation injury of myocardial cells.5. Orientin could inhibit phosphorylation of Bcl-2 and Bcl-XL, then enhance the interaction between Beclin 1 and Bcl-2 in ER to prevent Beclin 1 mediated myocardial cell excessive autophagy in H/R.