| Objective In recent years,there is an urgent need to seek safe and effective drugs for the treatment of myocardial ischemia-reperfusion injury.Bawei Chenxiang Powder is a traditional Chinese Tibetan patent medicine.It is mainly used in Tibetan medicine to treat coronary heart disease.Animal experiments have found that it has anti-myocardial ischemia damage effects,but there is a lack of exploration and analysis of cellular and molecular mechanisms.Therefore,the serum pharmacology method was used in this experiment to study the effect and molecular mechanism of Bawei Chenxiang Powder against myocardial hypoxia/reoxygenation injury at the cellular level.Methods 1.Using the cell viability and LDH content in the cell supernatant as indicators,the orthogonal test was used to optimize the hypoxia/reoxygenation model of H9C2 cells under 2% oxygen concentration.2.The experiment was divided into blank group,model group and drug group.The drug group was divided into low-dose group(Bawei Chenxiang Powder was administered with 2.5 g/kg of serum of rats),and middle-dose group(Bawei Chenxiang Powder was administered with 8 g/kg).g/kg gavage of rat serum)and high-dose group(Bawei Chenxiang powder 12 g/kg gavage rat serum).Different doses of Bawei Chenxiang Powder containing medicated serum interfered with hypoxia/reoxygenation H9C2 cells.3.Cell viability was determined by CCK-8 method;LDH content(or activity)in cell supernatant was determined by biochemical method;CK,GSH-Px,SOD,CAT,Complex I and MDA content in cell supernatant was determined by ELISA method;Electron microscopy was used to observe cell morphology and ultrastructure;flow cytometry and fluorescence microscopy were used to determine the content of reactive oxygen species and apoptosis rate in cells;real-time quantitative PCR or Western blotting were used to determine oxidative stress-related factors(Ndufa10,Nrf2,Keap1).,HO-1 and Trx),apoptosis-related proteins(Bcl-2,Bax,Caspase-3 and Cytc)and the gene or protein expression of signaling pathway proteins PI3K/AKT/GSK3β.Results 1.It was determined by orthogonal test that under 2% oxygen concentration,20% blank serum,sugar-free medium,and hypoxia for 8 hours;under21% oxygen concentration,20% blank serum,high glucose medium,and reoxygenation for 4 hours as cardiomyocytes Optimal model conditions for hypoxia/reoxygenation.2.Compared with the model group,the cell survival rate in the drug group was significantly increased,and the contents of LDH,CK and MDA in the supernatant were significantly decreased;the contents of GSH-Px,SOD,Complex I and CAT were significantly increased.3.Compared with the model group,the number of dead cells under the light microscope and the degree of damage to the cell wall of the drug group were reduced;under the electron microscope,the cell morphology and structure were normal,only a few mitochondria were slightly swollen,and a large number of lipid droplets and a small amount of autogenous cells were seen in the cytoplasm.phagocytosis,and a small amount of mildly expanded rough endoplasmic reticulum was found in some cells;fluorescence microscopy and flow cytometry showed that the content of reactive oxygen species in the drug-administered group was significantly decreased,and the apoptosis rate was significantly decreased.4.Compared with the model group,the gene and protein expressions of oxidative stress-related factors Nrf2,Keap1,HO-1,Ndufa10 and Trx in the drug group were significantly decreased;the gene and protein expressions of apoptotic factors Bax,Caspase-3 and Cytc were significantly decreased,The gene and protein expression of the anti-apoptotic factor Bcl-2 was significantly increased.5.In the PI3K/AKT/GSK3 signaling pathway,the protein and gene expressions of PI3 K and AKT were significantly decreased,and the expression of GSK3 was significantly increased.Conclusion 1.The optimal hypoxia/reoxygenation model of cardiomyocytes is optimized.The optimized hypoxia/reoxygenation model is 20% rat blank serum,sugar-free medium,(2% oxygen concentration)hypoxia for 8 h;normal culture basal,normoxia(21% oxygen concentration)reoxygenation for 4 h.2.The detection results of cell viability,oxidative stress-related indexes in supernatant,mitochondrial microstructure,reactive oxygen species content,oxidative stress-related genes and proteins showed that the medicated serum of Bawei Chenxiang Powder had an effect on cardiomyocyte hypoxia.The oxidative stress injury after hypoxia/reoxygenation has a protective effect,and its effect may be exerted by reducing the oxidative stress injury after cardiomyocyte hypoxia/reoxygenation.3.The medicated serum of Bawei Chenxiang Powder can promote the gene and protein expression of anti-apoptotic factor Bcl-2,inhibit the gene and protein expression of apoptotic factors Bax,Caspase-3 and Cytc,thereby inhibiting hypoxia/reoxygenation of cardiomyocytes apoptosis,resulting in the protection of hypoxic/reoxygenated cardiomyocytes.4.The medicated serum of Bawei Chenxiang Powder can up-regulate the expression of PI3 K and AKT gene and protein,and down-regulate the gene and protein expression of GSK3β.Stimulates apoptosis through mitochondrial pathway,and produces anti-myocardial hypoxia-reoxygenation injury.5.The medicated serum of Bawei Chenxiang Powder reduces the contents of LDH,CK and MDA in H9C2 cell culture medium under hypoxia for 8 hours,and increases the content of GSH-PX,SOD,CAT and Complex I,indicating that the medicated serum of Bawei Chenxiang Powder can inhibit the lack of hypoxia.The peroxidation of oxygenated cardiomyocytes has a certain protective effect on the damage caused by hypoxia of cardiomyocytes. |
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