Study On The Cardiotoxicity And Mechanism Of Ophiopogon Japonicus D'

Shenmai injection is consisted by red ginseng and Ophiopogon japonicus.It is an important traditional Chinese medicine injection for clinical treatment of cardiovascular diseases.In recent years,many studies have been reported that Shenmai injection has significant adverse reactions,with the exception of allergic reactions.In the reports,we find Shenmai Injection may cause chest tightness,rapid heartbeat and other symptoms,but the specific mechanism of the adverse reactions has not been reported yet.It needs to be further explored.In this study,UPLC-TOF-MS was used to detect the contents of ginsenoside Rb,Rg and Re,and Ophiopogon japonicus D and D'in Shenmai injection,further toxicity screening tests in the cell.H9c2 cells were selected to discover the effects of ginsenosides Rb1,Rb2,Rb3,Rg1,Rg2,Rg3,Re,and Ophiopogon japonicus D,D'on the survival rate of the cells.Monomeric components in Shenmai Injection that have a potentially toxic effect on cardiomyocytes were unkown.The preliminary screening results showed that ginsenosides Rb1,Rb2,Rb3,Rg1,Rg2,Rg3,and Re did not have significantly affect cardiomyocyte viability at 800?mol·L^-1 on H9c2 cells.And Ophiopogon japonicus D?OPD?was administered at high dose(100?mol·L^-1)slightly decreased the viability of H9c2.Meanwhile,it was unexpectedly found that the Ophiopogon saponin D'?OPD'?decreased the viability of cardiomyocytes only at 2.5?mol·L^-1,and has obviously toxic effect on cardiomyocytes.Further determination of lactate dehydrogenase?LDH?was performed in H9c2 cells.Simultaneously,the high-content immunofluorescence technique detected the effect of saponins on the number of myocardial cell nuclei number.The results were consistent with the trend of cell viability,suggested that OPD'may be a key component of cardiac toxicity in Shenmai injection.The results provided important clues for further depth study.Therefore,in this study,OPD?as the research object,based on the initial screening results of its obvious cardiomyocyte cytotoxicity,the evidence and possible mechanism of myocardial cytotoxicity were further explored at the cellular level,and finally confirmed at the animal level to verify its cardiotoxicity.In vitro experiments selected rat H9c2 cardiomyocytes,human Ac16cardiomyocytes,and rat primary cardiomyocytes to verify the cytotoxicity of OPD'and to explore the toxicity of OPD'and its possible mechanism.The effect of different concentrations of OPD's on the morphology of three types of cells was observed under a fluorescence microscope.The viability of H9c2 cells was meared by MTS after the treatment of OPD'on H9c2 and the IC₅₀ was calculated.The intracellular LDH release were used by a lactate dehydrogenase kit to measure the effect of OPD?on cell damage.The degree of myocardial cell damage,flow cytometry was used to detected OPD?on the H9c2 reactive oxygen species?ROS?,mitochondrial membrane potential?MMP?,calcium ion content and apoptosis rate and other targets which were closely related to cytotoxicity.Real-time quantitative PCR technique was used to detect the expression of OPD?on the gene level of apoptotic pathway and endoplasmic reticulum stress pathway related factors,and Western Blot was used to detect the protein level of OPD?on apoptotic pathway and endoplasmic reticulum stress pathway-related protein.The effects of expression were examined by laser confocal scanning microscopy?LSCM?to record transient changes in total Ca²⁺concentration in primary cardiomyocytes.The results showed that OPD?at a lower concentration(5?mol·L^-1)can produce certain toxicity to cardiomyocytes,specifically by inhibiting the activity of cardiomyocytes and increasing the release of LDH,and the content of reactive oxygen species in the cells was significantly increased.The calcium ion content also increased,the mitochondrial membrane potential decreased or even disappeared,and the apoptosis rate increased significantly.The results of real-time fluorescence quantitative PCR showed that the expression of apoptosis-related genes P53,BAX,cytochrome C,CHOP,Caspase 3,Caspase 9 and endoplasmic reticulum stress-related genes ATF4 and GRP78 were all elevated 24h after OPD?Dtreatment.Western blot further detected the expression of related molecules at the protein level,and the results were consistent with the influence of mRNA.In addition to the effects on the above cell lines,OPD?treatment resulted in significant apoptosis of the primary rat neonatal cells,increased lactate dehydrogenase levels,and calcium transient results,indicating that high concentrations of OPD?(20?mol·L^-1)were used.The cells died instantly and further verified the cytotoxicity of OPD'.In vivo experimental study,SD rats were injected intravenously for 10 days.The weight changes of the rats were recorded and the visceral coefficient of major organs was calculated.The effects of OPD'on cardiac function of rats were measured by echocardiography and electrocardiogram.Pathological sections were used to further analyze the toxic effects of OPD'.Blood biochemical indicators were analyzed.The results of in vivo experiments showed that OPD'reduced body weight,increased organ coefficient,and edema of major organs.Electrocardiographic examination revealed that OPD'caused arrhythmia in rats,impaired cardiac function,and increased release of inflammatory factors.OPD?causes white blood cell aggregation,necrosis of myocardial cells,peripheral arterial hyperplasia,thinning of the arterial wall,rupture of the blood vessels,extensive bleeding of the heart,and disruption of normal heart function.In summary,this study firstly identified cardiomyocyte cytotoxicity of the main effective saponin component OPD?in Shenmai injection.The cytotoxicity of OPD?was verified from the cellular and animal levels,respectively,and the toxic mechanism of OPD?was preliminarily explored.It may be that the intracellular endoplasmic reticulum stress response is initiated by overloading intracellular calcium ions,which activates apoptosis pathway-related factors and induces apoptosis,suggesting that the major saponin component OPD'in Chinese herbal Ophiopogon japonicus has potential cardiotoxicity.The clinical application should pay attention to the dosage of Ophiopogon japonicus medicinal materials.At the same time,the production of traditional Chinese medicine injections containing Ophiopogon japonicus should strictly control the content of OPD'.