Effect Of Acute Macleaya Total Alkaloid Poisoning On The Expression Of HCN4 In Sinus Node

Objective:To observe the manifestations of macleaya total alkaloid poisoning in rabbits and the morphological changes of the sinus node tissues,and to explore the changes of HCN4（hyperpolarization activated cyclic nucleotide gated cation channel 4）expression in the sinus node during acute macleaya total alkaloid poisoning in rabbits.Qualitative detection of the main components of Macleaya alkaloids and comparison of the cytotoxicity of different components of Macleaya alkaloids on primary sinoatrial node cells and primary myocardial cells.Methods:In the acute toxicity test of rabbits,20 healthy and clean rabbits,half male and half female,were randomly divided into blank control group（n=5）and experimental group（n=20）.The maximum dose volume of 0.1%sodium carboxymethyl cellulose（0.1%CMC-Na）solution and 0.1%CMC-Na solution of macleaya total alkaloids with gradient concentration were administered by gavage respectively.Observe the general signs of rabbits in the experimental group within 24h,make statistics on the death situation and time of rabbits,calculate the median lethal dose(LD₅₀),and use LD₅₀ and2LD₅₀ to dissolve the total base of macleaya in 0.1%CMC-Na solution of the maximum administration volume to gavage rabbits again until the number of dead rabbits in each group is 4.Systematically dissect the rabbits,and use hematoxylin eosin（HE）staining method Masson trichrome staining was used to observe the histopathological changes of some organs and sinoatrial node.Immunohistochemical staining and Western Blot were used to detect the expression of HCN4.Statistical analysis was used to calculate whether the difference was statistically significant,and further understand the effect of acute macleaya alkaloids poisoning on the level of HCN4 in rabbit sinoatrial node.High performance liquid chromatography（HPLC）was used to qualitatively detect the main components of Macleaya alkaloids.The primary sinoatrial node cells and primary myocardial cells of SD rats were cultured.Cell counting reagent-8（CCK-8）was used to calculate the 50%inhibitory concentration IC₅₀ and its 95%confidence limit of different components on primary sinoatrial node cells and primary myocardial cells.The difference of cytotoxicity of different components in Macleaya alkaloids was compared.Results:1.According to the results of acute toxicity test,the LD₅₀ of acute macleaya alkaloids poisoning in rabbits was 510.62mg/kg,with a 95%confidence interval of（255.31-1021.24）mg/kg.2.The results of HE staining in rabbits with acute macleaya intoxication showed that P cells in sinoatrial node tissues were vacuolated,cytoplasm was lightly stained,and small blood vessels in liver,spleen,kidney and other organs were congestion,without obvious specific pathological changes;3.Western Blot and immunohistochemical methods were used to confirm that the HCN4 expression in rabbits with acute poisoning was mainly located in the cytoplasm and membrane of P cells in the sinus node.Compared with the control group,the HCN4 expression in the sinus node of rabbits and SD rats with acute macleaya alkaloids poisoning decreased（P<0.05）,and there was no significant difference between the high dose group and the low dose group in the HCN4 expression in the sinus node of rabbits with acute poisoning by macleaya alkaloids.4.The main components of Macleaya alkaloids were sanguinarine and chelerythrine by HPLC;5.IC₅₀ of sanguinarine on primary sinoatrial node cells of SD rats=1.619μM.95%confidence interval is（0.7915-1.988）μM.IC₅₀=1.626 for primary myocardial cells of SD ratsμM.95%confidence interval is（1.445-1.777）μM;6.Effect of chelerythrine on primary sinoatrial node cells IC50=1.903μM.95%confidence interval is（1.548-2.515）μM;Effect of chelerythrine on primary myocardial cells IC₅₀=1.997μM.95%confidence interval is（1.862-2.148）μM。Conclusion:1.The LD₅₀ of acute macleaya total alkaloid poisoning in rabbits was 510.62mg/kg,with a 95%confidence interval of（255.31-1021.24）mg/kg.The main target organs of the poisoning had no characteristic histopathological changes,2.The expression of HCN4 in the sinoatrial node of the poisoned group was lower than that of the control group;3.The main components of Macleaya alkaloids are sanguinarine and chelerythrine;4.The cytotoxicity of sanguinarine to primary sinoatrial node cells was stronger than that to primary myocardial cells;The cytotoxicity of chelerythrine to primary sinoatrial node cells was stronger than that to primary myocardial cells;5.The toxicity of sanguinarine to primary cardiomyocytes and primary sinoatrial node cells is stronger than that of chelerythrine to primary cardiomyocytes and primary sinoatrial node cells.