Substance Basis And Mechanism Of Pseudoallergy Reaction Caused By Sodium Aescinate For Injection

OBJECTIVE: The aim of this study was to investigate the pharmacodynamic,toxicological effects,and the related mechanism of sodium aescinate for injection(SAI)and its active components.Through experiments in vivo and in vitro to explore the main active ingredients and mechanism that caused clinical adverse reactions,in order to provide a certain basis for the rationality and safety of clinical medication.METHODS:(1)The rats were divided into sham operation group,brain injury model group and SAI group(3.5 mg/kg/d),8 SD rats in each group.Behavioral changes,brain water content,TTC staining,pathological changes and related gene expression were observed.(2)After stimulating rat basophilic leukemia cells(RBL-2H3)with SAI and its active ingredients,cell morphology changes were observed,cell viability,histamine,β-hexosaminidase,oxidative stress indicators,and apoptosis were detected to discuss the main toxic components that cause adverse reactions to SAI.(3)To explore whether SAI(3.84,7.68,15.36 mg/kg)can induce anaphylactoid reaction in mice and its related mechanism,we observed the blue staining in mice ears,blood biochemical factors and pathological changes by injecting Evans blue(EB)into mice tail vein.(4)To observe the effect of loratadine pretreatment on RBL-2H3 cell viability,β-hexosaminidase,apoptosis and intracellular calcium induced by SAI.RESULTS:(1)After 7 days of treatment,the behavioral score of brain injury rats in SAI group gradually tended to be normal;the degree of brain injury was alleviated compared with the model group;q RTPCR results showed that SAI could inhibit the expression of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α,inhibit the occurrence of brain edema mediated by aquaporin 4(AQP4),so as to reduce brain edema and nerve injury of brain injury rats.(2)SAI above 60 μg/m L concentration could lead to cell apoptosis and necrosis of RBL-2H3 cells,oxidative stress,increasing of intracellular calcium concentration as well as the increase of extracellular histamine and β-hexosaminidase.After treatment of SAI and its four main active components(60 μg/m L)with RBL-2H3 cells,SA-B and SA-D could lead to changes in cell morphology from spindle to round,and a large number of cell fragmentation,thus promote the release of intracellular particulate matter,increased the intracellular calcium concentration as well as cell apoptosis and necrosis.In addition,the concentrations of extracellular histamine and β-hexosaminidase increased.The sequence of toxicity of SAI and its four active components to RBL-2H3 cells was as follows: SA-B > SA-D > SAI > SA-A > SA-C.(3)After injection of SAI for 30 min,the concentration dependent blue staining was observed in the ears of mice.Histamine and vascular endothelial growth factor(VEGF)in serum increased in a dose-dependent manner,but Ig E did not change significantly.Pathological changes were observed in the ears,lungs and kidneys of mice to a certain extent.q RT-PCR results showed that the expression of Rho A,ROCK,MYPT and MIC genes were up-regulated.(4)5 μg/m L loratadine preincubated with RBL-2H3 cells alleviated the decrease of cell viability,increase of intracellular calcium concentration cell apoptosis and necrosis.and the decrease of extracellular β-hexosaminidase content of RBL-2H3 cells induced by SAI,which indicated that loratadine could inhibit the degranulation of RBL-2H3 cells induced by SAI.CONCLUSIONS: SAI can inhibit AQP4 mediated brain edema by inhibiting the expression of IL-1β,IL-6 and TNF-α,which can protect the brain injured rats and alleviate brain edema.In vivo and in vitro experiments have shown that SAI can trigger pseudoallergy reactions,and SA-B and SA-D may be the main toxic substances that trigger pseudoallergy reactions.The use of antihistamines can reduce the occurrence of its adverse reactions,which provides a certain reference for improving the safety and effectiveness of its clinical application.