Effects Of Qingnaoxuanqiao Dropping Pills (QNXQDP) On The Inflammatory Reaction Of Ischemia-reperfusion Rats

Ischemic cerebral vascular disease(ICVD) is well known with its high incidence,high death rate,high relapse rate and high mutilation rate.In recent years,it is proved that inflammatory reaction plays an important role in ischemic damage.It has been recognized that cytokines, adhesion molecules,inflammatory mediums,transcription factors and leukocyte infiltration are associated with ICVD and intimately related to each other,moreover,5-LOX,CysLT1,CysLT2 and their receptors which are reported to express in the brain,may be involved in the inflammatory pathogenesis of cerebral ischemia.All the factors mentionded above and their effect is the so-called inflammatory reaction cascade.Therefore,further research about the mechanism of ischemic cerebrovascular disease and the action targets of the pathological Changes have profound significance in modern medicine.Qingnaoxuanqiao Prescription is an experienced prescription of Academician Wang Yongyan,which is widely used in clinical.This prescription consists of Fructus.Gardeniae, Radix notoginseng and Borneolum syntheticum.Nowadays it has been processed into deropping pill with modern technology.Early results show that the deropping pill has good efficacy on treating acute and early convalescence period of ischemic apoplexy.In this study,by means of biochemical assay,radioimmunoassay(RIA),enzyme-linked immunosorbent assay(ELISA),immunohistochemistry(IHC) and molecular biology,the QNXQDP on the inflammatory reaction in rats with middle cerebral artery occlusion(MCAO) are investigated.Meanwhile the mechanisms are further investigated from following aspects:cytokine production,leukocyte infiltrate,adhesion molecules and activation from nuclear transcription factors to the t expression level of CysLTs and CysLTs receptorsThe chief results are as follows.1.Protective effects of QNXQDP on cerebral ischemic injury in rats with MCAOOBJECTIVE:To study the effects of QNXQDP on nerve symptoms,infarction size, pathomorphology changes,content of neurone specific enolase(NSE) in serum and Ca²⁺ in brain cell.METHODS:nerve symptoms were scored at different time-points(ischemia 2 hours, reperfusion 4 hours and reperfusion 22 hours),After 22 hours of reperfusion,TTC staining of brain tissue was made to determine the size of cerebral infarction,HE staining of brain was also performed to observe morphological changes,the content of NSE was assayed by RIA,the content of Ca²⁺ was assayed using Flow Cytometry.RESULTS:After MCAO,nerve symptoms, the size of cerebral infarction,the.content of NSE and Ca²⁺ rose greatly,QNXQDP decreased significantly.In model group,a number of neurons appeared degeneration,necrosis or disappearance,with leukocyte infiltration.In QNXQDP group,the status of pathomorphology was improved.CONCLUSION:QNXQDP can treat cerebral ischemic injury.2.Effects of QNXQDP on inflammatory cascade in rats with MCAO2.1 Effects of QNXQDP on content of inflammatory cytokines in rats with MCAOOBJECTIVE:To research the effects of QNXQDP on the content of inflammatory cytokines after ischemia-reperfusion in rats with MCAO.METHODS:After ischemia 2 hours and reperfusion 22 hours,the contents of IL-1β,IL-8,IL-6,TNF-α,IL-4,IL-10 and TGF-βwere asssyed by ELISA.RESULTS:After MCAO,the contents of IL-1β,IL-8,IL-6,TNF-α,IL-4,IL-10 and TGF-βincreased significantly in contrast to sham group,QNXQDP can decrease them remarkably.CONCLUSION:QNXQDP can reduce the contents of cytokines in rats with MCAO to alleviate cerebral ischemic injury.2.2 Effects of QNXQDP on content of nitric oxide(NO) and prostaglandin analogs,Activity of nitric oxide synthase(NOS) in rats with MCAOOBJECTIVE:To research the effects of QNXQDP on the nitric oxide,nitric oxide synthase and prostaglandin analogs after ischemia-reperfusion in rats with MCAO.METHODS: ischemia 2 hours and reperfusion 22 hours,determining of nitrate reductase NO content in brain homogenate,cNO and iNO activity;measuring of plasma TXB2 and 6 - keto -PGF1αcontent with RIA.RESULTS:â‘ After MCAO,content of NO and activity of TNO,nNO, iNO were significantly higher than sham group in brain.â‘¡After MCAO,QNXQDP decreasing content of NO and activity of TNO,nNO and iNO,also decreasing contents of TXB2 and 6 - keto-PGF1αin plasma.CONCLUSION:QNXQDP can decreased content of NO and activity of TNO,nNO and iNO,regulating TXB2 and 6 - keto-PGF1αto relieve cerebral ischemic injury.2.3 Effects of QNXQDP on leukocyte and endothelial cells adhesion chemotaxis and infiltration in rats with MCAOOBJECTIVE:To research the effects of QNXQDP on the expression of cell adhesion molecules(ICAM-I) and Macrophage inflammatory protein-1a(MIP-1a),changes of myoloperoxidase(MPO) content in brain tissue.METHODS:The expression of ICAM-â… and MIP-1a protein was assayed by IHC.MPO was assayed by biochemisty.RESULTS:After MCAO,the expressing positive area and integral optical density of ICAM-â… and MIP-1a, content of MPO increased significantly in model group,QNXQDP 180mg/kg and 90 mg/kg can inhibit the positive area and integral optical density of ICAM-â… and MIP-1a on cortex and hippocampus,reduce the content of MPO in brain tissue.CONCLUSION:QNXQDP can decrease leukocyte and endothelial cells adhesion,chemotaxis and infiltration in rats with MCAO.2.4 Effects of QNXQDP on nuclear transcription factors in rats with MCAO OBJECTIVE:To investigate the effects of QNXQDP on the expression of nuclear factor-κB (NF-κB) in brain tissue.METHODS:The expression of NF-κB protein was assayed by IHC. RESULTS:After MCAO,the expression of NF-κB increased significantly in model group, and cortex and hippocampus showed nuclear translocation.QNXQDP 180mg/kg and 90 mg/kg could inhibit the nuclear translocation and decreace the positive area and integral optical density of NF-κB in cortex and hippocampus.CONCLUSION:QNXQDP could inhibit the expression of transcription factors in brain.3.Effects of QNXQDP on 5 - lipoxygenase / leukotriene pathway in rats with MCAOOBJECTIVE:To investigate the effects of QNXQDP on the content of 5-LOX,LTB4 and CysLTs,CysLT1 and CysLT2 expression in brain tissue.METHODS:ischemia 2 hours and reperfusion 6,12,24,36,48h hours,the content of 5-LOX,LTB4 and CysLTs was assayed by ELISA.RESULTS:ischemia 2 hours and reperfusion 6,12,24,36,48h hours,the content of 5-LOX,LTB4 and CysLTs were increased in model group compare with shame group, expression of CysLT1 and CysLT2 in cortex and hippocampus also increased in cortex and hippocampus.QNXQDP can decrease the content of 5-LOX,LTB4 and CysLTs in brain tissue,CONCLUSION:QNXQDP could decrease the contents of 5-LOX,LTB4 and CysLTs in brain,also could inhibit the expression of CysLT1 and CysLT2 in cortex and hippocampus.CONCLUSIONQNXQDP can alleviate neuronal injury and the inflammatory reaction after of ischemia-reperfusion in rats,whose its main mechanisms of action may be associated with:â‘ inhibition of neuron-specific enolase(NSE) levels and calcium overload;â‘¡regulation of inflammatory cytokines,reducing the NO content and inhibiting NOS activity;â‘¢inhibiting leukocyte and endothelial cells adhesion,chemotaxis and infiltration in brain tissue;â‘£reducing the content of 5-LOX,LTB4 and CysLTs,inhibiting the expression of cysteinyl leukotriene receptor(CysLT1 and CysLT2) in cortex and hippocampus.Its prevention and treatment inflammatory injury after cerebral ischemia-reperfusion.In addition,CysLT1 and CysLT2may be targets for inhibition.In this experiment study,we observed the effects of QNXQDP on the expression of protein CysLT1 and CysLT2 in cortex and hippocampus of ischemia-reperfusion rats,which has not been reported by now.The reports about the dynamic changes of the content 5-LOX, LTB4 and CysLTs after cerebral ischemia-reperfusion within 48 hours are also very few. Therefore,our study is innovative to a certain some extent.