Study On The Effect Of Tongluo Qingnao Recipe On Acute Cerebral Edema In Ischemic Stroke And The Mechanism Of Astrocyte HMGB1/TLR4

Ischemic stroke is one of the leading diseases with high morbidity and mortality worldwide.Cerebral edema is one of the greatest complications of ischemic stroke and is the main cause of acute deterioration and death in patients with large ischemic strokes.Causes.Astrocytes are important cells involved in cerebral edema,which are closely related to changes in cerebrovascular function and altered activity of water transport proteins and plays an important role in the steady state of cerebral edema.High-mobility group box 1(HMGB1)/The Toll-like receptor 4(TLR4)pathway acts as an inflammatory Marker pathways play a very important role in the development of cerebral edema in early ischemic stroke.HMGB 1 is the inflammatory A potent inducer of the cascade,expressed predominantly on glial cells,causes neuroedema and induces macrophage synthesis of tumor necrosis factor(tumor necrosis factor,TNF-),interleukin-6(inter-leukin-6,IL-6),interleukin 1(inter-leukin-,IL-1β).leads to the accumulation and infiltration of inflammatory cells,while inducing inflammation and a range of secondary tissue damage.Thus,in studies of ischemic brain edema,the astrocyte HMGB1/TLR4 pathway affects AQP4 and blood-brain barrier The impact of the Tong LuoQingNao(TLQN)is a preliminary study conducted by our laboratory to treat ischemic A new Chinese medicine for acute cerebral edema in stroke,which is mainly composed of Panax Notoginseng(Panax notoginseng).Saponins,PNS),gardenia glycosides(Geniposide,GE),baicalein((Baicalin,BA)consists of three active ingredients that have a significant inhibitory effect on the inflammatory response to cerebral ischemia and have the ability to relieve the Cerebral edema,which decreases the expression of water channel protein(Aquaporin-4,AQP4).In this study,we investigated the effects of TLQN and its active components on the astrocyte HMGB 1/TLR4 inflammatory pathway on AQP4 and the blood-brain barrier,so as to investigate the mechanism of TLQN inhibiting cerebral edema in ischemic stroke.Part Ⅰ.Effects of Tongluoqingnao formula on cerebral edema and astroglial HMGB1/TLR4 pathway and blood-brain barrier in ischemia-reperfused rats.OBJECTIVE:To observe the effects of TLQN and its active components on acute phase edema and the effects on astrocyte HMGB1/TLR4 pathway and blood-brain barrier in ischemia-reperfusion rats using a rat cerebral ischemia-reperfusion model.METHODS:A rat cerebral ischemia-reperfusion model was replicated by wire embolization and randomly divided into a sham-operated group(Sham),a reperfusion group(CIR),Tongluoqingnao(TLQN)42mg/kg group(where baicalein:gardenia saponin:panax ginseng total saponin=1:12:6),panax ginseng total saponin(PNS)13.3mg/kg group,gardenia saponin+baicalein(BA+GE)In the 26.5mg/kg+2.2mg/kg group,administered on the day of surgery,2 d by tail vein injection,1 day by tail vein injection.Reperfusion for 24 h and 48 h was established as the observation period.1.observe the effect of TLQN on cerebral ischemia reperfusion by weight,neurological function score,dry and wet brain weight,and functional MRI.Effects of acute cerebral edema in perfused rats.2.Biochemical detection of IL-6,IL-1,TNF-,IL-1 and TNF-in the ischemic side cortex and blood of ischemia-reperfused rats.Concentration content.3.Western blot and immunofluorescence methods were used to detect the effects of TLQN and its active components on ischemia rejuvenation.Perfused rats 24h,48h cortical AQP4,HMGB1,TLR4,NF-B,p-NF-B expression and changes in protein levels.4.using transmission electron microscopy(TEM)and immunohistochemistry to detect the effects of TLQN and its active components on cell swelling and protein levels in ischemia-reperfused rats.Blood-brain barrier effects.RESULTS:1.Body weight and neurological function scores showed significant neurological deficit symptoms in reperfused rats.The TLQN group significantly decreased the neurological deficit score at 24 h,48 h(P<0.05),while the PNS group and the BA+GE group showed a significant reduction in neurological deficit score in 48 h rats(P<0.05).2.the combination of brain water content and MRI results showed that,compared with the Sham group,the 24 h ischemia-reperfusion rats.Brain water content increased significantly(P<0.05);brain water content decreased significantly after 24 h of reperfusion in TLQN rats.(P<0.05);reperfusion for 48 h.Brain water content in TLQN,BA+GE and PNS groups.Significantly reduced(P<0.05).The ADC signals in the hippocampus,corpus callosum and anterior cortex of the CIR group were significantly reduced(P<0.01),and then the ADC signals in the hippocampus,corpus callosum and anterior cortex of the CIR group were significantly reduced(P<0.05).The anterior cortex and corpus callosum ADCs were significantly higher in the TLQN,PNS and BA+GE groups than in the PNS and BA+GE groups for 24 h of perfusion.CIR group(P<0.05),while there was a tendency to increase in the hippocampal region(P>0.05).3.Biochemical assays showed that IL-6,IL-1,and TNF-were significantly increased in the ischemic side of the cerebral cortex of CIR group rats.Ascending(P<0.01);after administration of TLQN,PNS,BA+GE interventions,and reperfusion for 24 and 48h In the ischemic side of the cerebral cortex,IL-6,IL-1,and TNF-were significantly decreased in rats(P<0.05).4.Immunofluorescence results were similar to those of Western-Blots,and the CIR group compared to the Sham group.AQP4 concentration was significantly increased(p<0.01)and GFAP activation(p<0.01)in the ischemic side of the cerebral cortex of rats.And HMGB1,TLR4,and p-NF-B also increased significantly(p<0.01);giving TLQN.After PNS,BA+GE interventions and reperfusion for 24h and 48h,cortical AQP4 concentrations in the ischemic side of the rat cerebral cortex Significantly reduced(P<0.05);GFAP received significant inhibition(P<0.05),and HMGB1,TLR4,and The p-NF-B content was also significantly down-regulated(p<0.05).5.The TLQN and its associated immunohistochemical Claudin-5 were shown in transmission electron microscopy,GFAP perimeter,and immunohistochemical Claudin-5 assays.The active component significantly reduced endothelial astrocyte swelling in ischemia-reperfused rats and improved the blood-brain barrier.The tight junctions of Oxygen-Glucose Deprivation/Reperfusion(OGD/R)astrocytes allow their barrier function to be repaired.Part Ⅱ Effects of Tongluoqingnao formula and its active ingredients on Oxygen-glucose deprivation/Reperfusion(OGD/R)astrocyte neuroinflammation and HMGB1/TLR4 pathway.OBJECTIVE:To apply an in vitro co-culture experimental method with human brain astrocytes and astrocytes/microvascular endothelial cells to observe the effects of TLQN and monomers on the HMGB1/TLR4 pathway and endothelial cell tight junctions in OGD/R-injured astrocytes.Methods:1.Normal astrocytes were used to detect TLQN and each of its active components(PNS,BA,GE)The drug toxicity concentration was used as the basis for the subsequent efficacy experiments.The CCK-8 method was used to detect the viability of astrocytes and observe the cell morphology.2.2.construct OGD/R cell model,oxygenose deprivation for 6 h/reoxy-complexose for 12 h(OGD/R),and then test the viability of astrocytes by CCK-8 method.6/12),astrocytes were divided into normal(Control)and OGD/R groups,and the drug group(TLQN,PNS,BA,GE)after 2.5-50 g/ml of intervention,respectively,to explore the effects of TLQN and monomer on the Drug efficacy and drug delivery concentration of OGD/R-injured astrocytes.The CCK-8 method was used to detect the viability of astrocytes and observe the cell morphology.3.3.biochemical method to detect the content of IL-6,IL-1,TNF-in the cell culture medium.Western Blot,Immunocytochemical fluorescence assay for cellular GFAP,AQP4,HMGB1,...TLR4,p-NF-B expression and protein level changes.4.using immunofluorescence and transmission electron microscopy techniques,it was found in HA/BMECs co-culture system that after detecting OGD/R Survival of BMECs by culture medium secreted by HA cells reduces the intercellular tight junction protein Claudin-5,ZO-1 and effects within cells.RESULTS:1.Drug toxicity combined with pharmacokinetic experiments showed that TLQN and its active ingredients PNS,BA,GE No significant toxicity was observed at concentrations ranging from 2.5 to 50 g/ml.2.compared with HA cells after OGD/R,where TLQN(5 μg/m L)and PNS(10 μg/m L)were not significantly toxic.g/mL)intervention,the cell viability was significantly enhanced(P<0.05)and had the best effect,which could significantly improve the cell status.3.immunofluorescence results were similar to those of Western-Blots:1)Compared with the Control group.The AQP4 concentration of HA cells in the OGD/R group was significantly increased(p<0.01),and HMGB1,TLR4,p-NF-B were also significantly increased(P<0.01);administration of TLQN(5 mg/kg)and Significant reduction in HA cell AQP4 concentration after PNS(10 mg/kg)intervention(p<0.05).HMGB1,TLR4,and p-NF-B contents were also significantly down-regulated(P<0.01),and the addition of ethyl pyruvate(EP)after inhibition of OGD/R group HA cells AQP4,HMGB1,TLR4,p-NF-B Expressed.4.CCK8 results showed a significant reduction in the survival of BMECs by HA extracellular fluid after GD/R(P<(0.05);immunofluorescence results showed that the tight junction proteins Claudin-5,ZO-,and ZO-between BMECs cells were not significantly different from each other.1 had a significant decrease.After TLQN and PNS intervention,the survival of BMECs increased,and intercellular Claudin-5,ZO-1 increased in number,the above results are consistent with the trend after EP intervention,indicating that TLQN has an indirect inhibitory effect on endothelial cells function of the injury.CONCLUSION:In vivo and in vitro experimental studies have shown that Tongluoqingnao the brain can reduce acute cerebral edema in ischemia-reperfusion rats,especially Cortical edema acts.The mechanism is inhibition of AQP4 expression and astrocyte swelling via the astrocyte HMGB1/TLR4 pathway.It also decreases the expression of edema-related inflammatory factors IL-1β,IL-6,and TNF-α,and the Tongluoqingnao formula can be used to clear the brain by The astrocyte HMGB1/TLR4 pathway indirectly increases the tight junctions of endothelial cells and improves blood-brain barrier function.The active component of its action is the blood-activating group(Panax ginseng total saponin).