Research On Effect Of HMGB1/TLR4/MMP9 Signaling Pathway To Injure Blood Brain Barrier In Acute Intracerebral Hemorrhage Rats And On Protective Mechanism Of Rehmannia And Rhubarb Decoction

Research on effect of HMGB1/TLR4/MMP9 signaling pathway to injure blood brain barrier in acuteintracerebral hemorrhage rats and on protective mechanism of Rehmannia and Rhubarb Decoction Background: Intracerebral hemorrhage is a common critical disease in neurology with high mortality and morbidity,causing heavy burden to society and family.However,whether surgical or medical treatment in recent 20 years both failed to achieve significant progress.Traditional Chinese medicine(TCM)has a long history in the treatment of acute hemorrhagic stroke to own rich clinical experience.Because of its multilevel and multitarget effect,TCM has became a new hot spot.Rehmannia and Rhubarb Decoction(RRD)firstly shown in volume 18 of Sun Simiao’s《Qian Jin Yi Fang》in Tang dynasty,consisting of rehmannia and raw rhubarb,can clear heat and nourish Yin as well as stop bleeding and promote blood circulation.RRD treats various hemorrhagic diseases well,which has a clear clinical efficacy.Blood brain barrier(BBB)as an important guard of the central nervous system is firstly damaged after ICH occurs.Matrix metalloproteinase 9(MMP9)has been elucidated as an important molecular causing BBB damage.To clarify its upstream regulatory mechanism has a great significance.RRD could down-regulate expression of MMP9 in acute ICH rats based on the early experiment.However the exact mechanism is unclear.In this experiment,we used autologous blood injection to make ICH rat model and evaluated behavior evaluation,structure and function of BBB,expressions of relative protein after intervention of all groups.Further we used HMGB1-induced astrocyte model in vitro and evaluated expressions of relative protein and mRNA as well as MMP9 activity after intervention of all groups.So as to make clear the effect of HMGB1/TLR4/MMP9 signaling pathway on BBB injury in acute ICH rats and protective mechanism of RRD,providing theoretical basis for the treatment of ICH.Part Ⅰ Research on effect of HMGB1/TLR4/MMP9 signaling pathway to injure blood brain barrier in acuteintracerebral hemorrhage rats Objective: To observe the effect of HMGB1/TLR4/MMP9 signaling pathway to injure blood brain barrier in acute intracerebral hemorrhage rats.Method: 68 spontaneous hypertensive rats were randomly divided into SHAM group,ICH group,TLR4 antagonist TAK-242 group and HMGB1 inhibitor ethyl pyruvate(EP)group,with 17 rats in each group.Needling without blood transfusion was done in sham group and the other three groups were established into ICH models by autologous blood injection.TLR4 antagonist TAK-242 was intraperitoneally injected at 3mg/kg/d and HMGB1 inhibitor ethyl pyruvate was intraperitoneally injected at 80mg/kg/d.The samples were taken after 3 days of continuous administrations.Neurological defect scores were performed in each group.Structure of BBB was observed by electron microscopy and immunofluorescence staining.Function of BBB was tested by brain water content and Evans blue assay.Expressions of HMGB1,TLR4 and MMP9 protein around brain hematoma were tested by Western blot.Results: Neurological deficit: compared with SHAM group,neurological deficit scores in each ICH model groups were higher(P<0.05),which were lower in TAK-242 group and EP group compared with ICH group(P<0.05).Structure of BBB: compared with SHAM group,structure of BBB in each ICH model groups was obviously damaged by electron microscope.Tight junction protein Claudin-5 and astrocyte were reduced by immunofluorescence staining.Compared with ICH group,TAK-242 group and EP group showed different degrees of remission on destruction of BBB structure by electron microscope,and showed a certain increase expressions of claudin-5 and astrocytes by immunofluorescence staining.Function of BBB: compared with SHAM group,brain water content and permeability of BBB were increased in each ICH model groups,which were lower in TAK-242 group and EP group compared with ICH group(P<0.05).Expression of protein: compared with SHAM group,expressions of HMGB1,TLR4 and MMP9 protein around brain hematoma were increased in each ICH model groups,which were lower in TAK-242 group and EP group compared with ICH group(P<0.05).Conclusion: BBB structure and function was destructed in acute ICH rats,which may be related to expressions of HMGB1/TLR4/MMP9 signaling pathway.Part Ⅱ Mechanism on Rehmannia and Rhubarb Decoction to protect blood brain barrier via HMGB1/TLR4/ MMP9 signaling pathway in acute intracerebral hemorrhage rats Objective: To observe protective effect of Rehmannia and Rhubarb Decoction on blood brain barrier via HMGB1/TLR4/MMP9 signaling pathway in acute intracerebral hemorrhage rats.Method: 100 spontaneous hypertensive rats were randomly divided into SHAM group,ICH group,RRD group,TAK-242+RRD group and EP+RRD group,with 20 rats in each group.Needling without blood transfusion was done in sham group and the other three groups were established into ICH models by autologous blood injection.Administration of TAK-242 and EP were as before.RRD was given 18.5g/kg/d by gavage while SHAM group and ICH group were received the same volume of pure water.The samples were taken after 3 days of continuous administrations.Neurological defect scores were performed in each group.Structure of BBB was observed by electron microscopy and immunofluorescence staining.Function of BBB was tested by brain water content,Evans blue assay and MRI.The pathological change around brain hematoma were tested by TUNEL staining and Nissl staining.Expressions of HMGB1,TLR4 and MMP9 protein and Claudin-5,Occludin,Zo-1 around brain hematoma were tested by Western blot.Results: Neurological deficit: compared with SHAM group,neurological deficit scores in each ICH model groups were higher(P<0.05);which were lower in RRD group,TAK-242+RRD group and EP+RRD group compared with ICH group(P<0.05).Structure of BBB: compared with SHAM group,structure of BBB in each ICH model groups was obviously damaged by electron microscope.Tight junction protein Claudin-5 and astrocyte was reduced by immunofluorescence staining.Compared with ICH group,RRD group,TAK-242+RRD group and EP+RRD group showed different degrees of remission on destruction of BBB structure by electron microscope,and showed a certain increase expressions of claudin-5 and astrocyte by immunofluorescence staining.Function of BBB: compared with SHAM group,brain water content,permeability of BBB and brain edema were increased in each ICH model groups,which were lower in RRD group,TAK-242+RRD group and EP+RRD group compared with ICH group(P<0.05).The pathological change: compared with sham group,apoptosis were increased and Nissl body were decreased around brain hematoma in each ICH model groups were higher(P<0.05),which were improved in RRD group,TAK-242+RRD group and EP+RRD group compared with ICH group(P<0.05).Expressions of protein: compared with SHAM group,expressions of HMGB1,TLR4 and MMP9 protein around brain hematoma were increased and tight junction protein Claudin-5 Occludin zo-1were decreased in each ICH model groups.Compared with ICH group,expressions of HMGB1,TLR4 and MMP9 protein were decreased and Claudin-5,Occludin,and Zo-1were increased in RRD group,in TAK-242+RRD group and EP+RRD group(P<0.05).Conclusion: Rehmannia and Rhubarb Decoction could protect blood brain barrier in acute intracerebral hemorrhage rats,improve injury of blood brain barrier structure and function,which may be related to the mechanism of its down-regulation of HMGB1/TLR4/MMP9 signaling pathway.Part Ⅲ Mechanism on Rehmannia and Rhubarb Decoction to HMGB1/TLR4/MMP9 signaling pathway in astrocyte Objective: To observe the effect of Rehmannia and Rhubarb Decoction to HMGB1/TLR4/MMP9 signaling pathway in astrocytes.Method: The astrocyte model induced by HMGB1 was randomly divided into control group,HMGB1 model group,RRD group,TLR4 antagonist group and TLR4 antagonist+RRD group.After co-culture for 6 hours,expressions of TLR4 and MMP9 protein were tested by Western blot,expressions of TLR4 and MMP9 mRNA were tested by RT-PCR,and MMP9 activity was tested by gelatinase assay.Result: Expressions of protein: compared with control group,expressions of TLR4 and MMP9 protein were increased in each HMGB1 model groups,which were inhibited extently in RRD group,TLR4 antagonist group and TLR4 antagonist+RRD group compared with HMGB1 model group.Expressions of mRNA: compared with control group,expressions of TLR4 and MMP9 mRNA were up-regulated in each HMGB1 model groups(P<0.05),which were significantly reduced in RRD group,TLR4 antagonist group and TLR4 antagonist+RRD group compared with HMGB1 model group(P<0.05).MMP9 activity: compared with control group,MMP9 activity increased in each HMGB1 model groups(P<0.05),which was significantly inhibited in RRD group,TLR4 antagonist group and TLR4 antagonist+RRD group compared with HMGB1 model group(P<0.05).Conclusion: Rehmannia and Rhubarb Decoction could significantly inhibit MMP9 activity of HMGB1-induced astrocyte,and the mechanism may be related to its downregulation of TLR4 MMP9 mRNA expression...