Protective Effect Of Astragalus Injection On Systemic Inflammatory On Brain Injury LPS Induced In C57 Mice

BackgroundAmong multiple organ injuries caused by severe systemic inflammation,complications of the nervous system can cause complications that are more complicated than inflammation itself and other organ damage.Serious lesions can result in permanent illness or even death.Clinically,Astragalus are widely used in hypertension,cardiovascular and cerebrovascular diseases,diabetes and its complications,nephrotic syndrome and other diseases.Astragalus injection has a positive inotropic effect on the heart,enhance myocardial contractility,increase coronary blood flow,can protect myocardial cells,improve cardiovascular function.The protection of systemic inflammatory on brain injury has not been reported.This study was to investigate the protective effect of Astragalus injection on the systemic inflammatory on brain injury LPS induced in C57 mice.And explore the regulation effects of Astragalus injection based on miR-155-5p and its downstream signals.It changes some protein of the blood-brain barrier to protect the structure and function of the blood-brain barrier.Our study can provide a theoretical basis for the treatment of cerebrovascular inflammatory diseases.This study was supported by the project of NSFC-8107308-4,NSFC-No.81572678 and XDJK2017D154.Objectives1.Observe the protective effect of Astragalus injection on LPS-induced systemic inflammatory injury in mice.Clarify whether Astragalus injection can protect LPS-induced systemic inflammatory brain injury.2.Observe the effects of Astragalus injection on LPS-induced systemic inflammatory injury in mice at the 6th day and 17 th day.Observe the protective effect of Astragalus injection on systemic inflammatory brain injury.3.Observe the effects of Astragalus injection on the structure and function of the blood-brain barrier in mice brain injury of LPS-induced systemic inflammatory.Based on the regulation of miR-155 and downstream signals,further explore the possible mechanism of Astragalus injection in protecting the blood-brain barrier.Methods1.Intraperitoneal injection of 2 mg/kg LPS for three days was repeated to induce systemic inflammation in mice.There are 6 groups that are control group,model group(LPS),positive control(dexamethasone 30 mg/kg)and Astragalus injection group(2,10,20 g/kg).All these groups were daily treated for 17 days beginning at 3 days ahead of LPS administration.Observe the following indicators at the 6th and 17 th day respectively:(1)Water intake,body temperature,body weight,etc.in mice were used to evaluate the improvement of the survival state of mice by Astragalus injection;(2)Organ coefficient in brain,heart,liver,spleen,lung and kidney of mice were used to evaluate the protective effect of Astragalus injection on inflammatory lesions of other organs in mice;(3)H&E staining and nissl staining were used to observe the improvement of brain pathological changes in mice treated by Astragalus injection.2.New object recognition experiment and open field test were used to evaluate the improvement of cognitive of mice treated by Astragalus injection.3.(1)By Evans Blue heart perfusion,observe the effects of Astragalus injection on the permeability of the blood-brain barrier induced by LPS.Transmission electron microscopy was used to observe the ultrastructural of blood-brain barrier.The double-labeled immunofluorescence technique was used to observe the expression of tight junction proteins Claudin-5,ZO-1,Occludin and CD31 in mice brain.The protective effects on the blood-brain barrier of Astragalus injection were obersved in structure.4.Claudin-5,ZO-1,Occludin,α-actinin1,Vinculin,cateninα1,mir-155-5p and its downstream genes mRNA expression were detected by qPCR.The tight junction protein Claudin-5,ZO-1,Occludin and RhoA,ROCK2 signaling pathway related proteins were detected by WB.The mechanism of protection of Astragalus injection on blood-brain barrier induced by LPS was preliminary studied.Results 1.Protective effects of Astragalus injection on systemic inflammation injury in C57 mice induced by LPSAstragalus injection can reduce the damage of brain tissue caused by LPS.It can improve weight,food and water intake,maintain body temperature,and increase survival rate.It can also reduce the infiltration of inflammatory cells,neuron swelling,and neuronal necrosis in brain.On the 6th day,Astragalus injection can decrease the organ coefficient increase of brain,heart,and lung caused by LPS.On the 17 th day,each organ coefficientis were restored.2.Protective effects of Astragalus injection on cognitive decline of C57 mice induced by LPSAfter 3 days of LPS administration,the crawling number and the number of erection of the model group mice were significantly decreased compared with the normal group(P<0.01).On the 17 th day,the new object recognition coefficient of the model group mice was significantly decreased(P <0.05),the number of crawling cells decreased significantly(P <0.001),the number of erections decreased,while the number of modifications,the number of fecal pellets,and the retention time of the central lattice increased.Compared with the model group,the new object recognition coefficient of the Astragalus injection 10 g/kg group was significantly higher(P <0.05),dexamethasone can significantly increase the number of crawling(P <0.01),increase the number of uprights and decorations,decrease the number of feces pellets and the retention time in the central lattice.3.Protective effects of Astragalus injection on blood-brain barrier induced by LPSThe C57 mice of model group showed that the whole brain were severely stained with blue dye and the fluorescence signal of the whole cerebral cortex red fluorescence signal was stronger and there was a large amount of EB leakage in the brain tissue.Astragalus injection can reduce the EB leakage in the brain tissue.Astragalus injection 10 g/kg group obviously antagonized LPS-induced astrocyte swelling and endothelial cell junction opening.4.Effect of Astragalus injection on the expression of tight junction between brain vascular endothelial cells induced by LPSOn the 6th day,compared with control,LPS significantly decreased the mRNA expression of Claudin-5(P<0.01),α-actinin1(P<0.01),Vinculin(P<0.01),cateninα1(P<0.01),Rho(P<0.01),ROCK2(P<0.01)and mir-155-5p(P<0.001),increased the mRNA expression of SHIP1(P<0.001)and BCL6(P<0.01).Compared with LPS group,Astragalus injection 10 g/kg group significantly increased the mRNA expression of ZO-1(P<0.001),α-actinin1(P<0.05),Vinculin(P<0.05),cateninα1(P<0.001),ROCK2(P<0.05),PKCβ(P<0.05)and mir-155-5p(P<0.001),decreased the mRNA expression of Claudin-5(P<0.001),Rho(P<0.05),RhoA,SHIP1(P<0.001),BCL6(P<0.001)and SOCS1(P<0.001).On the 17 th day,compared with control,LPS significantly decreased the mRNA expression of Claudin-5(P<0.01),Vinculin(P<0.01),cateninα1(P<0.01)and mir-155-5p(P<0.001),increased the mRNA expression of RhoA(P<0.05)and PKCβ(P<0.01).Compared with LPS group,Astragalus injection 10 g/kg group significantly increased the mRNA expression of Claudin-5(P<0.001),α-actinin1(P<0.05),Vinculin(P<0.01),cateninα1(P<0.05),BCL6(P<0.001)and mir-155-5p(P<0.01),decreased the mRNA expression of Rho(P<0.05)、RhoA(P<0.01)、PKCβ(P<0.05)、SHIP1(P<0.05).On the 6th day,compared with control,LPS significantly decreased the protein expression of Claudin-5,ZO-1,Occludin,ROCK2(P <0.01).Compared with LPS group,Astragalus injection 10 g/kg group significantly increased the protein expression of Occludin,ZO-1,Claudin-5,RhoA,ROCK2.On the 17 th day,compared with control,LPS significantly decreased the protein expression of RhoA(P <0.05).Compared with LPS group,Astragalus injection 10 g/kg group significantly increased the protein expression of RhoA and ZO-1(P <0.05),reduced the protein expression of ROCK2(P <0.01).ConclusionAstragalus Injection has a protective effect on LPS-induced systemic inflammatory injury,cognitive decline,the function and structure of the blood-brain barrier destruct.Its protective mechanism is related to miR-155-5p.It can up-regulate the expression of brain vascular endothelial cell connexin,and then protects the blood-brain barrier caused by systemic inflammation.