| Compound angelica injection(CAI)is a compound preparation made from three Chinese medicines of angelica,Chuanxiong,and safflower,having a good effect of promoting blood circulation and remove stasis pain.The previous study of the research group found that CAI has a potential protective effect on cerebral ischemia stroke caused by middle cerebral artery occlusion(MCAO)in rats,but further mechanisms remain to be elucidated.With the deepening of studies on cerebral ischemic stroke,more and more evidences show that inflammatory response is an important factor leading to the occurrence and development of cerebral ischemic stroke,and Janus-activated kinase2/Signal transducer and activator of transcripti3(JAK2/STAT3)is an important inflammatory signaling pathway,and the study also confirmed its involvement in the occurrence and development of cerebral ischemic stroke.Therefore,based on the JAK2/STAT3 signaling pathway,this study will explore the possible role of CAI in the inflammatory response of cerebral ischemic stroke,so as to reveal the mechanism of the protective effect of CAI on ischemic strokeObjective:The effect of CAI in the inflammatory reaction of cerebral ischemia stroke based on JAK2/STAT3 signaling pathway.Establishing permanent MCAO model of rat,the effect of CAI on cerebral ischemia stroke model of rats were observed from neurologic function,volume of cerebral infarction and pathological morphology of the cerebral cortex,the effect of CAI in the inflammatory reaction of on cerebral ischemia stroke model of rats were studied from interleukin-6(IL-6)and JAK2/STAT3 signaling pathway.Using the conditioned culture medium of Brain microvascular endothelial cells(BMECs)acted by CAI to intervene the Microglia(MG)cells caused by oxygen and glucose deprivation(OGD),the effect of CAI on the OGD model of MG cells were observed from cell morphology and activity,the effect of CAI in the inflammatory reaction of the OGD model of MGs were studied from migration and JAK2/STAT3 signaling pathway.Methods:1.To study the effect of CAI on neurologic function,volume of cerebral infarction and pathological morphology of the cerebral cortex in cerebral ischemia stroke model of rats:A total of rats were randomly divided into normal group,sham operation group and modeling group.Cerebral ischemia stroke model of rats with cerebral ischemia stroke were reproduced by MCAO,modeling group rats were divided into model group(3 mL·kg-1 normal saline),CAI group(5 mL·kg-1 CAI)and edaravone group(5 mL·kg-1 Edaravone injection)continuing 7 days.Neurologic function was detected by neuroethology,volume of cerebral infarction was detected by triphenyl tetrazolium chloride(TTC)method,and the pathological morphology of the cerebral cortex was observed by hematoxylin eosin(HE)staining in each group.2.To Study on the effect of CAI on the IL-6 and JAK2/STAT3 signaling pathway in cerebral ischemia stroke model of rats:The Enzyme linked immunosorbent assay(ELISA)was used to detect contents of IL-6 in plasma of each group,and the Western blot was used to detect the protein expression of p-JAK2,JAK2,p-STAT3,STAT3 of JAK2/STAT3 signaling pathway in each group.3.To study the effect of conditioned culture medium of BMECs acted by CAI on the morphology and activity of MG cells caused by the OGD:The BMECs were primary cultured,the purity of BMECs were determined by immunofluorescence assay.BMECs are cultured in the third generation,the third generation of BMECs were divided into normal group,model group,low-dose CAI group(1.25 ?L·mL-1),middle-dose CAI group(2.5 ?L·mL-1)and high-dose CAI group(5 ?L·mL-1).The model group and CAI group were modeled by the Oxygen and glucose deprivation(OGD)method.The conditioned medium of each group were collected as Normal-Conditioned medium(N-CM),Ischemic-Conditioned medium(I-CM),Ischemia CAI-Conditioned medium Low?Middle?High(IC-CML?M?H).Among them,1.25 ?L·mL-1CAI acted on the I-CM of BMECs as the IC-CML,2.5 ?L·mL-1 CAI acted on the I-CM of BMECs as the IC-CMM,and 5 ?L·mL-1 CAI acted on the I-CM of BMECs as the IC-CMH.The MG cells were primary cultured,the purity of MG cell were determined by immunofluorescence assay,and MGs are cultured in the third generation.The third generation of MG cells were divided into N-CM group,I-CM group,IC-CML group,IC-CMM group,IC-CMH group.Add BMECs conditioned medium with CAI in the third generation of MG cells.Besides the N-CM group,the I-CM group and three of IC-CM group were modeled by the OGD.The inverted optical microscope and CCK-8 method were used to observe MG cells morphology and activity in each group.4.To Study on the effect of conditioned culture medium of BMECs acted by CAI on the cells migration and JAK2/STAT3 signaling pathway in MG cells caused by the OGD:The cell migration system was used to observe MG cells migration in each group of MG cells caused by OGD,and the Western blot was used to detect the protein expression of p-JAK2,JAK2,p-STAT3,STAT3 of JAK2/STAT3 signaling pathway in each group of MG cells.Results:1.CAI has protective effect on cerebral ischemia stroke model of rats,which can improve neurological function,relieve pathological morphology damage of the cerebral cortex,and at the same time,it tends to decrease th e volume of cerebellar infarction.The result of neurological function score showed that CAI group was significantly reduced compared with the model group(P<0.01).The result of TTC staining showed that volume of cerebral infarction in CAI group tended to decrease compared with the model group.The results of HE staining showed that,in the model group,the cell in the cerebral cortex were sparse and disordered,the number of cells was reduced and the nucleus was constricted.Compared with model group,the cell were arranged in an orderly manner,and the number of cells undergoing necrosis was significant reduced in CAI group.2.CAI can alleviate inflammatory response of cerebral ischemia stroke model of rats by inhibiting the expression of IL-6 and the abnormal activation of JAK2/STAT3 signaling pathway.The results of ELISA showed that compared with the sham group,the concentration of IL-6 in the blood plasma of model group increased significantly(P<0.01)compared with the sham group,and the concentration of IL-6 in the blood plasma of CAI group was significantly reduced compared with the model group(P<0.01).The results of Western blot showed that the expression of p-JAK2/JAK2 and p-STAT3/STAT3 protein in the brain tissue of model group increased significantly(P<0.01)compared with the sham group,and the expression of p-JAK2/STAT3 and p-STAT3/STAT3 protein in the brain tissue of CAI group decreased significantly(P<0.01)compared with the model group3.BMECs conditioned medium of CAI can relieve the damage of morphology and viability of MG cells caused by the OGD.The results of immunofluorescence staining showed that the purity of BMECs and MG cells in the third generation of primary culture were above 95%,which could be used in subsequent experiments.The result of invert optical microscope observation showed that each dose of IC-CM group could relieve the damage of the MG cells caused by OGD,make MG cells volume normal,cell spacing decreased,and increase the number of viable MG cells increased.The result of CCK-8 showed that each dose of IC-CM group could increase the viability of MG cells,and IC-CMM group and IC-CML group significantly increased(P<0.05,P<0.01).4.BMECs conditioned medium of CAI can alleviate inflammatory response of MG cells caused by the OGD by inhibiting the migration of MG and the abnormal activation of JAK2/STAT3 signaling pathway.The results of cell migration system showed that each dose of:IC-CM group could significantly inhibit MG migration(P<0.05).The results of Western blot showed that the expression of p-JAK2/JAK2 and p-STAT3/STAT3 protein in the I-CM group significant increased(P<0.01)compared with the N-CM group,and the expression of p-JAK2/STAT3 and p-STAT3/STAT3 protein in the each of IC-CM group tended to decrease compared with the I-CM group,and IC-CMM group and IC-CML group significantly decreased(P<0.01).Conclusion:1.CAI has neuroprotective effect on cerebral ischemia stroke model of rats,which can improve neurological function and relieve pathological morohology damage of the cerebral cortex.The protective mechanism may be related to reduce the inflammatory response by inhibiting the expression of IL-6 and the abnormal activation of JAK2/STAT3 signaling pathway.2.BMECs conditioned medium of CAI has protective effect on MG caused by the OGD,which can relieve the damage of cell morphology,improve cell viability.The mechanism may be related to reduce the inflammatory response by inhibiting the migration of MG and the abnormal activation of JAK2/STAT3 signaling pathway.3.JAK2/STAT3 signaling pathway may be one of the therapeutic targets of CAI to reduce the inflammatory response and exert the neuroprotective effect of cerebral ischemic stroke. |
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