黄连解毒汤通过调节外泌体内趋化因子表达对MCAO小鼠脑内中性粒细胞浸润的影响OA

Objective:To investigate whether Huanglian Jiedutang can inhibit neutrophil infiltration in the brains of middle cerebral artery occlusion(MCAO)mice by regulating the expression of neutrophil-related chemokines in exosomes,thereby achieving therapeutic effects.Methods:A total of 130 male specific pathogen-free(SPF)C57BL/6J mice were randomly divided into four groups:Sham-operated group,MCAO model group,Huanglian Jiedutang group(6 g·kg-1),and Ginaton group(21.6 mg·kg-1),with 10 mice in the Ginaton group and 40 mice in each of the remaining three groups.Mice in the Huanglian Jiedutang group and the Ginaton group were administered the corresponding drugs by oral gavage once daily at a volume of 0.15 mL·(10 g)-1 for 7 consecutive days,while the sham-operated and model groups received an equal volume of saline via the same route.After 7 days,MCAO surgery was performed.The distal and proximal ends of the right common carotid artery(CCA)were ligated,a small incision was made between the two ligatures,and a silicone rubber-coated monofilament with a rounded tip was inserted into the lumen to occlude the CCA.The filament was left in place for 1 h to establish a focal cerebral ischemia model.At 24 h after modeling,mice were evaluated.Neurological function was assessed using the Longa score.Cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Cerebral blood flow was observed by laser speckle imaging.Hematoxylin and eosin(HE)staining and Nissl staining were used to observe pathological changes in brain tissues.Exosomes were isolated from mouse plasma and brain tissues by ultracentrifugation and molecular size exclusion and identified by electron microscopy,particle size analysis,and protein blotting.Long-chain RNA libraries of exosomes were constructed and sequenced.Real-time quantitative reverse transcription polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of inflammatory factors and neutrophil-related chemokines in exosomes from plasma and brain tissues of each group.Enzyme-linked immunosorbent assay(ELISA)was used to detect the protein expression of inflammatory factors and neutrophil-related chemokines in exosomes from brain tissues of each group.Immunohistochemistry was used to detect the expression of the neutrophil-specific protein myeloperoxidase(MPO)in the brains of mice in each group.Results:Compared with the sham-operated group,the model group showed decreased neurological function scores(P＜0.01),obvious cerebral infarction(P＜0.01),reduced cerebral blood flow(P＜0.01),neuronal necrosis in the brain,and decreased numbers of Nissl bodies(P＜0.01).The mRNA expression levels of IL-1β,MPO,CXCL1,CXCL2,CXCL3,CXCL10,CCL2,and CCL3 in exosomes from plasma and brain tissues were significantly increased(P＜0.05,P＜0.01).The protein expression levels of IL-1β,MPO,CXCL2,and CXCL10 in exosomes from brain tissues were increased(P＜0.05,P＜0.01),and MPO-positive rates and mean optical density values in brain tissues were elevated(P＜0.01).Compared with the model group,the Huanglian Jiedutang group and the Ginaton group showed increased neurological function scores(P＜0.05),reduced cerebral infarct volume(P＜0.01),restored cerebral blood flow(P＜0.01),reduced necrotic cells in the brain,and increased numbers of Nissl bodies(P＜0.01).In the Huanglian Jiedutang group,the mRNA expression levels of IL-1β,MPO,CXCL1,CXCL2,CXCL3,CXCL10,CCL2,and CCL3 in exosomes from plasma and brain tissues were decreased(P＜0.05,P＜0.01).The protein expression levels of IL-1β,MPO,CXCL2,and CXCL10 in exosomes from brain tissues were reduced(P＜0.05,P＜0.01),and MPO-positive rates and mean optical density values in brain tissues were decreased(P＜0.01).Conclusion:Huanglian Jiedutang can effectively regulate the expression of neutrophil-related chemokines in exosomes from plasma and brain tissues of MCAO mice,thereby reducing neutrophil infiltration in the brain and achieving therapeutic effects.