Bone marrow mesenchymal stem cell-derived exosomes promote microglia/macrophage M2 polarization in acute cerebral ischemia rats and inhibit inflammatory response / 中国药科大学学报

@#The aim of the present study was to investigate the effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on the polarization of M1/M2 microglia/macrophages in rats with acute cerebral ischemia.Ultrahigh-speed centrifugation was employed to isolate and identify exosomes; a middle cerebral artery occlusion (MCAO) model was prepared in rats using the intraluminal filament technique; Longa scoring and corner tests were used to evaluate the neurological function of rats; 2, 3, 5-triphenyltetrazole chloride (TTC) staining was used to assess the infarct volume in rat brains; immunofluorescence double-labeling of CD16/32/Iba1 and CD206/Iba1 was performed to detect M1/M2 phenotypes of microglia/macrophages; RT-qPCR was employed to measure the mRNA expression of CD86, inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), arginase-1 (Arg-1), interleukin-10 (IL-10), and transforming growth factor beta (TGF-β) in the ischemic penumbra of rat brains.The experimental results showed that BMSC-Exos reduced the number of CD16/32+/Iba1+ positive cells in the ischemic penumbra (P < 0.01) while increasing the number of CD206+/Iba1+positive cells (P < 0.01), and decreased the mRNA expression of iNOS, CD86, and TNF-α, while increasing the mRNA expression of Arg-1, TGF-β, and IL-10 (P < 0.05 or P < 0.01).This research suggests that BMSC-Exos can regulate M1/M2 polarization of microglia/macrophages in rats with acute cerebral ischemia, alleviate neuroinflammation, and improve ischemic brain injury.

Chinese medicine Buyang Huanwu decoction promotes neurogenesis and angiogenesis in ischemic stroke rats by upregulating miR-199a-5p expression / 浙江大学学报·医学版

OBJECTIVE@#To investigate the mechanism of Chinese medicine Buyang Huanwu decoction (BYHWD) promoting neurogenesis and angiogenesis in ischemic stroke rats.@*METHODS@#Male SD rats were randomly divided into sham operation group, model group, BYHWD group, antagonist group and antagonist control group with 14 rats in each. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 min with intraluminal filament and reperfusion for 14 d in all groups except sham operation group. BYHWD (13 g/kg) was administrated by gastrogavage in BYHWD group, antagonist group and antagonist control group at 24 h after modeling respectively, and BrdU (50 mg/kg) was injected intraperitoneally in all groups once a day for 14 consecutive days. miR-199a-5p antagomir or NC (10 nmol) was injected into the lateral ventricle at d5 after ischemia in antagonist and antagonist control groups, respectively. The neurological deficits were evaluated by the modified neurological severity score (mNSS) and the corner test, and the infract volume was measured by toluidine blue staining. Neurogenesis and angiogenesis were detected by immunofluorescence double labeling method. The expression level of miR-199a-5p was tested by real-time RT-PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were determined by Western blotting.@*RESULTS@#BYHWD treatment significantly promoted the recovery of neurological function (@*CONCLUSIONS@#Buyang Huanwu decoction promotes neurogenesis and angiogenesis in rats with cerebral ischemia, which may be related to increased protein expression of VEGF and BDNF through upregulating miR-199a-5p.

Astragaloside Ⅳ inhibits inflammation after cerebral ischemia in rats through promoting microglia/macrophage M2 polarization / 浙江大学学报·医学版

OBJECTIVE@#To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on microglia/macrophage M1/M2 polarization and inflammatory response after cerebral ischemia in rats.@*METHODS@#Forty eight male SD rats were randomly divided into sham operation control group, model control group and AS-Ⅳ group with 16 rats in each. Focal cerebral ischemia model was induced by occlusion of the right middle cerebral artery (MCAO) using the intraluminal filament. After ischemia induced, the rats in AS-Ⅳ group were intraperitoneally injected with 40 mg/kg AS-Ⅳ once a day for 3 days. The neurological functions were evaluated by the modified neurological severity score (mNSS) and the corner test on d1 and d3 after modelling. The infarct volume was measured by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining on d3 after ischemia. The expression of M1 microglia/macrophage markers CD86, inducible nitric oxide synthase (iNOS) and pro-inflammatory factors TNF-α, IL-1β, IL-6, M2 microglia/macrophages markers CD206, arginase-1 (Arg-1), chitinase-like protein (YM1/2) and anti-inflammatory factors interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) was detected by real-time RT-PCR. The expression of CD16/32/Iba1 and CD206/Iba1 was determined by double labeling immunefluorescence method in the peripheral area of cerebral ischemia.@*RESULTS@#Compared with model control group, AS-Ⅳ treatment improved neurological function recovery and reduced infarct volume after ischemia (@*CONCLUSIONS@#The findings suggest that AS-Ⅳ ameliorates brain injury after cerebral ischemia in rats, which may be related to inhibiting inflammation through promoting the polarization of the microglia/macrophage from M1 to M2 phenotype in the ischemic brain.

Bone marrow mesenchymal stem cell transplantation enhances angiogenesis and functional recovery after cerebral ischemia in rats / 中华行为医学与脑科学杂志

Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on angiogenesis and functional recovery after focal cerebral ischemia in rats.Methods BMSCs were isolated and cultured using the whole bone marrow adherent method,and conducted phenotypic identification using flow cytometry analysis of surface positive antigen of CD29,CD90 and the negative antigen of CD34,CD45.Rats were subjected to middle cerebral artery occlusion (MCAO) for 90 minutes,and divided into three groups randomly,the sham group,model group and BMSCs group.24 hours after cerebral ischemia,rats were injected with 1 ml BMSCs solution (1 × 106 cells/ml) or PBS via the tail vein.The modified neurological severity score(mNSS) test,the corner test and the adhesive tape test were used to evaluate sensorimotor function on the 1,7,14 and 28 days after ischemia.Infarcted volume was detected by toluidine blue staining,and the numbers of vWF positive microvessels and vascular endothelial growth factor (VEGF) positive cells in the ischemic boundary were determined by immunofluorescence.Results By flow cytometric analysis,the cell phenotype of passage 3 BMSCs showed that CD29,CD90,CD34 and CD45 were 98.3％,97.4％,0.2％ and 4.8％,respectively.Compared with the model group,BMSCs significantly reduced the score of mNSS(P＜0.01),the number of right turn of corner test(P＜0.05),latency of removal adhesive tape(P＜0.05) and the infarcted volume (P＜0.01).The numbers of vWF positive vesscls and the VEGF positive cells were (42.97±8.64)/mm2 and (54.83± 10.66)/mm2 at the boundary zone in model group 14 days after ischemia,respectively.BMSCs significantly increased the numbers of vWF positive vessels ((69.43± 7.29)/mm2) and VEGF positive cells ((78.70±6.16)/mm2,P＜0.01).Conclusion BMSCs can improve the functions of cerebral lesions after cerebral ischemia,which may be associated with the enhanced angiogenesis and VEGF expression in the ischemic boundary.

Effect of Buyanghuanwu decoction on the expression of growth-associated protein 43 and synaptophysin after focal cerebral ischemia in mice / 中华行为医学与脑科学杂志

Objective To investigate the effect of Buyanghuanwu decoction(BYHWD) on the expression of growth-associated protein 43(GAP-43) and synaptophysin(SYP) after focal cerebral ischemia in mice.Methods Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion followed by reperfusion.BYHWD (20 g/kg) was administered orally 24 h after ischemia and once a day.The neurological score and the corner test were used to evaluate sensorimotor function on 1,3,7,and 14 days after ischemia.The expression of GAP-43 and SYP in the ischemic boundary zone was determined by immunohistochemistry 14 days after ischemia.Results Compared with the model group,BYHWD significantly ameliorated neurological dysfunction at 3,7 and 14 days and reduced the number of right turn at 7 and 14 days after ischemia(P ＜ 0.05).The average optical density of GAP-43 and SYP was 0.217 ±0.012 and 0.278 ±0.019 at the boundary zone in model group 14 days after ischemia,respectively.BYHWD also significantly increased the immunoreactivity of GAP-43 (0.250 ± 0.013)and SYP(0.323 ± 0.017) (P＜ 0.01).Conclusion BYHWD promotes axonal regeneration and synaptic plasticity in the ischemic boundary zone,which may contribute to functional recovery after focal cerebral ischemia.

Effect of buyanghuanwu decoction on angiogenesis and Ang-1/Tie-2 expression after focal cerebral ischemia in mice / 中华行为医学与脑科学杂志

Objective To investigate the effect of Buyanghuanwu decoction(BYHWD) on angiogenesis and Ang-1/Tie-2 expression after focal cerebral ischemia in mice. Methods Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion followed by reperfusion. BYHWD (20 g/kg) was administered orally 24 h after ischemia and once a day. The neurological score and the corner test were used to evaluate sensorimotor function during 28 days after ischemia. The microvessels density and the expression of Ang-1/Tie-2 in the ischemic boundary zone were determined by immunohistochemistry on 28 days after ischemia. Results Compared with the model group, BYHWD significantly ameliorated neurological dysfunction and reduced the number of right turn during 3 to 28 days after ischemia(P＜0.05 ). In addition,the numbers of CD31 ,Ang-1 and Tie-2 immunopositive cells were (437 ±59) ,(389 ±61 ) and (251 ±42) at the boundary zone in model group 28 days after ischemia,respectively. However, BYHWD treatment significantly increased the numbers of CD31(609 ± 68 ), Ang-1 (551 ±66) and Tie-2 (342 ±46) immunopositive cells(P＜0. 01 ). Conclusion BYHWD promotes angiogenesis,which may contribute to recovery of neurological function after focal cerebral ischemia,and Ang-1/Tie-2 pathway appears to mediate BYHWD-induced angiogenesis.

Effects of Buyanghuanwu Decoction on Infiltration of Neutrophils and Expression of ICAM-1 after Middle Cerebral Artery Occlusion in Rats / 浙江中医药大学学报

Objective To investigate the effects of Buyanghuanwu decoction on infiltration of neutrophils and expression of ICAM-1 after middle cerebral artery occlusion (MCAO) in rats. Methods Rats were pretreated with Buyanghuanwu decoction(13 g/kg and 26 g/kg) for 7d and then subjected to cerebral ischemia/reperfusion injury induced by an MCAO. After a 90min ischemia and a 24h reperfusion, the neurological deficit and the survival neurons were determined. The effects of Buyanghuanwu decoction on infiltration of neutrophils and expression of ICAM-1 by immunohistochemistry are evaluated. Results Buyanghuanwu decoction significantly ameliorated the neurological deficit and increased survival neurons . The myeloperoxidase (MPO) positive cells and the ICAM-1 positive vessels in the vehicle-treated rats were increased significantly (P