Treatment with β-sitosterol ameliorates the effects of cerebral ischemia/reperfusion injury by suppressing cholesterol overload, endoplasmic reticulum stress, and apoptosis

β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.

A feasible strategy for focal cerebral ischemiareperfusion injury: remote ischemic postconditioning

It is difficult to control the degree of ischemic postconditioning in the brain and other isch- emia-sensitive organs. Remote ischemic postconditioning could protect some ischemia-sensitive organs through measures on terminal organs. In this study, a focal cerebral ischemia-reperftlsion injury model was established using three cycles of remote ischernic postconditioning, each cycle consisted of 10-minute occlusion of the femoral artery and 10-minute opening. The results showed that, remote ischemic postconditioning significantly decreased the percentage of the in- farct area and attenuated brain edema. In addition, inflammatory nuclear factor-KB expression was significantly lower, while anti-apoptotic Bcl-2 expression was significantly elevated in the ce- rebral cortex on the ischemic side. Our findings indicate that remote ischemic postconditioning attenuates focal cerebral ischemia/reperfusion injury, and that the neuroprotective mechanism is mediated by an anti-apoptotic effect and reduction of the inflammatory response.

Ischemic postconditioning enhances glycogen synthase kinase-3β expression and alleviates cerebral ischemia/reperfusion injury

The present study established global brain ischemia using the four-vessel occlusion method. Following three rounds of reperfusion for 30 seconds, and occlusion for 10 seconds, followed by reperfusion for 48 hours, infarct area, the number of TUNEL-positive cells and Bcl-2 expression were significantly reduced. However, glycogen synthase kinase-3β activity, cortical Bax and caspase-3 expression significantly increased, similar to results following ischemic postconditioning. Our results indicated that ischemic postconditioning may enhance glycogen synthase kinase-3β activity, a downstream molecule of the phosphatase and tensin homolog deleted on chromosome 10/phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which reduces caspase-3 expression to protect the brain against ischemic injury.

A molecular probe carrying anti-tropomyosin 4 for early diagnosis of cerebral ischemia/reperfusion injury

In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.

Preconditioning and postconditioning reduce hepatic ischemia-reperfusion injury in rats

BACKGROUND: Ischemia-reperfusion injury occurs when ischemic tissues or organs suffer from further functional and structural damage when their blood supply recovers. This study aimed to contrast the protective effects of ischemic preconditioning and ischemic postconditioning in hepatic ischemia-reperfusion injury in rats. METHODS: Thirty-two healthy male Wistar rats were randomly divided into four groups: sham-operated (SO), ischemia-reperfusion (IR), ischemic preconditioning (I-pre), and ischemic postconditioning (I-post). Blood samples and hepatic tissue were taken from all groups after the experiments. RESULTS: There were significant differences between the IR, I-pre and I-post groups in alanine aminotransferase and aspartate aminotransferase levels, NF-kappa B p65 expression, apoptosis index and superoxide dismutase activity in hepatic tissue. There were no significant differences between the I-pre and I-post groups. CONCLUSIONS: Ischemic postconditioning and ischemic preconditioning reduce hepatic ischemia-reperfusion injury, but in clinical practice the former is a more appropriate choice.

Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis:nicotiflorin and JAK2/STAT3 pathway

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius.Previous studies have shown its cerebral protective effect,but the mechanism is undefined.In this study,we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway.The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion.Nicotiflorin（10 mg/kg） was administered by tail vein injection.Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay.Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining.Additionally,p-JAK2,p-STAT3,Bcl-2,Bax,and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay.Nicotiflorin altered the shape and structure of injured neurons,decreased the number of apoptotic cells,down-regulates expression of p-JAK2,p-STAT3,caspase-3,and Bax,decreased Bax immunoredactivity,and increased Bcl-2 protein expression and immunoreactivity.These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

Buyanghuanwu decoction promotes angiogenesis after cerebral ischemia/reperfusion injury：mechanisms of brain tissue repair

Buyanghuanwu decoction has been shown to protect against cerebral ischemia/reperfusion injury,but the underlying mechanisms remain unclear.In this study,rats were intragastrically given Buyanghuanwu decoction,15 m L/kg,for 3 days.A rat model of cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion.In rats administered Buyanghuanwu decoction,infarct volume was reduced,serum vascular endothelial growth factor and integrin αvβ3 levels were increased,and brain tissue vascular endothelial growth factor and CD34 expression levels were increased compared with untreated animals.These effects of Buyanghuanwu decoction were partially suppressed by an angiogenesis inhibitor（administered through the lateral ventricle for 7 consecutive days）.These data suggest that Buyanghuanwu decoction promotes angiogenesis,improves cerebral circulation,and enhances brain tissue repair after cerebral ischemia/reperfusion injury.

Limb remote ischemic postconditioning protects integrity of the blood-brain barrier after stroke

Integrity of the blood-brain barrier structure is essential for maintaining the internal environment of the brain.Development of cerebral infarction and brain edema is strongly associated with blood-brain barrier leakage.Therefore,studies have suggested that protecting the blood-brain barrier may be an effective method for treating acute stroke.To examine this possibility,stroke model rats were established by middle cerebral artery occlusion and reperfusion.Remote ischemic postconditioning was immediately induced by three cycles of 10-minute ischemia/10-minute reperfusion of bilateral hind limbs at the beginning of middle cerebral artery occlusion reperfusion.Neurological function of rat models was evaluated using Zea Longa’s method.Permeability of the blood-brain barrier was assessed by Evans blue leakage.Infarct volume and brain edema were evaluated using 2,3,5-triphenyltetrazolium chloride staining.Expression of matrix metalloproteinase-9 and claudin-5 m RNA was determined by real-time quantitative reverse transcription-polymerase chain reaction.Expression of matrix metalloproteinase-9 and claudin-5 protein was measured by western blot assay.The number of matrix metalloproteinase-9-and claudin-5-positive cells was analyzed using immunohistochemistry.Our results showed that remote ischemic postconditioning alleviated disruption of the blood-brain barrier,reduced infarct volume and edema,decreased expression of matrix metalloproteinase-9 m RNA and protein and the number of positive cells,increased expression of claudin-5 m RNA and protein and the number of positive cells,and remarkably improved neurological function.These findings confirm that by suppressing expression of matrix metalloproteinase-9 and claudin-5 induced by acute ischemia/reperfusion,remote ischemic postconditioning reduces blood-brain barrier injury,mitigates ischemic injury,and exerts protective effects on the brain.

Neuroprotective effect of ischemic postconditioning on sciatic nerve transection

Ischemic preconditioning or postconditioning has been shown to have neuroprotective effect on cerebral ischemia, but it has not been studied in peripheral nerve injury. In this study, a rat model of sciatic nerve transection was established, and subjected to three cycles of ischemia for 10 minutes ＋ reperfusion for 10 minutes, once a day. After ischemic postconditioning, serum insulin-like growth factor 1 expression increased; sciatic nerve Schwann cell myelination increased; sensory function and motor function were restored. These findings indicate that ischemic postconditioning can effectively protect injured sciatic nerve. The protective effect is possibly associated with upregulation of insulin-like growth factor 1.

Neuroprotective effect of ischemic preconditioning in focal cerebral infarction: relationship with upregulation of vascular endothelial growth factor

Neuroprotection by ischemic preconditioning has been confirmed by many studies, but the precise mechanism remains unclear. In the present study, we performed cerebral ischemic pre- conditioning in rats by simulating a transient ischemic attack twice （each a 20-minute occlusion of the middle cerebral artery） before inducing focal cerebral infarction （2 hour occlusion-reper- fusion in the same artery）. We also explored the mechanism underlying the neuroprotective effect of ischemic preconditioning. Seven days after ocdusion-reperfusion, tetrazolium chloride staining and immunohistochemistry revealed that the infarct volume was significantly smaller in the group that underwent preconditioning than in the model group. Furthermore, vascular endothelial growth factor immunoreactivity was considerably greater in the hippocampal CA3 region of preconditioned rats than model rats. Our results suggest that the protective effects of ischemic preconditioning on focal cerebral infarction are associated with upregulation of vascu- lar endothelial growth factor.

Brain-derived neurotrophic factor inhibits glucose intolerance after cerebral ischemia

Brain-derived neurotrophic factor is associated with the insulin signaling pathway and glucose me- tabolism. We hypothesized that expression of brain-derived neurotrophic factor and its receptor may be involved in glucose intolerance following ischemic stress. To verify this hypothesis, this study aimed to observe the changes in brain-derived neurotrophic factor and tyrosine kinase B receptor expression in glucose metabolism-associated regions following cerebral ischemic stress in mice. At day 1 after middle cerebral artery occlusion, the expression levels of brain-derived neurotrophic factor were significantly decreased in the ischemic cortex, hypothalamus, liver, skeletal muscle, and pancreas. The expression levels of tyrosine kinase B receptor were decreased in the hypothalamus and liver, and increased in the skeletal muscle and pancreas, but remained unchanged in the cortex Intrahypothalamic administration of brain-derived neurotrophic factor （40 ng） suppressed the de- crease in insulin receptor and tyrosine-phosphorylated insulin receptor expression in the liver and skeletal muscle, and inhibited the overexpression of gluconeogenesis-associated phosphoenolpy- ruvate carboxykinase and glucose-6-phosphatase in the liver of cerebral ischemic mice. However, serum insulin levels remained unchanged. Our experimental findings indicate that brain-derived neurotrophic factor can promote glucose metabolism, reduce gluconeogenesis, and decrease blood glucose levels after cerebral ischemic stress. The low expression of brain-derived neurotrophic factor following cerebral ischemia may be involved in the development of glucose intolerance.

By regulating IP3/PKC/TRPV4 pathway hyperoside induces endothelium-dependent vasodilatation in rat basilar artery following four vessel occlusion ischemia reperfusion

OBJECTIVE To investigate regulatory effects of hyperoside(Hyp)on IP3/PKC/TRPV4 pathway in rat cerebral basilar artery(CBA)subjected to global cerebral ischemia-reperfusion(I/R).METHODS The model of global cerebral I/R in rats was established by four-vessel occlusions methods.The treated rats were administrated with Hyp(50 mg·kg^-1)group,Hyp(50 mg·kg^-1)+HC-067047(10 mg·kg^-1),Hyp(50 mg·kg^-1)+2APB(2 mg·kg^-1),Hyp(50 mg·kg^-1)+BisI(2.5 mg·kg^-1),Hyp(50 mg·kg^-1)+2APB(2 mg·kg^-1)+BisI(2.5 mg·kg^-1).Hematoxylin-eosin(HE)and Nissl staining were performed and the contents of methane dicarboxylic aldehyde(MDA),neuron-specific enolase(NSE),S100β and the activity of lactic dehydrogenase(LDH)in serum were measured by enzyme-linked immunosorbnent assay(ELISA).The specific blocker N-nitro-L-arginine-methyl-ester(L-NAME)and indomethacin(Indo)were used to delete the prostacyclin(PGI2)and nitric oxide(NO)dependent relaxation.The protein expression level of TRPV4 was detected by Western blotting.Ca2+intensity in vascular smooth muscle cells was measured by confocal laser scanning microscope and flow cytometry was performed to observe the apoptosis of CBA endothelial cells after in vivo administration.RESULTS Hyp induced a dose-dependent relaxation of CBA in IR rats via a PGI2 and NO independent manner,as evidenced by alleviated patho⁃logical changes and up-regulated expression of TRPV4 protein in the endothelial cells from cerebral vessels.Hyp signifi⁃cantly reduced the contents of MDA,NSE,S100βand the activity of LDH in serum and decreased the fluorescence intensity of Ca2+in cerebral vascular smooth muscle cells by in vivo administration.The apoptotic rate of endothelial cells in Hyp treated group was significantly less than that in IR group.CONCLUSION Hyp does in fact ameliorate I/R injury by regulatingIP3/PKC/TRPV4 pathway.

N-乙酰半胱氨酸联合缺血后处理减轻糖尿病小鼠心肌缺血再灌注后肺损伤的作用研究

目的研究N-乙酰半胱氨酸(NAC)联合缺血后处理(IPostC)对糖尿病小鼠心肌缺血再灌注后肺损伤的作用。方法选择15周龄雄性db/db糖尿病小鼠30只,分为假手术组(D-SO组,n=10)、心肌缺血/再灌注组(D-I/R组,n=10)和NAC联合缺血后处理组(D-NAC+IPostC组,n=10)。D-SO组小鼠开胸后不做任何处理;D-I/R组小鼠干预为冠状动脉左前降支结扎60 min,后复灌15 min。D-NAC+IPostC组在结扎冠状动脉左前降支前30 min腹腔注射NAC150 mg/kg,缺血后处理的干预方式为小鼠缺血60 min后即刻进行3个周期再灌注/缺血,然后再灌注15 min。于再灌注结束后颈动脉取血,检测血清C反应蛋白(CRP)、肿瘤坏死因子-α(TNF-α)、单核细胞趋化蛋白-1(MCP-1)水平,处死小鼠,取肺组织,检测湿干重(W/D)比值,光镜下观察肺组织病理形态学变化,计算肺损伤评分,测定肺组织氧化应激相关标志物谷胱甘肽(GSH)、超氧化物歧化酶(SOD)及丙二醛(MDA)水平,采用Western Blot法检测肺组织缺氧诱导因子1α(HIF-1α)和血管内皮生长因子(VEGF)的表达水平。结果与D-SO组比较,D-I/R组小鼠光镜下病理学损伤严重(P<0.05),肺W/D比值增加(P<0.05),血清TNF-α、CRP水平降低(P<0.05),MCP-1水平升高(P<0.05),肺组织MDA含量增加(P<0.05),SOD及GSH活性降低(P<0.05),肺组织HIF-1α及VEGF表达上调(P<0.05)。与D-I/R组比较,D-NAC+IPostC组肺组织镜下病理学损伤明显减轻(P<0.05),肺W/D比值降低(P<0.05),血清TNF-α、MCP-1水平降低(P<0.05),CRP水平升高(P<0.05),肺组织氧化应激因子MDA含量降低(P<0.05),抗氧化应激因子SOD及GSH活性升高(P<0.05),肺组织HIF-1α、血管内皮生长因子(VEGF)水平表达增高(P<0.05)。结论NAC联合IPostC可减轻糖尿病心肌缺血再灌注小鼠肺损伤,其机制可能与HIF-1α/VEGF信号通路相关。

杜鹃花总黄酮通过抑制TNF-α/caspase-8/caspase-3信号通路减轻大鼠脑缺血再灌注损伤的作用

目的探索杜鹃花总黄酮(TFR)通过抑制TNF-α/caspase-8/caspase-3信号通路保护脑缺血再灌注(I/R)损伤的机制。方法采用大脑中动脉闭塞术(MCAO)建立大鼠I/R模型,将造模成功大鼠随机分为假手术(Sham)、脑缺血再灌注(MCAO)和脑缺血再灌注术后TFR 200 mg/kg干预(TFR 200 mg/kg)组,制备MCAO大鼠模型,在脑缺血再灌注损伤手术后,TFR 200 mg/kg组连续14 d给予TFR(200 mg/kg)药物溶液。术后14 d,依据大鼠神经功能评分,脑血流图观察脑血流情况,处死大鼠,苏木精-伊红(HE)染色观察脑组织细胞形态学变化,试剂盒检测大鼠血清中乳酸脱氢酶(LDH或LD)和神经特异性烯醇化酶(NSE)两种酶活性;ELISA试剂盒检测白细胞介素-1(IL-1)和IL-6水平,Western blot和免疫组化同时检测大鼠脑组织中裂解型胱天蛋白酶3(cleaved caspase-3)、caspase-8、肿瘤坏死因子(TNF)-α蛋白的表达水平。结果脑缺血再灌注处理后,MCAO导致了大鼠神经功能异常,神经功能评分指数显著升高,脑组织病理形态学和脑血流量变化明显,脑组织中cleaved caspase-3、caspase-8、TNF-α蛋白的表达水平显著增加,血清中LDH、NSE、IL-1、IL-6水平明显升高;TFR 200 mg/kg组大鼠神经功能评分明显降低,脑组织病理损伤显著改善,脑组织中cleaved caspase-3、caspase-8、TNF-α蛋白的表达以及血清中LDH、NSE、IL-1、IL-6水平降低。结论TFR可能通过抑制TNF-α/caspase-8/caspase-3信号通路减轻脑缺血缺氧性损伤。

Protective effect of ischemic postconditioning on lung ischemia-reperfusion injury in rats and the role of heme oxygenase-1

Objective： To investigate the effect of ischemic postconditioning （1PO） on acute lung ischemia-reperfusion （I/R） injury and the protein expression of haeme oxygenase-1 （HO-1）, a cytoprotective defense against oxidative injury. Methods： After being anesthetized with chloralhydrate, forty-eight healthy SD rats were randomly divided into 6 groups （8 in each）： sham operation group （S group）; I/R group： left lung hilum was clamped for 40 minutes followed by 105 minutes of reperfusion; IPO group： left lung hilum was clamped for40 minutes and postconditioned by 3 cycles of 30 seconds of reperfusion and 30 seconds of reocclusion; Heroin （HM）＋ I/R group： heroin, an inducer of HO-1 was injected intraperitoneally at 40 μmol·kg^-1·day^-1 for two consecutive days prior to 40 minutes clamping of left lung hilum; ZnPPIX＋IPO group： zinc protoporphyrin IX, an inhibitor of HO-1 was injected intraperitoneally at 20 mg·kg^-1 24 hours prior to 40 minutes clamping of left lung hilum; and HM＋S group： HM was administered as in the HM＋I/R group without inducing lung I/R. Arterial partial pressure of oxygen （PaO2） and malondialdehyde （MDA） content in serum were assessed. The left lung was removed for determination of wet/dry lung weight ratio and expression of HO-1 protein by immuno-histochemical technique and for light microscopic examination. Results： The PaO2 was significantly lower in all the experimental groups compared with sham group （90 roan Hg ±11 mmHg）. However, the values of PaO2in IPO （81 mm Hg±7 mm Hg） and HM＋I/R （80 mm Hg±9 mm Hg） were higher than that in I/R （63 mm Hg±9 mm Hg） and ZnPPIX＋IPO （65 mm Hg±8 mm Hg） groups （P〈0.01）. The protein expression of HO- 1 in lung tissue was significantly increased in I/R group compared with S group （P〈0.01）. While the HO-1 protein expression was higher in IPO and HM＋I/R groups as compared with I/R group （P〈0.05, P〈0.01 ）. The lung wet/ dry （W/D） weight ratio and MDA content in serum were significantly increased in I/R group as compared with S or HM＋S groups （P〈0.01）, accompanied by severe lung tissue histological damage, which was attenuated either by IPO or by HM pretreatment （P〈0.01, IPO or HM＋I/R vs. I/R）. The protective effect of IPO was abolished by ZnPPIX. Conclusion： Ischemic postconditioning can attenuate the lung ischemia-reperfusion injury through upregulating the protein expression of HO-I that leads to reduced postischemic oxidative damage.

Synergistic Protection of Danhong Injection (丹红注射液) and Ischemic Postconditioning on Myocardial Reperfusion Injury in Minipigs

Objective： To explore the synergistic protection of Danhong Injection （丹红注射液,DHI） and ischemic postconditioning on myocardial reperfusion injury in minipigs.Methods： Acute myocardial infarction model was made by balloon occlusion in left anterior descending coronary artery （LAD） of minipigs,and then postconditioning was simulated through inflation/deflation of the angioplasty balloon.Minipigs were divided into four groups： the sham operation group （SH group）,the ischemia/reperfusion group （I/R group）,the ischemic postconditioning group （POC group） and DHI combined with ischemic postconditioning group （PAD group,DHI 20 mL through ear vein）,six in each group.After 24-h continuous observation,myocardial infarction size was assessed by triphenyltetrazolium staining （TTC）.Morphological changes of ischemic myocardium were observed by light microscopy,and cardiomyocyte ultrastructure was studied with electron microscopy.The superoxide dismutase （SOD） and malondialdehyde （MDA） activity in heart homogenates were measured by a biochemical method.Results： The myocardial infarction size was smaller in the POC group than in the I/R group （0.26±0.02 vs.0.37±0.09,P〈0.05）,and the PAD group （0.14±0.08） displayed a significantly reduced infarction size relative to the I/R group （P〈0.01） and POC group （P〈0.05）.The damage of myocardial tissue was severe in the I/R group shown by light and electron microscopy： myocardial fibers disorder,sarcoplasmic dissolution,myofilament fracture,mitochondria swelling and even vacuolization formation and a large number of inflammatory cell infiltrations.Compared with the I/R group,reduction of reperfusion injury in the PAD group included more orderly arranged myocardial fibers,less infiltration of inflammatory cells and maintenance of mitochondrial integrity.Compared with the I/R group,the damage of myocardial tissue in the POC group was improved,but not as significant as that in the PAD group.SOD levels in the POC group and the PAD group were significantly higher than those in the I/R group （96.96±13.43,112.25±22.75 vs.76.32±10.63,P〈0.05）,and MDA was significantly lower in the POC group and the PAD group compared to the I/R group （1.27±0.19,1.09±0.21 vs.1.47±0.16,P〈0.05）.Conclusion： DHI and ischemic postconditioning show a synergistic cardioprotection on myocardial reperfusion injury in minipigs.

温阳复元方通过调控miRNA-137/线粒体铁死亡通路保护大鼠脑缺血再灌注损伤机制研究

目的探讨温阳复元方对miRNA-137/线粒体铁死亡通路的调控作用,阐明该方对脑缺血再灌注损伤(CIRI)的神经保护作用机制。方法线栓法制备大鼠CIRI模型,将144只SD大鼠随机分为假手术(SO)组、模型(I/R)组、补阳还五汤对照(BYHW)组、温阳复元方(WYFY)组、温阳复元方+miRNA-137模拟物(WYFY+mimic)组、温阳复元方+miRNA-137抑制物(WYFY+Inhibitor)组,每组按再灌注时间点分为3个亚组。采用Zea-Longa评分法进行神经功能缺损评分(NFS);TTC染色测量脑梗死体积;qRT-PCR和Western blot法观察miRNA-137、膜铁转运蛋白(FPN)和二价金属离子转运体1(DMT1)的mRNA及其蛋白表达水平。结果(1)NFS结果:I/R组NFS较SO组显著增加(P<0.01),与I/R组相比,BYHW组NFS仅在12 h时评分降低(P<0.05),而WYFY组在24 h和3 d的NFS明显降低(P<0.05或P<0.01),WYFY+mimic组NFS显著下降(P<0.01),WYFY+Inhibitor组在3 d时NFS降低(P<0.05);与WYFY组相比,WYFY+mimic组NFS仅在12 h时明显降低(P<0.05)。(2)TTC染色结果:I/R组梗死体积较SO组显著增大(P<0.01),BYHW组和WYFY组较I/R组明显减小(P<0.05或P<0.01),WYFY组梗死体积较BYHW组进一步减小(P<0.01);相较于WYFY组,WYFY+mimic组能进一步减小其梗死体积(P<0.05),而WYFY+Inhibitor组梗死灶明显增大(P<0.05)。(3)miRNA-137 mRNA表达水平:I/R组miRNA-137mRNA的表达较SO组显著下降(P<0.01),WYFY治疗后其表达量显著增加(P<0.01),且WYFY组其表达水平较BYHW组进一步增高(P<0.01);与WYFY组同期比较,WYFY+mimic组miRNA-137 mRNA表达量均进一步升高(P<0.01),WYFY+Inhibitor组抑制该表达(P<0.01)。(4)FPN mRNA及蛋白表达水平:I/R组FPN mRNA和蛋白表达水平较SO组均降低(P<0.01),WYFY组能显著增加其表达(P<0.01),该组在12 h和3 d时的表达量较BYHW组进一步增加(P<0.05或P<0.01);与WYFY组同期对比,WYFY+mimic组其表达水平在24 h及3 d时增加(P<0.01),而WYFY+Inhibitor组其表达水平均降低(P<0.01)。(5)DMT1 mRNA及蛋白表达水平:I/R组DMT1mRNA和蛋白表达量较SO组均显著增加(P<0.01);与I/R组比较,WYFY能降低其表达水平(P<0.01),且WYFY组在3 d时其表达量较BYHW组进一步降低(P<0.05);与WYFY组同期相比,WYFY+mimic组其表达水平在24 h和3 d时均降低(P<0.01),而WYFY+Inhibitor组其表达水平均显著增加(P<0.01)。结论温阳复元方可能通过上调miRNA-137的表达,进而上调铁转运蛋白FPN的表达,下调DMT1的表达,调节铁代谢,最终抑制线粒体铁死亡,发挥其神经保护作用,其疗效优于补阳还五汤。

壮药双路通脑方对脑缺血再灌注损伤大鼠神经元凋亡的影响

目的 基于氧化应激和炎症反应探讨壮药双路通脑方对脑缺血再灌注损伤大鼠神经元凋亡的影响。方法 将大鼠随机分为假手术组,模型组,壮药双路通脑方低、中、高剂量组(9.0、18.0、36.0 g/kg),依达拉奉组(3.0 mg/kg),每组18只,采用线栓法构建大脑中动脉闭塞/再灌注模型以模拟脑缺血再灌注损伤,给予相应药物干预6 d,采用Zeal Longa评分法对大鼠进行神经功能缺损评分,TTC染色检测脑梗死面积,HE染色和TUNEL染色检测脑组织缺血半暗带海马CA1区神经元病理损伤和细胞凋亡情况,试剂盒检测脑组织SOD活性和GSH、MDA、IL-6、IL-1β、TNF-α水平,Western blot法检测大鼠脑组织Bax、Bcl-2、caspase-3、cleaved-caspase-3、TLR4、NF-κB p65、Nrf2、HO-1蛋白表达。结果 与模型组比较,壮药双路通脑方中、高剂量组和依达拉奉组大鼠神经功能缺损评分,脑梗死率,神经元凋亡率,脑组织缺血半暗带IL-6、IL-1β、TNF-α、MDA水平,Bax、TLR4蛋白表达,cleaved-caspase-3/caspase-3、p-NF-κB p65/NF-κB p65比值降低(P<0.05),GSH水平、SOD活性和Bcl-2、Nrf2、HO-1蛋白表达升高(P<0.05)。结论 壮药双路通脑方可抑制氧化应激和炎症反应,抑制神经元凋亡,改善神经功能,减轻脑缺血再灌注损伤大鼠的脑损伤,其机制可能与抑制TLR4/NF-κB信号通路和激活Nrf2/HO-1信号通路有关。

双下肢缺血后处理保护再灌注心肌时效性及对线粒体通路调控的研究

目的:观察双下肢缺血后处理(即远隔器官缺血后处理,remote ischemic postconditioning,RIpostC)保护缺血再灌注小鼠心肌的时效性及对心肌线粒体依赖性凋亡和坏死通路的调控。方法:成年雄性C57BL/6J野生型小鼠被随机分为假手术(sham)组、心肌缺血再灌注(myocardial ischemia/reperfusion,MI/R)组、缺血后处理组、RIpostC组及RIpostC延迟1、5、10、15、30和60 min组。阻断左冠脉45 min,再灌注24 h,建立MI/R模型;气囊袖带阻断双下肢血流5 min,再灌注5 min,实施RIpostC。再灌注24 h后,Evans blue和TTC染色观察心肌梗死面积与血清心肌钙蛋白I变化。TUNEL和高迁移率族盒蛋白1(high mobility group box protein 1,HMGB1)染色观察心肌凋亡和坏死;线粒体水肿实验观察心肌线粒体膜电位变化;Western blot观察心肌细胞凋亡和线粒体膜通透性转换孔(mitochondrial permeability transition pore,mPTP)相关蛋白表达。结果:与MI/R组比较,RIpostC及RIpostC延迟1、5、10和15 min组心肌梗死面积明显减少,RIpostC延迟30和60 min组则无明显改变。缺血后处理与RIpostC对缺血再灌注心肌有类似保护效应。RIpostC减少缺血再灌注心肌细胞凋亡和坏死发生。RIpostC降低缺血再灌注心肌亲环蛋白D(cyclophilin D,CypD)、Bax和Bak蛋白表达水平。结论:心肌再灌注后15 min内实施RIpostC能够减少心肌梗死面积,其保护作用与缺血后处理类似;RIpostC通过调控线粒体依赖性凋亡和坏死通路减轻MI/R损伤。

针刺干预脑缺血再灌注损伤的氧化应激机制

缺血性脑卒中是一种发病率、致残率及复发率很高的神经系统疾病,是患者、家属和临床工作者面临的共同难题。脑卒中发生后病理进程复杂,其中氧化应激在脑卒中后关键病理环节脑缺血再灌注损伤中通过对大脑中蛋白质、DNA和RNA等造成氧化损伤,对疾病预后极其不利。近期研究证明针刺可以显著抑制脑卒中后缺血级联反应中的氧化应激,本研究深入探讨了针刺干预脑缺血再灌注后氧化应激的机制,为针刺作为一种多通路抑制氧化应激而改善脑卒中患者预后提供理论依据,推进针刺在临床中的合理应用。