雷公藤甲素诱导肝细胞选择性自噬的形态学观察

摘要 目的：观察雷公藤甲素诱导肝细胞选择性自噬的水平。方法：在雷公藤甲素给药处理小鼠的尾静脉高压注射GFP-LC3质粒，制备肝细胞自噬示踪模型，观察雷公藤甲素在动物体内诱导肝细胞自噬的水平。在GFP-LC3稳定表达的L02细胞株中转入RFP-P62质粒，用活细胞工作站和荧光显微镜动态观察雷公藤甲素诱导L02细胞选择性自噬的轮廓，同时也观察细胞自噬复合体LC3-P62的变化。结果：动物实验结果表明，雷公藤甲素可显著诱导肝细胞自噬体的形成，Western blot结果显示P62蛋白和LC3II蛋白的表达趋势一致。活细胞动态观察及免疫荧光双标记实验结果表明LC3-P62存在共定位，提示雷公藤甲素可诱导肝细胞自噬流的形成。结论：雷公藤甲素可诱导肝细胞选择自噬，其生物学意义可能与肝细胞损伤后修复相关。     

Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemia

Recent reports indicate that autophagy serves as a stress response and may participate in pathophysiology of cerebral ischemia. Nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin), the rate-limiting enzyme in mammalian NAD⁺ biosynthesis, protects against ischemic stroke through inhibiting neuronal apoptosis and necrosis. This study was taken to determine the involvement of autophagy in neuroprotection of Nampt in cerebral ischemia. Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation (OGD) in cultured cortical neurons were performed. Nampt was overexpressed or knocked-down using lentivirus-mediated gene transfer in vivo and in vitro. Immunochemistry (LC3-II), electron microscope and immunoblotting assays (LC3-II, beclin-1, mammalian target of rapamycin [mTOR], S6K1 and tuberous sclerosis complex-2 [TSC2]) were performed to assess autophagy. We found that overexpression of Nampt increased autophagy (LC3 puncta immunochemistry staining, LC3-II/beclin-1 expression and autophagosomes number) both in vivo and in vitro at 2 hours after MCAO. At the early stage of OGD, autophagy inducer rapamycin protected against neuronal injury induced by Nampt knockdown, whereas autophagy inhibitor 3-methyladenine abolished the neuroprotective effect of Nampt partly. Overexpression or knockdown of Nampt regulated the phosphorylation of mTOR and S6K1 signaling pathway upon OGD stress through enhancing phosphorylation of TSC2 at Ser1387 but not Thr1462 site. Furthermore, in cultured SIRT1-knockout neurons, the regulation of Nampt on autophagic proteins LC3-II and beclin-1 was abolished. Our results demonstrate that Nampt promotes neuronal survival through inducing autophagy via regulating TSC2-mTOR-S6K1 signaling pathway in a SIRT1-dependent manner during cerebral ischemia.     

Induction of autophagy contributes to the neuroprotection of nicotinamide phosphoribosyltransferase in cerebral ischemia

Recent reports indicate that autophagy serves as a stress response and may participate in pathophysiology of cerebral ischemia. Nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin), the rate-limiting enzyme in mammalian NAD (+) biosynthesis, protects against ischemic stroke through inhibiting neuronal apoptosis and necrosis. This study was taken to determine the involvement of autophagy in neuroprotection of Nampt in cerebral ischemia. Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation (OGD) in cultured cortical neurons were performed. Nampt was overexpressed or knocked-down using lentivirus-mediated gene transfer in vivo and in vitro. Immunochemistry (LC3-II), electron microscope and immunoblotting assays (LC3-II, beclin-1, mammalian target of rapamycin [mTOR], S6K1 and tuberous sclerosis complex-2 [TSC2]) were performed to assess autophagy. We found that overexpression of Nampt increased autophagy (LC3 puncta immunochemistry staining, LC3-II/beclin-1 expression and autophagosomes number) both in vivo and in vitro at 2 hours after MCAO. At the early stage of OGD, autophagy inducer rapamycin protected against neuronal injury induced by Nampt knockdown, whereas autophagy inhibitor 3-methyladenine abolished the neuroprotective effect of Nampt partly. Overexpression or knockdown of Nampt regulated the phosphorylation of mTOR and S6K1 signaling pathway upon OGD stress through enhancing phosphorylation of TSC2 at Ser1387 but not Thr1462 site. Furthermore, in cultured SIRT1-knockout neurons, the regulation of Nampt on autophagic proteins LC3-II and beclin-1 was abolished. Our results demonstrate that Nampt promotes neuronal survival through inducing autophagy via regulating TSC2-mTOR-S6K1 signaling pathway in a SIRT1-dependent manner during cerebral ischemia.     

Inhibition of Autophagy Contributes to Ischemic Postconditioning-Induced Neuroprotection against Focal Cerebral Ischemia in Rats

Background

Ischemic postconditioning (IPOC), or relief of ischemia in a stuttered manner, has emerged as an innovative treatment strategy to reduce programmed cell death, attenuate ischemic injuries, and improve neurological outcomes. However, the mechanisms involved have not been completely elucidated. Recent studies indicate that autophagy is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. This study aims to determine the role of autophagy in IPOC-induced neuroprotection against focal cerebral ischemia in rats.

Methodology/Principal Findings

A focal cerebral ischemic model with permanent middle cerebral artery (MCA) occlusion plus transient common carotid artery (CCA) occlusion was established. The autophagosomes and the expressions of LC3/Beclin 1/p62 were evaluated for their contribution to the activation of autophagy. We found that autophagy was markedly induced with the upregulation of LC3/Beclin 1 and downregulation of p62 in the penumbra at various time intervals following ischemia. IPOC, performed at the onset of reperfusion, reduced infarct size, mitigated brain edema, inhibited the induction of LC3/Beclin 1 and reversed the reduction of p62 simultaneously. Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effects induced by IPOC. Conversely, autophagy inhibitor 3-methyladenine (3-MA) attenuated the ischemic insults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.

Conclusions/Significance

The present study suggests that inhibition of the autophagic pathway plays a key role in IPOC-induced neuroprotection against focal cerebral ischemia. Thus, pharmacological inhibition of autophagy may provide a novel therapeutic strategy for the treatment of stroke.     

Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke

Circular RNAs (circRNAs) are highly expressed in the central nervous system and are involved in the regulation of physiological and pathophysiological processes. However, the potential role of circRNAs in stroke remains largely unknown. Here, using a circRNA microarray, we showed that circular RNA Hectd1 (circHectd1) levels were significantly increased in ischemic brain tissues in transient middle cerebral artery occlusion (tMCAO) mouse stroke models and further validated this finding in plasma samples from acute ischemic stroke (AIS) patients. Knockdown of circHectd1 expression significantly decreased infarct areas, attenuated neuronal deficits, and ameliorated astrocyte activation in tMCAO mice. Mechanistically, circHECTD1 functions as an endogenous MIR142 (microRNA 142) sponge to inhibit MIR142 activity, resulting in the inhibition of TIPARP (TCDD inducible poly[ADP-ribose] polymerase) expression with subsequent inhibition of astrocyte activation via macroautophagy/autophagy. Taken together, the results of our study indicate that circHECTD1 and its coupling mechanism are involved in cerebral ischemia, thus providing translational evidence that circHECTD1 can serve as a novel biomarker of and therapeutic target for stroke.

Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AIS: acute ischemic stroke; AS: primary mouse astrocytes; BECN1: beclin 1, autophagy related; BMI: body mass index; circHECTD1: circRNA HECTD1; circRNAs: circular RNAs; CBF: cerebral blood flow; Con: control; DAPI: 4?,6-diamidino-2-phenylindole; ECA: external carotid artery; FISH: fluorescence in situ hybridization; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Gdna: genomic DNA; GFAP: glial fibrillary acidic protein; GO: gene ontology; HDL: high-density lipoprotein; IOD: integrated optical density; LDL: low-density lipoprotein; LPA: lipoprotein(a); MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MIR142: microRNA 142; mNSS: modified neurological severity scores; MRI: magnetic resonance imaging; NIHSS: National Institute of Health Stoke Scale; OGD-R: oxygen glucose deprivation-reperfusion; PCR: polymerase chain reaction; PFA: paraformaldehyde; SQSTM1: sequestosome 1; TIPARP: TCDD inducible poly(ADP-ribose) polymerase; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; UTR: untranslated region; WT: wild type     

Autophagy Activation Contributes to the Neuroprotection of Remote Ischemic Perconditioning Against Focal Cerebral Ischemia in Rats

Remote ischemic perconditioning (RIPer) has been proved to provide potent cardioprotection. However, there are few studies on neuroprotection of RIPer. This study aims to clarify the neuroprotective effect of RIPer and the role of autophagy induced by RIPer against cerebral ischemia reperfusion injury in rats. Using a transient middle cerebral artery occlusion (MCAO) model in rats to imitate focal cerebral ischemia. RIPer was carried out 4 cycles of 10 min ischemia and 10 min reperfusion, with a thin elastic band tourniquet encircled on the bilateral femoral arteries at the start of 10 min after MCAO. Autophagy inhibitor 3-methyladenine (3-MA) and autophagy inducer rapamycin were administered respectively to determine the contribution of autophagy in RIPer. Neurologic deficit scores, infarct volume, brain edema, Nissl staining, TUNEL assay, immunohistochemistry and western blot was performed to analyze the neuroprotection of RIPer and the contribution of autophagy in RIPer. RIPer significantly exerted neuroprotective effects against cerebral ischemia reperfusion injury in rats, and the autophagy-lysosome pathway was activated by RIPer treatment. 3-MA reversed the neuroprotective effects induced by RIPer, whereas rapamycin ameliorated the brain ischemic injury. Autophagy activation contributes to the neuroprotection by RIPer against focal cerebral ischemia in rats.     

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T₃-S₁ level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L4-5 level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.     

In vivo optical imaging of motor neuron autophagy in a mouse model of amyotrophic lateral sclerosis

Autophagy is involved in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). We have generated a novel double transgenic (DTg) mouse line by mating a green fluorescent protein (GFP)-fused microtubule-associated protein 1 light chain 3 (LC3) transgenic (LC3-Tg) mouse and a G93A mutant human Cu/Zn superoxide dismutase (mSOD1) transgenic (mSOD1-Tg) mouse. In vivo imaging of autophagy with these novel DTg mice was conducted at 10 (presymptomatic), 17 (early symptomatic) and 19 (late symptomatic) weeks of age. Fluorescence imaging analysis revealed a strong fluorescent signal in vivo over the T?-S? level at 17 and 19 weeks of age only in the DTg mice. Ex vivo autophagy imaging of spinal cord sections (20 μm) also showed a progressive increase of the fluorescence signal from 17 to 19 weeks in DTg mice in the anterior horn at the L?-? level, and the fluorescence signals were clearly observed in the gray matter of the spinal cord with a progressive increase of the signal and decreases in large motor neurons. Protein gel blot analysis revealed maximum LC3-I and LC3-II expressions at 19 weeks, consistent with the results from the in vivo autophagy imaging experiment. This method could also be applied as a unique tool for clarifying the role of autophagy, and to monitor the pathologic processes involving autophagy not only in ALS, but also other neurological diseases.     

Orexin-A protects against cerebral ischemia-reperfusion injury by inhibiting excessive autophagy through OX1R-mediated MAPK/ERK/mTOR pathway

Orexin A (OXA) is a neuroprotective peptide that exerts protective effects on multiple physiological and pathological processes. Activation of autophagy is linked to the occurrence of cerebral ischemia–reperfusion injury (CIRI); however, its function remains incompletely understood. In this study, OXA was sought to exert its neuroprotective role by regulating autophagy in oxygen and glucose deprivation and reoxygenation (OGD/R) model and middle cerebral artery occlusion (MCAO) model of rats, and to elucidate the underlying molecular mechanisms. Acridine orange (AO) staining was used to evaluate autophagic vacuoles. Cell viability was measured by CCK8. The levels of p-ERK_1/2, t-ERK_1/2, p-mTOR, LC3B, Beclin 1, and p62 were evaluated by western blotting. Apoptosis rate was detected by Hoechst 33342 staining and Terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL). OXA treatment alleviated neuronal apoptosis and significantly inhibited autophagy activity. Mechanistically, OXA exerted its neuroprotective effects in vivo and in vitro by suppressing over-activated autophagy by modulating OX1R-mediated MAPK/ERK/mTOR pathway. The results of this study elucidate the roles of autophagy in CIRI and the mechanisms underlying the neuroprotective action of OXA. Our findings could facilitate the development of novel therapeutics for ischemic stroke.     

Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia

The present study evaluated autophagy activation in astrocytes and its contribution to astrocyte injury induced by cerebral ischemia and hypoxia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. In vitro hypoxia in cultured primary astrocytes was induced by the oxygen-glucose deprivation (OGD). Alterations of astrocytes were evaluated with astroglia markers glial fibrillary acidic protein (GFAP). The formation of autophagosomes in astrocytes was examined with transmission electron microscopy (TEM). The expression of autophagy-related proteins were examined with immunoblotting. The role of autophagy in OGD or focal cerebral ischemia-induced death of astrocytes was assessed by pharmacological inhibition of autophagy with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). The results showed that GFAP staining was reduced in the infarct brain areas 3-12 h following pMCAO. Cerebral ischemia or OGD induced activation of autophagy in astrocytes as evidenced by the increased formation of autophagosomes and autolysosomes and monodansylcadaverine (MDC)-labeled vesicles; the increased production of microtubule-associated protein 1 light chain 3 (LC3-II); the upregulation of Beclin 1, lysosome-associated membrane protein 2 (LAMP2) and lysosomal cathepsin B expression; and the decreased levels of cytoprotective Bcl-2 protein in primary astrocytes. 3-MA inhibited OGD-induced the increase in LC3-II and the decline in Bcl-2. Furthermore, 3-MA and Baf slightly but significantly attenuated OGD-induced death of astrocytes. 3-MA also significantly increased the number of GFAP-positive cells and the protein levels of GFAP in the ischemic cortex core 12 h following pMCAO. These results suggest that ischemia or hypoxia-induced autophagic/lysosomal pathway activation may at least partly contribute to ischemic injury of astrocytes.     

Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways

It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema, and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.     

Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways

It has been reported that ischemic insult increases the formation of autophagosomes and activates autophagy. However, the role of autophagy in ischemic neuronal damage remains elusive. This study was taken to assess the role of autophagy in ischemic brain damage. Focal cerebral ischemia was introduced by permanent middle cerebral artery occlusion (pMCAO). Activation of autophagy was assessed by morphological and biochemical examinations. To determine the contribution of autophagy/lysosome to ischemic neuronal death, rats were pretreated with a single intracerebral ventricle injection of the autophagy inhibitors 3-methyl-adenine (3-MA) and bafliomycin A1 (BFA) or the cathepsin B inhibitor Z-FA-fmk after pMCAO. The effects of 3-MA and Z-FA-fmk on brain damage, expression of proteins involved in regulation of autophagy and apoptosis were assessed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunoblotting. The results showed that pMACO increased the formation of autophagosomes and autolysosomes, the mRNA and protein levels of LC3-II and the protein levels of cathepsin B. 3-MA, BFA and Z-FA-fmk significantly reduced infarct volume, brain edema and motor deficits. The neuroprotective effects of 3-MA and Z-FA-fmk were associated with an inhibition on ischemia-induced upregulation of LC3-II and cathepsin B and a partial reversion of ischemia-induced downregulation of cytoprotective Bcl-2. These results demonstrate that ischemic insult activates autophagy and an autophagic mechanism may contribute to ischemic neuronal injury. Thus, autophagy may be a potential target for developing a novel therapy for stroke.     

Phosphorylation of JNK Increases in the Cortex of Rat Subjected to Diabetic Cerebral Ischemia

Previous studies have demonstrated that the c-Jun N-terminal kinase (JNK) pathway plays an important role in inducing neuronal apoptosis following cerebral ischemic injury. JNK signaling pathway in activated during cerebral ischemic injury. It participates in ischemia-induced neuronal apoptosis. However, whether JNK signaling is involved in the process of neuronal apoptosis of diabetes-induced cerebral ischemia is largely unknown. This study was undertaken to evaluate the influence of cerebral ischemia–reperfusion injury on phosphorylation of JNK in diabetic rats. Twenty-four adult streptozotocin induced diabetic and 24 adult non-diabetic rats were randomly subjected to 15 min of forebrain ischemia followed by reperfusion for 0, 1, 3, and 6 h. Sixteen sham-operated diabetic and non-diabetic rats were used as controls. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Protein expression of phospho-JNK was examined by immunohistochemistry and Western blot. The numbers of TUNEL-positive cells and phospho-JNK protein expression in the cerebral cortices after 1, 3 and 6 h reperfusion was significantly higher in diabetic rats compared to non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). Western blot analysis showed significantly higher phospho-JNK protein expression in the cerebral cortices of the diabetic rats after 1 and 3 h reperfusion than that was presented in non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). These findings suggest that increased phosphorylation of JNK may be associated with diabetes-enhanced ischemic brain damage.     

大鼠局灶性脑缺血再灌注中nNOS来源的NO对细胞凋亡的影响

目的探讨大鼠局灶性脑缺血再灌注早期nNOS来源的NO对细胞凋亡的影响.方法闭塞大鼠左侧大脑中动脉造成局灶性脑缺血模型,给予选择性nNOS抑制剂-7硝基吲唑,应用原位末端标记法及流式细胞术检测缺血2h再灌注6h细胞凋亡的变化.结果 50mg/kg、25mg/kg剂量的7硝基吲唑可使1、NO含量显著降低.2、NT阳性细胞荧光强度及阳性细胞百分比显著减少.3、TUNEL阳性细胞明显减少.4、细胞凋亡百分率降低,AP峰降低.结论 nNOS来源的NO参与介导脑缺血再灌注早期的细胞凋亡.     

Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke

Rapamycin has been demonstrated to exhibit neuroprotective functions via the activation of autophagy in a cerebral ischemia model. However, the involvement of mitophagy in this process and its contribution to the protection of mitochondrial function remains unknown. The present study explored the characteristics of mitophagy after cerebral ischemia and the effect of rapamycin on mitochondrial function. Male Sprague–Dawley rats underwent transient middle cerebral artery occlusion (tMCAO). Neurological deficits scores; infarct volumes; mitophagy morphology; and the levels of malondialdehyde (MDA), adenosine triphosphate (ATP) and mitochondrial membrane potentials (Δψm) were examined. The expression of LC3, Beclin-1 and p62 in the mitochondrial fraction combined with transmission electronic microscopy were used to explore mitophagic activity after ischemia. We also blocked autophagosome formation using 3-methyladenine (3-MA) to check the linkage between the mitochondrial protective effect of rapamycin and enhanced mitophagy. We observed that rapamycin significantly enhanced mitophagy, as evidenced by the increase in LC3-II and Beclin-1 expression in the mitochondria and p62 translocation to the mitochondria. Rapamycin reduced infarct volume, improved neurological outcomes and inhibited mitochondrial dysfunction compared with the control animals (p < 0.05). However, these protective effects were reversed by 3-methyladenine treatment after rapamycin. The present study indicates that rapamycin treatment attenuates mitochondrial dysfunction following cerebral ischemia, which is linked to enhanced mitophagy.     

CD38 exacerbates focal cytokine production, postischemic inflammation and brain injury after focal cerebral ischemia

Background

Converging evidence suggests that inflammatory processes significantly influence brain injury and clinical impairment in ischemic stroke. Although early studies suggested a key role of lymphocytes, recent data has emphasized the orchestrating function of innate immunity, i.e., macrophages and microglia. The bifunctional receptor and ectoenzyme CD38 synthesizes calcium-mobilizing second messengers (e.g., cyclic ADP-ribose), which have been shown to be necessary for activation and migration of myeloid immune cells. Therefore, we investigated the dynamics of CD38 in stroke and the impact of CD38-deficiency on cytokine production, inflammation and cerebral damage in a mouse model of cerebral ischemia-reperfusion.

Methodology/Principal Findings

We show that the local expression of the chemokine MCP-1 was attenuated in CD38-deficient mice compared with wildtype mice after focal cerebral ischemia and reperfusion. In contrast, no significant induction of MCP-1 expression was observed in peripheral blood after 6 hours. Flow cytometry analysis revealed less infiltrating macrophages and lymphocytes in the ischemic hemisphere of CD38-deficient mice, whereas the amount of resident microglia was unaltered. An up-regulation of CD38 expression was observed in macrophages and CD8⁺ cells after focal cerebral ischemia in wildtype mice, whereas CD38 expression was unchanged in microglia. Finally, we demonstrate that CD38-deficiency decreases the cerebral ischemic injury and the persistent neurological deficit after three days of reperfusion in this murine temporary middle cerebral artery occlusion (tMCAO) model.

Conclusion/Significance

CD38 is differentially regulated following stroke and its deficiency attenuates the postischemic chemokine production, the immune cell infiltration and the cerebral injury after temporary ischemia and reperfusion. Therefore CD38 might prove a therapeutic target in ischemic stroke.     

人表皮角质形成细胞自噬功能细胞模型的构建及鉴定

目的: 建立稳定表达GFP-LC3的人永生化角质形成细胞HaCaT细胞系。方法: 将构建的pcDNA3.1-GFP-LC3 真核表达载体转入HaCaT细胞,经G418筛选稳定表达的细胞系。HaCaT细胞中GFP-LC3的表达分别用荧光显微镜与Western blot方法检测,并利用该稳定表达的细胞系观察验证Rapamycin对细胞发生自噬透射电镜超微结构的变化。结果: 获得了3株转染并经G418反复筛选的HaCaT细胞系,在倒置荧光显微镜下观察可见绿色荧光细胞的表达率在95% 以上,Western blot结果证实了GFP-LC3融合蛋白的表达。Western blot和激光共聚焦显微镜均证明Rapamycin可以诱导自噬的发生。透射电镜细胞超微结构的观察表明Rapamycin可以有效地诱导HaCaT-LC3细胞自噬的发生。结论: 成功构建GFP-LC3稳定表达的HaCaT系,该细胞系可以作为研究人角质形成细胞自噬功能的一种细胞模型。     

Lack of functional P2X7 receptor aggravates brain edema development after middle cerebral artery occlusion

Effective therapeutic measures against the development of brain edema, a life-threatening complication of cerebral ischemia, are necessary to improve the functional outcome for the patient. Here, we identified a beneficial role of purinergic receptor P2X7 activation in acute ischemic stroke. Involvement of P2X7 in the development of neurological deficits, infarct size, brain edema, and glial responses after ischemic cerebral infarction has been analyzed. Neurologic evaluation, magnetic resonance imaging, and immunofluorescence assays were used to characterize the receptor’s effect on the disease progress during 72 h after transient middle cerebral artery occlusion (tMCAO). Sham-operated animals were included in all experiments for control purposes. We found P2X7-deficient mice to develop a more prominent brain edema with a trend towards more severe neurological deficits 24 h after tMCAO. Infarct sizes, T2 times, and apparent diffusion coefficients did not differ significantly between wild-type and P2X7^?/? animals. Our results show a characteristic spatial distribution of reactive glia cells with strongly attenuated microglia activation in P2X7^?/? mice 72 h after tMCAO. Our data indicate that P2X7 exerts a role in limiting the early edema formation, possibly by modulating glial responses, and supports later microglia activation.