N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats

AIM:To observe the effects of N-acetylserotonin(NAS)administration on retinal ischemia-reperfusion(RIR)injury in rats and explore the underlying mechanisms involving the high mobility group box 1(HMGB1)/receptor for advanced glycation end-products(RAGE)/nuclear factor-kappa B(NF-κB)signaling pathway.METHODS:A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye.Eighty male Sprague Dawley were randomly divided into five groups:sham group(n=8),RIR group(n=28),RIR+NAS group(n=28),RIR+FPS-ZM1 group(n=8)and RIR+NAS+FPS-ZM1 group(n=8).The therapeutic effects of NAS were examined by hematoxylin-eosin(H&E)staining,and retinal ganglion cells(RGCs)counting.The expression of interleukin 1 beta(IL-1β),HMGB1,RAGE,and nod-like receptor 3(NLRP3)proteins and the phosphorylation of nuclear factorkappa B(p-NF-κB)were analyzed by immunohistochemistry staining and Western blot analysis.The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats.With NAS therapy,the HMGB1 and RAGE expression decreased significantly,and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression.Additionally,NAS exhibited an anti-inflammatory effect by reducing IL-1βexpression.The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression,so as to the IL-1βexpression and retinal edema,accompanied by an increase of RGCs in RIR rats.CONCLUSION:NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway,which may be a useful therapeutic target for retinal disease.

PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons

PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases.Although our previous lite rature mining study suggested that PANoptosis might occur in neuronal ischemia/repe rfusion injury,little experimental research has been reported on the existence of PANoptosis.In this study,we used in vivo and in vitro retinal neuronal models of ischemia/repe rfusion injury to investigate whether PAN optosis-like cell death(simultaneous occurrence of pyroptosis,apo ptosis,and necroptosis)exists in retinal neuronal ischemia/repe rfusion injury.Our results showed that ischemia/repe rfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo.Ischemia/repe rfusion inju ry also significantly upregulated caspase-1,caspase-8,and NLRP3 expression,which are important components of the PANoptosome.These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.

The Protective Effect of Compound Danshen Dripping Pills on Oxidative Stress after Retinal Ischemia/Reperfusion Injury in Rats

Objective: To investigate the effect of Compound Danshen Dripping Pills (CDDP) on oxidative stress after ischemia/reperfusion (I/R) injury in the rat retina. Methods: Adult male SD rats were randomly divided into 3 groups: sham (group A), I/R (group B), and I/R plus CDDP (group C). Retinal ischemia/reperfusion injury (RIRI) was introduced by increasing the intraocular pressure (IOP) to 110 mmHg for 60 min via cannulation into the anterior chamber. Right after the insult, CDDP was administered intragastrically (450 mg/kg/d) for 7 days. The levels of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the retinal tissues were determined on d1 and d7 after the ischemic insult. Results: Following ischemia, the MDA levels in group B and group C were significantly higher than those in group A (p < 0.01). CDDP significantly lowered MDA levels in group C when compared with group B (p < 0.01). The activities of SOD, GSH-Px and CAT were higher in group A than in group B and group C (p < 0.01). CDDP could increase the activities of SOD, GSH-Px and CAT remarkably in group C when compared with group B (p < 0.01). Conclusion: CDDP can protect the retina from I/R injury through reducing oxidative stress, and thus may be a promising method for the treatment of ischemic retinal disorders.

microRNA-455-5p alleviates neuroinflammation in cerebral ischemia/reperfusion injury

Neuroinflammation is a major pathophysiological factor that results in the development of brain injury after cerebral ischemia/reperfusion.Downregulation of microRNA(miR)-455-5p after ischemic stroke has been considered a potential biomarker and therapeutic target for neuronal injury after ischemia.However,the role of miR-455-5p in the post-ischemia/reperfusion inflammatory response and the underlying mechanism have not been evaluated.In this study,mouse models of cerebral ischemia/reperfusion injury were established by transient occlusion of the middle cerebral artery for 1 hour followed by reperfusion.Agomir-455-5p,antagomir-455-5p,and their negative controls were injected intracerebroventricularly 2 hours before or 0 and 1 hour after middle cerebral artery occlusion(MCAO).The results showed that cerebral ischemia/reperfusion decreased miR-455-5p expression in the brain tissue and the peripheral blood.Agomir-455-5p pretreatment increased miR-455-5p expression in the brain tissue,reduced the cerebral infarct volume,and improved neurological function.Furthermore,primary cultured microglia were exposed to oxygen-glucose deprivation for 3 hours followed by 21 hours of reoxygenation to mimic cerebral ischemia/reperfusion.miR-455-5p reduced C-C chemokine receptor type 5 mRNA and protein levels,inhibited microglia activation,and reduced the production of the inflammatory factors tumor necrosis factor-αand interleukin-1β.These results suggest that miR-455-5p is a potential biomarker and therapeutic target for the treatment of cerebral ischemia/reperfusion injury and that it alleviates cerebral ischemia/reperfusion injury by inhibiting C-C chemokine receptor type 5 expression and reducing the neuroinflammatory response.

2-(2-Benzofuranyl)-2-imidazoline treatment within 5 hours after cerebral ischemia/reperfusion protects the brain

We previously demonstrated that administering 2-（2-benzofuranyl）-2-imidazolin（2-BFI）, an imidazoline I2 receptor agonist, immediately after ischemia onset can protect the brain from ischemic insult. However, immediate administration after stroke is difficult to realize in the clinic. Thus, the therapeutic time window of 2-BFI should be determined. Sprague-Dawley rats provided by Wenzhou Medical University in China received right middle cerebral artery occlusion for 120 minutes, and were treated with 2-BFI（3 mg/kg） through the caudal vein at 0, 1, 3, 5, 7, and 9 hours after reperfusion. Neurological function was assessed using the Longa＇s method. Infarct volume was measured by 2,3,5-triphenyltetrazolium chloride assay. Morphological changes in the cortical penumbra were observed by hematoxylin-eosin staining under transmission electron microscopy. The apoptosis levels in the ipsilateral cortex were examined with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling（TUNEL） assay. The protein expression of Bcl-2 and BAX was detected using immunohistochemistry. We found the following： Treatment with 2-BFI within 5 hours after reperfusion obviously improved neurological function. Administering 2-BFI within 9 hours after ischemia/reperfusion decreased infarct volume and alleviated apoptosis. 2-BFI administration at different time points after reperfusion alleviated the pathological damage of the ischemic penumbra and reduced the number of apoptotic neurons, but the protective effect was more obvious when administered within 5 hours. Administration of 2-BFI within 5 hours after reperfusion remarkably increased Bcl-2 expression and decreased BAX expression. To conclude, 2-BFI shows potent neuroprotective effects when administered within 5 hours after reperfusion, seemingly by up-regulating Bcl-2 and down-regulating BAX expression. The time window provided clinical potential for ischemic stroke by 2-BFI.

Puerarin inactivates NLRP3-mediated pyroptotic cell death to alleviate cerebral ischemia/reperfusion(I/R)injury through modulating the LncRNA DUXAP8/miR-223-3p axis

NLRP3 inflammasome-mediated cell pyroptosis aggravates the development of cerebral ischemia/reperfusion(I/R)injury,and the aim of this study is to investigate the potential utilization of the Chinese medicine,Puerarin,in treating this disease.Through conducting in vitro and in vivo experiments,the present study illustrated that Puerarin regulated LncRNA double homeobox A pseudogene 8(DUXAP8)/miR-223-3p axis to inactivate NLRP3-mediated pyroptotic cell death,resulting in the attenuation of I/R injury.Specifically,the cerebral I/R injury in rat models and hypoxia/reoxygenation(H/R)in primary hippocampus neuron(PHN)cells were inducted,which were subsequently exposed to Puerarin treatment.As expected,we validated that Puerarin suppressed cell pyroptosis and rescued cell viability in I/R rat hippocampus tissues and H/R PHN cells.Next,through bioinformatics analysis,we noticed that miR-223-3p targeted both LncRNA DUXAP8 and NLRP3 mRNA,and both LncRNA DUXAP8 ablation and miR-223-3p overexpression inactivate NLRP3-mediated cell pyroptosis to rescue cell viability in H/R PHN cells.Interestingly,we evidenced that Puerarin restrained LncRNA DUXAP8 expressions,but upregulated miR-223-3p in I/R rat tissues and H/R PHN cells,and the protective effects of Puerarin on H/R PHN cells were abrogated by overexpressing LncRNA DUXAP8 and silencing miR-223-3p.Collectively,we concluded that Puerarin regulated LncRNA DUXAP8/miR-223-3p/NLRP3 signaling cascade to attenuate I/R injury.

Resveratrol prevents hypoxia-induced retinal ganglion cell death related with ErbB2

AIM: To confirm the changes in proteins related with hypoxia-induced retinal cell death and to assess the effects of resveratrol(Res).METHODS: The therapeutic effect of Res was verified using an ischemic/reperfusion(I/R) model in vivo and a hypoxia modelin retinal ganglion cells(RGCs) in vitro.Death of RGCs were confirmed by TUNEL assay.Protein expression was confirmed by Western blotting and immunohistochemistry.In addition, flow cytometric analysis was used to confirm the response in the cell unit to obtain more accurate data.RESULTS: ErbB2 expression and apoptosis in the ganglion cell layer(GCL) increased after I/R injury.Treatment of Res rescued I/R-induced ganglion cell death, downregulated apoptosis and ErbB2 protein expression in the retina.In subsequent in vitro models, Res affects apoptosis by regulating the phosphorylation and expression of mouse double minute 2 homolog(MDM2), along with those of ErbB2.These results suggest that Res reverses GCL-specific apoptosis via downregulation of ErbB2 in ischemic injury.CONCLUSION: In light of Res favorable properties, it should be evaluated in the treatment of RGC death and related retinal disease characterized by ErbB2 and MDM2 expression.Therefore, Res is appropriate therapeutic agent for treating ischemic injury-related eye diseases by targeting the expression of ErbB2 and MDM2.

The novel chalcone analog L2H17 protects retinal ganglion cells from oxidative stress-induced apoptosis

Chalcone is a plant metabolite widely found in fruits,vegetables,spices and tea,and has anti-tumor,anti-inflammation,immunomodulation,antibacterial and anti-oxidation activities,as well as many other pharmacological and biological effects.Our team has shown that its analogs have antioxidant activity,and oxidative stress is a pathological hallmark of retinal ischemia/reperfusion injury that can lead to retinal damage and visual loss.This investigation aims to identify a chalcone that protects retinal ganglion cells in vitro from the effects of oxidative stress and examine its mechanism.Rat retinal ganglion cell-5 cells were pretreated with chalcones and then exposed to tert-butyl hydroperoxide that causes oxidative damage.Controls received dimethyl sulfoxide only or tert-butyl hydroperoxide in dimethyl sulfoxide.Only（E）-3,4-dihydroxy-2′-methylether ketone（L2 H17）,of the five chalcone analogs,markedly increased the survival rate of oxidatively injured RGC-5 cells.Thus,subsequent experiments only analyzed the results of the L2 H17 intervention.Cell viability and apoptosis were measured.Intracellular superoxide dismutase and reactive oxygen species levels were used to assess induced oxidative stress.The mechanism of action by L2 H17 was explored by measuring the ER stress/UPR pathway and the expression and localization of Nrf2.All results demonstrated that L2 H17 could reduce the apoptosis of oxidatively injured cells,inhibit caspase-3 activity,increase Bcl-2 expression,decrease Bad expression,increase the activity of superoxide dismutase,inhibit the production of reactive oxygen species,increase Nrf2 immunoreactivity,and reduce the activating transcription factor 4,phospho-eukaryotic initiation factor 2 and CHOP expression.L2 H17 protects retinal ganglion cells induced by oxidative stress by regulating Nrf2,which indicates that it has the potential to become a drug for retinal ischemia/reperfusion.

Protection of retinal ganglion cells against optic nerve injury by induction of ischemic preconditioning

AIM： To explore if ischemic preconditioning （IPC） can enhance the survival of retinal ganglion cells （RGCs） after optic nerve axotomy. METHODS： Twenty-four hours prior to retinal ischemia 60min or axotomy, IPC was applied for ten minutes in groups of （n=72） animals. The survival of RGCs, the cellular expression of heat shock protein 27 （HSP27） and heat shock protein 70 （HSP70） and the numbers of retinal microglia in the different groups were quantified at 7 and 14d post-injury. The cellular expression of HSP27 and HSP70 and changes in the numbers of retinal microglia were quantified to detect the possible mechanism of the protection of the IPC. RESULTS： Ten minutes of IPC promoted RGC survival in both the optic nerve injury （IPC-ONT） and the retinal ischemia 60min （IPC-IR60） groups, examined at 7d and 14d post-injury. Microglial proliferation showed little correlation with the extent of benefit effects of IPC on the rescue of RGCs. The number of HSP27-positive RGCs was significantly higher in the IPC-ONT group than in the sham IPC-ONT group, although the percentage of HSP27-positive RGCs did not significantly differ between groups. For the IPC-IR60 group, neither the number nor the percentage ofthe HSP27-positive RGCs differed significantly between the IPC and the sham-operated groups. The number of HSP70-positive RGCs was significantly higher for both the IPC-ONT and the IPC-IR60 experimental groups, but the percentages did not differ. CONCLUSION： The induction of IPC enhances the survival of RGCs against both axotomy and retinal ischemia.

Early activation of caspase-1 after retinal ischemia and reperfusion injury in mice

Background Caspases are important in the signaling pathway of cellular apoptosis Caspase-3 protein expression has been shown to increase and parallel to neuronal apoptosis in retinal ischemia injury This study was to determine whether caspase-1 is involved in neuronal cell death or in retinal ischemia and reperfusion injury Methods In twenty-one adult mice, ischemia was induced by increasing the intraocular pressure The animals were sacrificed at 1 hour, 3 hours, 6 hours, 1 day, 3 days and 7 days after reperfusion Frozen sections were used for caspase-1 immunostaining and TUNEL labeling Results In normal retina, no caspase-1 positive cells were seen One hour after ischemia, numerous positive cells were noted in the ganglion cell layer (GCL) and inner side of inner nuclear layer (INL) At 3 hours, caspase-1 positive cells continued to increase and peaked at 6 hours, then decreased significantly at 1 day TUNEL positive cells were detected at 3 hours and peaked at 1 day after ischemia Double labeling of caspase-1 and TUNEL only showed few cells with co-localization after ischemia Conclusion Caspase-1 immunoreactivity preceds to the TUNEL labeling in the GCL and INL after retinal ischemia and reperfusion injury and its early activation may play an important role in the initiation of neuronal apoptosis

丹蒌片通过Keap1-Nrf2/HO-1信号通路缓解视网膜缺血-再灌注损伤

目的:探讨丹蒌片对小鼠视网膜缺血-再灌注损伤(RIRI)的保护作用及其机制。方法:将40只ApoE^(-/-)小鼠给予高脂饲料喂养6 wk,通过前房灌注加压法建立RIRI模型,分为对照组(给予生理盐水灌胃8 wk)、RIRI模型组(给予生理盐水灌胃8 wk)及丹蒌片低、中、高剂量组[分别给予丹蒌片溶液1、2、4 g/(kg·d)灌胃8 wk]。采用苏木精-伊红(HE)染色观察各组小鼠视网膜组织形态学变化情况,TUNEL染色检测各组小鼠视网膜细胞凋亡情况,Western-blot法检测各组小鼠视网膜组织中Kelch样环氧氯丙烷相关蛋白-1(Keap1)、转录因子核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、超氧化物歧化酶(Sod2)蛋白表达情况。结果:与对照组比较,RIRI模型组小鼠视网膜萎缩,厚度变薄,细胞凋亡增加,视网膜组织中Sod2蛋白表达下调,Keap1蛋白表达上调(均P<0.01);与RIRI模型组比较,丹蒌片中、高剂量组小鼠视网膜厚度增加(均P<0.01),丹蒌片低、中、高剂量组小鼠视网膜细胞凋亡降低(均P<0.05),丹蒌片低剂量组小鼠视网膜组织中Keap1和HO-1蛋白表达水平无明显差异(P>0.05),丹蒌片中、高剂量组小鼠视网膜组织中Sod2、Nrf2、HO-1蛋白表达上调,Keap1蛋白表达下调(均P<0.05)。结论:丹蒌片能缓解RIRI小鼠模型视网膜萎缩变薄,减少视网膜细胞凋亡,其作用是通过调控Keap1-Nrf2/HO-1信号通路降低RIRI氧化应激反应来实现。

Cardioprotective Effect of Angiotensin n Receptor Antagonist on Perfused Ischemic Reperfusion Injury of Whole Isolated Rat Hearts

Objectives Investigated the cardioprotective and mechanisms of losartan on whole isolated ischemic reperfused rat heart. Methods Langendorff perfused systems was used to investigate losartan effect on whole isolated rat hearts in CPK, LDH, MDA, SOD, ang II and arrhythmia. Results Losartan decreased incidence of arrhythmia, improved atrial ventricular block recovery in reperfu-sion period, during ischemic period, CPK and LDH in I/R group increased significantly compared with control group, 51. 33±27. 02 vs 22. 42 ± 13. 33, 31. 80 ±4.56 vs 22. 28 ± 15. 96, respectively, but greatly decreased in losartan group compared with I/R group, 23. 90±21.74 vs 51. 33 ±27. 02 and 11. 50 ±13. 20 vs 31. 80 ±4. 56, respectively. During reperfusion period CPK, LDH increased significantly in I/R group compared with control group, 49. 11 ± 20. 63 vs 12. 14 ±5.92 and 28. 70±4. 69 vs 23. 10±21. 38, respectively, but decreased greatly in losartan group compared with I/R group, 39. 40 ± 9. 60 vs 49. 11 ± 20.63 and 14. 50 ±13. 75 vs 28. 70±4. 69. The content of MDA, ang II in I/R group myocytes is higher than control group's , 26. ± 9. 25 vs 17. 2 ± 3. 37 and 8. 43 ± 3. 81 vs 4. 80 ± 0. 20. However the content of SOD in two groups has no significantly change, 148. 20 ± 8. 72 vs 145. 08±6. 82. the content of MDA in losartan group myocardial tissue is much lower than control group, 15.92±4.05 vs 26. 80 ± 9. 25 and the content of ang II in losartan group myocardial tissue is much higher than I/R group, 12. 44 ± 6. 09 vs 8. 43 ± 3. 21. The department of cardiology of second hospital of Tianjin medical u-niversity Tianjin 300211 However, SOD has no significant change in two groups, 143. 47±7. 91 vs 145. 08 ± 6. 82. Conclusions Losartan against is-chemic - reperfusion injury of whole isolated rat hearts, those beneficial effects are mediate primarily by the inhibited of angiotensin II binding with its receptor and inhibited oxygen free radical scavenging potential.

视网膜缺血再灌注损伤大鼠视网膜中白细胞介素-23和白细胞介素-17的表达及意义

目的通过建立视网膜缺血再灌注损伤(retinal ischemia-reperfusion injury,RIRI)模型,观察白细胞介素-23(interleukin-23,IL-23)和白细胞介素-17(interleukin-17,IL-17)在Sprague-Dawley(SD)大鼠视网膜上的表达,探讨其在RIRI中的可能作用机制。方法采用随机数字表法将75只健康无眼疾SD大鼠随机分为正常对照组和模型组。正常对照组不作任何处理,模型组采用前房灌注生理盐水升高眼压法建立RIRI模型。分别在建模后12 h、24 h、72 h、144 h处死SD大鼠,免疫组织化学染色法检测IL-23和IL-17蛋白在视网膜上的表达及分布情况;Western blot和ELISA检测分析各时间点视网膜IL-23和IL-17的表达水平和变化规律。结果免疫组织化学染色法显示IL-23和IL-17蛋白在正常对照组视网膜中表达极少,而在模型组表达明显增多。两者主要表达于视网膜神经节细胞层和内核层细胞的细胞浆。Western blot法检测发现IL-23和IL-17蛋白在RIRI视网膜中的表达明显增强,IL-23蛋白在建模后24 h表达最强,而IL-17蛋白在建模后72 h表达达到最高峰。ELISA法结果显示建模后12 h、24 h、72 h、144 h,模型组视网膜IL-23和IL-17蛋白的表达水平均较正常对照组明显升高,差异均有显著统计学意义(均为P<0.01)。结论 IL-23和IL-17在RIRI模型SD大鼠视网膜中表达明显增强,IL-23/IL-17通路作为致病因素,通过加重视网膜炎症反应参与RIRI的病理损伤。

低氧预适应对缺血再灌注损伤大鼠视网膜促红细胞生成素及活性Caspase-3蛋白表达的影响

目的观察低氧预适应(hypoxic preconditioning,HPC)后视网膜缺血再灌注损伤(retinal ischemia-reperfusion injury,RIRI)大鼠视网膜促红细胞生成素(erythropoietin,EPO)蛋白及活性Caspase-3蛋白表达的动态变化,探讨HPC预防RIRI的机制,为RIRI的防治提供有效的治疗方案。方法取健康Sprague Dawley成年大鼠随机分为正常对照组(CON组),RIRI组与HPC+缺血再灌注损伤组(HPC组,HPC后采用高眼压法制成RIRI模型),每组根据RIRI时间分为6h、12h、24h及72h4个亚组。采用免疫组织化学法动态观察HPC对RIRI大鼠视网膜细胞EPO蛋白、活性Caspase-3蛋白表达的影响。结果 CON组大鼠视网膜仅见极少量的EPO+细胞;RIRI后6h,HPC组EPO+细胞数开始增加;RIRI后12h,HPC组EPO+细胞数(171±21)mm-2进一步增加,显著高于RIRI组[(149±20)mm-2,P<0.05];RIRI后24h,HPC组EPO+细胞数(287±24)mm-2达较高水平并显著高于RIRI组[(238±22)mm-2,P<0.01];RIRI后72h,HPC组EPO+细胞数开始下降,但仍显著高于RIRI组(P<0.05)。RIRI后6h,RIRI组活性Caspase-3+细胞数(60±5)mm-2开始增加,显著多于HPC组[(53±5)mm-2,P<0.05];RIRI后12h,RIRI组与HPC组活性Caspase-3+细胞数进一步增加,HPC组活性Caspase-3+细胞显著少于RIRI组(P<0.01);RIRI后24h,RIRI组与HPC组活性Caspase-3+细胞数达较高水平,HPC组活性Caspase-3+细胞数(578±26)mm-2显著少于RIRI组[(624±25)mm-2,P<0.01];RIRI后72h,HPC组与RIRI组活性Caspase-3+细胞数均开始下降,HPC组Caspase-3+细胞数仍少于RIRI组(P<0.05)。结论 HPC可促进RIRI大鼠视网膜细胞EPO蛋白的表达,减轻活性Caspase-3蛋白的表达,从而减轻细胞凋亡,具有神经保护作用。

N-乙酰-5-羟色胺(NAS)对视网膜缺血再灌注损伤(RIRI)大鼠视网膜活性Caspase-3、Bcl-2、Bax表达的影响

目的探讨N-乙酰-5-羟色胺(N-acetylserotonin,NAS)对视网膜缺血再灌注损伤(retinal ischemia-reperfusion injury,RIRI)大鼠视网膜活性Caspase-3、Bcl-2、Bax表达的影响。方法取健康成年Sprague-Dawley大鼠66只,采用随机数字表法随机分为正常对照组(6只)、缺血再灌注组(30只)与药物组(30只),药物组于造模前30 min腹腔注射5 mg·kg-1NAS,缺血再灌注组腹腔注射同等剂量的生理盐水,缺血再灌注组与药物组按RIRI后时间,分为6 h、12 h、24 h、48 h、72 h五个亚组。采用HE染色法观察各组视网膜形态学变化,免疫组织化学染色检测各组大鼠视网膜活性Caspase-3、Bcl-2及Bax蛋白表达。结果HE染色显示正常对照组大鼠视网膜结构清晰,各层细胞排列紧密;缺血再灌注组大鼠RIRI后6 h、12 h视网膜各层高度水肿,24 h后水肿逐渐减轻,神经节细胞逐渐减少,分布较紊乱,随着时间延长,视网膜神经节细胞大片缺失;药物组6 h、12 h较缺血再灌注组细胞水肿明显减轻,24 h、48 h、72 h组较缺血再灌注组细胞排列规整,神经节细胞数量减少减轻。缺血再灌注组视网膜活性Caspase-3阳性细胞数在灌注后6 h开始表达增加,阳性细胞数为(561.15±37.19)个·mm^(-2),24 h达到较高水平,阳性细胞数为(1522.61±84.36)个·mm^(-2),随后逐渐下降,药物组视网膜各时间点活性Caspase-3阳性细胞数均显著少于缺血再灌注组,差异均有统计学意义(均为P<0.05)。正常对照组视网膜可见大量Bcl-2阳性细胞;缺血再灌注组RIRI后6 h Bcl-2阳性细胞开始下降,12 h后继续减少,24 h降至较低水平;药物组各时间点Bcl-2阳性细胞数均显著多于缺血再灌注组,差异均有统计学意义(均为P<0.05)。正常对照组大鼠视网膜几乎未见Bax阳性细胞;缺血再灌注组RIRI后6 h神经节细胞层及内核层可见Bax阳性细胞,24 h达到较高水平,48 h开始下降;药物组各时间点Bax阳性细胞均显著少于缺血再灌注组,差异均有统计学意义(均为P<0.05)。结论 NAS可促进RIRI大鼠视网膜Bcl-2蛋白的表达,抑制Bax蛋白的表达,降低活性Caspase-3蛋白表达,减轻细胞凋亡,具有神经保护作用。

白藜芦醇对大鼠视网膜缺血再灌注损伤后视网膜组织中Caspase-3和Bcl-2表达的影响

目的:研究白藜芦醇对大鼠视网膜缺血再灌注损伤(RIRI)后视网膜组织中Caspase-3和Bcl-2表达的影响,探讨白藜芦醇对RIRI的治疗作用及其机制。方法:选取SD大鼠90只,随机分为假手术组、模型组和治疗组,每组30只,通过对前房加压建立大鼠RIRI模型,模型组和治疗组大鼠缺血再灌注1、6、12、24和48h,治疗组大鼠用微量注射器于大鼠玻璃体腔内注射0.5nmol·L-1的白藜芦醇5μL。采用倒置显微镜观察视网膜组织结构,采用免疫组织化学法和Western blotting法检测各组大鼠视网膜组织中Caspase-3和Bcl-2的阳性细胞数和蛋白表达水平。结果:模型组大鼠造模后视网膜组织水肿,可见神经节细胞空泡样变性,细胞层排列呈现疏松状,视网膜神经节细胞数目大量减少,边界模糊,神经纤维层明显变薄;治疗组大鼠视网膜组织结构、损伤程度及神经节细胞变性程度均轻于模型组。免疫组织化学检测,与模型组比较,治疗组大鼠视网膜组织中Caspase-3和Bcl-2阳性细胞数明显升高(P<0.05)。Western blotting法检测,与模型组比较,治疗组大鼠各时间点视网膜组织中Bcl-2蛋白表达水平升高,24和48h时差异有统计学意义(P<0.05);治疗组各时间点大鼠视网膜组织中Caspase-3蛋白表达水平均明显降低(P<0.05)。结论:白藜芦醇对RIRI大鼠的视网膜神经节细胞结构有改善作用,其作用机制可能与降低视网膜组织中Caspase-3表达水平及升高视网膜组织中Bcl-2表达水平有关。

E-64d对大鼠视网膜缺血再灌注损伤中Calpain和Caspase-3表达的调控作用

目的探讨E-64d(半胱氨酸蛋白酶抑制剂及钙激活中性蛋白酶抑制剂)在大鼠视网膜缺血再灌注损伤(RIRI)中对Calpain/Caspase-3表达的调节作用。方法将80只SD大鼠随机分为正常组、视网膜缺血再灌注组、对照组和E-64d治疗组,并分为1、3、6、24、72h5个时间段。通过前房穿刺加压法制成RIRI动物模型,采用Western-blot以及RT-PCR方法测定在大鼠RIRI模型中的Calpian、Caspase-3蛋白和mRNA表达情况。结果缺血再灌注24hm-Calpain、Caspase-3蛋白的表达缺血再灌注组较正常组上升,而E-64d组表达与正常组相似,比缺血再灌注组下降。E-64d能下调视网膜缺血再灌注后m-Calpain/Calpastatin的mRNA比值,在24h与缺血组有显著统计学差异。结论E-64d能降低视网膜缺血再灌注时m-Calpain、Caspase-3蛋白的表达,调控m-Calpain和Calpastatin的mRNA比值。

缺血后适应对视网膜缺血-再灌注损伤的保护作用

背景研究证明，缺血后适应（IPC）对多种组织器官的缺血缺氧损伤均有一定的抵抗作用，但其对视网膜缺血缺氧的作用仍受到关注。目的探讨IPC对大鼠视网膜缺血-再灌注损伤（RIRI）后视网膜结构和功能的保护作用。方法将36只健康雄性Wistar大鼠以随机数字表法分为正常对照组、伪手术组、缺血-再灌注组、IPC组。利用前房灌注生理盐水升高眼压至100mmHg（1mmHg=0．133kPa）维持60min的方法制备RIRI大鼠模型，实施IPC处理鼠亚分为再灌注后即刻、1min、10min组（即IPCⅠ组、IPCⅡ组、IPCⅢ组），分别于实验后1d、7d行大鼠视网膜电图（ERG）检测，然后用过量麻醉法处死大鼠并制备视网膜切片，行苏木精-伊红染色，对各组大鼠视网膜厚度的变化和视网膜形态进行观察。采用SPSS13．0统计学软件的单因素方差分析对各组大鼠ERG各波振幅恢复率和视网膜厚度值的差异进行比较。结果实验后1d，与正常对照组大鼠比较，伪手术组大鼠视网膜结构接近正常，而缺血-再灌注组及IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠视网膜均出现水肿，可见空泡变性，主要在内丛状层（IPL）及内核层（INL）。缺血-再灌注组及IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠视网膜全层、INL、IPL及视网膜外层厚度值均明显高于正常对照组，差异均有统计学意义（均P〈0．05）。再灌注后7d，缺血-再灌注组大鼠视网膜全层厚度值明显低于正常对照组，差异均有统计学意义（均P〈0．05），尤以INL、IPL显著。IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠视网膜全层、INL、IPL及视网膜外层厚度值均明显高于缺血-再灌注组，差异均有统计学意义（均P〈0．05）。再灌注后7d，缺血-再灌注组、IPC各组大鼠ERG a波、b波和OPs振幅恢复率明显低于伪手术组和正常对照组大鼠，差异均有统计学意义（均P〈0．05）；而IPCⅠ组、IPCⅡ组、IPCⅢ组大鼠ERG a波、b波和OPs振幅恢复率明显高于缺血-再灌注组，差异均有统计学意义（均P〈0，05）。结论IPC对RIRI具有保护作用，在大鼠模型中，这种保护作用在再灌注后即刻至1min时最强。

微小RNA-181a与视网膜节细胞在视网膜缺血-再灌注中的关系及其作用机制探讨

背景视网膜缺血-再灌注（RIR）损伤是眼科常见病理改变之一，但其发病机制尚未明确。目的探讨大鼠RIR模型中微小RNA-181a（miR-181a）与视网膜神经节细胞（RGCs）凋亡之间的关系及可能的靶向机制。方法构建68只SD大鼠RIR模型，按随机数字表法随机分为对照组和造模后即刻组、造模后24h组及造模后72h组，每组17只，根据MiRanda、Targetscan和miRBase3个靶基因数据库的共同预测结果，选取肿瘤坏死因子-α（TNF-α）作为miR-181a的下游靶基因研究对象，通过免疫荧光标记法Westernblot及实时荧光定量PCR法观察miR-181a、TNF·仅在各组的表达情况及其与RGCs凋亡的关系。结果RGCs计数在造模后24h和72h显著减少，与对照组比较差异有统计学意义（P〈O．001）。各造模组miR-181a表达量随造模时间明显下降，与对照组比较差异有统计学意义（P〈0．05）。此外，随RIR时间的延长，miR-181a的表达量及RGCs的数量均逐渐减少，两者呈正相关（r=0．995，P=0．005）。TNF-α主要在内层视网膜表达，与miR-181a分布重叠；RIR即刻至24h之间TNF-α表达明显升高，与RGCs计数及miR-181a表达的变化趋势相反，但总体相关性分析无统计学意义。结论在RIR模型中，miR-181a可能参与RGCs凋亡的调控，TNF-α是其可能的下游靶基因之一，RIR24h前是重要的干预时机。深入研究miR-181a及其靶向基因有助于探寻新的神经保护治疗靶点。

Edaravone对视网膜缺血-再灌注损伤大鼠Bcl-2和Bax表达的影响

目的研究Edaravone对视网膜缺血-再灌注损伤(retinal ischem ia-reperfusion injury,RIRI)大鼠视网膜神经节细胞Bc-l2和Bax表达的影响。方法健康Wistar大鼠36只随机分为3组:空白对照组、模型组和Edaravone治疗组。空白对照组不做任何处理,模型组和Edaravone治疗组均采用前房加压法建立大鼠RIRI模型,Edaravone治疗组于缺血前30min行腹腔注射Edaravone(3mg.kg-1),恢复灌注30min后再行腹腔注射Edaravone(3mg.kg-1)。免疫组织化学法检测各组大鼠视网膜缺血-再灌注后24h视网膜神经节细胞Bc-l2和Bax的表达情况。结果空白对照组未见Bc-l2和Bax表达。再灌注后24h,模型组Bc-l2表达为0.406±0.022,Bax表达为0.661±0.034,Bc-l2/Bax为0.609±0.015;Edaravone治疗组Bc-l2表达为0.581±0.031,Bax表达为0.491±0.017,Bc-l2/Bax为1.104±0.094。与模型组比较,Edaravone治疗组Bc-l2表达明显增强(P<0.05),Bax表达明显减弱(P<0.05),Bc-l2/Bax比值升高(P<0.05)。结论 Edaravone对RIRI大鼠视网膜神经节细胞有明显的保护作用。