电针刺激能影响对脑缺血后大脑皮层神经元及JAK2-STAT3信号转导的通路

1、NEURAL REGENERATION RESEARCHVolume 7, Issue 6, February 2012Cite this article as: Neural Regen Res. 2012;7(6):457-462.Electroacupuncture effects on cortical neurons, as well as Janus kinase 2-signal transducer andactivator of transcription 3 signal transduction pathway, in a rat mod

2、el of cerebral ischemia* Rong Liu, Nenggui Xu, Wei Yi, Kangbai HuangGuangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, ChinaAbstractThe present study established a model of focal cerebral ischemia through heat-coagulationinduced occlusion of the middle cerebral artery. F

3、ollowing electroacupuncture at Baihui (GV20)and Dazhui (GV14), or intracerebroventricular infusion of AG490, a Janus kinase 2phosphorylation inhibitor, the amount of necrotic or degenerated neurons in the ischemiccerebral cortex decreased, neuronal swelling was ameliorated, and expression ofphosphor

4、ylated Janus kinase 2 and signal transducer and activator of transcription 3 decreased.Results confirmed that electroacupuncture promoted neuronal repair in the cerebral cortex byreducing expression of phosphorylated Janus kinase 2 and signal transducer and activator oftranscription 3, as well as we

5、akening the phosphorylated activation, thereby blocking abnormalactivation of the Janus kinase 2- signal transducer and activator of transcription 3 signaltransduction pathway.Key Words: cerebral ischemia; JAK2-STAT3; neurons; electroacupuncture; AG490unchanged following ischemic reperfusion.INTRODU

6、CTION AG490 is an inhibitor of JAK2 receptor, andapplication of AG490 has been shown toThe Janus kinase-signal transducer and inhibit JAK2 activation, resulting inactivator of transcription (JAK-STAT) system decreased STAT3 phosphorylated levels5-6.consists of Janus kinase (JAK) and signal AG490 has

7、 also been shown to reduce thetransducer and activator of transcription amount of neuronal apoptosis7.(STAT). Following cerebral ischemia, a large Electroacupuncture plays an important roleamount of cytokines and growth factors are in neuroprotection in the cerebral striatumreleased, which could act

8、ivate the following cerebral ischemia, and theseJAK-STAT signal transduction system1-3. protective mechanisms could contribute toThe JAK-STAT signal transduction system, anti-oxidative stress and gene expressionalin particular the JAK2-STAT3 signal changes due to electroacupuncture8.transduction pat

9、hway, plays an important Electroacupuncture has been shown torole in regulating neuronal apoptosis increase Bcl-2 protein expression, whichfollowing cerebral ischemia4. inhibits neuronal apoptosis, in the ischemicIntracerebroventricular infusion of AG490, a penumbra of rats with cerebral ischemia.Ja

10、nus kinase 2 phosphorylation inhibitor, in This suggests that acupuncture providesrats prevents post-ischemic JAK2 and neuroprotection following cerebralSTAT3 phosphorylation and significantly ischemia9. However, recent studies havereduces infarct volume, the number of not elucidated the functional

11、mechanisms ofapoptotic cells, and neurological deficits4. the JAK2-STAT3 signal transductionIntracerebral injection of siRNA specific for pathway affected by acupuncture followingSTAT3 leads to reduced STAT3 mRNA cerebral ischemia. In the present study, theexpression and phosphorylation, as well as

12、effects of electroacupuncture and AG490 ondecreased infarct volume, fewer apoptotic neuronal ultrastructure in the ischemiccells, and improved neurological function cortex of rats with focal cerebral ischemiafollowing transient middle cerebral artery were observed by transmission electronocclusion4.

13、 In addition, microscope. In addition, p-JAK2 andphosphorylated-JAK2 (p-JAK2) and p-STAT3 expression was observed in the p-STAT3 protein expression remains ischemic cortex by fluorescent Rong Liu, Studying for doctorate, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province,

14、 China Corresponding author: Nenggui Xu, Ph.D., Researcher, Doctoral supervisor, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Wei Yi, Ph.D., Professor, Masters supervisor, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China

15、. cn; yw8013 Received: 2011-10-27 Accepted: 2012-01-30 (N20110706001/YJ) Liu R, Xu NG, Yi W, Huang KB. Electroacupuncture effects on cortical neurons, as well as Janus kinase 2-signal transducer and activator of transcription 3 signal transduction pathway, in a rat model of cer

16、ebral ischemia. Neural Regen Res. 2012;7(6):457-462. doi:10.3969/j.issn.1673-5374.2012.06.009457Liu R, et al. / Neural Regeneration Research. 2012;7(6):457-462.immunohistochemistry and laser confocal scanning microscopy to evaluate the significance of JAK2 and STAT3 activation in f

17、ocal ischemia-induced neuronal damage. The mechanisms of action ofelectroacupuncture will provide further insight into treatment paradigms for ischemic cerebral diseases.RESULTSQuantitative analysis of experimental animals A total of 200 Sprague Dawley rats were randomly assigned to sham-surgery, mo

18、del, electroacupuncture, AG490, and electroacupuncture + AG490 groups, with 40 animals in each group. Except for the sham-surgery group, focal cerebral ischemia was established in the remaining groups. AG490 group rats were subjected to an intracerebroventricular infusion of AG490 20 minutes prior t

19、o middle cerebral artery occlusion. The electroacupuncture group was treated withelectroacupuncture at Baihui (GV20) and Dazhui (GV14) 1 hour post-middle cerebral artery occlusion. The electroacupuncture + AG490 group was intracerebroventricularly injected with AG490 20minutes prior to middle cerebr

20、al artery occlusion and electroacupuncture at Baihui and Dazhui 1 hour post-middle cerebral artery occlusion. All rats were included in the final analysis.Electroacupuncture and AG490 improved neuronal ultrastructure in the ischemic cortexIn the sham-surgery group, neurons were normal without swelli

21、ng. Euchromatin was plentiful and evenlydistributed, and few heterochromatins were observed in the nuclei. The nuclear membranes were clear, nucleoli were regular, and there were many cytoplasmicorganelles. Mitochondriae were normal with complete cristae (Figure 1A). At 2 hours post-ischemia, neuron

22、al nuclei were swollen and deformed, chromatins were unevenly distributed, and mitochondriae and cytoplasm458were swollen. At 1 and 3 days post-cerebral ischemia, the model group exhibited cytoplasmic swelling, loss of organelles, significantly distended mitochondriae, loss ofpart of the mitochondri

23、al cristae, and expanded rough endoplasmic reticulum with serious degranulation. Some neurons exhibited chromatin condensation, as well as aggregation at the periphery of the nuclei and nuclear condensation. The electroacupuncture, AG490, and electroacupuncture + AG490 groups exhibited similar chang

24、es in neuronal ultrastructure in the ischemic cortex at the same time points. Compared with the model group, there were less degenerative and necrotic neurons in the remaining three groups. In addition, there were less swollen neurons than in the model groups, as well as loss of a small part of mito

25、chondrial cristae and some slightly irregular nuclei (Figures 1B-F). At 7 days post- cerebral ischemia, there were less degenerative and necrotic neurons in the electroacupuncture, AG490, and electroacupuncture + AG490 groups compared with the model groups. Some nuclei were slightly irregular, and p

26、arts of the heterochromatins aggregated at the nuclar periphery. At 21 days, except for the sham-surgery group, changes of neuronal ultrastructure were not obvious. In addition, the neurons were slightly distended, the cell outline was clear, several mitochondrial cristae integrated after fracturing

27、, and there was sufficient organelles. TheseLiu R, et al. / Neural Regeneration Research. 2012;7(6):457-462.results suggested that post-cerebral ischemia, expression decreased in the electroacupuncture and electroacupuncture and AG490 intervention ameliorated AG490 groups (P < 0.05). Compared wit

28、h the modelneuronal swelling, increased the number of mitochondriae, groups, p-JAK2 expression significantly decreased in the and promoted neuronal repair in the cerebral cortex. electroacupuncture + AG490 group at the same ischemic Electroacupuncture and AG490 reduced p-JAK2 and time (P < 0.05).

29、 In the electroacupuncture group,p-STAT3 expression in the ischemic cortex p-STAT3 significantly decreased compared with model Immunofluorescence results showed that p-JAK2 group at 1 and 3 days post-ischemia (P < 0.01,expression was primarily in the neuronal cytoplasm of respectively). In the AG

30、490 group, p-STAT3 expression the ischemic cortex, with no obvious expression in the significantly decreased compared with the model group nuclei. p-STAT3 was primarily expressed in neuronal at 2 hours, as well as 1 and 3 days, post-ischemia (P < nuclei, with little expression in the cytoplasm. A

31、t different 0.01, respectively). Except for the 21-day subgroup,times in the sham-surgery group, there was little p-JAK2 p-STAT3 expression was less in the electroacupuncture + or p-STAT3 expression. However, in the model group, AG490 group at same time point than in with model some p-JAK2 and p-STA

32、T3 expression was detected in group (P < 0.05; Figure 2, Tables 1, 2). p-JAK2 andthe cerebral cortex at 2 hours post-ischemia. p-JAK2 and p-STAT3 overexpression following cerebral ischemia, as p-STAT3 expression peaked at 1 day post-ischemia, but well as abnormal activation of the JAK2-STAT3 sign

33、al decreased by 3 days. Compared with the sham-surgery transduction pathway, could serve as a mechanism of group at the same time point of ischemia, p-JAK2 and aggravated cerebral injury. Electroacupuncture treatment p-STAT3 expression in the model group was significantly was shown to down-regulate

34、p-JAK2 and p-STAT3 greater (P < 0.01). Similar changes existed between the expression following cerebral ischemia, as well as electroacupuncture and AG490 groups, with exception to decrease phosphorylated activation, thereby blocking the 2-hour and 21-day groups. Compared with the model abnormal

35、activation of the JAK2-STAT3 signalgroup at the same time point of ischemia, p-JAK2 transduction pathway.459Liu R, et al. / Neural Regeneration Research. 2012;7(6):457-462.cerebral ischemic cortex in the electroacupuncture, DISCUSSION AG490, and electroacupuncture + AG490 groups was less than in the

36、 model groups, and the degree ofCellular apoptosis due to cerebral ischemia is related to neuronal swelling was also less than in the model group. several cytokines, such as interleukin, caspase, c-fos, These results suggested that following cerebraland tumor necrosis factor-. The JAK-STAT signal is

37、chemia, electroacupuncture and AG490 intervention transduction system plays an important role in signal ameliorated neuronal swelling, increased the number of10transduction induced by these cytokines. This signal mitochondriae, and promoted neuronal repair in thetransduction system and the regulated

38、 mechanisms cerebral cortex.following cerebral ischemia have been shown to inhibit Immunofluorescence results revealed no p-JAK2 and the inflammatory course and improve neuronal injury in p-STAT3 expression in the sham-surgery group, which the ischemic region, as well as provide a novel method sugge

39、sted that p-JAK2 and p-STAT3 remained in10-11for treating cerebral ischemia. inactive or weak. However, following cerebral ischemia,The intracerebroventricular infusion of rats with AG490 p-JAK2 and p-STAT3 expression increased, and these significantly reduces infarct volume following cerebral chang

40、es occurred almost simultaneously in post-focal ischemia, as well as significantly reduces cellular cerebral ischemia. These results demonstrated that4apoptosis and ameliorates neurological deficits. expression and activation of p-JAK2 and p-STAT3Intervention of acupuncture on the JAK-STAT signal fo

41、llowing cerebral ischemia played an important role in transduction system following cerebral neuronal death induced by cerebral ischemia. In addition, ischemia/reperfusion results in up-regulated STAT3 electroacupuncture functional mechanisms on JAK2- protein expression in the hippocampus, as well a

42、s STAT3 at different times after cerebral ischemia were increased activation and nuclei translocation of STAT3, analyzed and compared with intracerebroventricular12thereby providing neuroprotection. STAT1 mRNA infusion of AG490. Results showed thatexpression increases at 3 days post-cerebral ischemi

43、a, electroacupuncture effectively inhibited over-expression and electroacupuncture has been shown to inhibit this of p-JAK2 and p-STAT3, as well as abnormal activation13expression. Increased STAT1 could be a critical of the JAK2-STAT3 signal transduction pathway. Thissignal for inducing apoptosis, a

44、nd inhibition by could be one of the critical mechanisms by whichelectroacupuncture could be serve as an important electroacupuncture protects against cerebral ischemia pathway for reducing apoptosis and improving cerebral and inhibits neuronal apoptosis.13function following cerebral ischemia. The m

45、arginalzone of the ischemic neuronal ultrastructure, which MATERIALS AND METHODSincluded mitochondrial swelling, cristae destruction, and degeneration of ribosomes, was less in the Designelectroacupuncture group than in the control groups. A randomized, controlled, animal experiment.These results su

46、ggested that electroacupuncture Time and settingreduced cerebral infarct size and marginal zone The experiment was performed at the Laboratory Animal neuronal apoptosis, thereby protecting ischemic Center and Laboratory of Electron Microscopy,14neurons. Guangzhou University of Chinese Medicine, Chin

47、a from In the present study, transmission electron microscope March 2010 to May 2011.was used to observe the effects of electroacupuncture Materialsand AG490 on neuronal ultrastructure in the ischemic A total of 200 adult, male, specific pathogen-free,cortex of rats with focal cerebral ischemia. Res

48、ults Sprague Dawley rats, aged 42-49 days and weighing showed significantly swollen neurons, loss of organelles, 180-240 g, were provided by the Laboratory Animal and unevenly distributed nuclear chromatins. The Center of Guangzhou University of Chinese Medicine amount of neuronal degeneration and n

49、ecrosis in the (license No. SCXK (Yue) 2008-0020). The rats were 460Liu R, et al. / Neural Regeneration Research. 2012;7(6):457-462.housed at 22 ± 2°C, with 65% relative humidity and free access to food, and were exposed daily to 12 hours of light. All experimental procedures were performe

50、d in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China15. MethodsEstablishment of a focal cerebral ischemia model Heat coagulation-induced middle cerebral artery occlusion was used to establish a model o

51、f focalcerebral ischemia. Rats were initially intraperitoneally anesthetized with a 10% chloral hydrate injection (330 mg/kg) and were fixed in a right upper lateral position on the surgical table. An incision was made at the median position between the ear and eye to isolate the temporal muscle. Th

52、e middle cerebral artery was exposed and occluded using a heated stainless steel wire. In the sham-surgery group, the middle cerebral arteries were exposed without occlusion. Intracerebroventricular infusion of AG490AG490 (Merck, Darmstadt, German), 250 m in 3% dimethyl sulphoxide, was continuously

53、infused into the lateral ventricles of rat cohorts. The drugs were filled into 25-L osmotic minipumps that contained 10 L solution. Each pump was connected to an Alzet brain infusion stainless steel cannula via peristaltic tubing and was primed overnight at 37°C to ensure immediate delivery pos

54、t-implantation. The cannula wasstereotaxically implanted into the lateral ventricle (bregma, 0.8 mm posterior, -4.8 mm dorsoventral, -1.5 mm lateral)16 and secured to the skull with a needle. The pump was placed in the skin fold on the neck. The cannula and pump implantation wasconducted under chlor

55、al hydrate injection anesthesia. The intracerebroventricular infusion lasted for 10minutes, and the needle was maintained for 2 minutes. Penicillin was used for disinfection following infusion. The middle cerebral artery of the AG490 group and electroacupuncture + AG490 group was occluded for 20 min

56、utes following intracerebroventricular infusion4. Electroacupuncture treatmentRats were electroacupunctured at 1 hour post-focal cerebral ischemia. The acupoints of Baihui and Dazhui in the Du meridian were selected, as previously described17. Baihui is located in the median of the parietal bone, an

57、d Dazhui is located in the median of the back, below the spinous process of the seventh cervical and first thoracic vertebrae. A 0.3 mm × 25 mm needle (Suzhou Medical Equipment Factory, Suzhou, China) was inserted approximately 15 mm ventral to Baihui and inserted vertically into Dazhui at a de

58、pth of 7.5 mm. Acupoints were connected to a G-6805 electric acupuncture apparatus (QingdaoXinsheng Medical Instrument Factory, Qingdao, China; 4/20 Hz, 1-2 mA for 30 minutes, once daily). Sample collection and transmission electron microscope analysisExperimental animals were sacrificed after 2 hou

59、rs,and 1, 3, 7, and 21 days post-surgery. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. The chest was opened to expose the heart for left ventricle aortic cannulation. A total of 200 mL normal saline and 300 mL 4% paraformaldehyde/phosphate- buffered saline (pH 7.4) was perfused. Brain tissues from two rats in each group were collected, a