丙泊酚對(duì)腦缺血再灌注大鼠腦組織Homer-1a表達(dá)的影響及其機(jī)制

沈大川，董斌，溫超

(1鐵嶺市中心醫(yī)院，遼寧鐵嶺112001；2大連醫(yī)科大學(xué)附屬第一醫(yī)院)

丙泊酚對(duì)腦缺血再灌注大鼠腦組織Homer-1a表達(dá)的影響及其機(jī)制

沈大川1，董斌2，溫超2

(1鐵嶺市中心醫(yī)院，遼寧鐵嶺112001；2大連醫(yī)科大學(xué)附屬第一醫(yī)院)

目的 觀(guān)察丙泊酚對(duì)腦缺血再灌注大鼠腦組織Homer-1a表達(dá)的影響，并探討其機(jī)制。方法 雄性SD大鼠120只，隨機(jī)分為假手術(shù)組、造模組、丙泊酚組，每組40只。模型組、丙泊酚組線(xiàn)栓法建立腦缺血再灌注模型，假手術(shù)組僅行血管分離處理不插入線(xiàn)栓；造模后30 min，丙泊酚組腹腔注射丙泊酚10 mg/100 g，假手術(shù)組和對(duì)照組均腹腔注射等體積生理鹽水。給藥處理3 h、24 h、72 h、7 d分別進(jìn)行神經(jīng)功能評(píng)分，采用Real-time PCR法檢測(cè)腦組織Homer-1a mRNA，應(yīng)用酶聯(lián)免疫吸附法檢測(cè)血清S-100β。結(jié)果 與假手術(shù)組比較，模型組和丙泊酚組4個(gè)時(shí)間點(diǎn)神經(jīng)功能評(píng)分、血清S-100β水平均增加(P均<0.05)，以模型組增高更顯著(P均<0.05)。與假手術(shù)組相比，模型組在3、24 h時(shí)間點(diǎn)腦組織Homer-1a mRNA降低(P均<0.05)；丙泊酚組4個(gè)時(shí)間點(diǎn)腦組織Homer-1a mRNA均較其他兩組升高(P均<0.05)。在所有大鼠樣本中，腦組織Homer-1a mRNA表達(dá)與血清S-100β呈負(fù)相關(guān)(r=-0.939，P<0.01)。結(jié)論 丙泊酚麻醉可通過(guò)誘導(dǎo)腦組織Homer-1a mRNA表達(dá)發(fā)揮對(duì)腦缺血再灌注大鼠的腦保護(hù)作用，該作用可能與其降低血清S-100β水平有關(guān)。

缺血再灌注損傷；腦組織；丙泊酚；Homer-1a基因；S-100β；大鼠

研究發(fā)現(xiàn)，麻醉藥通過(guò)擴(kuò)張腦血管、降低腦組織代謝、抑制自由基合成、減緩脂質(zhì)過(guò)氧化反應(yīng)、調(diào)控神經(jīng)遞質(zhì)等不同途徑，發(fā)揮抗腦缺血再灌注損傷的作用[1～3]。丙泊酚作為一種新型靜脈麻醉藥，具有起效快、輸注后無(wú)蓄積、蘇醒迅速而完全、不良反應(yīng)少等特點(diǎn)，已廣泛應(yīng)用于臨床。研究發(fā)現(xiàn)，丙泊酚具有一定的腦保護(hù)作用[4,5]，但其作用機(jī)制尚未完全闡明。Homer蛋白屬于突觸后密度蛋白質(zhì)家族，其中Homer-1a是該家族中第1個(gè)被確認(rèn)的蛋白，經(jīng)神經(jīng)元興奮、突觸形成或重塑性增加等神經(jīng)活動(dòng)誘導(dǎo)而快速短暫表達(dá)[6,7]。已有研究表明，Homer蛋白可抑制缺血后神經(jīng)細(xì)胞損傷[8]。2013年9月～2014年5月，本研究觀(guān)察丙泊酚對(duì)腦缺血再灌注大鼠腦組織Homer-1a表達(dá)的影響，并探討其機(jī)制。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物 雄性SD大鼠120只，體質(zhì)量200～250 g，由大連醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。

1.2 方法

1.2.1 分組及處理方法 按隨機(jī)數(shù)字法將大鼠分為假手術(shù)組、模型組、丙泊酚組，每組40只。模型組、丙泊酚組大鼠術(shù)前禁食過(guò)夜，應(yīng)用2%戊巴比妥鈉按40 mg/kg的劑量腹腔注射麻醉。手術(shù)分離頸總動(dòng)脈、頸外動(dòng)脈、頸內(nèi)動(dòng)脈，于頸總動(dòng)脈分叉處下方1 mm處向頸內(nèi)動(dòng)脈方向插入直徑0.25 mm的線(xiàn)栓，插入長(zhǎng)度約18 mm。線(xiàn)栓插入過(guò)程中有阻力感，并且大鼠出現(xiàn)呼吸頻率及心率加快、尿失禁等表現(xiàn)，可認(rèn)定出現(xiàn)腦缺血。缺血1 h后拔出線(xiàn)栓，恢復(fù)大腦中動(dòng)脈血供，即再灌注。假手術(shù)組僅進(jìn)行血管分離處理，不插入線(xiàn)栓。造模后30 min，丙泊酚組腹腔注射丙泊酚10 mg/100 g(Sigma-Aldrich)，假手術(shù)組和模型組均腹腔注射等體積生理鹽水。

1.2.2 神經(jīng)功能評(píng)分 分別于藥物處理3 h、24 h、72 h、7 d參照Z(yǔ)ea Longa等[9]方法對(duì)各組大鼠進(jìn)行神經(jīng)功能評(píng)分：無(wú)明顯異常表現(xiàn)計(jì)0分，左側(cè)Honer征計(jì)1分，左側(cè)前爪不能完全伸直計(jì)2分，向左側(cè)轉(zhuǎn)圈計(jì)3分，向左側(cè)傾倒計(jì)4分，死亡計(jì)5分。

1.2.3 血清S-100β檢測(cè) 分別于藥物處理3 h、24 h、72 h、7 d眼眶靜脈毛細(xì)管取血2 mL，分離血清，-80 ℃冰凍保存，1周內(nèi)檢測(cè)。采用S-100β酶聯(lián)免疫檢測(cè)試劑盒(上海拜力生物科技有限公司)檢測(cè)血清S-100β水平，操作步驟按說(shuō)明書(shū)進(jìn)行。

1.2.4 腦組織Homer-1a mRNA檢測(cè) 分別于藥物處理3 h、24 h、72 h、7 d斷頭法急性處死各組大鼠，開(kāi)顱后切取術(shù)側(cè)水腫腦組織，-80 ℃冰凍保存，1周內(nèi)檢測(cè)。應(yīng)用TRIzol試劑(Invitrogen)提取腦組織總RNA；應(yīng)用TaKaRa RNA PCR Kit(AMV)ver 3.0試劑盒(大連寶生物工程有限公司)對(duì)RNA樣本進(jìn)行逆轉(zhuǎn)錄獲得cDNA；參照試劑盒說(shuō)明書(shū)進(jìn)行Real-time PCR擴(kuò)增。Homer-1a上游引物5′-CAAACACTGTTTATGGACTG-3′、下游引物5′-TGCTGAATTGAA-TGTGTACC-3′，β-actin上游引物5′-GCAGAAGGAG-ATCACTGCCCT-3′、下游引物5′-GCTGATCCACATC-TGCTGGAA-3′。反應(yīng)條件為：95 ℃，5 min；95 ℃、30 s，60 ℃、45 s，72 ℃、20 s，40個(gè)循環(huán)。實(shí)驗(yàn)至少重復(fù)3次，采用2-ΔΔCT法對(duì)腦組織Homer-1a mRNA進(jìn)行統(tǒng)計(jì)分析。

2 結(jié)果

腦缺血再灌注大鼠模型建立過(guò)程中，8只大鼠死亡，將剩余72只造模成功大鼠隨機(jī)分入模型組和丙泊酚組，每組36只，每個(gè)時(shí)間點(diǎn)取9只進(jìn)行實(shí)驗(yàn)。假手術(shù)組40只大鼠全部存活，每個(gè)時(shí)間點(diǎn)取10只進(jìn)行實(shí)驗(yàn)。

2.1 各組神經(jīng)功能評(píng)分比較 見(jiàn)表1。

注：與假手術(shù)組同時(shí)點(diǎn)比較，*P<0.05；與模型組同時(shí)點(diǎn)比較，#P<0.05。

2.2 各組血清S-100β比較 見(jiàn)表2。

注：與假手術(shù)組同時(shí)點(diǎn)比較，*P<0.05；與模型組同時(shí)點(diǎn)比較，#P<0.05。

2.3 各組腦組織Homer-1a mRNA比較 見(jiàn)表3。

注：與假手術(shù)組同時(shí)點(diǎn)比較，*P<0.05；與模型組同時(shí)點(diǎn)比較，#P<0.05。

2.4 腦組織Honer-1a mRNA表達(dá)與血清S-100β的關(guān)系 相關(guān)性分析結(jié)果顯示，在所有大鼠樣本中，腦組織中Homer-1a mRNA表達(dá)與血清S-100β水平呈負(fù)相關(guān)(r=-0.939，P<0.01)。

3 討論

腦組織具有高代謝率的特點(diǎn)，缺血導(dǎo)致缺氧可影響神經(jīng)元代謝，造成嚴(yán)重神經(jīng)組織損傷。近年來(lái)，越來(lái)越多的麻醉藥物被用于缺血缺氧性腦損傷的治療，發(fā)揮了良好的腦保護(hù)作用。本研究結(jié)果也進(jìn)一步驗(yàn)證了丙泊酚具有抗腦缺血性損傷的腦保護(hù)作用。對(duì)于麻醉藥物的抗腦缺血性神經(jīng)損傷的保護(hù)作用機(jī)制，早期研究認(rèn)為與其減少生理電活動(dòng)從而降低代謝率有關(guān)。但近年的研究發(fā)現(xiàn)，與麻醉藥物阻止缺氧時(shí)鈉、鈣離子變化，抑制谷氨酸的毒性損傷能力及其抗腦缺血缺氧損傷的保護(hù)作用相關(guān)。

Homer-1a屬于短片段Homer家族蛋白。研究[10～12]發(fā)現(xiàn)，敲除Homer-1a基因的小鼠表現(xiàn)出神經(jīng)精神功能異常；大鼠腦挫傷后Homer-1a蛋白表達(dá)降低，Homer-1a蛋白參與腦損傷修復(fù)過(guò)程；說(shuō)明Homer-1a在缺血性腦損傷的發(fā)生、發(fā)展進(jìn)程中發(fā)揮重要作用。本研究發(fā)現(xiàn)，丙泊酚可提高缺血再灌注大鼠腦組織Homer-1a mRNA表達(dá)，說(shuō)明丙泊酚的腦保護(hù)作用可能與其調(diào)節(jié)Homer-1a水平有關(guān)。

研究發(fā)現(xiàn)，通過(guò)與mGluR、IP3R和NMDA受體等Homer相關(guān)蛋白結(jié)合，Homer-1a參與代謝性氨基酸受體信號(hào)轉(zhuǎn)導(dǎo)調(diào)節(jié)以及多巴胺信號(hào)通路的調(diào)節(jié)，從而發(fā)揮其作為負(fù)向調(diào)節(jié)蛋白的作用[13～16]。但在腦保護(hù)過(guò)程中，Homer-1a的作用機(jī)制尚未完全闡明。本研究同步檢測(cè)血清S-100β水平。S-100β是公認(rèn)的早期腦組織缺血缺氧性損傷的金標(biāo)準(zhǔn)[17]。結(jié)果發(fā)現(xiàn)模型組、丙泊酚組血清S-100β水平均較假手術(shù)組顯著升高，但丙泊酚組升高幅度不及模型組；在所有大鼠樣本中Homer-1a與S-100β呈負(fù)相關(guān)。以上結(jié)果說(shuō)明Homer-1a可能通過(guò)拮抗S-100β的作用而發(fā)揮其腦保護(hù)作用。

總之，丙泊酚麻醉可通過(guò)誘導(dǎo)腦組織Homer-1a mRNA表達(dá)發(fā)揮對(duì)腦缺血再灌注大鼠的腦保護(hù)作用，該作用可能與其降低外周血S-100β水平有關(guān)。

[1] Wise-Faberowski L, Raizada MK, Sumners C. Oxygen and glucose deprivation-induced neuronal apoptosis is attenuated by halothane and isoflurane[J]. Anesth Analg, 2001,93(5):1281-1287.

[2] Nishizawa Y. Glutamate release and neuronal damage in ischemia[J]. Life Sci, 2001,69(4):369-381.

[3] Warner DS. Isoflurane neuroprotection: a passing fantasy, again[J]. Anesthesiology, 2000,92(5):1226-1228.

[4] 賈東林,褚曉玉,張利萍.丙泊酚對(duì)自由基引起的大鼠腦皮質(zhì)線(xiàn)粒體損傷的保護(hù)作用[J].中國(guó)臨床藥理學(xué)與治療學(xué),2007,12(7):763-765.

[5] Wu XJ, Zheng YJ, Cui YY, et al. Propofol attenuates oxidative stress-induced PC12 cell injury via p38 MAP kinase dependent pathway[J]. Acta Pharmacol Sin, 2007,28(8):1123-1128.

[6] Xiao B, Tu JC, Patralia RS, et al. Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins[J]. Neuron, 1998,21(4):707-716.

[7] Foa L, Gasperini R. Development roles for Homer: more than just a pretty scaffold[J]. J Neurochem, 2009,108(1):1-10.

[8] Szumlinski KK, Ary AW, Lominac KD. Homers regulate drug-induced neuroplasticity: implications for addiction[J]. Biochem Pharmcol, 2008,75(1):112-133.

[9] Zea Longa E, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989,20(1):84-91.

[10] Szumlinski KK, Lominac KD, Kleschen MJ, et al. Behavioral and neurochemical phenotyping of Homer 1 mutant mice: possible relevance to schizophrenia[J]. Genes Brain Behav, 2005,4(5):273-288.

[11] Huang WD, Fei Z, Zhang X, et al. Traumatic injury induced homer-1a gene expression in cultured corticed neurons of rat[J]. Neurosci Lett, 2005,389(1):46-50.

[12] Shiraishi Y, Furuichi T. The Homer family proteins[J]. Genome Biology, 2007,8(2):206.

[13] Hu SJ, Xing JL. An experimental model for chronic compression of dorsal root ganglion produced by intervertebral foramen stenosis in the rat[J]. Pain, 1998,77(1):15-23.

[14] Lominac KD, Oleson EB, Pava M, et al. Distinct roles for different Homer 1 isoforms in behaviors and associated prefrontal cortex function[J]. J Neurosci, 2005,25(50):11586-11594.

[15] Bottai D, Guzowski JF, Schwarz MK, et al. Synaptic activity-induced conversion of intronic to exonic sequence in Homer 1 immediate early gene expression[J]. J Neurosci, 2002,22(1):167-175.

[16] Yamada H, Kuroki T, Nakahara T, et al. The dopamine D1 receptor agonist, but not the D2 receptor agonist, induces gene expression of Homer 1a in rat striatum and nucleus accumbens[J]. Brain Res, 2007,1131(1):88-96.

[17] Pankova TM, Starostina MV, Shtark MB, et al. Neuroprotective effect of ultra-low doses of antibodies against S100 protein in neuroblastoma culture during oxygen and glucose deprivation[J]. Bull Exp Biol Med, 2007,144(3):288-290.

Effect of propofol on expression of Homer-1a in cerebral ischemia reperfusion rats and the mechanism

SHENDa-chuan1,DONGBin,WENChao

(1TielingCentralHospital,Tieling112001,China)

Objective To observe the effect of propofol on the expression of Homer-1a in the brain tissues of cerebral ischemia reperfusion rats, and to explore its mechanism. Methods A total of 120 male SD rats were randomly divided into the sham operation group, model group and propofol group, 40 rats in each group. Cerebral ischemia reperfusion rat models were established in the model group and propofol group by using the suture method; sham operation group only

vascular separation processing, without line bolt insertion. Thirty min after modeling, rats in the propofol group were given propofol 10 mg/100 g by intraperitoneal injection, while rats in the sham operation and control groups were injected the same amount of normal saline. After the drug treatment for 3 h, 24 h, 72 h and 7 d, neurological function score was evaluated, Homer-1a mRNA level in the brain tissues was detected by using the real-time PCR, and S-100β in the peripheral blood was detected by enzyme linked immunosorbent assay. Results Compared with the sham operation group, the neurological function scores and S-100β levels of peripheral blood were increase at the four time points in the model and propofol groups (allP<0.05), and the model group increased more significantly (allP<0.05). Compared with the sham operation group, the level of Homer-1a mRNA decreased in the model group at the time points of 3 h and 24 h (allP<0.05). The Homer-1a mRNA level of the propofol group was higher than those in the other two groups at the four time points (allP<0.05). In all rat samples, Homer-1a mRNA expression level and S-100β level was negatively correlated (r=-0.939,P<0.01). Conclusion Propofol anesthesia has a protective effect on the brain tissues in the cerebral ischemia reperfusion rats by inducing Homer-1a mRNA expression, and this effect may be related to the decrease of S-100β level in the peripheral blood.

ischemia reperfusion injury; brain tissues; propofol; Homer-1a gene; S-100β; rats

國(guó)家自然科學(xué)基金資助項(xiàng)目(81371454)。

沈大川(1971-)，男，副主任醫(yī)師，研究方向小兒麻醉。E-mail: 934637173@qq.com

10.3969/j.issn.1002-266X.2015.12.004

R743.3

A

1002-266X(2015)12-0014-03

2014-11-30)