蒙藥額日敦烏日勒對(duì)大鼠周圍神經(jīng)損傷早期c-fos表達(dá)及神經(jīng)功能的影響

蒙藥額日敦烏日勒對(duì)大鼠周圍神經(jīng)損傷早期c-fos表達(dá)及神經(jīng)功能的影響

額爾敦朝魯，包春生，周鐵寶，趙福全，胡玉榮，李立

(內(nèi)蒙古民族大學(xué)附屬醫(yī)院，內(nèi)蒙古 通遼 028007)

摘要：目的觀察額日敦烏日勒對(duì)大鼠坐骨神經(jīng)損傷早期c-fos表達(dá)及神經(jīng)功能的影響，探討其療效及作用機(jī)理。方法將64只SD大鼠隨機(jī)分為假手術(shù)組、模型組、陽性對(duì)照組、額日敦烏日勒組。除了假手術(shù)組外建立坐骨神經(jīng)夾持損傷模型，陽性對(duì)照組大鼠每日給予維生素B1和維生素B12各3 mg/kg、10 μg/kg；額日敦烏日勒組大鼠每日給予額日敦烏日勒0．3 g/kg，灌胃體積均為2 mL/100 g，觀察造模后1周、4周每組大鼠坐骨神經(jīng)功能指數(shù)(SFI)、神經(jīng)傳導(dǎo)速度(NCV)及坐骨神經(jīng)中c-fos表達(dá)變化。結(jié)果治療1周后，假手術(shù)組、陽性對(duì)照組、額日敦烏日勒組SFI值較模型組差異有統(tǒng)計(jì)學(xué)意義(P<0．05),且額日敦烏日勒組優(yōu)于陽性對(duì)照組 (P<0．05) ；額日敦烏日勒組、陽性對(duì)照組與模型組比較，NCV值有所上升(P<0．05)；額日敦烏日勒組、陽性對(duì)照組與模型組比較，c-fos蛋白表達(dá)灰度值均明顯增高(P<0．05)。治療4周后，模型組、假手術(shù)組、陽性對(duì)照組、額日敦烏日勒組SFI值差異無統(tǒng)計(jì)學(xué)意義(P> 0．01)，額日敦烏日勒組、陽性對(duì)照組與模型組比較，NCV值明顯提高(P<0．05)；額日敦烏日勒組、陽性對(duì)照組與模型組比較,c-fos蛋白表達(dá)灰度值均明顯增高(P<0．05)。結(jié)論額日敦烏日勒對(duì)周圍神經(jīng)損傷有一定的療效，其作用機(jī)制可能是通過抑制c-fos表達(dá),從而起到促進(jìn)坐骨神經(jīng)感覺和運(yùn)動(dòng)功能恢復(fù)的作用。

關(guān)鍵詞：額日敦烏日勒；坐骨神經(jīng)損傷；坐骨神經(jīng)功能指數(shù)；免疫組化；c-fos蛋白

DOI:10.13463/j.cnki.jlzyy.2015.11.022

中圖分類號(hào)：R722.14文獻(xiàn)標(biāo)志碼： A

基金項(xiàng)目：內(nèi)蒙古自治區(qū)衛(wèi)生廳項(xiàng)目(2005061)。

作者簡介：額爾敦朝魯(1963-)，男,醫(yī)學(xué)碩士，教授，主任醫(yī)師，主要從事神經(jīng)系統(tǒng)感染免疫性疾病研究。

收稿日期:(責(zé)任編輯：張瑞彬2014-11-14)

Erdunwurile on C-fos expression and nerve function of early peripheral nerve injury in rats

Eerdunchaolu, BAO Chunsheng, ZHOU Tiebao, ZHAO Fuquan, HU Yurong, LI Li

(Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028007,

Inner Mongolia Autonomous Region,China)

Abstract：ObjectiveThe effects of Eridunwurile on c-fos expression and the neural function of Early stage following sciatic nerve injury in rats are observed through experiments, to explore the effects and mechanism of it. MethodsThe 64 SD rats were randomly divided into sham-operated group,model group, positive control group and Eridunwurile group. In addition to sham-operated group was established sciatic nerve injury model,positive control group's rats was given 3 mg/kg,10 μg/kg of vitamin B1,and vitamin B12;Erdunwurile group’s rats was given 0.3 g/kg,2 mL/100 g.At 1st and 4th weeks after treatment, 8 rats from each group detected sciatic nerve function index(SFI), and nerve conduction velocity (NCV) and grey value of c-fos positive expression. ResultsSFI: At 1st week after treatment, the recovery of Eridunwurile low-dose group, Eridunwurile high-dose group and positive control group was obviously superior to the model group,there is statistically significant (P<0．05); Eridunwurile low-dose group was significantly different from positive control group (P<0．05). TWL: At 1st week after treatment, compared with model group, TWL of Eridunwurile low-dose group were significantly prolonged. (P<0．05). NCV: At 1th and 4th weeks after treatment, compared with the model group, the NCV of Eridunwurile low-dose group and positive control group were obviously accelerated(P<0．05). After 4 weeks treatment Model group, control group, positive control group, the forehead, SFI value, le group no significant statistically(P> 0．01), Grey value of C-FOS positive expression：At 1st and 4th weeks after treatment, Gray value of Eridunwurile low-dose group, Eridunwurile high-dose group and positive control group was significantly higher than the model group (P<0．05); Gray value of Eridunwurile low-dose group was significantly higher than the positive control group(P<0．05). ConclusionErdunwurle to be effective treat sciatic nerve injury in rats, its mechanism is improve the conductive function, analgesia and recovery of sciatic nerve functional by inhibited c-fos expression.

Keywords：Eridunwurile;sciatic nerve injury; thermal withdrawal latency;sciatic nerve cduction velocity;c-fos protein

周圍神經(jīng)損傷是因直接或間接因素(如外傷、感染、免疫、代謝障礙等)引起神經(jīng)干或其分支損傷而導(dǎo)致神經(jīng)功能缺失。周圍神經(jīng)損傷再修復(fù)是一個(gè)復(fù)雜的過程，研究表明c-fos 表達(dá)參與軸突對(duì)傷害性刺激的反應(yīng)，對(duì)神經(jīng)受到外界刺激后能夠迅速作出反應(yīng)而進(jìn)行表達(dá)[1]。蒙藥額日敦烏日勒治療周圍神經(jīng)損傷療效確切，具有清毒熱、殺粘疫、開竅、調(diào)理體質(zhì)、清血熱等功效，前期研究已證實(shí)額日敦烏日勒對(duì)坐骨神經(jīng)損傷后的修復(fù)及神經(jīng)功能的恢復(fù)具有明顯的促進(jìn)作用[2]。本研究觀察藥物干預(yù)下坐骨神經(jīng)中c-fos表達(dá)變化，為蒙藥治療周圍神經(jīng)損傷提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物與分組實(shí)驗(yàn)動(dòng)物采用SD大鼠64只，體質(zhì)量170～230 g，雌雄各半，購自北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司，許可證號(hào):SCXK(北京)2006-0009。應(yīng)用EXCEL軟件將動(dòng)物按體質(zhì)量隨機(jī)分組，分為假手術(shù)組、模型組、陽性對(duì)照組、額日敦烏日勒組等4組，每組16只。

1.2模型制作大鼠稱重后，用新配制的10%水合氯醛 (10 mL/kg)腹腔注射麻醉，剪去手術(shù)區(qū)域的毛，俯臥位固定在自制的鼠板上，并消毒手術(shù)區(qū)域。消毒完后于右股骨后外側(cè)縱行切口，鈍性分離肌肉組織，暴露坐骨神經(jīng)，用特制的止血鉗擠壓坐骨神經(jīng)(扣到止血鉗的第一扣)10 s后松解止血鉗并關(guān)閉切口，注射青霉素0．15×106U/(kg·d)防止感染。假手術(shù)組僅暴露坐骨神經(jīng)，不造模。術(shù)后觀察各組大鼠一般情況。

1.3處理方法額日敦烏日勒組灌胃額日敦烏日勒0．3 g/kg，陽性對(duì)照組灌胃維生素B13 mg/kg+維生素B1210 μg/kg，灌胃體積為2 mL/100 g， 2次/d灌胃，連續(xù)灌胃4周。假手術(shù)組和模型組灌胃同體積蒸餾水。

1.4標(biāo)本制作分別在造模1周、4周時(shí)，將各組大鼠隨機(jī)選取8只，用10%水合氯醛 (10 mL/kg)腹腔注射麻醉，將含有4%多聚甲醛溶液和0．9%氯化鈉溶液的灌注針插入左心室，打開灌流器用0．9%氯化鈉溶液進(jìn)行灌注，同時(shí)剪開右心耳，當(dāng)肝臟發(fā)白時(shí)將其關(guān)閉，打開含有4%多聚甲醛溶液進(jìn)行灌注，待大鼠全身變硬后結(jié)束灌注。剪開大鼠右側(cè)臀股交界處皮膚，鈍性分離肌肉、筋膜等組織，取出損傷區(qū)1 cm的坐骨神經(jīng)。

1.5行為學(xué)檢測自制長59．5 cm、寬10 cm、高10 cm的大鼠行走箱，箱底鋪一張標(biāo)記好的白紙后將大鼠雙側(cè)后肢沾上墨水放進(jìn)行走箱內(nèi)。將印有大鼠雙后肢足印的白紙取出后選取最清楚的足印，用0．02 mm精度的游標(biāo)卡尺測量雙后肢1～5、2～4足趾的間距和足印長度。用Bain[3-4]公式計(jì)算SFI。

1.6免疫組化檢測分別在1周、4周時(shí)，各組取8只大鼠坐骨神經(jīng)置于4%多聚甲醛固定液中固定24 h。常規(guī)脫水透明后石蠟包埋組織，切4 μm厚的切片。用SP免疫組化染色方法檢測坐骨神經(jīng)干中c-fos蛋白的表達(dá)情況。在200倍鏡下每張切片連續(xù)選取5個(gè)視野進(jìn)行灰度值測定，并取其平均值。

2結(jié)果

2.1坐骨神經(jīng)功能指數(shù)(SFI)檢測治療1周后，與假手術(shù)組比較其他3組SFI均明顯降低 (P<0.05) ；與模型組比較，額日敦烏日勒組和陽性對(duì)照組SFI恢復(fù)明顯優(yōu)于模型組(P<0．05) ；額日敦烏日勒組優(yōu)于陽性對(duì)照組(P<0．05)。治療4周后，各組大鼠坐骨神經(jīng)功能基本恢復(fù)正常，各組間差異無統(tǒng)計(jì)學(xué)意義(P>0．05)。見表1。

表1　各組SFI結(jié)果比較

注：與假手術(shù)組比較，#P<0．05；與模型組比較，△P<0．05；與陽性對(duì)照組比較，▲P<0．05

2.2坐骨神經(jīng)傳導(dǎo)速度(NCV)檢測治療1周，與假手術(shù)組比較，模型組和陽性對(duì)照組NCV明顯減慢(P<0．05)；與模型組比較，額日敦烏日勒組NCV明顯加快 (P<0．05) ；與陽性對(duì)照組比較，額日敦烏日勒組NCV明顯加快 (P<0．05)。治療4周，與假手術(shù)組比較，模型組和陽性對(duì)照組NCV明顯減慢 (P<0．05)；與模型組比較，額日敦烏日勒組NCV明顯加快 (P<0．05)；與陽性對(duì)照組比較，額日敦烏日勒組NCV也明顯加快(P<0．05)。見表2。

表2　各組NCV結(jié)果比較

注：與假手術(shù)組比較，##P<0．01；與模型組比較，△△P<0．01；與陽性對(duì)照組比較，▲P<0．05

2.3免疫組化檢測治療1周后，與假手術(shù)組比較，額日敦烏日勒組差異無統(tǒng)計(jì)學(xué)意義(P>0．05)，而其他2組灰度值明顯降低(P<0．05)；與模型組比較，額日敦烏日勒組和陽性對(duì)照組灰度值明顯增高(P<0．05)；與陽性對(duì)照組比較，額日敦烏日勒組灰度值也明顯增高(P<0．05)。治療4周后，各組間的比較與造模1周相同。見表3，圖1、圖2。

表3　各組坐骨神經(jīng)c-fos表達(dá)灰度值比較 ± s)

注：與假手術(shù)組比較，#P<0．05；與模型組比較，△P<0．05；與陽性對(duì)照組比較，▲P<0．05

(400倍，A：假手術(shù)組，B：模型組，C：陽性對(duì)照組，D：額日敦烏日組) 圖1　治療1周后c-fos免疫組化結(jié)果

(400倍，A：假手術(shù)組，B：模型組，C：陽性對(duì)照組，D：額日敦烏日組) 圖2　治療4周后c-fos免疫組化結(jié)果

3討論

周圍神經(jīng)通過神經(jīng)元將信息沿著感覺和運(yùn)動(dòng)纖維傳遞，參與軀體與內(nèi)臟的運(yùn)動(dòng)、感覺和植物功能調(diào)節(jié)[5]。體內(nèi)外環(huán)境發(fā)生劇烈變化時(shí)可使其受到損傷，神經(jīng)損傷段的近、遠(yuǎn)端出現(xiàn)一系列復(fù)雜的病理生理變化，其神經(jīng)元及微環(huán)境發(fā)生改變，從而發(fā)生運(yùn)動(dòng)和感覺功能的障礙[6]。c-fos作為在原癌基因家族中快速反應(yīng)基因，參與神經(jīng)纖維對(duì)傷害性刺激的反應(yīng)，啟動(dòng)c-fos的靶基因使NGFmRNA 轉(zhuǎn)錄增加，促進(jìn)NGF的合成，從而促進(jìn)損傷神經(jīng)的修復(fù)[1]。

本實(shí)驗(yàn)研究結(jié)果表明，治療1周后，額日敦烏日勒組、陽性對(duì)照組與模型組比較，SFI值高于模型組(P<0．05)；額日敦烏日勒組、陽性對(duì)照組與模型組比較，NCV值有所上升(P<0．05)；額日敦烏日勒組、陽性對(duì)照組與模型組比較，c-fos蛋白表達(dá)灰度值均明顯增高(P<0．05)。治療4周后，模型組、假手術(shù)組、陽性對(duì)照組、額日敦烏日勒組SFI值無顯著差異(P>0．01)；額日敦烏日勒組、陽性對(duì)照組與模型組比較，NCV值明顯提高(P<0．05)；額日敦烏日勒組、陽性對(duì)照組與模型組比較，c-fos蛋白表達(dá)灰度值均明顯增高(P<0．05)，表明額日敦烏日勒具有抑制c-fos蛋白表達(dá)的作用，從而促進(jìn)損傷神經(jīng)的修復(fù)，提高神經(jīng)傳導(dǎo)速度，促進(jìn)坐骨神經(jīng)損傷大鼠運(yùn)動(dòng)功能的恢復(fù)。

參考文獻(xiàn)：

[1] NARITA M, OZAKI S, NARITA M, et al Change in the expression of c-fos in the rat brain following sciatic nerve legation [ J] . Neu rosci Lett, 2003, 352: 231.

[2] 烏查日拉圖,喜杰.蒙藥珍寶丸對(duì)大鼠坐骨神經(jīng)擠壓損傷的修復(fù)作用研究[J].中國民族醫(yī)藥雜志,2011,4(4):48-50.

[3] BAIN J R, MACKINNON S E, HUNTER D A. Functional evaluation of complete sciatic, peroneal and posterior tibial nerve lesions in the rat[J]. Plast Reconstrsurg, 1989, 83(1): 129.

MN-DE LA GARZA.Celecoxib accelerates functional recovery after sciatic nerve crush in the rat[J].Journal of Brachial Plexus and Peripheral Nerve Injury ,2008,3:25.

[5] 張立新,佟曉杰,賈樺,等.超短波對(duì)大鼠周圍神經(jīng)缺損修復(fù)術(shù)后神經(jīng)再生的影響[J].中國康復(fù)醫(yī)學(xué)雜志,2009,24(8):695-698.

[6] TOM V J,SANDROW-FEINBERG H R,MILLER K,et al.Combining peripheral nerve grafts and chondroitinase promotes functional axonal regeneration in the chronically injured spinal cord[J].J Neurosis,2009,29(47):14881-14890.