解小麗,周青山,程周
(1.中山市人民醫(yī)院麻醉科,廣東中山528403;2.武漢大學(xué)人民醫(yī)院麻醉科,湖北武漢430060)
阿魏酸鈉對(duì)神經(jīng)病理性疼痛大鼠脊髓Bcl-2和Bax的影響
解小麗1,周青山2,程周1
(1.中山市人民醫(yī)院麻醉科,廣東中山528403;2.武漢大學(xué)人民醫(yī)院麻醉科,湖北武漢430060)
目的觀察阿魏酸鈉對(duì)神經(jīng)病理性疼痛大鼠脊髓中Bcl-2和Bax表達(dá)的影響,探討阿魏酸鈉對(duì)脊髓的保護(hù)作用。方法成年雄性Wistar大鼠隨機(jī)分為兩組:A組(n=30)為生理鹽水組,結(jié)扎左側(cè)坐骨神經(jīng)后每天腹腔注入2ml生理鹽水;B組(n=30)為阿魏酸鈉組,結(jié)扎坐骨神經(jīng)后向腹腔注入2ml阿魏酸鈉,100mg·kg-1· d-1,連續(xù)7 d。分別在術(shù)前及術(shù)后6 h、1 d、3 d、7 d進(jìn)行行為學(xué)測(cè)定,記錄縮足閾值,檢測(cè)脊髓中Bcl-2和Bax的表達(dá),采用TUNEL法觀察脊髓神經(jīng)元凋亡。結(jié)果兩組大鼠坐骨神經(jīng)結(jié)扎后均可誘發(fā)出神經(jīng)病理性疼痛;凋亡神經(jīng)元和Bcl-2和Bax陽性細(xì)胞均于術(shù)后6 h開始出現(xiàn),并于術(shù)后1 d開始迅速增加,術(shù)后3 d達(dá)峰值。與生理鹽水組相比較,阿魏酸鈉組大鼠脊髓組織中神經(jīng)細(xì)胞凋亡指數(shù)和Bax表達(dá)水平顯著降低,而Bcl-2的表達(dá)水平卻顯著增加(P<0.05)。結(jié)論阿魏酸鈉可能通過促進(jìn)Bcl-2表達(dá),抑制Bax表達(dá),從而抑制了大鼠脊髓神經(jīng)元凋亡,對(duì)脊髓有保護(hù)作用。
阿魏酸鈉;脊髓保護(hù);Bcl-2;Bax
阿魏酸鈉是阿魏酸的鈉鹽,有增強(qiáng)免疫、清除自由基、抗氧化,抗炎、解痙鎮(zhèn)痛等作用,能夠抑制細(xì)胞毒性物質(zhì)的表達(dá),還能改善受損內(nèi)皮細(xì)胞功能,改善炎癥及血管生成,已在神經(jīng)變性、心血管、糖尿病及腫瘤疾病方面得到應(yīng)用,臨床上經(jīng)常應(yīng)用于治療周圍神經(jīng)病變[1-2]。在本研究中,我們給神經(jīng)病理性疼痛大鼠腹腔注射阿魏酸鈉,觀察其對(duì)大鼠脊髓中Bcl-2和Bax表達(dá)的影響,現(xiàn)報(bào)道如下:
1.1 實(shí)驗(yàn)材料及分組阿魏酸鈉購于上海試劑一廠,Bcl-2和Bax購自武漢博士德生物工程有限公司,TUNEL試劑盒購于Roche公司,SP試劑盒購于北京中山生物技術(shù)有限公司,Von Frey纖維購自美國Stoelting公司。由武漢大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供健康成年雄性Wistar大鼠60只,隨機(jī)分為兩組:A組(n=30)為生理鹽水組,結(jié)扎左側(cè)坐骨神經(jīng)后每天腹腔注入2 ml生理鹽水,連續(xù)7 d;B組(n=30)為阿魏酸鈉組,結(jié)扎左側(cè)坐骨神經(jīng)后向腹腔注入阿魏酸鈉100 mg·kg-1·d-1(用2ml生理鹽水稀釋),連續(xù)7 d。
1.2 檢測(cè)指標(biāo)
1.2.1 機(jī)械性觸誘發(fā)痛的測(cè)定于術(shù)前及術(shù)后6 h、1 d、3 d、7 d固定時(shí)間段測(cè)定,使用Von Frey纖維刺激大鼠左后肢足底,觀察是否出現(xiàn)縮足反應(yīng)。大鼠出現(xiàn)快速的縮足反應(yīng),記為陽性反應(yīng),而身體活動(dòng)所引起的縮足反應(yīng)不記作陽性反應(yīng)。每隔5 s測(cè)一次,連測(cè)10次,誘發(fā)4~6次縮足的閾值作為50%反應(yīng)率。
1.2.2 Bcl-2和Bax的檢測(cè)實(shí)驗(yàn)動(dòng)物分別于術(shù)前及術(shù)后6 h、1 d、3 d、7 d麻醉后心臟插管灌流固定,然后取L4-6段脊髓組織固定、石蠟包埋后連續(xù)切片。取各時(shí)間點(diǎn)標(biāo)本做免疫組化染色檢測(cè)Bcl-2和Bax表達(dá),觀察Bcl-2和Bax在細(xì)胞內(nèi)表達(dá)及計(jì)算陽性率。
1.2.3 TUNEL法檢測(cè)脊髓神經(jīng)元凋亡取各時(shí)間點(diǎn)石蠟切片用TUNEL試劑盒檢測(cè)凋亡神經(jīng)元,具體操作方法按試劑盒說明書進(jìn)行,分析脊髓凋亡神經(jīng)元并計(jì)算凋亡指數(shù)(AI=凋亡細(xì)胞核數(shù)/總細(xì)胞核數(shù)×100%)。
2.1 坐骨神經(jīng)結(jié)扎后的痛覺變化如表1所示,A組及B組大鼠于術(shù)后6 h即出現(xiàn)機(jī)械性觸誘發(fā)痛,形成神經(jīng)病理性疼痛;A組大鼠坐骨神經(jīng)結(jié)扎后各時(shí)間點(diǎn)疼痛閾值持續(xù)降低,與術(shù)前比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05);而B組疼痛閾值在術(shù)后3 d時(shí)達(dá)到最低值,之后緩慢回升,至術(shù)后7 d與術(shù)前已無明顯差別。由此可以觀察出阿魏酸鈉能夠減輕一定程度的疼痛。
表1 各組大鼠左側(cè)坐骨神經(jīng)結(jié)扎后疼痛閾值的變化(g,)
表1 各組大鼠左側(cè)坐骨神經(jīng)結(jié)扎后疼痛閾值的變化(g,)
注:與術(shù)前相比較,*P<0.05;B組與A組相比較,▼P<0.05。
A組B組14.60±0.55 14.67±0.58 10.33±1.53*10.90±0.98*5.00±1.00*6.80±1.37*2.28±0.99*5.65±1.23*▼1.36±0.36*13.51±1.10▼
2.2 神經(jīng)元凋亡變化坐骨神經(jīng)結(jié)扎后,A組和B組大鼠脊髓組織中凋亡神經(jīng)元顯著增多,而且均于術(shù)后3 d達(dá)到高峰,之后逐漸降低,但是B組各個(gè)時(shí)間點(diǎn)陽性神經(jīng)細(xì)胞明顯少于A組,兩組間比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05),由此可見阿魏酸鈉可抑制坐骨神經(jīng)結(jié)扎后脊髓神經(jīng)元的凋亡,見表2。。
2.3 脊髓Bcl-2和Bax表達(dá)的變化見表3和表4。坐骨神經(jīng)結(jié)扎后,A組和B組大鼠脊髓組織中Bcl-2和Bax表達(dá)均增加,于術(shù)后3 d達(dá)到高峰,之后逐漸降低,但是B組Bax陽性神經(jīng)細(xì)胞明顯少于A組,兩組間比較差異有統(tǒng)計(jì)學(xué)意義(P<0.05),而B組Bcl-2陽性神經(jīng)細(xì)胞明顯多于A組,表明阿魏酸鈉抑制了坐骨神經(jīng)結(jié)扎后大鼠脊髓中Bax的表達(dá),而促進(jìn)了Bcl-2的表達(dá)。
表2 各組大鼠坐骨神經(jīng)結(jié)扎后脊髓神經(jīng)元凋亡指數(shù)(%,)
表2 各組大鼠坐骨神經(jīng)結(jié)扎后脊髓神經(jīng)元凋亡指數(shù)(%,)
注:與術(shù)前相比較,*P<0.05;B組與A組相比較,▼P<0.05。
A組B組8.24±1.69 8.56±2.53 16.70±0.75*10.51±1.62*▼30.54±4.44*20.05±3.68*▼75.12±3.91*35.18±3.26*▼12.78±2.59*8.60±2.17▼
表3 各組大鼠坐骨神經(jīng)結(jié)扎后脊髓Bcl-2的變化(%,)
表3 各組大鼠坐骨神經(jīng)結(jié)扎后脊髓Bcl-2的變化(%,)
注:與術(shù)前相比較,*P<0.05;B組與A組相比較,▼P<0.05。
A組B組28.90±3.51 27.12±5.25 27.63±6.11 38.58±8.63*▼36.35±5.11*50.27±8.46*▼50.81±8.63b*68.74±9.26*▼25.73±8.33 35.72±8.24*▼
表4 各組大鼠坐骨神經(jīng)結(jié)扎后脊髓Bax的變化(%,)
表4 各組大鼠坐骨神經(jīng)結(jié)扎后脊髓Bax的變化(%,)
注:與術(shù)前相比較,*P<0.05;B組與A組相比較,▼P<0.05。
A組B組14.64±6.52 15.25±6.27 26.53±7.42*15.98±4.67c▼35.75±9.63*20.23±6.53*▼40.52±7.89*22.74±5.47*▼17.96±6.61 14.28±5.36
在本研究中,我們觀察到結(jié)扎大鼠坐骨神經(jīng)后可造成神經(jīng)病理性疼痛,其脊髓中有一定數(shù)量的神經(jīng)元凋亡,而腹腔注射阿魏酸鈉可以減輕神經(jīng)病理性疼痛,減少大鼠脊髓神經(jīng)元的凋亡,并且可以促進(jìn)Bcl-2的表達(dá)和抑制Bax的表達(dá),對(duì)脊髓有保護(hù)作用。
阿魏酸鈉抗神經(jīng)細(xì)胞凋亡的機(jī)制比較復(fù)雜,有研究[3-5]表明,神經(jīng)細(xì)胞凋亡是受基因調(diào)控的,已知與神經(jīng)元凋亡相關(guān)的基因有:Bcl-2基因家族、p53基因家族、早期快速反應(yīng)基因家族(c-fos和c-jun)等,各種基因在細(xì)胞凋亡中的作用各不相同:如p53基因野生型對(duì)神經(jīng)細(xì)胞凋亡有促進(jìn)作用,c-fos可誘導(dǎo)其他基因(遲發(fā)性基因)表達(dá)增加并參與對(duì)神經(jīng)元凋亡的調(diào)控。Bcl-2家族可以分為兩類:一類是抗細(xì)胞凋亡基因,其代表是Bcl-2基因,可抑制脊髓損傷后各種途徑的凋亡[6],可使實(shí)驗(yàn)動(dòng)物過度表達(dá)Bcl-2而減輕神經(jīng)損傷,提高組織對(duì)抗損傷的功能,促進(jìn)軸索向外生長及損傷的中樞神經(jīng)系統(tǒng)組織修復(fù),并有效阻止神經(jīng)細(xì)胞凋亡[7-8];另一類是促細(xì)胞凋亡基因,其代表是Bax基因,它是Bcl-2的拮抗基因,通過編碼相應(yīng)功能蛋白而起作用[9],Bax存在于胞漿中,接受凋亡信息后能夠被激活,之后通過末端疏水結(jié)構(gòu)域錨定在線粒體膜上,使線粒體通透性發(fā)生改變致使細(xì)胞色素C、凋亡誘導(dǎo)因子和凋亡蛋白酶激活因子-1釋放,進(jìn)而激活caspase系統(tǒng),促使細(xì)胞發(fā)生凋亡[10]。有研究證明[11]Bcl-2與Bax的表達(dá)強(qiáng)度直接或間接影響脊髓神經(jīng)細(xì)胞凋亡,且凋亡程度與其比值相關(guān)。在本研究中我們觀察出,阿魏酸鈉促進(jìn)抗細(xì)胞凋亡基因Bcl-2的表達(dá),同時(shí)還抑制了促細(xì)胞凋亡基因Bax的表達(dá),從而抑制了脊髓神經(jīng)元的凋亡。
總之,阿魏酸鈉可抑制大鼠坐骨神經(jīng)結(jié)扎后脊髓神經(jīng)元的凋亡,減輕神經(jīng)病理性疼痛,對(duì)脊髓具有保護(hù)作用,其調(diào)控機(jī)制可能與Bcl-2基因家族有關(guān),其具體機(jī)制有待進(jìn)一步研究。
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Influence of sodium ferulate on the expression of Bcl-2 and Bax in the spinal cord neurons in rats with neuropathic pain.
XIE Xiao-li1,ZHOU Qing-shan2,CHEN Zhou1.1.Department of Anesthesiology,People's Hospitalof Zhongshan City,Zhongshan 528403,Guangdong,CHINA;2.Department of Anesthesiology,Wuhan University Renmin Hospital,Wuhan 430060,Hubei,CHINA
ObjectiveTo observe the influence of sodium ferulate(SF)on the expression of Bcl-2 and Bax in spinal cord neurons in rats with neuropathic pain,and to study the protective effects of SF on the spinal cord neurons in rats.MethodsSixty healthy adult male wistar rats were simply randomized into two groups:group A(n=30) and group B(n=30).The left sciatic nerves of the rats were ligated under chloral hydrate anesthesia,then the rats were given 2 ml normal saline(group A)and 100 mg·kg-1·d-1SF(group B),which was dissolved in 2 ml normal saline respectively after operation.All the patients were tested for the time of paw withdraw by using the von Frey test before surgery,6 hours,1 day,3 days,and 7 days after surgery.Apoptosis of spinal cord neurons was detected with the TUNEL method.Changes of Bcl-2 and Bax expression in the spinal cord were detected through the immunohistochemical methods and image analysis.ResultsMechanical allodynia developed at 6 hours after sciatic nerve ligation.The expression of neuronal apoptosis,Bcl-2 and Bax were first detected in spinal cord at 6 hours after injury and thereafter,reaching the peak at 3 days.The spinal neuronal apoptotic index and the expression of Bax in group B was significantly lower than that in group A.However,the expression of Bcl-2 in group B was significantly higher than that in group A(P<0.05).ConclusionThrough promoting the expression of Bcl-2 and inhibiting the expression of Bax,sodium ferulate can effectively inhibit spinal neuronal apoptosis and protect spinal cord neurons in rats,
Sodium ferulate;Spinal cord protection;Bcl-2;Bax
R-332
A
1003—6350(2012)08—016—03
10.3969/j.issn.1003-6350.2012.08.007
2011-09-06)
解小麗(1979—),女,湖北省棗陽市人,主治醫(yī)師,碩士,主要從事臨床疼痛診療工作。