鞘內注射甘珀酸劑量依賴地緩解腰5脊神經切斷大鼠的痛覺高敏

李雪飛,許　倩,王　芬,鄭　曼,劉清珍,李偉彥

(1. 南京中醫藥大學附屬醫院麻醉科，江蘇 南京　210029；2. 南京軍區總醫院麻醉科，江蘇 南京　210002)

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鞘內注射甘珀酸劑量依賴地緩解腰5脊神經切斷大鼠的痛覺高敏

李雪飛1,許倩1,王芬1,鄭曼1,劉清珍2,李偉彥2

(1. 南京中醫藥大學附屬醫院麻醉科，江蘇 南京210029；2. 南京軍區總醫院麻醉科，江蘇 南京210002)

摘要:目的觀察鞘內注射甘珀酸(carbenoxolone, CBX)對腰5脊神經切斷(L5 spinal nerve transaction ，SNT)大鼠的鎮痛作用及其相關機制。方法60只♂ SD大鼠，隨機分成5組(n=12)：Ⅰ.假手術組，Ⅱ.模型組，Ⅲ.SNT+CBX(0.05 μg)，Ⅳ.SNT+CBX(0.5 μg)，Ⅴ.SNT+CBX(5 μg)。Ⅰ組僅暴露腰5脊神經，Ⅱ至Ⅴ組行SNT。術后10 d，Ⅰ和Ⅱ組鞘內注射生理鹽水10 μL，Ⅲ至Ⅴ組注射CBX 0.05 μg(10 μL)、0.5 μg(10 μL)和5 μg(10 μL)，分別于術前1 d，術后1、3、5、7、10 d各組注射生理鹽水和CBX，前1 h和給藥后1、2、4、6 h測雙側機械痛閾值(mechanical withdrawl thresholds MWT)，免疫組化及ELISA法觀察給藥后2h后脊髓背角膠質纖維酸性蛋白(glial fibrillary acidic protein, GFAP)和TNF-α、IL-1β表達的變化。結果與術前相比，Ⅱ至Ⅴ組雙側MWT明顯降低；與給藥前1 h相比，Ⅱ和Ⅲ組給藥后1、2、4、6 h雙側MWT均無明顯差異；Ⅳ組損傷側給藥后1、2、4 h MWT無明顯差異，而對側MWT明顯提高，同時該側GFAP和TNF-α、IL-1β的表達也降低；Ⅴ組給藥后1、2、4 h雙側MWT明顯提高，雙側GFAP和TNF-α、IL-1β的表達也明顯降低。結論鞘內注射CBX可緩解SNT大鼠雙側MWT，其機制可能與其抑制脊髓背角星形膠質細胞的活化及TNF-α和IL-1β釋放減少有關。

關鍵詞:甘珀酸；神經病理性疼痛；痛覺高敏；脊髓背角；星形膠質細胞； TNF-α；IL-1β

越來越多的證據表明，單側神經的損傷會導致雙側神經的細胞和分子結構的變化，并導致雙側痛覺高敏，把這種現象稱為鏡像痛(mirror image-pain, MIP)，它是神經病理性痛(neuropathic pain, NP)中的一種特殊現象[1]。

到目前為止，MIP的發生機制尚不清楚，隨著對膠質細胞研究的不斷深入，膠質細胞在NP中的作用越來越受到關注，神經的損傷或炎癥可導致膠質細胞的活化及增生，這些膠質細胞會釋放一些物質，反過來調控傷害性神經元的興奮性[2]。研究發現，星形膠質細胞在MIP的發生中可能扮演著更重要角色。星形膠質細胞之間信號傳遞的一個重要機制是縫隙連接(gap junction, GJ)，損傷側的傷害性信號可以通過GJ以鈣波的形式傳遞到對側脊髓，從而促進疼痛的傳導[3-4]。

甘珀酸(carbenoxolone, CBX)是一種GJ阻滯劑，使用CBX阻滯GJ可以抑制多種持續性疼痛動物模型中的痛覺高敏。

本實驗通過鞘內注射不同劑量的CBX來觀察對腰5脊神經切斷(SNT)大鼠的雙側機械痛閾(mechanical withdraw threshold, MWT)的影響，以及雙側脊髓背角膠質纖維酸性蛋白(glial fibrillary acidic protein, GFAP)和TNF-α、IL-1β表達的變化，來探討星形膠質細胞及炎性介質參與大鼠MIP的可能機制。

1材料與方法

1.1動物模型

體質量160～180 g的成年♂ Sprague-Dawley大鼠60只(由南京軍區總醫院動物實驗中心提供，清潔級)，單籠飼養。動物飼養室溫為(23±3)℃，周期光照8 ∶00～20 ∶00，大鼠自由進食，飲水。所有實驗均在光照期間完成。以2%的戊巴比妥(Sigma公司，美國)50 mg·kg-1腹腔注射麻醉，根據Kim[5]方法創建模型。

1.2藥物及分組

甘珀酸(Sigma,美國)；兔抗大鼠和膠原纖維酸性蛋白(anti-glial fibrillary acidic protein, GFAP)抗體(Sigma, 美國)；ELISA試劑盒。隨機分成5組(n=12)，Ⅰ. 假手術組 (Sham+NS)，Ⅱ.模型組(SNT+NS)，Ⅲ. SNT+CBX(0.05 μg)，Ⅳ. SNT+CBX(0.5 μg)，Ⅴ. SNT+CBX(5 μg)。

1.3鞘內注射

參照Mestre等[6]所建立的方法。大鼠麻醉后用連有PE-10導管的稍鈍針頭經腰5(L5)和腰4(L4)椎間隙行椎管穿刺，以動物出現突然地側向甩尾運動，作為穿刺成功的標志，鞘內注射采用微量進樣器進行。術后10 d，Ⅰ組和Ⅱ組鞘內注射生理鹽水10μL，Ⅲ至Ⅴ組鞘內注射CBX 0.05 μg(10 μL)、0.5 μg(10 μL)和5 μg(10 μL)，CBX濃度的選擇參考既往文獻。

1.4機械痛閾的測定

5組大鼠，每組隨機取6只固定進行行為學測定，分別于術前1 d，術后1 、3、5、7、10 d及給藥后1、2、4、6 h測定雙側MWT。MWT測定：將大鼠分別放置于金屬篩網上的有機玻璃箱里，安靜15 min，以Von Frey 纖維垂直刺其后肢足底中部皮膚，持續≤4 s，大鼠出現抬足、舔足、躲避等反應時，讀Electrovon Frey 讀數器上顯示的最大值(g)，每只大鼠重復測量5次，間隔5 min，去除最大和最小值，計算3 次的平均值即為大鼠的MWT值。

1.5免疫組織化學染色

5組大鼠術后10 d鞘內注射CBX 2 h后，隨機取3只按以下方法取材檢測。2%的戊巴比妥鈉50 mg·kg-1經腹腔注射麻醉，開胸后經左心室插管至升主動脈，依次灌注生理鹽水250 mL、4%多聚甲醛(pH 7.4)0.1 mL·L-1、PBS緩沖液250 mL，約1 h后取大鼠脊髓腰膨大，于上述固定液中固定24 h。經梯度酒精脫水，二甲苯透明石蠟包埋，進行連續切片，片厚40 μm，每個蠟塊連續切5片。以ABC法作GFAP免疫組化染色。染片用半自動圖像分析儀進行圖像分析，計算各切片GFAP陽性細胞數表示星形膠質細胞表達的強度。胞質出現棕黃色顆粒沉積為GFAP免疫組化陽性反應細胞。

1.6TNF-α和IL-1β表達檢測

ELISA測定雙側脊髓背角TNF-α和IL-1β濃度。按照試劑盒說明書操作,并在波長為450 nm,參考波長為 620 nm處,用酶標儀(美國 Bio-Rad公司)測定光密度,根據標準曲線算出TNF-α和IL-1β濃度。

1.7統計分析

2結果

2.1MWT測定結果

與Ⅰ組相比，Ⅱ至Ⅴ組損傷側術后1、3、5、7、10 d MWT均明顯下降(P<0.05)，術后5 d降至最低，持續到10 d，而對側術后7 d降至最低，持續到10 d；與術后10 d給藥前1 h相比，Ⅱ和Ⅲ組給藥后1、2、4、6 h雙側MWT均無明顯差異(P>0.05)；Ⅳ組給藥后1、2、4、6 h損傷側MWT無明顯差異(P>0.05)，而對側給藥后1 h MWT即開始提高(P<0.05)，2 h時達高峰(P<0.05)，6 h時與給藥前無明顯差異(P>0.05)；Ⅴ組給藥后1、2、4 h雙側MWT均明顯提高(P<0.05)，2 h時達高峰(P<0.05)，6 h時與給藥前無明顯差異(P>0.05)。見Fig 1。

Fig 1　The bilateral MWT of rats in five groups at each time point

compared with group Ⅰ, the bilateral MWT in group Ⅱ～Ⅴ was significantly decreased.vsthe MWT 1 h before intrathecal administration, the values at 1,2,4,6 h after administration of group Ⅱ and Ⅲ showed no marked difference. The ipsilateral MWT in group Ⅳ showed no significant difference at 1,2,4,6 h after administration, while the contralateral MWT was significantly increased at 1,2,4 h. In group Ⅴ the bilateral MWT was significantly improved at 1,2,4 h after administration.*P<0.05vsgroup Ⅰ;#P<0.05vsMWT 1h before intrathecal administration.

2.2GFAP測定結果

與Ⅰ組相比，Ⅱ和Ⅲ組雙側脊髓背角GFAP的染色明顯增強，Ⅳ組損傷側脊髓背角GFAP的染色明顯增強；與Ⅱ組相比，Ⅳ組對側脊髓背角GFAP的染色明顯減弱，損傷側無明顯變化，Ⅴ組雙側脊髓背角GFAP的染色均明顯減弱。GFAP陽性細胞數的比較，與Ⅰ組相比，Ⅱ和Ⅲ組雙側脊髓背角GFAP的陽性細胞數均明顯增多(P<0.05)；且Ⅱ、Ⅲ及Ⅳ組損傷側增加最明顯；與Ⅱ組相比，Ⅲ組及Ⅳ組損傷側脊髓背角GFAP陽性細胞數無明顯變化(P>0.05)，而Ⅳ組對側脊髓背角GFAP的陽性細胞數明顯減少(P<0.05)，Ⅴ組雙側脊髓背角GFAP陽性細胞數均明顯降低(P<0.05)。見Fig 2，3。

Fig 2　GFAP expression in bilateral dorsal horns of the spinal cord in rats

compared with group Ⅰ(A), the bilateral expressions of GFAP in group Ⅱ(B) and Ⅲ(C) were significantly enhanced. In group Ⅳ(D), the ipsilateral expressions of GFAP were significantly enhanced; compared with group Ⅱ (B), the bilateral expressions of GFAP in group Ⅴ(E) were significantly decreased. In group Ⅳ (D), the contralateral expression was abviously reduced.

2.3脊髓背角TNF-α和IL-1β的表達

與Ⅰ組相比，Ⅱ～Ⅴ組雙側脊髓背角TNF-α和IL-1β的水平均明顯提高(P<0.05)；與Ⅱ組相比，Ⅲ和Ⅳ組損傷側脊髓背角TNF-α和IL-1β的水平無明顯變化(P>0.05)，對側脊髓背角TNF-α和IL-1β的水平明顯降低(P<0.05)，且Ⅳ組降低的更明顯；Ⅴ組雙側脊髓背角TNF-α和IL-1β的水平均明顯降低(P<0.05)。見Tab 1。

Fig 3　Number of GFAP-positive cells in bilateral spinal dosal horns of spinal cord in five±s)

compared with group Ⅰ，the number of GFAP-positive cells in the bilateral dosal horns of the spinal cord was significantly increased in group Ⅱ～Ⅴ; compared with group Ⅱ, the contralateral spinal dorsal horn of GFAP positive cells was significantly reduced in group Ⅳ, the bilateral spinal cord dorsal horn of GFAP-positive cells was significantly decreased in group Ⅴ.*P<0.05vsgroup Ⅰ;#P<0.05vsgroup Ⅱ.

3結論

在臨床和動物模型中都發現有MIP的現象，臨床上如復雜區域疼痛綜合癥(complex regional pain syndrome, CRPS)、類風濕關節炎、纖維肌痛和NP；動物模型如神經損傷性疼痛、炎癥性疼痛及癌性疼痛[7-8]。MIP的典型特點是鏡像側的機械痛覺高敏，所以本研究主要觀察SNT大鼠的MWT值，研究發現，大鼠單側神經損傷后，損傷側術后1 d即出現明顯的MWT值升高，對側術后5 d表現出明顯的MWT值升高。目前對MIP的機制仍然不清楚，被廣泛接受的有三大主要假說：體液學說、神經學說及膠質細胞學說，近年來，中樞神經系統膠質細胞的激活，通過GJ、鈣波及促炎性因子的釋放3個途徑在MIP的發生機制中受到越來越多的關注。GJ是相鄰細胞膜之間的連接通道，允許離子和小分子如cAMP、IP3、ATP和小分子肽類在細胞間自由通過[9-11]。 在中樞神經系統內，GJ廣泛分布于星形膠質細胞之間，星形膠質細胞通過GJ廣泛偶聯形成一種星形膠質細胞網絡。有學者發現星形膠質細胞可以對因各種刺激而激活的神經元細胞做出快速電反應，表現為沿GJ傳播的Ca2+波[12]，傷害性刺激信號可能通過鈣波的形式在星形膠質細胞GJ網絡中傳遞，引起疼痛的擴散和傳播，進而導致遠處的神經膠質細胞和神經元的活化，導致域外和鏡像效應的產生。本實驗發現，Ⅱ組大鼠術后10 d脊髓背角雙側GFAP的表達明顯增加，而鞘內注射大劑量CBX后，脊髓背角雙側GFAP的表達明顯下降，提示GJ可能參與GFAP的激活。除此之外，促炎性因子在不同的NP模型及誘發或促進NP中的重要作用，在許多實驗研究中都已經被證實[13-14]，之前我們的研究也發現，SNT大鼠術后損傷側脊髓背角TNF-α、IL-1β均明顯增高，鞘內注射5 μg的CBX后MWT值明顯提高，同時TNF-α、IL-1β的表達也降低[15]。然而，大多數的研究都主要集中在神經損傷側的神經炎癥反應，神經損傷對側的炎癥反應的研究卻比較少，所以本實驗就觀察了大鼠雙側的神經炎癥反應情況，結果發現，SNT大鼠術后10 d雙側MWT明顯降低，同時雙側脊髓背角TNF-α和IL-1β的表達也明顯升高，鞘內給予低劑量、中劑量及大劑量的CBX后，對側脊髓背角TNF-α和IL-1β的表達都降低，且隨著劑量的增加，炎性因子降低的越明顯，因此我們推測神經損傷引起損傷側星形膠質細胞的活化及增生，傷害性刺激信號通過GJ以鈣波的形式傳遞到對側，激活對側的星形膠質細胞，引起對側促炎性細胞因子的釋放，這些細胞因子可以作為第二信使，進一步作用于激活的神經元和膠質細胞，導致持續的痛覺過敏和異常性疼痛。

Tab 1　Expression of TNF-α and IL-1β in bilateral dorsal horns of spinal cord in five±s,pg·mg-1)

vsgroup Ⅰ, the expression of TNF-α and IL-1β at 2 h after administration in group Ⅱ,Ⅲ,Ⅳ and Ⅴ was significantly increased;vsgroup Ⅱ, the expression of TNF-α and IL-1β on the contralateral side in group Ⅲ and Ⅳ was significantly reduced, while the ipsilateral side in groupⅣ had no significant changes. The bilateral expressions of GFAP, TNF-α and IL-1β in group Ⅴ were significantly decreased.*P<0.05vsgroup Ⅰ;#P<0.05vsgroup Ⅱ.

本實驗發現鞘內注射CBX可逆性地緩解大鼠雙側MWT，結合星形膠質細胞細胞和促炎性細胞因子在NP中的作用，因此，我們推測鞘內注射CBX后抑制了細胞之間的GJ，從而阻斷傷害性信號向對側脊髓背角的傳遞，抑制對側膠質細胞的激活及TNF-α和IL-1β等炎性細胞因子的釋放來逆轉痛覺高敏。

(致謝：本實驗在南京軍區南京總醫院比較醫學科麻醉科實驗室完成，在此衷心感謝我的導師李偉彥教授的耐心培養，實驗室劉清珍老師的細心指導，許倩等各位同學的合作和幫助！)

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Effect of intrathecal injection of carbenoxolone on hyperalgesia in rats with L5 spinal nerve transaction

LI Xue-fei1,XU Qian1, WANG Fen1, ZHENG Man1, LIU Qing-zhen2,LI Wei-yan2

(1.DeptofAnesthesiology,AffiliatedHospitalofNanjingUniversityofTraditionalChineseMedicine,Nanjing210029,China;2.DeptofAnesthesiology,NanjingGeneralHospitalofNanjingMilitaryCommand,Nanjing210002,China)

Abstract:AimTo investigate the antagonistic effect of intrathecal injection of carbenoxolone(CBX) on neuropathic pain and its underlying mechanism.MethodsSixty male Sprague-Dawley rats were randomly divided into five groups(n=12): group I received sham surgery then treated with saline; group Ⅱ received SNT then treated with saline; group Ⅲ received SNT then treated with 0.05 μg CBX; group Ⅳ received SNT then treated with 0.5 μg CBX; group Ⅴ received SNT then treated with 5 μg CBX. Treatment was undertaken with 10 μl volume as a single intrathecal injection on postoperative day 10. Mechanical withdrawl thresholds were measured 1d before operation, 1, 3, 5, 7 and 10 d after surgery, 1 h before intrathecal administration, and 1, 2, 4, 6 h after intrathecal administration. Lumbar spinal cord was obtained 2 h after intrathecal administration to determine the expressions of GFAP by immunohistology and TNF-α,IL-1β by ELISA in bilateral spinal dorsal horns. ResultsCompared with the sham group, the bilateral MWT in group Ⅱ～Ⅴ was significantly decreased. Compared with the MWT 1 h before intrathecal administration on day 10, the values at 1, 2, 4, 6 h after administration of group Ⅱ and Ⅲ had no marked difference. The ipsilateral MWT in group Ⅳ had no significant difference at 1, 2, 4 h after administration, the contralateral MWT was significantly increased, whereas GFAP and TNF-α,IL-1β was significantly decreased in the spinal cord . In group Ⅴ the bilateral MWT was significantly improved at 1, 2, 4 h after administration, whereas GFAP and TNF-α,IL-1β were significantly decreased in the spinal cord.ConclusionsIntrathecal CBX can inhibit the development of bilateral MWT. The analgesic effect of CBX is implemented partly via suppressing the actation of GFAP and the realease of TNF-α,IL-1β in the spinal doral horn.

Key words:carbenoxolone；neuropathic pain；hyperalgesia；spinal cord dorsal horn；astrocyte；TNF-α；IL-1β

收稿日期:2016-02-01,修回日期:2016-03-25

基金項目：國家自然科學基金資助項目(No 30901399)

作者簡介：李雪飛(1984-)，女，碩士，住院醫師，研究方向：星形膠質細胞與神經病理性疼痛，E-mail:xuefei9704@126.com;

doi:10.3969/j.issn.1001-1978.2016.06.024

文獻標志碼：A

文章編號：1001-1978(2016)06-0863-05

中國圖書分類號：R-332；R322.85；R392.12；R441.1；R741.041

網絡出版時間：2016-5-25 15:39網絡出版地址：http://www.cnki.net/kcms/detail/34.1086.R.20160525.1539.048.html

李偉彥(1965-)，男，博士，碩士生導師，研究方向：神經病理性疼痛，E-mail:weiyanlee@yahoo.com.cn