鹽酸小檗堿對(duì)顱腦創(chuàng)傷模型小鼠雙側(cè)丘腦繼發(fā)性損傷的神經(jīng)保護(hù)作用

黃樹宣 朱飛奇 裴中鄧 旭輝楊 志朱瑾 陳淳淳 林偉豐

·基礎(chǔ)研究·

鹽酸小檗堿對(duì)顱腦創(chuàng)傷模型小鼠雙側(cè)丘腦繼發(fā)性損傷的神經(jīng)保護(hù)作用

黃樹宣 朱飛奇 裴中鄧 旭輝楊 志朱瑾 陳淳淳 林偉豐

目的探討鹽酸小檗堿對(duì)顱腦創(chuàng)傷（TBI）模型小鼠雙側(cè)丘腦繼發(fā)性損傷（炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失）的神經(jīng)保護(hù)作用。方法采用自由落體撞擊法制備顱腦創(chuàng)傷模型，鹽酸小檗堿組小鼠予以鹽酸小檗堿50 mg/（kg·d）灌胃21 d，TBI組予等量生理鹽水灌胃21 d，對(duì)照組不予自由落體撞擊。免疫組織化學(xué)染色計(jì)數(shù)雙側(cè)丘腦誘導(dǎo)型一氧化氮合酶（iNOS）、環(huán)氧合酶?2（COX?2）、8?羥基脫氧鳥苷（8?OHdG）和神經(jīng)元核抗原（Neu N）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目，免疫熒光染色計(jì)數(shù)雙側(cè)丘腦膠質(zhì)纖維酸性蛋白（GFAP）陽性星形膠質(zhì)細(xì)胞和離子鈣結(jié)合蛋白1（Iba1）陽性小膠質(zhì)細(xì)胞數(shù)目。結(jié)果3組小鼠顱腦創(chuàng)傷同側(cè)丘腦iNOS（P=0.015）、COX?2（P=0.022）、8?OHdG（P=0.000）和Neu N（P=0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目以及GFAP陽性星形膠質(zhì)細(xì)胞數(shù)目（P=0.024）和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目（P= 0.000）差異均有統(tǒng)計(jì)學(xué)意義，其中，TBI組iNOS（P=0.005）、COX?2（P=0.011）和8?OHdG（P=0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目以及GFAP陽性星形膠質(zhì)細(xì)胞數(shù)目（P=0.011）和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目（P= 0.000）均高于對(duì)照組，而Neu N陽性神經(jīng)元數(shù)目低于對(duì)照組（P=0.000）；鹽酸小檗堿組iNOS（P=0.031）、COX?2（P=0.024）和8?OHd G（P=0.008）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目以及GFAP陽性星形膠質(zhì)細(xì)胞數(shù)目（P=0.031）和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目（P=0.012）均低于TBI組，僅8?OHdG陽性神經(jīng)元數(shù)目（P= 0.014）和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目（P=0.024）仍高于對(duì)照組，而Neu N陽性神經(jīng)元數(shù)目高于TBI組（P= 0.016）、仍低于對(duì)照組（P=0.027）。3組小鼠顱腦創(chuàng)傷對(duì)側(cè)丘腦僅COX?2（P=0.029）和8?OHd G（P= 0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目差異有統(tǒng)計(jì)學(xué)意義，其中，TBI組COX?2（P=0.011）和8?OHd G（P= 0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目高于對(duì)照組，鹽酸小檗堿組COX?2（P=0.047）和8?OHd G（P=0.010）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目低于TBI組，僅8?OHd G陽性神經(jīng)元數(shù)目仍高于對(duì)照組（P=0.004）。結(jié)論顱腦創(chuàng)傷可以引起雙側(cè)丘腦繼發(fā)性損傷，尤以同側(cè)丘腦顯著，對(duì)側(cè)丘腦僅出現(xiàn)炎癥反應(yīng)和氧化損傷；鹽酸小檗堿通過抑制顱腦創(chuàng)傷后雙側(cè)丘腦炎癥反應(yīng)和氧化損傷而發(fā)揮神經(jīng)保護(hù)作用。

小檗堿；顱腦損傷；丘腦；炎癥；氧化性應(yīng)激；神經(jīng)元；疾病模型，動(dòng)物

【Abstract】ObjectiveTo investigate the protective effect of berberine chloride on secondary damage(inflammation,oxidative damage and neuron loss)in bilateral thalami of traumatic brain injury(TBI) model mice.MethodsMice were randomly divided into 3 groups:control group(N=6),TBI group(N=6) and berberine group(N=6).TBI model was established by a free?falling hitting device.In control group, mice were not given free?falling hitting.Mice in berberine group were given a gavage of berberine chloride [50 mg/（kg·d)]for 21 d,while mice in TBI group were given the same dosage of normal saline for 21 d. Immunohistochemistry was used to count the number of neurons or gliocytes positive for inducible nitric oxide synthase(iNOS),cyclooxygenase?2(COX?2),8?hydroxy deoxyguanosine(8?OHdG)and neuronal nuclei (NeuN),the number of astrocytes positive for glial fibrillary acidic protein(GFAP)and the number of microglias positive for ionized calcium?binding adaptor molecule 1(Iba1).ResultsThe number of neurons or gliocytes positive for iNOS(P=0.015),COX?2(P=0.022),8?OHdG(P=0.000)and Neu N(P= 0.000),the number of astrocytes positive for GFAP(P=0.024)and microglias positive for Iba1(P=0.000) in TBI ipsilateral thalamus were significantly different among 3 groups.In TBI group,the number of neurons or gliocytes positive for iNOS(P=0.005),COX?2(P=0.011)and 8?OHd G(P=0.000),the number of astrocytes positive for GFAP(P=0.011)and microglias positive for Iba1(P=0.000)were significantly higher than those in control group,while the number of neurons positive for Neu N(P=0.000)was significantly lower than that in control group.In berberine group,the number of neurons or gliocytes positive for iNOS(P=0.031),COX?2(P=0.024)and 8?OHdG(P=0.008),the number of astrocytes positive for GFAP(P=0.031)and microglias positive for Iba1(P=0.012)were significantly lower than those in TBI group,while the number of neurons positive for 8?OHd G(P=0.014)and microglias positive for Iba1(P= 0.024)were significantly higher than those in control group.The number of neurons positive for Neu N in berberine group was significantly higher than that in TBI group(P=0.016),while lower than that in control group(P=0.027).Additionally,number of neurons or gliocytes positive for COX?2(P=0.029)and 8?OHdG (P=0.000)in TBI contralateral thalamus were significantly different among 3 groups.The number of neurons or gliocytes positive for COX?2(P=0.011)and 8?OHd G(P=0.000)in TBI group was significantly higher than that in control group,while the number of neurons or gliocytes positive for COX?2(P=0.047) and 8?OHd G(P=0.010)in berberine group was significantly lower than that in TBI group.The number of neurons positive for 8?OHd G in berberine group was significantly higher than that in control group(P= 0.004).ConclusionsTBI could cause secondary damage of bilateral thalami,especially in ipsilateral thalamus,but only cause inflammation and oxidative damage in contralateral thalamus.Berberine chloride might exert neuroprotective effect on bilateral thalami after TBI by significantly suppressing inflammation and oxidative damage.

This study was supported by Natural Science Foundation of Guangdong Province,China(No. S2013010015786)and Science and Technology Plan Project of Shaoguan,Guangdong Province,China(No. 2010-01).

顱腦創(chuàng)傷（TBI）導(dǎo)致的繼發(fā)性腦損傷不僅可以發(fā)生在原發(fā)部位，還可以發(fā)生在遠(yuǎn)隔海馬、基底節(jié)和丘腦等其他部位［1?2］，是顱腦創(chuàng)傷高病殘率和高病死率的重要原因，也是影響患者預(yù)后的關(guān)鍵。丘腦是大腦重要而復(fù)雜的中繼站，與大腦皮質(zhì)存在廣泛纖維聯(lián)系，不僅與感覺傳導(dǎo)有關(guān)，而且與認(rèn)知和行為密切相關(guān)，丘腦損傷可以導(dǎo)致感覺障礙、視覺障礙、睡眠障礙和認(rèn)知功能障礙［3］。近年研究顯示，小檗堿（又稱黃連素）具有抗炎癥反應(yīng)、抗氧化應(yīng)激、降低膽固醇等多種藥理學(xué)作用，在中樞神經(jīng)系統(tǒng)疾病中發(fā)揮重要神經(jīng)保護(hù)作用［4?5］。本研究制備顱腦創(chuàng)傷小鼠模型，觀察顱腦創(chuàng)傷后小鼠雙側(cè)丘腦炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失情況，并予以鹽酸小檗堿治療，探討該藥對(duì)顱腦創(chuàng)傷模型小鼠雙側(cè)丘腦繼發(fā)性損傷（炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失）的影響，以為臨床能夠更有效治療顱腦創(chuàng)傷提供理論依據(jù)。

材料與方法

一、實(shí)驗(yàn)材料

1.實(shí)驗(yàn)動(dòng)物無特定病原體（SPF）雌性C57BL/ 6小鼠共18只，月齡6個(gè)月，體重25～31 g、平均（27.67±1.88）g，由中山大學(xué)實(shí)驗(yàn)動(dòng)物中心提供［許可證號(hào)：SYXK（粵）2010-0107］，于室溫（22±2）℃、相對(duì)濕度（50±5）%、12 h晝-12 h夜循環(huán)照明環(huán)境中飼養(yǎng)，自由攝食、飲水。

2.試劑與儀器（1）主要試劑：鹽酸小檗堿（規(guī)格：500 mg）由美國Sigma?Aldrich公司提供。Ⅰ抗工作液中兔抗小鼠誘導(dǎo)型一氧化氮合酶（iNOS）多克隆抗體（1∶200）為美國Proteintech公司產(chǎn)品，兔抗小鼠環(huán)氧合酶?2（COX?2）多克隆抗體（1∶200）由美國Bioworld公司提供，山羊抗小鼠8?羥基脫氧鳥苷（8?OHd G）多克隆抗體（1∶200）為美國Calbiochem公司產(chǎn)品，兔抗小鼠神經(jīng)元核抗原（NeuN）單克隆抗體（1∶200）購自英國Abcam公司，兔抗小鼠膠質(zhì)纖維酸性蛋白（GFAP）單克隆抗體（1∶500）為英國Abcam公司產(chǎn)品，兔抗小鼠離子鈣結(jié)合蛋白1（Iba1）單克隆抗體（1∶500）由日本和光純藥工業(yè)株式會(huì)社提供。辣根過氧化物酶（HRP）標(biāo)記的驢抗山羊IgGⅡ抗（1∶500）購自美國Life公司，辣根過氧化物酶標(biāo)記的驢抗兔IgGⅡ抗（1∶500）由丹麥Dako公司提供，Alexa Fluor 555標(biāo)記的山羊抗兔熒光IgGⅡ抗（1∶500）購自美國Life公司。（2）主要儀器：顱腦創(chuàng)傷撞擊器為北京拜安吉科技有限公司產(chǎn)品，腦立體定位儀由深圳市瑞沃德生命科技有限公司提供，小鼠顱骨鉆為韓國Saeshin公司產(chǎn)品，光學(xué)顯微鏡購自日本Nikon公司，熒光顯微鏡購自日本Olympus公司，CM1950型冰凍切片機(jī)由德國Leica公司提供。

二、研究方法

1.動(dòng)物模型制備與分組（1）模型制備：采用自由落體撞擊法制備顱腦創(chuàng)傷小鼠模型，在電熱毯上以體積分?jǐn)?shù)為4.2%水合氯醛（0.10 ml/10 g）腹腔注射全身麻醉，顱骨正中切開一長約1.50 cm頭皮切口，矢狀縫旁左側(cè)、冠狀縫和人字縫之間開一直徑約5 mm骨窗，以20 g砝碼自高度為20 cm處垂直自由下落，以預(yù)先消毒的撞擊針頭撞擊骨窗。撞擊后以骨蠟封閉骨窗，消毒后縫合頭皮，將小鼠置于電熱爐旁觀察1 h，待蘇醒后放回籠中飼養(yǎng)。（2）動(dòng)物分組：采用隨機(jī)數(shù)字表法隨機(jī)分為對(duì)照組、顱腦創(chuàng)傷模型組（TBI組）和鹽酸小檗堿治療組（鹽酸小檗堿組），每組各6只小鼠。鹽酸小檗堿組于模型制備后予鹽酸小檗堿50 mg/（kg·d）溶于生理鹽水中灌胃（1次/d），連續(xù)21 d；TBI組于模型制備后予等量生理鹽水灌胃（1次/d），連續(xù)21 d；對(duì)照組僅開骨窗，不予自由落體撞擊。

2.腦組織灌注和冰凍切片制備模型制備后21 d，小鼠腹腔注射4.20%水合氯醛（0.10 ml/10 g）全身麻醉，仰臥位固定，剪開胸腔，顯露心臟，直視下將針頭插入左心室，剪開右心耳，先以4℃生理鹽水50 ml快速心臟灌注，再以4℃質(zhì)量分?jǐn)?shù)為4%多聚甲醛溶液50～100 ml緩慢灌注。斷頭切取腦組織，置重新配置的4%多聚甲醛溶液中12 h，再依次置于4℃、體積分?jǐn)?shù)為20%和30%的蔗糖溶液中脫水6 h至腦組織標(biāo)本沉淀。取出腦組織標(biāo)本，以光學(xué)相干斷層掃描術(shù)（OCT）包埋劑包埋，于恒冷冰凍切片機(jī)上行冠狀位連續(xù)切片，層厚10 μm，置?80℃冰箱中保存?zhèn)溆谩?/p>

3.免疫組織化學(xué)染色取出腦組織冰凍切片，0.01 mol/L磷酸鹽緩沖液（PBS）水化20 min，枸櫞酸微波中火修復(fù)5 min，體積分?jǐn)?shù)為0.3%Triton X?100破膜30 min，以0.01 mol/L磷酸鹽緩沖液沖洗5 min（×3次），體積分?jǐn)?shù)為3%過氧化氫滅活內(nèi)源性過氧化物酶15 min，抗原封閉液封閉1 h，滴加Ⅰ抗工作液中兔抗小鼠iNOS多克隆抗體、兔抗小鼠COX?2多克隆抗體、山羊抗小鼠8?OHd G多克隆抗體和兔抗小鼠Neu N單克隆抗體，4℃過夜，0.01 mol/L磷酸鹽緩沖液沖洗5 min（×3次），再滴加辣根過氧化物酶標(biāo)記的驢抗山羊IgGⅡ抗或辣根過氧化物酶標(biāo)記的驢抗兔IgGⅡ抗，常溫孵育1 h，0.01 mol/L磷酸鹽緩沖液沖洗5 min×（4次），二氨基聯(lián)苯胺（DAB）顯色1 min，蘇木素復(fù)染3 min，中性樹膠封片，于光學(xué)顯微鏡下觀察。每只小鼠取3張大致相同層面腦組織切片，觀察雙側(cè)丘腦iNOS、COX?2、8?Ohd G和Neu N陽性神經(jīng)元或膠質(zhì)細(xì)胞，隨機(jī)選取5個(gè)視野（× 400），計(jì)數(shù)陽性細(xì)胞數(shù)目。

4.免疫熒光染色取出冰凍切片，0.01 mol/L磷酸鹽緩沖液水化20 min，枸櫞酸微波中火修復(fù)約5 min，0.3%Triton X?100破膜30 min，以0.01 mol/L磷酸鹽緩沖液沖洗5 min（×3次），抗原封閉液封閉1 h，滴加Ⅰ抗工作液中兔抗小鼠GFAP單克隆抗體和兔抗小鼠Iba1單克隆抗體，4℃過夜，0.01 mol/L磷酸鹽緩沖液沖洗5 min（×3次），再滴加Alexa Fluor 555標(biāo)記的山羊抗兔熒光IgGⅡ抗，于37℃避光孵育1 h，以0.01 mol/L磷酸鹽緩沖液沖洗5 min（×4次），含4’，6?二脒基?2?苯基吲哚（DAPI）封片劑封片，于熒光顯微鏡下觀察。每只小鼠取3張大致相同層面腦組織切片，觀察雙側(cè)丘腦GFAP陽性星形膠質(zhì)細(xì)胞和Iba1陽性小膠質(zhì)細(xì)胞，隨機(jī)選取5個(gè)視野（×200），計(jì)數(shù)陽性細(xì)胞數(shù)目。

三、統(tǒng)計(jì)分析方法

采用SPSS 17.0統(tǒng)計(jì)軟件進(jìn)行數(shù)據(jù)處理與分析。計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，采用單因素方差分析，兩兩比較行LSD?t檢驗(yàn)。以P≤0.05為差異具有統(tǒng)計(jì)學(xué)意義。

表1 3組小鼠顱腦創(chuàng)傷同側(cè)丘腦炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失的比較Table 1.Comparison of inflammation,oxidative damage and neuron loss in TBI ipsilateral thalamus among 3 groups

表1 3組小鼠顱腦創(chuàng)傷同側(cè)丘腦炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失的比較Table 1.Comparison of inflammation,oxidative damage and neuron loss in TBI ipsilateral thalamus among 3 groups

TBI，traumatic brain injury，顱腦創(chuàng)傷；iNOS，inducible nitric oxide synthase，誘導(dǎo)型一氧化氮合酶；COX?2，cyclooxygenase?2，環(huán)氧合酶?2；8?OHd G，8?hydroxy deoxyguanosine，8?羥基脫氧鳥苷；NeuN，neuronal nuclei，神經(jīng)元核抗原；GFAP，glial fibrillary acidic protein，膠質(zhì)纖維酸性蛋白；Iba1，ionized calcium?binding adaptor molecule 1，離子鈣結(jié)合蛋白1。The same for table below

Group Control TBI Berberine F value P value N 6 6 6 iNOS 9.89±4.17 16.89±4.55 11.78±1.85 5.682 0.015 COX?2 8.04±4.92 16.88±6.88 9.23±3.48 4.945 0.022 8?OHdG 16.66±3.70 33.32±7.30 24.63±2.50 17.058 0.000 NeuN 22.56±3.00 16.52±1.39 19.69±1.16 13.369 0.000 GFAP 16.67±8.06 29.33±9.39 19.00±4.06 4.816 0.024 Iba1 11.30±3.69 28.92±6.76 19.53±6.11 14.469 0.000

結(jié)果

一、鹽酸小檗堿對(duì)顱腦創(chuàng)傷模型小鼠同側(cè)丘腦炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失的影響

3組小鼠顱腦創(chuàng)傷同側(cè)丘腦iNOS（P=0.015）、COX?2（P=0.022）、8?OHd G（P=0.000）和Neu N（P= 0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目差異均有統(tǒng)計(jì)學(xué)意義（表1）。TBI組iNOS（P=0.005）、COX?2（P= 0.011）和8?OHd G（P=0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目均高于對(duì)照組，Neu N陽性神經(jīng)元數(shù)目低于對(duì)照組（P=0.000）。鹽酸小檗堿組iNOS（P=0.031）、COX?2（P=0.024）和8?OHd G（P=0.008）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目均低于TBI組，其中iNOS和COX?2陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目降至正常水平（均P> 0.05）、8?OHd G陽性神經(jīng)元數(shù)目仍高于對(duì)照組（P= 0.014）；而Neu N陽性神經(jīng)元數(shù)目高于TBI組（P= 0.016），但仍低于對(duì)照組（P=0.027，圖1）。3組小鼠顱腦創(chuàng)傷同側(cè)丘腦GFAP陽性星形膠質(zhì)細(xì)胞數(shù)目（P=0.024）和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目（P=0.000）差異亦有統(tǒng)計(jì)學(xué)意義（表1）。對(duì)照組星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞數(shù)目較少，TBI組星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞數(shù)目明顯增加（P=0.011，0.000），鹽酸小檗堿組星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞增殖受到抑制，細(xì)胞數(shù)目少于TBI組（P=0.031，0.012），其中星形膠質(zhì)細(xì)胞數(shù)目降至正常水平（P>0.05）、小膠質(zhì)細(xì)胞數(shù)目仍高于對(duì)照組（P=0.024，圖2）。

二、鹽酸小檗堿對(duì)顱腦創(chuàng)傷模型小鼠對(duì)側(cè)丘腦炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失的影響

3組小鼠顱腦創(chuàng)傷對(duì)側(cè)丘腦COX?2（P=0.029）和8?OHd G（P=0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目差異均有統(tǒng)計(jì)學(xué)意義，而iNOS和Neu N陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目以及GFAP陽性星形膠質(zhì)細(xì)胞和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目差異均無統(tǒng)計(jì)學(xué)意義（P> 0.05，表2）。TBI組COX?2（P=0.011）和8?OHd G（P=0.000）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目均高于對(duì)照組；鹽酸小檗堿組COX?2（P=0.047）和8?OHd G（P= 0.010）陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目均低于TBI組，其中COX?2陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目降至正常水平（P>0.05）、8?OHd G陽性神經(jīng)元數(shù)目仍高于對(duì)照組（P=0.004，圖3）。

討論

顱腦創(chuàng)傷可在原發(fā)部位形成病灶，原發(fā)部位的繼發(fā)性腦損傷亦是當(dāng)前顱腦創(chuàng)傷的研究熱點(diǎn)，因此，大量關(guān)于顱腦創(chuàng)傷的研究易忽略遠(yuǎn)隔部位的繼發(fā)性改變。在本研究中，我們發(fā)現(xiàn)顱腦創(chuàng)傷第21天小鼠雙側(cè)丘腦均存在炎癥反應(yīng)和氧化損傷的繼發(fā)性改變，同側(cè)丘腦甚至出現(xiàn)神經(jīng)元缺失，與McAllister［6］報(bào)告的顱腦創(chuàng)傷可以導(dǎo)致遠(yuǎn)隔部位繼發(fā)性損傷的結(jié)果相一致。我們觀察到TBI組小鼠顱腦創(chuàng)傷同側(cè)丘腦星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞增殖活化、iNOS和COX?2表達(dá)、氧化損傷、神經(jīng)元缺失均較對(duì)照組明顯，且TBI組神經(jīng)細(xì)胞形態(tài)也發(fā)生改變，表現(xiàn)為細(xì)胞輪廓不清、軸突難辨、胞體變小、胞核固縮，推測(cè)這種神經(jīng)細(xì)胞形態(tài)改變是炎癥反應(yīng)和氧化損傷加重所致。小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)炎癥反應(yīng)的主要參與者［7?8］，前者可以釋放氧自由基、一氧化氮（NO）、炎性因子等加重神經(jīng)損傷［9?10］；后者不僅可以釋放炎性因子，還可以在損傷區(qū)域形成膠質(zhì)瘢痕，對(duì)軸索修復(fù)和再生產(chǎn)生機(jī)械性阻礙屏障［11］。iNOS和COX?2是炎癥反應(yīng)的關(guān)鍵酶，在炎癥反應(yīng)中起重要作用，減少iNOS和COX?2過表達(dá)可以發(fā)揮神經(jīng)保護(hù)作用［12?13］；氧化損傷在顱腦創(chuàng)傷中也可以通過氧自由基釋放、脂質(zhì)過氧化或刺激炎癥反應(yīng)以對(duì)神經(jīng)元產(chǎn)生損害作用［14?15］。在本研究中，我們還發(fā)現(xiàn)TBI組小鼠顱腦創(chuàng)傷對(duì)側(cè)丘腦COX?2和8?OHd G表達(dá)水平較對(duì)照組明顯增加，而星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞增殖活化、iNOS表達(dá)水平、神經(jīng)元缺失數(shù)目與對(duì)照組差異無統(tǒng)計(jì)學(xué)意義，這可能是由于顱腦創(chuàng)傷模型在原發(fā)部位的損傷較輕，也可能是由于損傷時(shí)間較長，對(duì)側(cè)丘腦繼發(fā)性炎癥反應(yīng)逐漸減輕，出現(xiàn)神經(jīng)修復(fù)和再生。

圖1 光學(xué)顯微鏡觀察，對(duì)照組顱腦創(chuàng)傷同側(cè)丘腦iNOS、COX?2和8?OHd G陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目較少、神經(jīng)元數(shù)目較多且形態(tài)完整，TBI組iNOS、COX?2和8?OHdG陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目增加、神經(jīng)元數(shù)目減少，鹽酸小檗堿組iNOS、COX?2和8?OHdG陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目明顯減少、神經(jīng)元數(shù)目增加且形態(tài)完整免疫組織化學(xué)染色（EnVision二步法）×400Figure 1Optical microscopy findings In control group,number of neurons or gliocytes positive for iNOS,COX?2 and 8?OHd G was small in TBI ipsilateral thalamus,the number of neurons was large,and the neurons were intact in morphology.In TBI group, the number of neurons or gliocytes was significantly increased,the number of neurons was decreased.In berberine group,the number of neurons or gliocytes was significantly reduced,the number of intact neurons was increased.Immunohistochemical staining(En Vision)×400

在本研究中，我們還觀察到鹽酸小檗堿對(duì)顱腦創(chuàng)傷模型小鼠雙側(cè)丘腦炎癥反應(yīng)和氧化損傷均有抑制作用，并增加同側(cè)丘腦神經(jīng)元存活數(shù)目。既往大量研究顯示，小檗堿在細(xì)胞水平或其他動(dòng)物模型上具有抗炎癥反應(yīng)、抗氧化應(yīng)激等作用：Chen等［16］通過腹腔注射鹽酸小檗堿以抑制神經(jīng)炎癥反應(yīng)，從而發(fā)揮神經(jīng)保護(hù)作用；Ye等［17］發(fā)現(xiàn)，在脂肪組織和脂肪細(xì)胞中，小檗堿可以抑制巨噬細(xì)胞激活、減少腫瘤壞死因子?α（TNF?α）和白細(xì)胞介素?1（IL?1）釋放，從而改善胰島素抵抗；有文獻(xiàn)報(bào)道，小檗堿通過腺苷酸活化蛋白激酶（AMPK）通路抑制小膠質(zhì)細(xì)胞活化，下調(diào)炎性因子iNOS和COX?2表達(dá)，從而發(fā)揮抑制神經(jīng)炎癥反應(yīng)的作用［18］；亦有研究顯示，小檗堿可以減弱酒精性肝病小鼠模型氧化應(yīng)激和脂質(zhì)過氧化［19］；在6?羥基多巴胺（6?OHDA）誘導(dǎo)的帕金森病動(dòng)物模型中，小檗堿通過抗氧化反應(yīng)減少多巴胺能神經(jīng)元缺失，并改善運(yùn)動(dòng)功能［20］。但在顱腦創(chuàng)傷模型中，小檗堿對(duì)雙側(cè)丘腦炎癥反應(yīng)、氧化損傷的影響尚未見諸報(bào)道，本研究證實(shí)鹽酸小檗堿能夠通過抑制炎癥反應(yīng)和氧化損傷來對(duì)雙側(cè)丘腦產(chǎn)生神經(jīng)保護(hù)作用。在本研究中，TBI組小鼠雙側(cè)丘腦均出現(xiàn)較嚴(yán)重的氧化損傷，且同側(cè)丘腦炎癥反應(yīng)較對(duì)側(cè)丘腦更嚴(yán)重，甚至出現(xiàn)神經(jīng)元缺失，因此我們推測(cè)，丘腦神經(jīng)損害作用可能是以炎癥反應(yīng)介導(dǎo)為主，而鹽酸小檗堿對(duì)顱腦創(chuàng)傷后丘腦的神經(jīng)保護(hù)作用可能是同時(shí)通過抗炎癥反應(yīng)和抗氧化損傷的作用來介導(dǎo)的。

圖2 熒光顯微鏡觀察顯示，對(duì)照組顱腦創(chuàng)傷同側(cè)丘腦GFAP陽性星形膠質(zhì)細(xì)胞和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目較少，TBI組GFAP陽性星形膠質(zhì)細(xì)胞和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目明顯增加，鹽酸小檗堿組GFAP陽性星形膠質(zhì)細(xì)胞和Iba1陽性小膠質(zhì)細(xì)胞數(shù)目明顯減少免疫熒光染色×200Figure 2Fluorescent microscopy findings The number of astrocytes positive for GFAP and microglias positive for Iba1 in TBI ipsilateral thalamus was small in control group,but was significantly increased in TBI group and significantly reduced in berberine group.Immunofluorescent staining×200

綜上所述，顱腦創(chuàng)傷后雙側(cè)丘腦均出現(xiàn)炎癥反應(yīng)、氧化損傷等繼發(fā)性腦損傷，甚至同側(cè)丘腦出現(xiàn)神經(jīng)元缺失，鹽酸小檗堿可以顯著抑制顱腦創(chuàng)傷后雙側(cè)丘腦炎癥反應(yīng)和氧化損傷，并增加同側(cè)丘腦神經(jīng)元存活數(shù)目，可能為未來更有效治療顱腦創(chuàng)傷提供新的方向。

表2 3組小鼠顱腦創(chuàng)傷對(duì)側(cè)丘腦炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失的比較Table 2. Comparison of inflammation,oxidative damage and neuron loss in TBI contralateral thalamus among 3 groups

表2 3組小鼠顱腦創(chuàng)傷對(duì)側(cè)丘腦炎癥反應(yīng)、氧化損傷和神經(jīng)元缺失的比較Table 2. Comparison of inflammation,oxidative damage and neuron loss in TBI contralateral thalamus among 3 groups

Group Control TBI Berberine F value P value N 6 6 6 iNOS 11.70±1.28 12.89±2.45 10.55±2.09 2.057 0.162 COX?2 9.34±3.63 15.19±4.60 10.79±1.67 4.508 0.029 8?OHdG 18.13±6.09 29.17±2.50 24.05±2.57 19.854 0.000 NeuN 23.29±2.95 22.42±1.80 20.00±2.35 2.982 0.081 GFAP 15.93±3.38 18.21±4.40 16.27±3.88 0.595 0.564 Iba1 12.80±2.63 14.97±2.11 15.67±2.37 2.378 0.127

圖3 光學(xué)顯微鏡觀察顯示，對(duì)照組顱腦創(chuàng)傷對(duì)側(cè)丘腦COX?2和8?OHd G陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目較為少見，TBI組COX?2和8?OHd G陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目明顯增加，鹽酸小檗堿組COX?2和8?OHd G陽性神經(jīng)元或膠質(zhì)細(xì)胞數(shù)目明顯減少免疫組織化學(xué)染色（EnVision二步法）×400Figure 3Optical microscopy findings The number of neurons or gliocytes positive for COX?2 and 8?OHdG in TBI contralateral thalamus was small in control group,but was significantly increased in TBI group and was significantly reduced in berberine group.Immunohistochemical staining(EnVision)×400

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Protective effect of berberine chloride on secondary damage of bilateral thalami in traumatic brain injury model mice

HUANG Shu?xuan1,ZHU Fei?qi1,PEI Zhong2,DENG Xu?hui1,YANG Zhi1,ZHU Jin?hua1,CHEN Chun?chun1,LIN Wei?feng1

1Department of Neurology,Yuebei People's Hospital Affiliated to Shantou University Medical College, Shaoguan 512026,Guangdong,China

2Department of Neurology,the First Affiliated Hospital,Sun Yat?sen University,Guangzhou 510080, Guangdong,China

Berberine;Craniocerebral trauma;Thalamus;Inflammation;Oxidative stress; Neurons;Disease models,animal

ZHU Fei?qi(Email:zfqzsu2004@aliyun.com)

2017?02?27）

10.3969/j.issn.1672?6731.2017.04.009

廣東省自然科學(xué)基金資助項(xiàng)目（項(xiàng)目編號(hào)：S2013010015786）；廣東省韶關(guān)市科技計(jì)劃項(xiàng)目［項(xiàng)目編號(hào)：韶科（衛(wèi)）2010-01］

512026韶關(guān)，汕頭大學(xué)醫(yī)學(xué)院附屬粵北人民醫(yī)院神經(jīng)內(nèi)科［黃樹宣（現(xiàn)在廣州醫(yī)科大學(xué)附屬第一醫(yī)院神經(jīng)內(nèi)科，郵政騙碼：510080），朱飛奇（現(xiàn)在廣東省深圳市羅湖區(qū)人民醫(yī)院神經(jīng)內(nèi)科，郵政編碼：518001），鄧旭輝，楊志，朱瑾華,陳淳淳,林偉豐］；510080廣州，中山大學(xué)附屬第一醫(yī)院神經(jīng)科（裴中）

朱飛奇（Email：zfqzsu2004@aliyun.com）