藏紅花對(duì)糖尿病大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡過(guò)程中線粒體途徑的影響

黨曉潔

(西安市第四醫(yī)院眼科,西安,710004)

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藏紅花對(duì)糖尿病大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡過(guò)程中線粒體途徑的影響

黨曉潔

(西安市第四醫(yī)院眼科,西安,710004)

目的:觀察藏紅花對(duì)糖尿病大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡過(guò)程中線粒體功能的影響。方法:采用隨機(jī)數(shù)字表法將72只8～10周齡健康無(wú)眼疾SD雄性大鼠隨機(jī)分為正常對(duì)照組、糖尿病模型組、藏紅花治療組,每組24只。正常對(duì)照組大鼠不作任何處理。其余組采用鏈脲佐菌素(SYZ)25 mg/(kg·d)連續(xù)腹腔注射3 d,1次/d,建立2型糖尿病模型,以血糖≥16.7 mmol/L為造模成功。模型成功建立后,藏紅花治療組50 mg/kg的劑量腹腔注射藏紅花素溶液,1次/d,連續(xù)3個(gè)月。并檢測(cè)3組大鼠視網(wǎng)膜醛糖還原酶(AR)、神經(jīng)細(xì)胞凋亡及視網(wǎng)膜中B細(xì)胞淋巴瘤/白血病-2(bcl-2)、bcl-2相關(guān)X蛋白(bcx)、細(xì)胞色素C(cyt-c)、半胱天冬酶-3(cespase-3)蛋白的表達(dá)。結(jié)果:糖尿病模型組大鼠視網(wǎng)膜中的AR活性明顯高于正常對(duì)照組和藏紅花治療組,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05);對(duì)照組RGCs層見(jiàn)少量TUNEL染色陽(yáng)性細(xì)胞,成棕褐色染色;B:糖尿病模型組RGCs、內(nèi)核層可見(jiàn)大量TUNEL染色陽(yáng)性細(xì)胞;C:藏紅花治療組RGCs、內(nèi)核層可見(jiàn)量TUNEL染色陽(yáng)性細(xì)胞,但數(shù)量遠(yuǎn)少于糖尿病模型組;糖尿病模型組大鼠視網(wǎng)膜中的bcl-2、bax、cyt-c及caspase-3蛋白表達(dá)均明顯低于正常對(duì)照組和藏紅花治療組,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。結(jié)論:藏紅花可通過(guò)抑制AR活性、上調(diào)凋亡抑制基因bcl-2的表達(dá)及下調(diào)凋亡促進(jìn)基因bax、cyt-c和caspase-3的表達(dá),抑制線粒體凋亡途徑活化,減少糖尿病大鼠視網(wǎng)膜神經(jīng)細(xì)胞的凋亡,發(fā)揮對(duì)糖尿病視網(wǎng)膜損傷的保護(hù)作用。

藏紅花;糖尿病;視網(wǎng)膜神經(jīng)節(jié);細(xì)胞凋亡;線粒體;功能

糖尿病視網(wǎng)膜病變(Diabetic Retinopathy,DR)發(fā)病機(jī)制既往認(rèn)為主要與多元醇、蛋白激酶C、糖基化終末產(chǎn)物、氨基己糖等途徑有關(guān)[1]。但單獨(dú)應(yīng)用針對(duì)這4條途徑的特異性阻斷劑不能取得滿意的治療效果,這4條途徑似乎彼此獨(dú)立[2-3]。Cheung N[4]提出活性氧(Reactive Oxygen,ROS)的過(guò)量產(chǎn)生與上述4條途徑關(guān)系密切。ROS主要生成于線粒體電子傳遞過(guò)程,可導(dǎo)致線粒體內(nèi)DNA的氧化損傷,影響線粒體呼吸功能、降低線粒體膜電位,導(dǎo)致細(xì)胞功能障礙。視網(wǎng)膜缺血再灌注損傷(RIR)可引起視神經(jīng)損傷,與細(xì)胞凋亡、炎性反應(yīng)及氧自由基密切相關(guān)[5]。藏紅花是鳶尾科植物,主要成分藏紅花素,通過(guò)抑制視網(wǎng)膜炎性反應(yīng)因子IL-lB、TNF-α的含量,起到抗炎作用[6]。我們觀察藏紅花素對(duì)RIR時(shí)大鼠視網(wǎng)膜組織結(jié)構(gòu)及線粒體途徑的影響,以探討藏紅花素對(duì)糖尿病大鼠視神經(jīng)損傷作用及其機(jī)制?，F(xiàn)將結(jié)果報(bào)道如下。

1 材料與方法

1.1 建立糖尿病大鼠模型、分組及視網(wǎng)膜取材 雄性SD大鼠72只,體重200～250 g,無(wú)特定病原體級(jí),醫(yī)藥大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。采用隨機(jī)數(shù)字表法將72只8～10周齡健康無(wú)眼疾SD雄性大鼠隨機(jī)分為正常對(duì)照組、糖尿病模型組、藏紅花治療組,每組24只。正常對(duì)照組大鼠不作任何處理。其余組采用鏈脲佐菌素(SYZ)25 mg/(kg·d)連續(xù)腹腔注射3 d,1次/d,建立2型糖尿病模型,以血糖≥16.7 mmol/L為造模成功。模型成功建立后,藏紅花治療組50 mg/kg的劑量腹腔注射藏紅花素溶液,1次/d,連續(xù)3個(gè)月。大鼠水合氯醛麻醉后,取得視網(wǎng)膜血管組織。

1.2 紫外分光光度測(cè)定視網(wǎng)膜AR活性 取液氮中保存的視網(wǎng)膜,按體積比1∶4加入預(yù)冷的(0.01 mmol/L PBS,pH 7.2～7.4)制成組織勻漿,4 ℃、3 000 r/min離心15 min,離心半徑13.5 cm,上清即為酶粗提取液,用BCA蛋白定量試劑盒對(duì)蛋白的表達(dá)進(jìn)行定量分析。AR活性測(cè)定反應(yīng)體系的組分(最終濃度)為:0.1 mol/L PBS[內(nèi)含0.4 mol/L(NH4)2SO4,pH 6.2],0.08 mmol/L NADPH,5 mmol/L DL-甘油醛,5 L酶粗提取液,反應(yīng)體積100 L。其中DL-甘油醛為反應(yīng)底物。反應(yīng)自加入底物開(kāi)始,37 ℃水浴反應(yīng)5 min,加入預(yù)冷的PBS終止反應(yīng),采用DU800型紫外分光光度計(jì)(美國(guó)Beckman Coulter公司)測(cè)定NADPH在340 nm處的吸光度(A340)值。AR活性單位定義為每毫克蛋白質(zhì)每分鐘消耗1 mol NADPH,以1 mol/(min·L)表示。

1.3 培養(yǎng)細(xì)胞的免疫組織化學(xué)法鑒定 RPE細(xì)胞在無(wú)菌條件下進(jìn)行分離,EDTA質(zhì)量分?jǐn)?shù)為0.02%和胰蛋白酶質(zhì)量分?jǐn)?shù)為0.25%進(jìn)行消化,培養(yǎng)瓶?jī)?nèi)接種入細(xì)胞密度為5×104個(gè)/mL,體積分?jǐn)?shù)5% CO:孵箱內(nèi)溫度37 ℃進(jìn)行培養(yǎng)。RPE細(xì)胞的活力采用錐蟲(chóng)藍(lán)染色法測(cè)定,首先預(yù)冷丙酮固定細(xì)胞爬片10 min。細(xì)胞鑒定采用角蛋白單克隆抗體,用1∶3 000稀釋的鼠抗人角蛋白抗體為一抗,羊抗小鼠lgG采用生物素標(biāo)記作為二抗,滴加辣根酶標(biāo)記鏈霉卵白素工作液,DAB顯色,充分沖洗脫水,封片,拍照。

1.4 Western blot法檢測(cè)視網(wǎng)膜中bcl-2、bax、cyt-c及caspase-3的蛋白表達(dá) 視網(wǎng)膜組織細(xì)胞總蛋白進(jìn)行RIPA裂解提取,并行蛋白定量檢測(cè),變性,上樣,電泳,轉(zhuǎn)至聚偏氟乙烯膜,質(zhì)量分?jǐn)?shù)5%脫脂奶粉封閉,加入相應(yīng)抗體(l%脫脂奶粉稀釋作為一抗和二抗,1∶300為一抗效價(jià)比,1∶1 000為二抗效價(jià)比),曝光洗片,膠片掃描后用Ipwin32圖像分析軟件分析結(jié)果,450 nm細(xì)胞吸光度作為測(cè)定波長(zhǎng),內(nèi)參基因?yàn)棣?actin,目的基因A值/β-actin A值作為各凋亡調(diào)控因子的相對(duì)表達(dá)量,每個(gè)檢測(cè)指標(biāo)樣本量為3。分別計(jì)算bcl-2、bax、cyt-c及caspsse-3條帶與B-actin條帶的灰度比值,以代表目的的蛋白的相對(duì)表達(dá)水平。

2 結(jié)果

2.1 視網(wǎng)膜組織中AR的活性 糖尿病模型組大鼠視網(wǎng)膜中的AR活性明顯高于正常對(duì)照組和藏紅花治療組,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)表1。

表1 3組大鼠視網(wǎng)膜AR活性和細(xì)胞AI的比較

注:與糖尿病模型組比較,*P<0.001;AR:醛糖還原酶,AI凋亡指數(shù)。

表2 3組大鼠視網(wǎng)膜中bcl-2、bax、cyt-c及caspase-3蛋白表達(dá)

注:與糖尿病模型組比較,*P<0.001。

2.2 視網(wǎng)膜組織的TUNEL染色 結(jié)果見(jiàn)圖1。

圖1 TUNEL法檢測(cè)到3組大鼠RGCs凋亡情況(×400)

注：A:對(duì)照組RGCs層見(jiàn)少量TUNEL染色陽(yáng)性細(xì)胞,成棕褐色染色;B:糖尿病模型組RGCs、內(nèi)核層可見(jiàn)大量TUNEL染色陽(yáng)性細(xì)胞;C:藏紅花治療組RGCs、內(nèi)核層可見(jiàn)量TUNEL染色陽(yáng)性細(xì)胞,但數(shù)量遠(yuǎn)少于糖尿病模型組;注:RGCs:視網(wǎng)膜神經(jīng)節(jié)細(xì)胞。

2.3 3組大鼠視網(wǎng)膜中bcl-2、bax、cyt-c及caspase-3蛋白表達(dá) 糖尿病模型組大鼠視網(wǎng)膜中的bcl-2、bax、cyt-c及caspase-3蛋白表達(dá)均明顯低于正常對(duì)照組和藏紅花治療組,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)表2。

3 討論

DR早期病變主要是視網(wǎng)膜血管病變。目前研究已證實(shí)糖尿病血管病變與細(xì)胞凋亡和黏附分子表達(dá)、炎性損傷有密切關(guān)系[7]。盡管發(fā)病機(jī)制尚十分清楚。糖尿病視網(wǎng)膜疾病的療法將會(huì)深刻影響遺傳性視網(wǎng)膜疾病患者的生活質(zhì)量[8]。正因如此,這個(gè)領(lǐng)域吸引了大量的研究去開(kāi)發(fā)相應(yīng)的療法[9-11]。有幾個(gè)因素使得眼睛成為基因替換療法的理想器官,包括易接近性、免疫豁免、體積小、組織分區(qū)和天然的對(duì)側(cè)對(duì)照。筆者觀察藏紅花對(duì)糖尿病大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡過(guò)程中線粒體功能的影響。相關(guān)研究認(rèn)為視網(wǎng)膜主要損傷在視網(wǎng)膜色素上皮細(xì)胞、Muller細(xì)胞及視網(wǎng)膜全層的線粒體[12-15]。線粒體疾病是指病變發(fā)生在細(xì)胞的線粒體內(nèi),是線粒體基因組(mtDNA)和/或核基因組(nDNA)編碼線粒體蛋白的基因變異引起的線粒體結(jié)構(gòu)和氧化磷酸化功能的損傷,而引起的疾病。線粒體病通常表現(xiàn)為ATP能量減少、活性氧自由基(ROS)增多和乳酸中毒等造成細(xì)胞損傷或細(xì)胞凋亡等[16-17]。人的部分生理結(jié)構(gòu)屬于自然凋亡,如人的有尾階段,尾部在發(fā)育過(guò)程中自動(dòng)凋亡。細(xì)胞凋亡在人神經(jīng)變性病中起重要作用[18-22]。在營(yíng)養(yǎng)因子匱乏的情況下,促凋亡分子Bax是交感神經(jīng)元和運(yùn)動(dòng)神經(jīng)元死亡所必需的。而且,成年Bax缺失性轉(zhuǎn)基因小鼠比其野生型副本具有更多的神經(jīng)元。這些發(fā)現(xiàn)表明,在許多神經(jīng)元集群中,Bax在發(fā)育過(guò)程中調(diào)控自然發(fā)生的細(xì)胞死亡。也觀察到,在胚胎期(Embryonic Life),Bax是外周和CNS神經(jīng)元自然發(fā)生死亡的關(guān)鍵調(diào)節(jié)劑(Davies,2000)。在一定的實(shí)驗(yàn)條件下,已知的抗凋亡蛋白Bel-2和Bcl-xL可中和Bax的活性。設(shè)想當(dāng)促凋亡蛋白的濃度超過(guò)抗凋亡蛋白的濃度時(shí),會(huì)刺激凋亡。此類(lèi)凋亡包括線粒體中的變化,其最終導(dǎo)致稱(chēng)為半胱天冬酶(Caspases)的絲氨酸蛋白酶家族的激活[23-25]。這導(dǎo)致死亡細(xì)胞從內(nèi)部被消化,這是一種細(xì)胞凋亡標(biāo)志對(duì)調(diào)節(jié)細(xì)胞凋亡的基因和蛋白特別是負(fù)調(diào)控(即抑制)細(xì)胞凋亡的那些蛋白的更多了解,可以設(shè)計(jì)出新的治療法,從而可防止細(xì)胞凋亡的不適當(dāng)激活[26-27]。藏紅花素是從藏紅花中提取的單體,通過(guò)腹腔注射及口服后可以吸收入血液,到達(dá)視網(wǎng)膜;靜脈注射藏紅花提取液可以減少腦缺血再灌注模型的腦損傷,說(shuō)明藏紅花提取液可以通過(guò)血腦屏障[28]。研究結(jié)果顯示糖尿病模型組大鼠視網(wǎng)膜中的AR活性明顯高于正常對(duì)照組和藏紅花治療組,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05);對(duì)照組RGCs層見(jiàn)少量TUNEL染色陽(yáng)性細(xì)胞,成棕褐色染色;B:糖尿病模型組RGCs、內(nèi)核層可見(jiàn)大量TUNEL染色陽(yáng)性細(xì)胞;C:藏紅花治療組RGCs、內(nèi)核層可見(jiàn)量TUNEL染色陽(yáng)性細(xì)胞,但數(shù)量遠(yuǎn)少于糖尿病模型組;糖尿病模型組大鼠視網(wǎng)膜中的bcl-2、bax、cyt-c及caspase-3蛋白表達(dá)均明顯低于正常對(duì)照組和藏紅花治療組,且差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。提示藏紅花可通過(guò)抑制AR活性、上調(diào)凋亡抑制基因bcl-2的表達(dá)及下調(diào)凋亡促進(jìn)基因bax、cyt-c和caspase-3的表達(dá),抑制線粒體凋亡途徑活化,減少糖尿病大鼠視網(wǎng)膜神經(jīng)細(xì)胞的凋亡,發(fā)揮對(duì)糖尿病視網(wǎng)膜損傷的保護(hù)作用。但由于視網(wǎng)膜損傷本身是個(gè)多因素協(xié)同的結(jié)果,有關(guān)其具體機(jī)制還有待進(jìn)一步拓展體內(nèi)外實(shí)驗(yàn)研究,觀察相關(guān)信號(hào)通路、視神經(jīng)血流改變及相關(guān)基因表達(dá)情況加以探討。

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(2015-11-05收稿 責(zé)任編輯：王明)

Effect of Saffron on Mitochondria Pathway during Apoptosis of Retinal Ganglion Cells in Diabetic Rats

Dang Xiaojie

(Xi′anNo.4Hospital,Xi′an710004,China)

Objective:To observe the effect of saffron on mitochondrial function of diabetic rat retinal ganglion cell apoptosis process.Methods:Using random number table method,72 SD male rats 8 ～ 10 weeks old with healthy eyes were randomly assigned to normal control group, diabetic model group, and saffron treatment group (n=24). Normally controlled rats experienced no treatment. Other rats were given streptozocin (SYZ) 25 mg / (kg / D) by intraperitoneal injection for 3 days,once daily, to establish the model of type 2 diabetes and their blood sugar would be more than or equal to 16.7 mmlo/L for a successful modeling. After the model was established, saffron treatment group was provided with intraperitoneal injection of crocin solution 50 mg / kg, once per day, for 3 consecutive months. And Retinal aldose reductase (AR), neural cell apoptosis and B cell lymphoma / leukemia-2 (Bcl-2), cell pigment and Bcl-2 associated X protein (BCX), cytochrome C (Cyt-C), cespase-3 protein expression were detected.Results:The AR activity in the retina of diabetic rats was significantly higher than that of the normal control group and the treatment group, and the difference was statistically significant (P<0.05); there was small amount of TUNEL positive cells in RCGs layers of the control group, brown staining; B: The RGCs and nuclear layer in diabetic model group had a number of TUNEL staining positive cells; C: there were TUNEL staining positive cells in RGCs and kernel layer in Saffron treatment group, but much less than the number of that of the diabetic model group; Retinal Bcl-2, Bax, Cyt-C and caspase-3 protein expression of diabetic rats were much lower than those of normal control group and treatment group, and the difference is statistically significant (P<0.05).Conclusion:Saffron inhibited AR activity and the activation of the mitochondrial apoptotic pathway, regulated expression of apoptosis inhibition of bcl-2 gene and apoptosis promoting gene Bax, Cyt-C and Caspase-3, reduced the apoptosis of nerve cells in the retina of diabetic rats and showed protective effect on diabetes optic omental injury.

Saffron; Diabetes mellitus; Retinal ganglion cells; Apoptosis; Mitochondrial; Function

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào):81273902)——藏紅花對(duì)改善線粒體功能抑制視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡的機(jī)制研究

黨曉潔(1980.09—),女,大學(xué)本科,主治醫(yī)師,研究方向:小兒眼科,斜弱視,E-mail:36934386@qq.com

R587.1;R285.5

A

10.3969/j.issn.1673-7202.2016.11.048