[摘要]目的 探討淫羊藿素(ICT)對脂多糖(LPS)誘導的原代皮質(zhì)星形膠質(zhì)細胞腫瘤壞死因子α(TNF-α)和誘導型一氧化氮合酶(iNOS)基因表達的影響及胰島素樣生長因子1受體(IGF-1R)阻斷劑JB-1的阻斷作用。方法 常規(guī)培養(yǎng)原代皮質(zhì)星形膠質(zhì)細胞,將其分為對照組、LPS組、ICT+LPS組和JB-1+ICT+LPS組。對照組和LPS組細胞分別給予體積分數(shù)0.01二甲基亞砜(DMSO)和1 mg/L的LPS處理,其余組在有或無JB-1(1 mg/L)預(yù)處理的情況下,給予ICT(10 μmol/L)預(yù)保護1 h,再加入1 mg/L的LPS共同作用6 h。應(yīng)用實時熒光定量PCR檢測各組細胞TNF-α和iNOS基因的表達。結(jié)果 與對照組比較,LPS組TNF-α和iNOS基因表達明顯上調(diào)(F=81.98、118.60,q=20.41、25.22,P<0.01);ICT預(yù)保護能明顯降低由LPS誘導的TNF-α和iNOS基因表達的上調(diào)(q=7.34、13.31,P<0.01),此作用可以被IGF-1R阻斷劑JB-1所阻斷(q=4.65、7.52,P<0.05)。結(jié)論ICT能夠抑制LPS誘導的原代皮質(zhì)星形膠質(zhì)細胞的炎性反應(yīng),其抗炎機制可能與IGF-1R途徑的激活有關(guān)。
[關(guān)鍵詞]淫羊藿素;脂多糖類;星形細胞;受體,IGF1型;腫瘤壞死因子α;一氧化氮合酶Ⅱ型
[中圖分類號]R338.2
[文獻標志碼]A
[文章編號]2096-5532(2021)02-0167-04
[ABSTRACT]Objective To investigate the effect of icaritin (ICT) on the gene expression of tumor necrosis factor-α (TNF-α) and inducible nitric-oxide synthase (iNOS) in primary cultured cortical astrocytes induced by lipopolysaccharide (LPS) and the blocking effect of the insulin-like growth factor-1 receptor (IGF-1R) antagonist JB-1."Methods Primary cultured cortical astrocytes were obtained by conventional methods and were then divided into control group, LPS group, ICT+LPS group, and JB-1+ICT+LPS group. The astrocytes in the control group and the LPS group were treated with volume fraction 0.01 dimethyl sulfoxide and 1 mg/L LPS, respectively, and those in the other groups were pretreated with ICT (10 μmol/L) for 1 hour with or without JB-1 (1 mg/L), followed by LPS (1 mg/L) treatment for another 6 hours. Quantitative real-time PCR was used to measure the gene expression of TNF-α and iNOS in each group."Results Compared with the control group, the LPS group had significantly upregulated gene expression of TNF-α and iNOS (F=81.98,118.60;q=20.41,25.22;Plt;0.01). Pretreatment with ICT significantly inhibited LPS-induced upregulation of TNF-α and iNOS (q=7.34,13.31;Plt;0.01), which was blocked by the IGF-1R antagonist JB-1 (q=4.65,7.52;Plt;0.05)."Conclusion ICT can inhibit LPS-induced inflammatory response in primary cultured cortical astrocytes, and its anti-inflammatory mechanism may be related to activation of the IGF-1R pathway.
[KEY WORDS]icaritin; lipopolysaccharides; astrocytes; receptor, IGF type 1; tumor necrosis factor-alpha; nitric oxide synthase type Ⅱ
星形膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)中一種多功能的膠質(zhì)細胞,與其他類型細胞相互作用發(fā)揮著多種生理功能[1-2]。近年來星形膠質(zhì)細胞介導的炎癥反應(yīng)在中樞神經(jīng)系統(tǒng)中的作用被廣泛報道,有研究顯示,阿爾茨海默病(AD)病人大腦皮質(zhì)淀粉樣斑塊周圍存在大量反應(yīng)性星形膠質(zhì)細胞[3-4],并伴有一些促炎細胞因子或炎癥標志物的表達增加[3]。炎性因子及β-淀粉樣蛋白能夠直接誘導星形膠質(zhì)細胞活化[5-6],形成有害的神經(jīng)炎癥循環(huán)。越來越多的研究表明,神經(jīng)炎癥貫穿于AD發(fā)生發(fā)展的始終[7-8]。因此,針對星形膠質(zhì)細胞介導的AD炎癥病理,開發(fā)有效的抗炎藥物,可能有助于減緩AD的發(fā)生發(fā)展。既往研究表明,傳統(tǒng)中藥淫羊藿的主要活性成分淫羊藿苷可以通過限制炎癥反應(yīng)、氧化應(yīng)激在AD病理中發(fā)揮神經(jīng)保護效應(yīng)[9-10],而淫羊藿素(ICT)作為淫羊藿苷的代謝衍生物,也具有很強的抗炎作用[11-12]。研究已顯示,ICT能夠與雌激素受體(ER)結(jié)合,發(fā)揮類雌激素樣作用[13]。胰島素樣生長因子1受體(IGF-1R)介導的信號途徑參與大腦發(fā)育、突觸傳遞等功能[14]。ER與IGF-1R在神經(jīng)元和神經(jīng)膠質(zhì)細胞中有廣泛的共表達[15-16]。有研究表明,IGF-1R可以與ER相互作用,協(xié)同促進人骨肉瘤細胞的增殖并抑制炎癥[17]。本實驗室前期的研究已經(jīng)證實,10 μmol/L的ICT可以通過ER信號途徑對抗脂多糖(LPS)誘導的原代中腦星形膠質(zhì)細胞的炎癥反應(yīng)[18],在整體動物水平,ICT能夠通過IGF-1R信號途徑抑制海馬炎癥反應(yīng)[19]。但是IGF-1R信號通路是否參與ICT對抗LPS誘導的原代皮質(zhì)星形膠質(zhì)細胞的炎癥反應(yīng),目前尚不清楚。本研究應(yīng)用LPS誘導原代皮質(zhì)星形膠質(zhì)細胞炎性反應(yīng),探討ICT對LPS誘導的細胞腫瘤壞死因子α(TNF-α)和誘導型一氧化氮合酶(iNOS)基因表達的影響以及JB-1的阻斷作用,以期為AD提供新的治療途徑。
1 材料與方法
1.1 主要材料
ICT購自上海同田生物公司,應(yīng)用二甲基亞砜(DMSO)配制成10 mmol/L的溶液;LPS及JB-1購自Sigma公司,用無菌生理鹽水配制成1 g/L的溶液;DMEM/F12培養(yǎng)液和胎牛血清購自BI公司;青霉素/鏈霉素儲存液購自新華制藥廠,分裝后置于-20 ℃冰箱保存?zhèn)溆?多聚-D-賴氨酸(poly-D)購自Sigma公司;新生SD大鼠(<24 h)購自青島大任富城畜牧有限公司;TRIzol購自Invitrogen公司;逆轉(zhuǎn)錄試劑盒以及SYBR Green購于Takara公司;引物由Takara公司設(shè)計并合成。
1.2 原代皮質(zhì)星形膠質(zhì)細胞培養(yǎng)及分組
在超凈工作臺中將新生SD大鼠斷頭,取腦,置于含有DMEM/F12基礎(chǔ)培養(yǎng)液的平皿中,在體式顯微鏡下分離大腦皮質(zhì),剝除腦膜和血管。用槍頭輕輕吹打,使腦組織呈離散狀態(tài),收集細胞懸液至大離心管中,以1 000 r/min離心5 min,棄上清,加入含有體積分數(shù)0.10胎牛血清、100 kU/L青霉素和100 mg/L鏈霉素混合雙抗的DMEM/F12培養(yǎng)液,吹打混勻。接種于20 g/L poly-D包被的150 cm2培養(yǎng)瓶中,置于含體積分數(shù)0.05 CO2的37 ℃細胞培養(yǎng)箱中培養(yǎng)7~10 d,期間每隔2 d更換1次培養(yǎng)液。待細胞鋪滿瓶底,呈現(xiàn)明顯分層時,置于37 ℃恒溫搖床上,以210 r/min震蕩16~18 h后,棄掉上清,用DMEM/F12基礎(chǔ)培養(yǎng)液清洗細胞3次,加2.5 g/L胰蛋白酶消化1~3 min,用含血清的完全培養(yǎng)液終止消化。將貼于培養(yǎng)瓶上的細胞輕柔吹下,收集于大離心管中,以1 000 r/min離心5 min后,加入完全培養(yǎng)液后吹打混勻。將星形膠質(zhì)細胞接種于6孔板中,在光鏡下觀察細胞融合度達到80%~90%時進行分組和加藥處理。將細胞隨機分為對照組(A組)、LPS組(B組)、ICT+LPS組(C組)以及JB-1+ICT+LPS組(D組)。對照組細胞給予體積分數(shù)0.01的DMSO處理;LPS組細胞則給予1 mg/L的LPS處理6 h;ICT+LPS組細胞給予LPS前先給予ICT(10 μmol/L)預(yù)保護1 h;JB-1+ICT+LPS組細胞先給予1 mg/L的JB-1作用1 h,繼而給予ICT(10 μmol/L)處理1 h,最后加入LPS共同作用6 h。
1.3 實時熒光定量PCR方法檢測TNF-α和iNOS的mRNA表達
應(yīng)用TRIzol提取細胞總RNA,按照Takara反轉(zhuǎn)錄試劑盒要求配制兩步反應(yīng)體系,經(jīng)過42 ℃變性2 min,37 ℃反轉(zhuǎn)錄15 min,然后升溫至85 ℃,作用5 s使反轉(zhuǎn)錄酶失活,于4 ℃冷卻,將mRNA反轉(zhuǎn)錄合成cDNA。采用SYBR Green染料法定量檢測目的基因TNF-α、iNOS及內(nèi)參照基因GAPDH表達,按照熒光定量PCR說明書配制PCR反應(yīng)體系,采用兩步法經(jīng)過40個循環(huán)完成擴增,采用2-△△CT法計算基因相對表達量。PCR擴增引物及其序列見表1。
1.4 統(tǒng)計學處理
應(yīng)用GraphPad Prism 5.0軟件進行統(tǒng)計學處理。實驗結(jié)果以x2±s形式表示,多組比較采用單因素方差分析(One-Way ANOVA),并繼以Tukey法進行兩兩比較。P<0.05表示差異有顯著性。
2 結(jié) 果
與對照組比較,LPS組原代皮質(zhì)星形膠質(zhì)細胞的TNF-α和iNOS基因表達明顯上調(diào)(F=81.98、118.60,q=20.41、25.22,P<0.01);ICT預(yù)保護能明顯降低由LPS誘導的TNF-α和iNOS基因表達的上調(diào)(q=7.34、13.31,P<0.01),此作用可以被IGF-1R阻斷劑JB-1所阻斷(q=4.65、7.52,P<0.05)。見表2。
3 討 論
越來越多的研究表明,小膠質(zhì)細胞和星形膠質(zhì)細胞的漸進激活以及隨之而來的促炎因子的過度產(chǎn)生,是神經(jīng)炎癥過程中的主要因素[20]。星形膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)中最豐富的膠質(zhì)亞型[21-22],可與神經(jīng)元等多種類型細胞相互作用[23]。在許多AD轉(zhuǎn)基因小鼠模型中,常常在淀粉樣斑塊和(或)神經(jīng)元纖維纏結(jié)這兩個病理學特征出現(xiàn)之前,觀察到激活的星形膠質(zhì)細胞在皮質(zhì)、海馬等受影響的腦區(qū)積聚[24-25]。并且體內(nèi)研究發(fā)現(xiàn),淀粉樣斑塊周圍的星形膠質(zhì)細胞增生[26],炎癥遞質(zhì)在β-淀粉樣斑塊和神經(jīng)原纖維纏結(jié)周圍高表達[27]。因此,抑制星形膠質(zhì)細胞的炎癥反應(yīng),或許可以減緩AD的病理進程。
傳統(tǒng)中藥淫羊藿的成骨作用以及調(diào)節(jié)性功能、調(diào)節(jié)免疫系統(tǒng)的作用曾被廣泛報道,但是近年來人們認識到了其具有神經(jīng)保護作用[28-29]。ICT是來源于淫羊藿的一種黃酮類化合物,其具有抗炎、抗氧化、抗凋亡等多種藥理活性[30]。已有研究結(jié)果顯示,在小鼠腹腔巨噬細胞炎癥和腹膜炎模型中,ICT可以顯著減少iNOS、白細胞介素6(IL-6)等炎性因子的產(chǎn)生[11],而在神經(jīng)系統(tǒng)中ICT可以抑制LPS誘導的C57BL/6J小鼠的海馬神經(jīng)炎癥[31]。為了探討ICT的抗炎神經(jīng)保護作用,本研究利用LPS誘導原代培養(yǎng)的皮質(zhì)星形膠質(zhì)細胞炎癥反應(yīng),觀察ICT對LPS誘導的細胞TNF-α和iNOS基因表達的影響。結(jié)果顯示,LPS可明顯上調(diào)星形膠質(zhì)細胞炎性因子TNF-α和iNOS的基因表達,應(yīng)用ICT預(yù)保護可明顯抑制兩種炎性因子的基因表達。
IGF-1R主要表達于神經(jīng)元和星形膠質(zhì)細胞,是IGF-1的直接作用靶點,并且在皮質(zhì)、海馬有較高水平的表達[32]。本課題組前期研究發(fā)現(xiàn),激活I(lǐng)GF-1R信號通路可以抑制神經(jīng)毒素對多巴胺能神經(jīng)元的損傷[33]。為進一步探討IGF-1R信號通路是否參與了ICT抗皮質(zhì)星形膠質(zhì)細胞的炎癥反應(yīng),本研究觀察了IGF-1R特異性阻斷劑JB-1對ICT的阻斷效應(yīng),結(jié)果顯示JB-1預(yù)處理部分阻斷了ICT的抗炎保護作用。提示ICT的抗炎機制可能與IGF-1R信號途徑的激活有關(guān),其他的信號途徑可能也參與了ICT的抗炎作用。
綜上所述,ICT能夠抑制LPS誘導的原代皮質(zhì)星形膠質(zhì)細胞TNF-α和iNOS的基因表達,其抗炎機制可能與IGF-1R信號通路的激活有關(guān)。本研究結(jié)果為ICT對抗神經(jīng)炎癥提供了實驗依據(jù)。
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(本文編輯 馬偉平)