目的 探討銀杏二萜內(nèi)酯（ginkgo diterpene lactone，GDL）對(duì)血小板活化因子（platelet-activating factor，PAF）誘導(dǎo)神經(jīng)元損傷的保護(hù)作用及相關(guān)機(jī)制。方法 構(gòu)建PAF誘導(dǎo)神經(jīng)元損傷的細(xì)胞模型與動(dòng)物模型，給予GDL或PERK激動(dòng)劑CCT020312進(jìn)行處理。分別使用CCK-8和Annexin V-FITC細(xì)胞凋亡檢測(cè)試劑盒測(cè)定小鼠海馬神經(jīng)元細(xì)胞系HT22細(xì)胞活力和凋亡；使用乳酸脫氫酶（lactate dehydrogenase，LDH）檢測(cè)試劑盒檢測(cè)HT22細(xì)胞培養(yǎng)上清液中LDH活性；采用Fluo-4鈣離子檢測(cè)試劑盒測(cè)定HT22細(xì)胞鈣離子水平；采用分子對(duì)接探究相關(guān)分子機(jī)制；使用Western blotting檢測(cè)HT22細(xì)胞與小鼠腦組織的細(xì)胞凋亡相關(guān)蛋白[半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）、cleaved Caspase-3、B細(xì)胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相關(guān)X蛋白（Bcl-2 associated X protein，Bax）]和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白[蛋白激酶R樣內(nèi)質(zhì)網(wǎng)激酶（protein kinase R-like ER kinase，PERK）、p-PERK、真核翻譯起始因子-2α（eukaryotic initiation factor-2α，eIF-2α）、p-eIF2α、轉(zhuǎn)錄激活因子4（activating transcription factor 4，ATF4）、C/EBP同源蛋白（C/EBP-homologous protein，CHOP）]的表達(dá)。結(jié)果 體外細(xì)胞實(shí)驗(yàn)結(jié)果顯示，與對(duì)照組比較，模型組細(xì)胞活性顯著降低（P＜0.001），LDH釋放率升高（P＜0.001），鈣離子水平升高（P＜0.001），凋亡細(xì)胞顯著增加（P＜0.001），cleaved Caspase-3、Bax蛋白表達(dá)水平顯著升高（P＜0.001），Bcl-2蛋白表達(dá)水平顯著下降（P＜0.001），內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白表達(dá)水平顯著升高（P＜0.05、0.001）；與模型組比較，GDL顯著升高了細(xì)胞活力（P＜0.05、0.001），顯著上調(diào)Bcl-2的表達(dá)（P＜0.05、0.001），顯著下調(diào)cleaved Caspase-3、Bax、p-PERK、p-eIF2α、ATF4、CHOP的表達(dá)（P＜0.05、0.01、0.001）；CCT020312處理后GDL的效果均被逆轉(zhuǎn)（P＜0.001）。體內(nèi)動(dòng)物實(shí)驗(yàn)結(jié)果顯示，與對(duì)照組比較，模型組小鼠腦組織Bax蛋白表達(dá)水平顯著升高（P＜0.001），Bcl-2蛋白表達(dá)水平顯著下降（P＜0.001），內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白表達(dá)水平顯著升高（P＜0.001）；與模型組比較，GDL顯著上調(diào)Bcl-2的表達(dá)（P＜0.01、0.001），顯著下調(diào)p-PERK、p-eIF2α、ATF4、CHOP、Bax的表達(dá)（P＜0.001）；CCT020312處理后GDL的效果均被逆轉(zhuǎn)（P＜0.001）。結(jié)論 PAF可以激活神經(jīng)元內(nèi)質(zhì)網(wǎng)應(yīng)激的PERK/ATF4/CHOP通路來(lái)誘導(dǎo)鈣超載，從而使神經(jīng)元凋亡；而GDL可以通過(guò)抑制PERK/ATF4/CHOP通路來(lái)抑制內(nèi)質(zhì)網(wǎng)應(yīng)激、鈣超載和凋亡，以減輕PAF誘導(dǎo)的神經(jīng)元損傷。

Objective To investigate the protective effect and related mechanism of ginkgo diterpene lactone (GDL) on platelet-activating factor (PAF)-induced neuronal injury. Methods A cellular model and an animal model of PAF-induced neuronal injury were constructed, GDL or PERK agonist CCT020312 were administered. The cell viability and apoptosis of HT22 cells were determined using CCK-8 and Annexin V-FITC apoptosis assay kits, respectively; Activity of lactate dehydrogenase (LDH) in HT22 cell culture supernatant was measured using a LDH assay kit, and calcium level in HT22 cells was determined using a Fluo-4 calcium assay kit; Molecular docking was used to explore the relevant molecular mechanisms; Western blotting was used to detect the expressions of apoptosis-related proteins [cystein-asparate protease-3 (Caspase-3), cleaved Caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax)] and endoplasmic reticulum stress-related proteins [protein kinase R-like ER kinase (PERK), p-PERK, eukaryotic initiation factor-2α (eIF-2α), p-eIF2α, activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP)] in HT22 cells and brain tissues of mice. Results The results of in vitro cell experiments showed that, compared with control group, cell activity was significantly reduced in model group (P < 0.001), release rate of LDH was increased (P < 0.001), level of calcium ions was increased (P < 0.001), number of apoptotic cells was significantly increased (P < 0.001), the expression levels of cleaved Caspase-3 and Bax proteins were significantly increased (P < 0.001), the expression level of Bcl-2 protein was significantly decreased (P < 0.001), and the expression levels of endoplasmic reticulum stress-related proteins were significantly increased (P < 0.05, 0.001). Compared with model group, GDL significantly increased cell viability (P < 0.05, 0.001), significantly up-regulated Bcl-2 expression (P < 0.05, 0.001), and significantly down-regulated the expression of cleaved Caspase-3, Bax, p-PERK, p-eIF2 α, ATF4 and CHOP (P < 0.05, 0.01, 0.001); The effect of GDL after CCT020312 treatment was reversed (P < 0.001). The results of in vivo animal experiments showed that compared with control group, the expression level of Bax protein in brain tissue of mice in model group was significantly increased (P < 0.001), the expression level of Bcl-2 protein was significantly decreased (P < 0.001), and the expression levels of endoplasmic reticulum stress-related proteins were significantly increased (P < 0.001); Compared with model group, GDL significantly up-regulated the expression of Bcl-2 (P < 0.01, 0.001), and significantly down-regulated the expressions of p-PERK, p-eIF2α, ATF4, CHOP and Bax (P < 0.001); The effect of GDL after CCT020312 treatment was reversed (P < 0.001). Conclusion PAF can active PERK/ATF4/CHOP pathway of neuronal endoplasmic reticulum stress to induce calcium overload and thus neuronal apoptosis, whereas GDL can inhibit endoplasmic reticulum stress, calcium overload and apoptosis by inhibiting PERK/ATF4/CHOP pathway to attenuate PAF-induced neuronal injury.

R285.5

江蘇省基礎(chǔ)研究計(jì)劃自然科學(xué)基金—前沿引領(lǐng)技術(shù)基礎(chǔ)研究專項(xiàng)（BK20232014）；連云港市“521”人才工程支持項(xiàng)目（LYG06521202221）