摘要
目的探讨黄芩苷在体内急性高眼压模型及体外氧糖剥夺/再灌注(OGD/R)模型视网膜神经节细胞(RGC)死亡中的作用及其机制。方法实验研究。取SPF级C57BL/6小鼠,通过前房灌注平衡盐溶液建立急性高眼压模型,于建模后不同时间点(分为对照组及急性高眼压后1、3、5、7 d组,每组取5只小鼠)取材评估视网膜损伤程度;同时联合荧光金上丘逆行标记RGC计数,评价尾静脉注射50 mg/kg黄芩苷对急性高眼压建模5 d时RGC损伤的作用。利用Western印迹、实时荧光定量PCR法检测小鼠急性高眼压模型视网膜炎性反应因子的表达。体外培养纯化的原代RGC,建立OGD/R模型,流式细胞术评价不同浓度(2.5、5.0、10.0μmol/L)黄芩苷对RGC存活率的影响。建立小鼠小胶质细胞BV2细胞和原代RGC共培养体系,在OGD/R模型下,酶联免疫吸附试验检测BV2细胞炎性反应因子的分泌,评估黄芩苷对BV2细胞的作用及RGC存活的影响。采用方差分析进行统计学分析。结果在小鼠急性高眼压模型中可观察到视网膜厚度变薄,建模后3 d厚度下降至87.32%±0.94%(t=6.73,P<0.01),5 d后厚度降至74.86%±2.43%(t=13.40,P<0.01),7 d后仅为63.53%±2.15%(t=19.46,P<0.01),差异有统计学意义。荧光金上丘逆行标记RGC计数同样观察到急性高眼压建模后5 d RGC存活率下降至61.32%±5.94%(t=6.59,P<0.01),给予尾静脉注射50 mg/kg黄芩苷预处理后,RGC的存活率提高至89.93%±10.08%(t=4.84,P<0.01)。Western印迹、实时荧光定量PCR检测急性高眼压建模后炎性反应因子肿瘤坏死因子α、白细胞介素1β、诱导性一氧化氮合酶的表达,均有不同程度的增高,黄芩苷干预后炎性反应因子的表达降低。同样,在体外OGD 2 h/R 6 h后原代RGC存活率下降至51.53%±1.36%(t=14.91,P<0.01),5.0、10.0μmol/L黄芩苷预处理后存活率提高至69.37%±7.09%、66.23%±4.25%(t=5.50,4.53;均P<0.01),差异有统计学意义。在BV2细胞和RGC共培养体系下,OGD 2 h/R 6 h后RGC的存活率为41.83%±3.55%,低于单纯RGC培养OGD 2 h/R 6 h后RGC的存活率51.33%±1.16%(t=4.96,P=0.042);5.0μmol/L黄芩苷预处理后,可减轻BV2细胞分泌白细胞介素1β,由(61.33±5.78)pg/ml降至(39.97±8.76)pg/ml(t=4.19,P=0.010),且与5.0μmol/L黄芩苷预处理后单纯RGC培养(69.37%±7.09%)比,可提高RGC存活率至73.00%±5.20%(t=2.82,P=0.048)。结论体内急性高眼压模型、体外单纯OGD/R模型下,黄芩苷可直接减轻视网膜及RGC损伤,并通过抑制视网膜炎症细胞炎性反应因子的表达,发挥保护RGC的作用。
Objective To explore the potential neuroprotection effects and associated mechanism of baicalin in a rodent acute hypertensive glaucoma model and oxygen-glucose deprivation/reperfusion (OGD/R) induced retinal ganglion cell (RGC) injury.Methods Experiment research. A rapid and substantial elevation of intraocular pressure was performed to establish an acute hypertensive glaucoma model, and retinal thickness was assessed at 1, 3, 5, and 7 days. The mice were then randomly divided into three groups: normal control group, hypertension group, and baicalin (50 mg/kg) for hypertension group. The effects of baicalin on the RGCs were evaluated by retrograde transporting of Fluoro-Gold. The mRNA levels of tumor necrosis factor-α, interleukine-1β (IL-1β), and inducible nitric oxide synthase were detected by real-time PCR, and the protein levels were measured by Western blot in the retina tissue of acute hypertensive glaucoma model. Purified primary RGC survival under OGD/R stress was measured by flow cytometry, which was also performed to measure the survival rate of RGCs pretreated by different doses of baicalin (2.5 μmol/L, 5.0 μmol/L, and 10.0 μmol/L). The effects of baicalin on primary RGCs co-cultured with mouse microglia cell line BV2 were evaluated by flow cytometry. The cytokine IL-1β in the culture supernatant was measured by immunochemical analyses. Statistical analysis was performed using analysis of variance.Results Retinal tissue injuries and RGC loss were observed both in vivo and in vitro. Retinal thickness was decreased to 87.32%±0.94% at 3 days (t=6.73, P<0.01), 74.86%±2.43% at 5 days (t=13.40, P<0.01), and 63.53%±2.15% at 7 days (t=19.46, P<0.01). Treatment of 50 mg/kg baicalin significantly promoted the RGC survival from 61.32%±5.94% to 89.93%±10.08% (t=4.84, P<0.01). Baicalin alleviated the retinal damages by suppressing the expression of inflammatory cytokines as revealed by Western blot and real-time PCR. In vitro the RGC survival under OGD/R stress was increased from 51.53%±1.36% to 69.37%±7.09% and 66.23%±4.25% with 5.0, 10.0 μmol/L baicalin administration (t=5.50, 4.53;both P<0.01). BV2 under OGD/R stress did extra damage to RGCs, and baicalin could reverse the damages and increase the survival from 69.37%±7.09% to 73.00%±5.20% (t=2.82, P=0.048) by reducing the release of IL-1β [(39.97±8.76) pg/ml vs. (61.33±5.78) pg/ml, t=4.19, P=0.010].Conclusion Baicalin could alleviate retina tissue injury directly and promote the survival of RGCs by downregulating the expression of inflammatory cytokines and protecting RGCs from ischemia reperfusion injury.
作者
张莹莹
李智冬
蒋楠
万沛星
邓彩彬
苏文如
卓业鸿
Zhang Yingying;Li Zhidong;Jiang Nan;Wan Peixing;Deng Caibin;Su Wenru;Zhuo Yehong(Zhongshan Ophthalmic Center,Sun Yat-sen University,State Key Laboratory of Ophthalmology,Guangzhou 510060,China)
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2020年第5期376-382,共7页
Chinese Journal of Ophthalmology
基金
国家自然科学基金面上项目(81870658)。
