目的 探讨枸杞多糖调控腺苷酸活化蛋白激酶(AMPK)/不协调的51类激酶1(ULK1)自噬途径对高渗诱导角膜上皮细胞凋亡的影响.方法 通过500 mOsm·L-1渗透压的饱和氯化钠溶液作用于角膜上皮细胞建立眼干燥症细胞模型,分为模型组、阳性对照组(0.3％玻璃酸钠滴眼液)、抑制剂组(1 mg·mL-1的枸杞多糖+10 μmol·L-1 compound C)和低、高剂量实验组(0.5、1.0 mg·mL-1的枸杞多糖),另取正常培养的CRL-11135细胞作为空白组(不进行任何处理).用流式细胞仪检测各组细胞的凋亡情况,用单丹磺酰戊二胺染色法检测各组细胞的自噬情况,用蛋白质印迹法检测各组细胞中磷酸化AMPK(p-AMPK)/AMPK和p-ULK1/ULK1的表达水平.结果 低、高剂量实验组和阳性对照组、抑制剂组、模型组、空白组的细胞凋亡率分别为(26.47±2.13)％、(13.68±2.21)％、(12.54±2.16)％、(18.73±2.12)％、(37.56±3.25)％和(6.35±2.14)％,自噬小体相对含量分别为(59.63±8.14)％、(89.89±9.04)％、(90.31±9.13)％、(62.75±7.26)％、(43.11±6.45)％和(100.00±0.00)％,p-AMPK/AMPK 分别 为 0.45±0.07、0.64±0.08、0.66±0.06、0.53±0.04、0.34±0.05 和 0.87±0.06,p-ULK1/ULK1 分别为0.54±0.06、0.75±0.05、0.77±0.03、0.65±0.04、0.46±0.04 和 0.92±0.08.低、高剂量实验组和阳性对照组的上述指标与模型组比较,在统计学上差异均有统计学意义(均P＜0.05);抑制剂组的上述指标与高剂量实验组比较,在统计学上差异均有统计学意义(均P＜0.05).结论 枸杞多糖可通过激活AMPK/ULK1自噬途径抑制高渗诱导的角膜上皮细胞凋亡.
Objective To investigate the influences of Lycium barbarum polysaccharide on apoptosis of corneal epithelial cells induced by hypertonicity through regulating adenosine monophosphate activated protein kinase(AMPK)/uncoordinated 51-like kinase 1(ULK1)autophagy pathway.Methods The ophthalmoxerosis cell model was established by osmotic pressure of 500 mOsm·L-1 on corneal epithelial cells.They were divided into model group,positive control group(0.3％sodium hyaluronate eye drops),inhibitor group(1 mg·mL-1 Lycium barbarum polysaccharide+10 μmol·L-1compound C),experimental-L,-H groups(0.5,1.0 mg·mL 1 Lycium barbarum polysaccharide),and normal cultured CRL-11135 cells were taken as blank group(no treatment was performed).Cell apoptosis was detected by flow cytometry.Autophagy was detected by MDC staining.Western blot was used to detect the expressions of p-AMPK/AMPK and p-ULK1/ULK1.Results The apoptosis rates of experimental-L,experimental-H,positive control,inhibitor,model and blank groups were(26.47±2.13)％,(13.68±2.21)％,(12.54±2.16)％,(18.73±2.12)％,(37.56±3.25)％and(6.35±2.14)％;the relative contents of autophagosomes were(59.63±8.14)％,(89.89±9.04)％,(90.31±9.13)％,(62.75±7.26)％,(43.11±6.45)％and(100.00±0.00)％;p-AMPK/AMPK were 0.45±0.07,0.64±0.08,0.66±0.06,0.53±0.04,0.34±0.05 and 0.87±0.06;p-ULKl/ULKl were 0.54±0.06,0.75±0.05,0.77±0.03,0.65±0.04,0.46±0.04 and 0.92±0.08,respectively.The above indexes in experimental-L,-H groups and positive control group were significantly different from those in model group(all P＜0.05);the above indexes in inhibitor group were significantly different from those in experimental-H group(all P＜0.05).Conclusion Lycium barbarum polysaccharide can inhibit the apoptosis of corneal epithelial cells induced by hypertonicity by activating the AMPK/ULK1 autophagy pathway.
田雯雯;段志学;蒲建中;王晶;律鹏
兰州大学第二医院健康管理中心,甘肃兰州 730000兰州大学第二医院眼科,甘肃兰州 730000
中医学
枸杞多糖;眼干燥症;腺苷酸活化蛋白激酶/不协调的51类激酶1;角膜上皮细胞;高渗;凋亡
Lycium barbarum polysaccharide;ophthalmoxerosis;adenosine monophosphate activated protein kinase/uncoordinated 51-like kinase 1;corneal epithelial cell;hypertonicity;apoptosis
《中国临床药理学杂志》 2024 (001)
42-46 / 5
甘肃省科技计划基金资助项目(21JR7RA422)
10.13699/j.cnki.1001-6821.2024.01.009
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