目的 探讨紫外线照射对人视网膜色素上皮细胞(ARPE-19细胞)活性和沉默信息调节因子6(SIRT6)表达的影响.方法 将对数生长期的ARPE-19细胞按照照射剂量随机分为对照组(0 mJ·cm-2)及7.5 mJ·cm-2、15.0 mJ·cm-2、30.0 mJ·cm-2、60.0 mJ·cm-2、120.0 mJ·cm-2、240.0 mJ·cm-2照射剂量组进行干预培养.照射后继续常规培养24 h,采用倒置荧光显微镜观察细胞形态并拍照,CCK-8法检测细胞存活率,Western blot检测各组细胞中SIRT6的表达情况,采用免疫荧光染色对对照组、30.0 mJ·cm-2照射剂量组和240.0 mJ·cm-2照射剂量组ARPE-19细胞中SIRT6进行定位检测.结果 对照组ARPE-19细胞呈梭形贴壁生长,边界清楚,连接紧密,细胞状态良好;30.0 mJ·cm-2照射剂量组ARPE-19细胞形态不规整,细胞数量略有减少,细胞间连接疏松.CCK-8法检测结果显示:与对照组相比,ARPE-19细胞在不同剂量紫外线照射后,细胞存活率显著降低且呈剂量依赖性(均为P<0.05).与对照组相比,除较低照射剂量组(7.5 mJ·cm-2、15.0 mJ·cm-2)外,其余各组SIRT6蛋白相对表达量均降低,且随着照射剂量的增加呈剂量依赖性逐渐降低(均为P<0.05).经过30.0 mJ·cm-2紫外线照射后,ARPE-19细胞随着再培养时间的延长,细胞中SIRT6蛋白的相对表达量逐渐下降(均为P<0.05).对照组、30.0 mJ·cm-2照射剂量组和240.0 mJ·cm-2照射剂量组SIRT6主要表达于ARPE-19细胞核内;与对照组相比,30.0 mJ·cm-2照射剂量组和240.0 mJ·cm-2照射剂量组ARPE-19细胞中SIRT6的荧光强度显著降低,且240.0 mJ·cm-2照射剂量组降低更明显.结论 紫外线照射能够降低ARPE-19细胞的增殖活性,同时也能下调细胞中SIRT6的表达.

ETHNOPHARMACOLOGICAL RELEVANCE:Lycium barbarum polysaccharide (LBP) extracted from the Lycium barbarum L. has been widely used to improve diabetes and its relative complications. However, the mechanisms have not fully understood. A recent study has demonstrated that LBP upregulates suituin 1 (SIRT1). OBJECTIVE:This study was to define the role of Sirt1 and its downstream signaling pathways in diabetic cataract using in vitro and in vivo models. MATERIALS AND METHODS:Human lens epithelial cell line SRA01/04 cells were cultured under high glucose (HG) medium with treatment of LBP or vehicle. Cell viability, apoptosis, protein and/or mRNA levels of Sirt1, BAX, Bcl-2, active-caspase-3, FOXO1, p27 and acetylated p53 were measured. SIRT1 upregulated- and knocked-down cells were generated and tested in high glucose culture. Diabetes mellitus was induced in rats by streptozotocin injection. Body weight, blood glucose levels, lens transparency and retinal function were assessed and SIRT1, as well as the aforementioned biomarkers were measured using Western blotting and qPCR in the animal lens samples. RESULTS:The results showed that HG decreased cell viability and LBP prevented the decrease. The reduced viability in HG cultured SRA01/04 cells was associated with increased levels of BAX, active caspase 3, FOXO1, p27, and p53 and decreased levels of SIRT1 and Bcl-2. Further experiments using sirt1 gene modulated cells showed that upregulation of Sirt1 improved viability, increase cell division as reflected by an increased proportion of S phase in the cell cycle, reduced the number of apoptotic cell death and suppressed p53 acetylation and caspase 3 activation. Opposite results were observed in SIRT1 knock-down cells. Treating diabetic animals with LBP reduced body weight loss and blood glucose content in diabetic animals. Similarly, LBP hindered the development of cataract in lenses and improved retinal function. The beneficial effect of LBP on diabetic cataract was associated with the supression of p53, caspase 3, FOXO1, BAX, p27 and elevation of SIRT1 and Bcl-2, which were consistent with the in vitro findings. CONCLUSION:Our findings showed that diabetes caused cataract is associated with suppression of SIRT1 and Bcl-2 and activation of other cell death related genes. LBP prevented diabetic cataract in animals by upregulating Sirt1 and Bcl-2 and suppressing cell death related genes.

目的 观察高浓度葡萄糖对人晶状体上皮细胞(SRA01/04)活性及沉默信号调节蛋白6(SIRT6)表达的影响.方法 将SRA01/04细胞在不同葡萄糖浓度(5.5 mmol·L-1、15.5 mmol·L-1、25.5 mmol·L-1、35.5 mmol·L-1、45.5 mmol·L-1、55.5 mmol·L-1、75.5 mmol·L-1)培养基中培养48 h后,用光镜观察SRA01/04细胞形态;CCK-8试剂盒检测各组细胞的活性;Western blot检测各组细胞SIRT6蛋白的表达水平.然后将SRA01/04细胞用55.5 mmol·L-1葡萄糖干预,培养不同时间(0 h、6 h、12 h、24 h、48 h、72 h)后,Western blot检测不同时间SRA01/04细胞SIRT6蛋白表达水平.免疫荧光技术检测对照组(5.5 mmol·L-1葡萄糖)和高糖组(55.5 mmol·L-1葡萄糖)细胞的SIRT6蛋白表达情况.结果 随着葡萄糖浓度增加,SRA01/04细胞的数量逐渐降低.CCK-8检测结果显示,与5.5 mmol·L-1葡萄糖组相比,随着葡萄糖浓度增加,SRA01/04细胞的活性逐渐降低,差异均有统计学意义(均为P<0.05).Western blot检测结果显示,SRA01/04细胞的SIRT6蛋白表达与葡萄糖呈浓度和时间依赖性,45.5 mmol·L-1、55.5 mmol·L-1、75.5 mmol·L-1葡萄糖组与5.5 mmol·L-1葡萄糖组比较,以及24 h组、48 h组和72 h组与0 h组比较,差异均有统计学意义(均为P<0.05).细胞免疫荧光结果显示,SRA01/04细胞中的SIRT6蛋白主要在细胞核表达,高糖组SRA01/04细胞中SIRT6蛋白表达较对照组显著降低(P<0.05),且高糖组细胞核出现明显的核固缩和核碎裂现象.结论 高浓度葡萄糖可以降低S RA01/04细胞的增殖活性,降低S I RT6蛋白的表达水平.

目的 观察枸杞多糖(LBP)对高糖诱导的人晶状体上皮细胞(SRA0 1/04)凋亡及SIRT1表达的影响.方法 将SRA (OVOA)细胞随机分为正常对照组、高糖组及高糖+LBP低、中、高3个剂量组.各条件下培养48 h后采用CCK-8法检测各组细胞的活性,流式细胞技术(FCM)检测细胞的凋亡率,蛋白免疫印迹法检测各组细胞中SIRT1蛋白的相对表达量.结果 CCK-8结果显示,与正常对照组相比,高糖组细胞活性降低;而与高糖组相比,LBP高剂量组细胞活性升高,差异有统计学意义(P＜0.05).流式细胞术显示,与正常对照组相比,高糖组细胞凋亡率显著上升;与高糖组相比,LBP组细胞凋亡率显著下降,差异有统计学意义(P＜0.05).蛋白印迹法结果显示,与正常对照组相比,高糖组细胞中的SIRT1蛋白表达降低;与高糖组相比,LBP组细胞中的SIRT1蛋白表达升高,差异有统计学意义(P＜0.05).结论 LBP可能通过调节SIRT1的表达对高糖环境下人晶状体上皮细胞凋亡产生抑制作用.

To observe the effect of sirt1 on retinal ganglion cells (RGC) with high glucose culture and to explore the role of sirt1 in the development of diabetic retinopathy. Method RGC was infected by sirt1 lentivirus overexpression vector pLV5-sirt1 and interference vector pLV3-si-sirt1. The normal control group and control virus vector group were set up at the same time. After 48 h of infection, the viability of RGC was detected by CCK8 kit, the apoptosis rate was detected by FCM analysis, and the protein expression of p53, FOXO3a, NF-κ B, caspase-3 was detected by Western blot. After RGC were infected with lentivirus, the cell viability of lentivirus overexpression vector pLV5-sirt1 was significantly higher than that of the high glucose group and the sirt1 overexpression control group, while the cell viability of interference vector pLV3-si-sirt1 was significantly lower than that of the high glucose group and the sirt1 interference control group (P < 0.05). At the same time, the apoptosis rate of RGC cells infected by lentivirus overexpression vector pLV5-sirt1 was lower than that of the high glucose group and the control virus vector group, while the apoptosis rate of the interference vector pLV3-si-sirt1 cells was significantly higher than that of the high glucose group and the control virus vector group (P < 0.05). The results of Western blotting showed that the expression of p53, FOXO3a, NF-κ B and caspase-3 in RGC cells decreased significantly after infection with pLV5-sirt1 compared with the high glucose group and the control virus vector group, while the expression of p53, FOXO3a, NF-κB and caspase-3 in RGC cells increased significantly after infection with pLV3-si-sirt1 (P < 0.05). Sirt1 can inhibit the apoptosis of RGCs through regulating the expression of some apoptotic cytokinessome, and it can be used as a candidate gene for the biotherapy of retinal diseases.