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目的探讨姜黄素对糖尿病早期大鼠视网膜Müller细胞的影响及作用机制。方法雄性SD大鼠腹腔注射链脲佐菌素诱导糖尿病(diabetes mellitus,DM)模型,实验分4组(每组各6只):对照组(SD大鼠腹腔单次注射柠檬酸缓冲液)、DM组(链脲佐菌素诱导的DM大鼠)、DMSO组(DM大鼠腹腔注射二甲基亚砜及生理盐水混合液,每天一次)、姜黄素组(DM大鼠腹腔注射姜黄素80mg·kg(-1),每天一次)。3个月后检测视网膜丙二醛(malondialdehyde,MDA)及还原型谷胱甘肽(reduced gluta-thione hormone,GSH)含量,视网膜胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫组织化学染色,Western-blot检测视网膜GFAP及谷氨酰胺合成酶(glutamine synthetase,GS)表达变化,高效液相色谱检测视网膜谷氨酸含量。结果对照组MDA、谷氨酸、GSH分别为(3.47±0.51)nmol·mg(-1)prot、(35.6±4.8)nmol·mg(-1)、(98.61±9.82)mg·g(-1)prot,DM组分别为(7.21±0.83)nmol·mg(-1)prot、(49.4±4.1)nmol·mg(-1)、(54.86±3.47)mg·g(-1)prot,DMSO组分别为(7.13±0.74)nmol·mg(-1)prot、(52.5±3.7)nmol·mg(-1)、(59.61±5.38)mg·g(-1)prot;DM组及DMSO组MDA及谷氨酸含量较对照组明显增加,GSH含量降低,差异均有统计学意义(均为P<0.05);姜黄素组MDA、GSH及谷氨酸含量分别为(3.91±0.43)nmol·mg(-1)prot、(86.93±7.84)mg·g(-1)prot、(38.4±3.7)nmol·mg(-1),与对照组相比差异均无统计学意义(均为P>0.05),而与DM组相比,姜黄素组MDA含量、谷氨酸含量均降低(均为P<0.05),GSH含量增加(P<0.05)。Western-blot检测结果显示,DM及DMSO组视网膜GFAP表达上调,GS表达下调,姜黄素组GFAP及GS表达均无明显变化。免疫组织化学染色结果显示,对照组视网膜Müller细胞未见明显GFAP阳性染色,DM组及DMSO组Müller细胞胞体及突起呈明显GFAP阳性,但姜黄素组Müller细胞GFAP阳性染色明显弱于DM组。结论姜黄素可上调DM早期大鼠视网膜GS表达,清除视网膜谷氨酸,保护Müller细胞,其机制可能与姜黄素抑制DM视网膜氧化应激有关。
Objective To investigate the effect of curcumin on retinal Müller cells in diabetic rats and its mechanism. Methods Male Sprague-Dawley rats were injected intraperitoneally with streptozotocin to induce diabetic mellitus (DM) model. The experiment was divided into 4 groups (6 in each group): the control group (single intraperitoneal injection of citrate buffer) DM group (streptozotocin-induced DM rats), DMSO group (DM rats, intraperitoneal injection of dimethyl sulfoxide and saline mixture once a day), curcumin group (DM rats intraperitoneal injection of curcumin 80mg Kg (-1) once daily). Three months later, the content of retinal malondialdehyde (MDA) and reduced glutathione (GSH), the content of glial fibrillary acidic protein (GFAP) immunohistochemical staining The changes of GFAP and glutamine synthetase (GS) in retina were detected by Western-blot. The content of glutamate in retina was detected by HPLC. Results The levels of MDA, glutamate and GSH in the control group were (3.47 ± 0.51) nmol · mg (-1) prot, 35.6 ± 4.8 nmol · mg -1 and 98.61 ± 9.82 mg · g -1 ) prot and DM groups were (7.21 ± 0.83) nmol · mg (-1) prot, (49.4 ± 4.1) nmol · mg (-1) and (54.86 ± 3.47) mg · g -1 prot DMSO groups (7.13 ± 0.74) nmol · mg (-1) prot, (52.5 ± 3.7) nmol · mg (-1) and (59.61 ± 5.38) mg · g -1 Prot; (P <0.05). The contents of MDA, GSH and glutamate in curcumin group were (3.91 ± 0.43) nmol · mg (P <0.05), and the content of glutamate in curcumin group was significantly higher than that in control group (P <0.05). Compared with the control group, there was no significant difference between the two groups (all P> 0.05) , While the contents of MDA and glutamic acid in curcumin group were lower than those in DM group (both P <0.05) and GSH content was increased (P <0.05). The results of Western-blot showed that the expression of GFAP and the expression of GS were down-regulated in the DM and DMSO groups. There was no significant change in the GFAP and GS expression in the curcumin group. Immunohistochemical staining showed that there was no obvious positive staining of GFAP in Müller cells in control group, but significant GFAP positive staining of Müller cells in DM group and DMSO group. However, GFAP positive staining of Müller cells in curcumin group was weaker than that in DM group. CONCLUSION: Curcumin can up-regulate the expression of GS in the retina of DM rats in early stage and remove the glutamate in the retina to protect the Müller cells. The mechanism may be related to the inhibition of oxidative stress induced by curcumin in diabetic retinopathy.