目的：探讨姜黄素抑制BN大鼠脉络膜新生血管(CNV)的机制。

方法：激光光凝诱导BN大鼠36只建立CNV模型，随机分为6组，每组6只。正常组BN大鼠正常饲养，不做干预； 实验组BN大鼠行532nm倍频激光光凝后，根据不同分组分别干预14d：模型组：生理盐水灌胃14d； 雷珠单抗组：光凝后第2d玻璃体腔注射雷珠单抗注射液(10mg/mL，0.2mL/支)1次5μL。姜黄素组：低\〖100mg/(kg·d)\〗、中\〖200mg/(kg·d)\〗、高\〖400mg/(kg·d)\〗剂量的姜黄素混悬液分别灌胃14d。光凝14d后进行眼底照相、FFA与ICGA检查。制作BN大鼠CNV眼组织病理学标本，采用HE染色法观察不同组CNV中央厚度。制作BN大鼠CNV眼组织病理学标本，采用免疫组织化学法观察不同组CNV眼组织中AKT/p-AKT/ HIF-1α/VEGF信号通路各因子蛋白的表达情况。采用RT-qPCR法检测CNV眼组织中AKT/HIF-1α/VEGF信号通路各因子mRNA的相对表达量。采用蛋白免疫印迹(Western blot)法检测CNV眼组织中AKT/p-AKT/HIF-1α/VEGF信号通路各因子蛋白的相对表达量。

结果：模型组，雷珠单抗组，姜黄素低、中、高剂量组CNV生成率分别为78.18%、73.21%、77.19%、75.86%、74.55%，均大于70%； 荧光素渗漏平均光密度值分别为182.12±6.59、119.22±8.03、166.45±8.33、164.34±5.69、149.22±6.45； 雷珠单抗组较模型组显著降低(P<0.05)； 姜黄素低、中、高剂量组较雷珠单抗组显著升高(P<0.05)，其中姜黄素高剂量组较模型组显著降低(P<0.05)。HE染色结果显示：正常组BN大鼠视网膜组织各层结构清晰、排列整齐。光凝后14d，雷珠单抗组CNV中央厚度较模型组显著减少(P<0.05)； 姜黄素高剂量组较模型组显著减少(P<0.05)，较雷珠单抗组显著增加(P<0.05)。免疫组化结果显示：正常组BN大鼠视网膜组织结构中AKT、p-AKT、HIF-1α、VEGF因子表达呈阴性，未见棕黄色反应物。光凝后14d，模型组AKT、p-AKT、HIF-1α、VEGF因子的表达高于正常组(P<0.05)； 雷珠单抗组低于模型组(P<0.05)； 姜黄素高剂量组的表达较模型组显著降低(P<0.05)，较雷珠单抗组显著增加(P<0.05)。mRNA结果显示：光凝后14d，模型组AKT、HIF-1α和VEGF mRNA相对表达量均高于正常组(P<0.05)； 雷珠单抗组均低于模型组(P<0.05)； 姜黄素高剂量组较模型组显著降低(P<0.05)，较雷珠单抗组显著增加(P<0.05)。Western blot结果显示：光凝后14d，AKT蛋白各实验组间比较差异无统计学意义。p-AKT蛋白相对表达量模型组较正常组显著增加(P<0.05)； 雷珠单抗组较模型组显著降低(P<0.05)； 姜黄素高剂量组较模型组显著降低(P<0.05)。HIF-1α和VEGF蛋白相对表达量模型组较正常组显著增加(P<0.05)，雷珠单抗组较模型组显著降低(P<0.05)，HIF-1α蛋白相对表达量姜黄素高剂量组较模型组显著降低(P<0.05)，较雷珠单抗组显著增加(P<0.05)。VEGF蛋白相对表达量姜黄素中、高剂量组较模型组显著降低(P<0.05)。

结论：400mg/(kg·d)的姜黄素具有抑制BN大鼠实验性CNV的作用。姜黄素抑制BN大鼠实验性CNV的机制可能与降低AKT/p-AKT/HIF-1α/VEGF信号通路的活性密切相关。

METHODS: CNV model of 36 BN rats was established through laser photocoagulation induction, and they were divided into 6 groups with 6 rats in each group. Normal group was fed normally with no intervention, while 532nm laser photocoagulation was used to establish a experimental CNV model in BN rats. Rats after modeling were respectively intervened for 14d and divided into model group, ranibizumab group, curcumin low \〖100mg/(kg·d)\〗, medium \〖200mg/(kg·d)\〗, and high \〖400mg/(kg·d)\〗 dose group. The model group was given intragastric administration of saline for 14d, ranibizumab(10mg/mL, 0.2mL/dose)was injected at 2d after photocoagulation with 5μL once for rats in ranibizumab group, and different concentrations of curcumin were intragastrically administrated to the rats in low, medium and high groups for 14d. Fundus photography, fundus fluorescein angiography(FFA)and indocyanine green angiography(ICGA)examination were performed at 14d after photocoagulation. Ocular histopathological specimens of rats with CNV were made, and the central thickness of CNV were observed by HE staining. Ocular histopathological specimens were made, and the expressions of AKT/p-AKT/HIF-1α/VEGF signaling pathway-related proteins were observed by immunohistochemistry. The mRNA relative expressions of AKT/HIF-1α/VEGF factor in CNV tissues were detected by RT-qPCR, and the protein expressions of AKT/p-AKT/HIF-1α/VEGF factor in CNV tissues were detected by Western-blot.

RESULTS: CNV generation rates in the model group, the ranibizumab group, and the low, medium and high-dose curcumin groups were 78.18%, 73.21%, 77.19%, 75.86%, 74.55%, respectively, which were higher than 70%. The average absorbance were 182.12±6.59, 119.22±8.03, 166.45±8.33, 164.34±5.69, 149.22±6.45, respectively; the ranibizumab group was significantly lower than the model group(P<0.05); the low-dose, medium-dose and high-dose groups were significantly higher than the ranibizumab group(P<0.05), and the curcumin high-dose group was significantly lower than the model group(P<0.05). HE staining showed that the retinal tissue structure of BN rats in normal group was clear and neatly arranged. The central thickness of CNV in the ranibizumab group was significantly reduced at 14d after photocoagulation compared with the model group(P<0.05); While the curcumin high-dose group was significantly reduced compared with the model group(P<0.05), but increased when compared with ranibizumab group(P<0.05). Immunohistochemistry results showed that AKT, p-AKT, HIF-1α, and VEGF factors were negatively expressed in the retinal tissue structure of BN rats in the normal group, and no brown-yellow reactants were found. The expression of AKT, p-AKT, HIF-1α, and VEGF factors in the model group were higher than that in the normal group at 14d after photocoagulation(P<0.05); the ranibizumab group was lower than the model group(P<0.05). While the expression of the curcumin high-dose group was significantly decreased compared with the model group(P<0.05), but significantly increased when compared with ranibizumab group(P<0.05). The mRNA results showed that the relative expression levels of AKT, HIF-1α and VEGF mRNA in the model group at 14d after photocoagulation were higher than those of the normal group(P<0.05); the ranibizumab group was lower than the model group(P<0.05). While curcumin high-dose group was significantly decreased compared with the model group(P<0.05), but significantly increased when compared with ranibizumab group(P<0.05). Western-blot results showed that there was no significant difference in the relative expression of AKT protein among each experimental groups at 14d after photocoagulation. The relative expression of p-AKT protein in the model group was significantly higher than that in the normal group(P<0.05); the ranibizumab group was significantly lower than the model group(P<0.05); the curcumin high-dose group was significantly lower than the model group(P<0.05). The relative expression levels of HIF-1α protein were significantly higher in the model group than in the normal group(P<0.05), and the ranibizumab group was lower than in the model group(P<0.05). The relative expression levels of HIF-1α protein was lower in the curcumin high-dose group than in the model group(P<0.05)but higher than ranibizumab group(P<0.05). The relative expression level of VEGF protein was significantly lower in the curcumin medium/high-dose group than in the model group(P<0.05).

CONCLUSION: Curcumin at 400mg/(kg·d)has an inhibitory effect on CNV in BN rats. The mechanism may be closely related to inhibiting the activation of AKT/p-AKT/HIF-1α/VEGF signaling pathways.