目的 探究紫草素（Shikonin, SHI）通过调节核因子-红细胞2型相关因子2/血红素加氧酶（Nrf2/HO-1）信号通路对肉芽肿性小叶性乳腺炎模型大鼠的影响及作用机制。 方法 建立肉芽肿性小叶性乳腺炎（GLM）大鼠模型，将大鼠分为对照组（Control组）、模型组（GLM组）、紫草素低剂量组（SHI-L组，17.5 mg?kg-1·d-1 SHI）、紫草素中剂量组（SHI-M组，35 mg?kg-1·d-1 SHI）、紫草素高剂量组（SHI-H组，70 mg?kg-1·d-1 SHI）和紫草素高剂量+Nrf2抑制剂ML385组（SHI-H+ML385组，70 mg?kg-1·d-1 SHI+14 mg?kg-1·d-1 ML385）。HE染色观察乳腺组织病理变化情况；ELISA检测乳腺组织中IL-1β、TNF-α、IL-8、T-AOC、SOD、GSH、MPO、NAGase和ROS水平；免疫荧光检测NLRP3表达；Western blotting检测Nrf2和HO-1蛋白表达。 结果 与Control组相比，GLM组大鼠乳腺小叶完全被破坏、大片结节样慢性肉芽肿炎性病灶生成，乳腺小叶组织边界不清，腺叶内出现空泡，伴有大量淋巴细胞及中性粒细胞浸润；IL-8、IL-1β、TNF-α、ROS、MPO和NAGase水平、NLRP3阳性表达率显著增加（P<0.05），T-AOC、SOD和GSH水平、Nrf2和HO-1蛋白表达显著降低（P<0.05）。与GLM组相比，SHI-L组、SHI-M组和SHI-H组乳腺组织病变逐渐减轻；IL-8、IL-1β、TNF-α、ROS、MPO和NAGase水平、NLRP3阳性表达率依次降低（P<0.05），T-AOC、SOD和GSH水平、Nrf2和HO-1蛋白表达依次升高（P<0.05）。与SHI-H组相比，SHI-H+ML385组乳腺组织病变加重；IL-8、IL-1β、TNF-α、ROS、MPO和NAGase水平、NLRP3阳性表达率显著增加（P<0.05），T-AOC、SOD和GSH水平、Nrf2和HO-1蛋白表达显著降低（P<0.05）。 结论 紫草素发挥抗炎抗氧化作用改善大鼠肉芽肿性小叶性乳腺炎，其机制可能与激活Nrf2信号通路，上调HO-1表达有关。
Abstract
Objective To explore the effect of shikonin (SHI) on the treatment of granuloma lobular mastitis in model rats by regulating nuclear factor-erythroid 2 related factor 2/heme oxygenase (Nrf2/HO-1) signaling pathway and its mechanism. Methods The rat model of granuloma lobular mastitis (GLM) was established. Rats were grouped into a control group, a model group (GLM group), a low-dose shikonin group (SHI-L group, 17.5 mg?kg-1·d-1 SHI), a medium-dose shikonin group (SHI-M group, 35 mg?kg-1·d-1 SHI), a high-dose shikonin group (SHI-H group, 70 mg?kg-1·d-1 SHI), and a high-dose shikonin+Nrf2 inhibitor ML385 group (SHI-H+ML385 group, 70 mg?kg-1·d-1 SHI+14 mg?kg-1·d-1 ML385). HE staining was applied to observe the pathological changes of breast tissue. ELISA was applied to detect the levels of IL-1β, TNF-α, IL-8, T-AOC, SOD, GSH, MPO, NAGas, and ROS in breast tissue. Immunofluorescence was applied to detect NLRP3 expression. Western blotting was used to detect the expression of Nrf2 and HO-1 proteins. Results Compared with the control group, the mammary lobule of rats in GLM group was completely destroyed, large nodular chronic granuloma inflammatory lesions were generated, the boundary of the mammary lobule tissue was unclear, vacuoles appeared in the glandular lobe, and accompanied by infiltration of a large number of lymphocytes and neutrophils. The levels of IL-8, IL-1β, TNF-α, ROS, MPO and NAGas, and the positive expression rate of NLRP3 were obviously increased (P<0.05), the levels of T-AOC, SOD, and GSH, and the expression of Nrf2 and HO-1 proteins were obviously reduced (P<0.05). Compared with the GLM group, the breast tissue lesions in the SHI-L, SHI-M, and SHI-H groups gradually decreased, the levels of IL-8, IL-1β, TNF-α, ROS, MPO and NAGas, the positive expression rate of NLRP3 were sequentially reduced (P<0.05), the levels of T-AOC, SOD, and GSH, and the expression of Nrf2 and HO-1 proteins were sequentially increased (P<0.05). Compared with the SHI-H group, the SHI-H+ML385 group had more severe breast tissue lesions, the levels of IL-8, IL-1β, TNF-α, ROS, MPO and NAGas, and the positive expression rate of NLRP3 were obviously increased (P<0.05), the levels of T-AOC, SOD, and GSH, and the expression of Nrf2 and HO-1 proteins were obviously reduced (P<0.05). Conclusions Shikonin can improve granulomatous lobular mastitis in rats by exerting anti-inflammatory and antioxidant effects, which may be related to the activation of Nrf2 signaling pathway and upregulation of HO-1 expression.
关键词
紫草素； /
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核因子-红细胞2型相关因子2/血红素加氧酶信号通路； /
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肉芽肿性小叶性乳腺炎
Key words
Shikonin; /
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Nuclear factor-erythroid 2 related factor 2/heme oxygenase signaling pathway /
Granuloma lobular mastitis
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基金
天津市自然科学基金（18JCYBJC93600）
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