杨梅素调节cAMP/PKA/CREB信号通路对炎症性肠病大鼠免疫功能的影响

Effect of myricetin on immune function in rats with inflammatory bowel disease by regulating the cAMP/PKA/CREB signaling pathway

关键词:  杨梅素  环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应成分结合蛋白信号通路  炎症性肠病  免疫功能

Key words:  Myricetin  Cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate responsive component binding protein signaling pathway  Inflammatory bowel disease  Immune function

摘要:

目的 探究杨梅素（Myr）调节环磷酸腺苷（cAMP）/蛋白激酶A（PKA）/环磷酸腺苷反应成分结合蛋白（CREB）信号通路对炎症性肠病（IBD）大鼠免疫功能的影响。方法 建立IBD大鼠模型，实验分为Control组、Model组、杨梅素低、中、高剂量（Myr-L、Myr-M、Myr-H，28、56、112mg/kg/d Myr）组和杨梅素高剂量+PKA抑制剂H89（Myr-H+H89，112mg/kg/d Myr+7mg/kg/d H89）组。对大鼠疾病活动指数（DAI）进行评分；测定免疫功能指标及结肠长度；试剂盒测定血清中IL-6、IL-17A、TNF-α、cAMP水平；HE染色观察结肠组织病理变化；流式细胞术测定Treg细胞比例；免疫组化检测结肠组织中MPO表达；Western blot测定cAMP/PKA/CREB信号通路相关蛋白。结果 与Control组相比，Model组结肠组织细胞排列紊乱、有大量炎性细胞浸润，出现严重溃疡现象，大量细胞坏死，粘膜水肿和结肠壁增厚，DAI评分、IL-6、TNF-α和IL-17A水平、脾脏系数、胸腺系数、MPO光密度值显著增加（P<0.05），结肠长度、Treg细胞比例、cAMP浓度、p-PKA/PKA和p-CREB/CREB水平显著降低（P<0.05）。与Model组相比，Myr-L、Myr-M和Myr-H组结肠组织细胞排列较整齐、粘膜水肿和结肠壁增厚减轻，炎性细胞浸润、细胞坏死及溃疡现象减少，DAI评分、IL-6、TNF-α和IL-17A水平、脾脏系数、胸腺系数、MPO光密度值逐渐降低（P<0.05），结肠长度、Treg细胞比例、cAMP浓度、p-PKA/PKA和p-CREB/CREB水平逐渐增加（P<0.05）。与Myr-H组相比，Myr-H+H89组结肠组织病理变化加重，DAI评分、IL-6、TNF-α和IL-17A水平、脾脏系数、胸腺系数、MPO光密度值显著增加（P<0.05），结肠长度、Treg细胞比例、cAMP浓度、p-PKA/PKA和p-CREB/CREB水平显著降低（P<0.05）。结论 杨梅素可能通过激活cAMP/PKA/CREB信号通路抑制机体炎症水平，调节免疫功能，对IBD大鼠发挥保护作用。

Abstract:

Objective: To investigate the effect of myricetin (Myr) on immune function in rats with inflammatory bowel disease (IBD) by regulating the cAMP/PKA/CREB signaling pathway. Methods: IBD rat models were established and separated into Control group, Model group, low, medium, and high dose myricetin (Myr-L, Myr-M, Myr-H, 28, 56, 112mg/kg/d Myr) groups, and high dose myricetin+PKA inhibitor H89 (Myr-H+H89112mg/kg/d Myr+7mg/kg/d H89) group. The Disease Activity Index (DAI) of rats was scored; immune function indicators and colon length were measured; reagent kits were applied to measure the levels of IL-6, IL-17A, TNF-α, and cAMP in serum; HE staining was applied to observe pathological changes in colon tissue; flow cytometry was applied to determine the proportion of Treg cells; immunohistochemistry was applied to detect the expression of MPO in colon tissue; Western blot was applied to determine cAMP/PKA/CREB signaling pathway related proteins. Results Compared with the Control group, the colon tissue cells in the Model group were disorderly arranged, with a large number of inflammatory cell infiltration, severe ulceration, a large number of cell necrosis, mucosal edema, and colon wall thickening, the DAI score, IL-6, TNF-α, and IL-17A levels, spleen coefficient, thymus coefficient, and MPO optical density values were obviously increased (P<0.05), the colon length, Treg cell ratio, cAMP concentration, p-PKA/PKA, and p-CREB/CREB levels were obviously reduced (P<0.05). Compared with the Model group, the arrangement of colon tissue cells in the Myr-L, Myr-M, and Myr-H groups was relatively neat, mucosal edema and colon wall thickening were reduced, and inflammatory cell infiltration, cell necrosis, and ulcer phenomenon were reduced, the DAI score, IL-6, TNF-α, and IL-17A levels, spleen coefficient, thymus coefficient, and MPO optical density values were gradually reduced (P<0.05), the colon length, Treg cell ratio, cAMP concentration, p-PKA/PKA, and p-CREB/CREB levels were gradually increased (P<0.05). Compared with the Myr-H group, the pathological changes in the colon tissue of the Myr-H+H89 group worsened, the DAI score, IL-6, TNF-α, and IL-17A levels, spleen coefficient, thymus coefficient, and MPO optical density values were obviously increased (P<0.05), the colon length, Treg cell ratio, cAMP concentration, p-PKA/PKA, and p-CREB/CREB levels were obviously reduced (P<0.05). Conclusion: Myricetin may inhibit inflammation levels, regulate immune function, and exert protective effects on IBD rats by activating the cAMP/PKA/CREB signaling pathway.