紫云英苷诱导炎性痛小鼠前扣带回皮质星形胶质细胞自噬

Astragalin induces autophagy of astrocytes in anterior cingulate cortex of inflammatory pain mice

目的:探讨紫云英苷(astragalin,AST)对完全弗氏佐剂(complete Freund's adjuvant,CFA)诱发的炎性痛模型小鼠前扣带回皮质内星形胶质细胞活化状态及自噬相关蛋白表达水平的影响。方法:选取24只6月龄雄性C57BL/6小鼠随机分为4组:对照(control)组、生理盐水(saline)组和CFA模型组、CFA+60 mg/kg AST给药组,每组各6只动物。AST给药组小鼠按体重进行腹腔注射给予60 mg/kg AST,连续给药21 d。采用von Frey纤维丝测定各组小鼠机械缩足反应阈值;利用多重免疫荧光染色法检测各组小鼠前扣带回皮质自噬相关因子LC3、p62、ATG12和beclin-1的表达以及星形胶质细胞活化情况;Western blot法检测各组小鼠前扣带回皮质内自噬相关蛋白LC3、p62、ATG12和beclin-1的表达水平。结果:行为学测试结果显示,AST显著提高CFA小鼠的机械疼痛阈值(P<0.05)。免疫荧光染色结果表明,AST显著增强CFA小鼠前扣带回皮质LC3(P<0.01)、ATG12(P<0.01)、beclin-1(P<0.05)的荧光强度,减弱p62(P<0.05)的荧光强度,并抑制星形胶质细胞活化。Western blot结果进一步表明,AST显著上调CFA小鼠前扣带回皮质内LC3(P<0.01)、ATG12(P<0.01)和beclin-1(P<0.01)的表达水平,下调p62(P<0.05)表达水平。结论:AST可缓解CFA小鼠的炎症性疼痛,其镇痛机制可能与抑制CFA小鼠前扣带回皮质星形胶质细胞活化,并促进其发生自噬有关。

AIM:To investigate the effects of astragalin(AST)on activation status of astrocytes and the expression level of autophagy-related proteins in the cortex of the anterior cingulate cortex of mice with a complete Freund's adjuvant(CFA)-induced inflammatory pain model.METHODS:Twenty-four 6-month-old male C57BL/6 mice were randomly divided into four groups:control group,saline group,CFA model group and CFA+60 mg/kg AST administration group,and six mice in each group.Mice in the AST administration group received 60 mg/kg AST by intraperitoneal injection on a body weight basis for 21 d.The paw withdrawal threshold in each group of mice was evaluated by the von Frey test.The expression levels of autophagy-related factors LC3,p62,ATG12 and beclin-1,and astrocyte activation were detected by multiplex immunofluorescence staining in the anterior cingulate cortex of mice in each group.Western blot was used to measure the levels of autophagy-related proteins LC3,p62,ATG12 and beclin-1 in the anterior cingulate cortex of mice in each group.RESULTS:Behavioural tests showed that AST significantly increased mechanical pain thresholds in CFA mice(P<0.05).The results from multiple immunofluorescent staining showed that AST significantly increased the fluorescence intensity of LC3(P<0.01),ATG12(P<0.01)and beclin-1(P<0.05),attenuated the fluorescence intensi‐ty of p62(P<0.05),and inhibited the activation of astrocytes in the anterior cingulate cortex of CFA mice.Western blot results further confirmed that AST significantly increased the expressions of LC3(P<0.01),ATG12(P<0.01),beclin-1(P<0.01),and decreased the expression of p62(P<0.05)in the anterior cingulate cortex of CFA mice.CONCLU⁃SION:AST relieves CFA-induced inflammatory pain of mice,and its analgesic mechanism may be related to the inhibi‐tion of activation of cortical astrocytes in the anterior cingulate cortex and the promotion of autophagy in CFA mice.