大建中汤调控小胶质细胞自噬治疗腹泻型肠易激综合征内脏痛和抑郁症共病的作用机制

Mechanism of Dajianzhong Decoction in treating visceral pain and depression associated with diarrhea irritable bowel syndrome through regulating microglial autophagy

目的 探讨大建中汤治疗腹泻型肠易激综合征内脏痛和抑郁症共病的作用机制。方法 采用慢性不可预知性刺激加葡聚糖硫酸钠方法建立腹泻型肠易激综合征内脏痛和抑郁症共病大鼠模型。大鼠随意分为正常对照组、模型组、大建中汤组[10.8 g/(kg·d)]、氟西汀组[0.01 g/(kg·d)]。治疗14 d后,采用强迫游泳评估各组大鼠的抑郁状况,腹壁撤退反应(AWR)评估各组大鼠肠道痛觉敏感性,酶联免疫吸附法检测各组大鼠前扣带回皮质(ACC)中白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、IL-10、IL-4表达;蛋白质印迹法检测ACC区髓鞘脂蛋白(PLP)、髓鞘少突胶质细胞糖蛋白(MOG)、髓鞘碱性蛋白(MBP)及微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、p62、Beclin-1,以及小胶质细胞相关蛋白精氨酸酶1(Arg1)和诱导型一氧化氮合酶(iNOS)蛋白表达;免疫荧光双标法观察ACC区自噬相关蛋白LC3-Ⅱ和MG离子钙结合衔接分子1(Iba1)共表达情况。结果 与正常组大鼠相比,共病大鼠强迫游泳时间减少(P<0.01),不动时间增加(P<0.01),在20、40、60 mmHg(1 mmHg≈0.133 kPa)3个压力下AWR分数上升(P<0.01),前扣带回皮质PLP、MOG、MBP蛋白表达下降(P<0.01),iNOS蛋白表达升高(P<0.01),促炎因子TNF-α、IL-1β升高(P<0.01),抗炎因子IL-10、IL-4水平有增加趋势,LC3-Ⅱ、Beclin-1蛋白表达下降(P<0.01),p62蛋白表达升高(P<0.01),LC3-Ⅱ、Iba1阳性细胞降低(P<0.01);与模型组大鼠比较,大建中汤组不动时间及各压力下AWR评分降低(P<0.01),ACC中PLP、MOG、MBP蛋白表达升高(P<0.01),Arg1蛋白表达升高(P<0.01)、iNOS蛋白表达下降(P<0.01),TNF-α、IL-1β下降(P<0.01),IL-10、IL-4升高(P<0.01),LC3-Ⅱ、Beclin-1蛋白表达升高(P<0.01),p62蛋白表达降低(P<0.01),LC3-Ⅱ、Iba1阳性细胞增加(P<0.01)。上述各指标,氟西汀组与大建中汤组差异均无统计学意义。结论 通过促进自噬以调控小胶质细胞清除髓鞘碎片,是大建中汤治疗腹泻型肠易激综合征内脏痛和抑郁症共病的可能作用机制。

Objective To explore the mechanism of Dajianzhong Decoction in treating visceral pain and depression associated with diarrhea irritable bowel syndrome.Methods Chronic unpredictable stimulation and dextran sodium sulfate were used to establish a rat model with visceral pain and depression associated with diarrhea irritable bowel syndrome. The rats were randomly divided into normal group, model group, Dajianzhong Decoction group [10.8 g/(kg·d)], and fluoxetine group [0.01 g/(kg·d)]. After 14 days of treatment, the depression level of the rats in each group was evaluated by forced swimming, the intestinal pain sensitivity of the rats in each group was evaluated by abdominal wall withdrawal reaction(AWR), the interleukin(IL)-1β, tumor necrosis factor-α(TNF-α), IL-10, and IL-4 expression in the anterior cingulate cortex(ACC) of the rats in each group were detected by enzyme linked immunosorbent assay. Western blotting was used to detect the expression of myelin sheath lipoprotein(PLP), myelin oligodendrocyte glycoprotein(MOG), myelin basic protein(MBP), microtubule associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ), p62, Beclin-1, microglial cell(MG)-associated protein Arg1, and inducible nitric oxide synthase(iNOS). The coexpression of autophagy related protein LC3-Ⅱ and MG calcium binding adaptor molecule 1(Iba1) in the ACC region was observed by the immunofluorescence double labeling method.Results Compared with the normal rats, the model rats showed shorter forced swimming times(P<0.01), and increased immobility times(P<0.01). The AWR fraction increased under pressures of 20, 40, and 60 mmHg. The protein levels of PLP, MOG, and MBP in the anterior cingulate cortex decreased(P<0.01), and iNOS protein increased(P<0.01). The proinflammatory factors TNF-α and IL-1β increased significantly(P<0.01). We observed an increasing trend in the levels of the anti-inflammatory factors IL-10 and IL-4;LC3-Ⅱ and Beclin-1 protein decreased significantly(P<0.01);p62 protein increased(P<0.01);and the number of cells that were positive for LC3-Ⅱ and Iba1 decreased(P<0.01). Compared with the model group, the immobility time and AWR scores under various pressures in the Dajianzhong Decoction group decreased(P<0.01). In the Dajianzhong Decoction group, the protein levels of PLP, MOG, MBP in the anterior cingulate cortex increased(P<0.01);Arg1 protein increased(P<0.01);iNOS protein decreased(P<0.01);TNF-α and IL-1β decreased(P<0.01);IL-10 and IL-4 increased(P<0.01);LC3-Ⅱ and Beclin-1 protein increased(P<0.01);p62 protein decreased(P<0.01);and LC3-Ⅱ, Iba1 positive cells increased significantly(P<0.01). There was no significant difference in the above indices between the fluoxetine group and the Dajianzhong Decoction group.Conclusion The mechanism of Dajianzhong Decoction in treating visceral pain and depression associated with diarrhea irritable bowel syndrome may involve promoting autophagy to regulate microglia to clear myelin sheath fragments.