1.上海中医药大学附属曙光医院消化内科(上海 201203)
王煜姣,女,博士研究生,主要从事中医药防治脾胃病研究工作
凌江红,教授,博士研究生导师; E-mail:459183870@qq.com
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王煜姣,王香香,杨珂鸣,等.柴胡疏肝散对功能性消化不良大鼠AMPK/SIRT3信号通路的影响[J].上海中医药杂志,2023,57(11):5-11.
WANG Yujiao,WANG Xiangxiang,YANG Keming,et al.Effects of Chaihu Shugan Powder on AMPK/SIRT3 signaling pathway in rats with functional dyspepsia[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(11):5-11.
王煜姣,王香香,杨珂鸣,等.柴胡疏肝散对功能性消化不良大鼠AMPK/SIRT3信号通路的影响[J].上海中医药杂志,2023,57(11):5-11. DOI: 10.16305/j.1007-1334.2023.2308049.
WANG Yujiao,WANG Xiangxiang,YANG Keming,et al.Effects of Chaihu Shugan Powder on AMPK/SIRT3 signaling pathway in rats with functional dyspepsia[J].Shanghai Journal of Traditional Chinese Medicine,2023,57(11):5-11. DOI: 10.16305/j.1007-1334.2023.2308049.
目的,2,基于氧化应激及腺苷一磷酸(AMP)活化蛋白激酶(AMPK)/沉默信息调节因子3(SIRT3)信号通路探讨柴胡疏肝散干预功能性消化不良(FD)大鼠的作用机制。,方法,2,将32只SD大鼠随机分为正常组、模型组、柴胡疏肝散组、多潘立酮组,每组8只。模型组、柴胡疏肝散组、多潘立酮组均用改良夹尾刺激法造模,造模的同时正常组和模型组予质量分数为0.9%的氯化钠溶液灌胃,柴胡疏肝散组和多潘立酮组分别予柴胡疏肝散水煎液(4.8 g/kg)、多潘立酮混悬液(4.5 mg/kg)灌胃,连续干预4周。干预结束后,检测大鼠胃排空率,苏木精-伊红(HE)染色法观察胃组织病理变化,透射电镜观察胃Cajal间质细胞(ICCs)线粒体超微结构,酶联免疫吸附试验(ELISA)法检测大鼠血清活性氧(ROS)、丙二醛(MDA)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平,实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法检测大鼠胃组织,AMPK,、,SIRT3,、锰超氧化物歧化酶(,MnSOD,)mRNA表达水平,Western blot法检测大鼠胃组织磷酸化AMPK(p-AMPK)、AMPK、SIRT3、MnSOD、微管相关蛋白轻链3(LC3)、P62蛋白表达。,结果,2,①与正常组比较,模型组大鼠胃排空率降低(,P,<,0.05),ICCs线粒体肿胀、形态欠规则,分支较多,呈空泡化改变,见自噬小体;与模型组比较,柴胡疏肝散组、多潘立酮组大鼠胃排空率升高(,P,<,0.05),ICCs线粒体结构损伤减轻。②与正常组比较,模型组大鼠血清ROS、MDA水平升高(,P,<,0.05),SOD、CAT、GSH-Px水平降低(,P,<,0.05);与模型组比较,柴胡疏肝散组、多潘立酮组大鼠血清ROS、MDA水平降低(,P,<,0.05),SOD、CAT、GSH-Px水平升高(,P,<,0.05)。③与正常组比较,模型组大鼠胃组织,AMPK,、,SIRT3,、,MnSOD, mRNA表达水平降低(,P,<,0.05);与模型组比较,柴胡疏肝散组、多潘立酮组大鼠胃组织,AMPK,、,SIRT3,、,MnSOD, mRNA表达水平升高(,P,<,0.05)。④与正常组比较,模型组大鼠胃组织LC3Ⅱ蛋白表达水平升高(,P,<,0.05),SIRT3、MnSOD、P62蛋白表达水平及p-AMPK/AMPK值降低(,P,<,0.05);与模型组比较,柴胡疏肝散组、多潘立酮组大鼠胃组织LC3Ⅱ蛋白表达水平降低(,P,<,0.05),SIRT3、MnSOD、P62蛋白表达水平及p-AMPK/AMPK值升高(,P,<,0.05)。,结论,2,柴胡疏肝散可有效促进FD大鼠胃动力,其作用与激活AMPK/SIRT3通路、减轻线粒体氧化应激、抑制ICCs自噬有关。
Objective,2,To investigate the mechanism of Chaihu Shugan Powder in treating functional dyspepsia (FD) in rats through exploring oxidative stress and the AMP-activated protein kinase/sirtuin 3(AMPK/SIRT3) signaling pathway.,Methods,2,Thirty-two SD rats were randomly divided into four groups: normal group, model group, Chaihu Shugan Powder group, and domperidone group, with eight rats in each group. The model group, Chaihu Shugan Powder group, and domperidone group were modeled using the modified tail-clamping stimulation method. The normal group and model group were administered 0.9% sodium chloride solution by gavage, while the Chaihu Shugan Powder group and domperidone group were administered Chaihu Shugan Powder aqueous decoction (4.8 g/kg) and domperidone suspension (4.5 mg/kg) by gavage for four consecutive weeks. After the intervention, the gastric emptying rate of the rats was detected, hematoxylin-eosin (HE) staining was used to observe the histopathological changes in the gastric tissue, transmission electron microscopy was used to observe the mitochondrial ultrastructure of gastric interstitial cells (ICCs), and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in the rat serum. The mRNA expression levels of ,AMPK,, ,SIRT3,, and manganese superoxide dismutase (,MnSOD,) in the rat gastric tissue were quantified using real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Additionally, Western blot assay was used to detect the protein expression levels of phosphorylated AMPK (p-AMPK), AMPK, SIRT3, MnSOD, microtubule-associated protein light chain 3 (LC3), and P62 in the rat gastric tissue.,Results,2,①The model group of rats exhibited a lower gastric emptying rate than the normal group (,P,<,0.05). Additionally, the mitochondria of ICCs in the model group displayed characteristics such as swelling, irregular morphology, increased branching, vacuolization, and the presence of autophagic vesicles. The gastric emptying rate was higher in both the Chaihu Shugan Powder group and the domperidone group than that in the model group (,P,<,0.05), and there was a reduction in damage to the ICCs mitochondrial structure in both groups. ②Compared with the normal group, the model group showed elevated serum levels of ROS and MDA (,P,<,0.05) and decreased levels of SOD, CAT, and GSH-Px (,P,<,0.05). However, compared with the model group, the Chaihu Shugan Powder group and the domperidone group exhibited decreased serum levels of ROS and MDA (,P,<,0.05) and increased levels of SOD, CAT, and GSH-Px (,P,<,0.05). ③The mRNA expression levels of ,AMPK,, ,SIRT3,, and ,MnSOD ,in the gastric tissues of rats were reduced in the model group compared with those levels in the normal group (,P,<,0.05). However, both the Chaihu Shugan Powder group and the domperidone group showed higher mRNA expression levels of ,AMPK,, ,SIRT3,, and ,MnSOD, in the rat gastric tissues than the model group (,P,<,0.05). ④The protein expression level of LC3Ⅱ in the rat gastric tissues was higher in the model group than that in the normal group (,P,<,0.05). Furthermore, the protein expression levels of SIRT3, MnSOD, P62, and the ratio of p-AMPK/AMPK were lowered (,P,<,0.05). Conversely, the protein expression level of LC3Ⅱ in the rat gastric tissues was lower in both the Chaihu Shugan Powder group and the domperidone group than that in the model group (,P,<,0.05), and the protein expression levels of SIRT3, MnSOD, P62, and the ratio of p-AMPK/AMPK were increased in both the Chaihu Shugan Powder group and the domperidone group (,P,<,0.05).,Conclusion,2,Chaihu Shugan Powder can effectively promote gastric motility in rats with FD by activating the AMPK/SIRT3 pathway, reducing mitochondrial oxidative stress, and inhibiting autophagy in ICCs.
功能性消化不良柴胡疏肝散经典名方中医药疗法作用机制氧化应激自噬大鼠
functional dyspepsiaChaihu Shugan Powderclassic Chinese prescriptionstraditional Chinese medicine therapymechanism of actionoxidative stressautophagyrat
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