柴胡疏肝散对功能性消化不良大鼠胃组织线粒体功能及线粒体自噬的影响  

Effect of Chaihu Shugansan on Mitochondrial Function and Mitophagy in Gastric Tissue of Rats with Functional Dyspepsia

作　　者：李莉[1] 贾庆玲 王煜姣 王香香 凌江红 LI Li;JIA Qing-ling;WANGYu-jiao;WANG Xiang-xiang;LING Jiang-hong(Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200021,China)

机构地区：[1]上海中医药大学附属曙光医院,上海200021

出　　处：《中国实验方剂学杂志》2021年第23期26-34,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81973774)。

摘　　要：目的观察柴胡疏肝散对功能性消化不良(FD)模型大鼠胃动力、胃组织线粒体功能及线粒体自噬的影响,揭示柴胡疏肝散防治FD的作用机制。方法将32只SPF级SD大鼠适应性喂养1周后随机分为正常组、模型组、柴胡疏肝散组(4.8 g·kg^(-1)),多潘立酮组(4.5 mg·kg^(-1)),每组8只。除正常组外,其余3组均采用改良夹尾刺激法复制FD大鼠模型。4周后,采用营养性半固体糊法观察FD大鼠胃排空率,酶联免疫吸附测定法(ELISA)检测血清柠檬酸合成酶(CS),胃动素(MTL),胃泌素(GAS)含量,苏木素-伊红(HE)染色观察胃组织病理变化,透射电镜观察线粒体特征,免疫荧光共定位法观察胃组织微管相关蛋白1轻链3(LC3)与电压依赖性阴离子通道蛋白1(VDAC1)表达,提取新鲜胃组织线粒体,生化试剂盒检测线粒体活性氧(ROS),丙二醛(MDA)水平,超氧化物歧化酶(SOD)含量,蛋白免疫印迹法(Western blot)检测线粒体LC3,自噬关键分子酵母Atg6同系物(Beclin1),p62蛋白表达。结果与正常组比较,模型组大鼠胃排空率显著降低(P<0.01),血清CS,MTL,GAS含量显著降低(P<0.01),HE染色可见各组大鼠胃组织未见糜烂、溃疡等病理改变,透射电镜观察胃组织线粒体肿胀、扩张,出现空泡病变,LC3和VDAC1免疫荧光共定位表达显著增加(P<0.01),线粒体ROS,MDA含量显著升高(P<0.01),SOD含量显著降低(P<0.01),LC3,Beclin1蛋白表达水平明显升高(P<0.05,P<0.01),p62蛋白表达水平明显下降(P<0.05);与模型组比较,柴胡疏肝散组和多潘立酮组大鼠胃排空率均明显升高(P<0.05),血清CS,MTL,GAS含量明显升高(P<0.05,P<0.01),透射电镜观察胃组织线粒体核膜完整,内部嵴形态清晰,嵴密度较高,部分存在线粒体分裂融合现象,LC3和VDAC1共定位表达显著减少(P<0.01),线粒体ROS,MDA含量均明显降低(P<0.05,P<0.01),SOD含量明显升高(P<0.05),LC3,Beclin1蛋白表达水平明显降低(P<0.05,P<0.01),p62蛋白表达水平显著升高(P<0.01)�Objective To observe the effects of Chaihu Shugansan on gastric motility and mitochondrial function and mitophagy in rats with functional dyspepsia(FD)and to preliminarily reveal its mechanism in preventing and treating FD.Method Thirty-two SD rats of SPF grade were randomly divided into the normal group,model group,Chaihu Shugansan(4.8 g·kg^(-1))group,and domperidone group(4.5 mg·kg^(-1))after one week of adaptive feeding,with eight rats in each group.Rats in all groups except for the normal group were exposed to modified tail-clamping stimulation for inducing FD.Four weeks later,the semi-solid nutritive medium was used to observe the gastric emptying rate of FD rats,and the serum citrate synthase(CS),motilin(MTL),and gastrin(GAS)were detected by enzyme-linked immunosorbent assay(ELISA).The pathological changes in gastric tissue were observed by hematoxylin-eosin(HE)staining,the mitochondrial characteristics by transmission electron microscopy,and the expression of microtubule-associated protein light chain 3(LC3)and voltage-dependent anion-selective channel 1(VDAC1)by immunofluorescence co-localization.The mitochondria were extracted from fresh gastric tissue for measuring the reactive oxygen species(ROS),malondialdehyde(MDA),and superoxide dismutase(SOD)levels using the biochemical kits.The mitochondrial LC3,yeast Atg6 homologous(Beclin1),and p62 protein expression was assayed by Western blot.Result Compared with the normal group,the model group exhibited significantly reduced gastric emptying rate(P<0.01)and serum CS,MTL,and GAS levels(P<0.01).HE staining showed no pathological changes like erosion or ulcer in the gastric tissue of rats in each group.However,the mitochondria in the gastric tissue were observed swollen and dilated under the transmission electron microscope,and vacuolar lesions appeared.The co-expression of LC3 with VDAC1 increased significantly(P<0.01).The mitochondrial ROS and MDA levels were elevated significantly(P<0.01),while SOD content was significantly reduced(P<0.01).The protein expr

关 键 词：柴胡疏肝散 功能性消化不良(FD) 胃动力 线粒体功能 氧化应激 线粒体自噬 

分 类 号：R2-0[医药卫生—中医学] R289