疏肝理气法通过PI3K/PDK1/Akt信号通路调控胃Cajal间质细胞凋亡的机制研究
|
摘要
目的探究疏肝理气法对胃Cajal间质细胞凋亡的调控作用及可能的作用机制。方法选用120只Wistar大鼠,平均分为三组进行处理:对照组,阳明腑实证组和疏肝理气法治疗组。阳明腑实证造模成功后,疏肝理气法治疗组每日灌服柴胡疏肝散水煎剂,连续灌胃处理5 d,阳明腑实证组和对照组每日灌服等量的0.9%氯化钠,连续处理5d。处理20 d后,观察各组大鼠的存活率,检测存活大鼠的平均体重;利用放射免疫法检测血浆P物质(SP)水平;分离各组大鼠胃Cajal间质细胞,利用流式细胞仪检测各组细胞凋亡情况;Western-blot检测胃Cajal间质细胞中凋亡相关蛋白和PI3K/PDK1/Akt信号通路相关蛋白表达情况。结果处理20 d后,阳明腑实证组大鼠存活率、存活大鼠平均体重、SP含量均显著低于对照组(P<0.05),疏肝理气法治疗组大鼠存活率、存活大鼠平均体重、SP含量显著高于阳明腑实证组(P<0.05)。阳明腑实证组细胞凋亡率及Bax、Cleaved-caspase3、Cleaved-caspase9蛋白表达显著高于对照组,Bcl-2、PI3K、PDK1、Akt、p-Akt蛋白表达显著低于对照组(P<0.05),疏肝理气法治疗组细胞凋亡率及Bax、Cleavedcaspase3、Cleaved-caspase9蛋白表达均显著低于阳明腑实证组,Bcl-2、PI3K、PDK1、Akt、p-Akt蛋白表达显著高于阳明腑实证组(P<0.05)。结论疏肝理气法可能通过PI3K/PDK1/Akt信号通路减少由阳明腑实证引起的胃Cajal间质细胞凋亡,有效改善胃肠功能。
Objective To investigate the effect of soothing liver and regulating the qi method on gastric Cajal interstitial cell apoptosis and its possible mechanism. Methods 120 Wistar rats were divided into three groups: control group,yangmingfushi syndrome group and soothing liver and regulating the qi method group. After the success of the model,the patients in the treatment group were treated with Chaihu Shugan San Decoction,and the rats in the control group and the control group were fed with the same amount of 0. 9% Sodium chloride,continuous treatment for5 d. After 20 days treatment,the survival rate of rats in each group was observed,and the average weight of the surviving rats was measured. Plasma substance P( SP) levels were measured by radioimmunoassay. The Cajal interstitial cells in the stomach of each group rats were separated,and the apoptosis of each group was detected by flow cytometry. The expression of apoptosis related proteins and PI3K/PDK1/Akt signaling pathway related proteins in stomach Cajal cells of was detected by Western-blot. Results After 20 days,the survival rate,the average body weight and SP content of the rats in the yangmingfushi group were significantly lower than those in the control group( P < 0. 05). The survival rate,the average body weight and SP content of the rats treated with soothing liver and regulating qi method were significantly higher than those of the yangmingfushi Group( P < 0. 05). The apoptosis rate and Bax,Cleaved-caspase3,Cleaved-caspase9 protein expression in the soothing liver and regulating the qi method group were significantly higher than those in the control group,and the expression of Bcl-2,PI3 K,PDK1,Akt and p-Akt protein was significantly lower than that in the control group( P < 0. 05); cell apoptosis rate and Bax,Cleaved-caspase3 and Cleaved-caspase9 protein expression in soothing liver and regulating the qi method group were significantly lower than that of yangmingfushi syndrome group,the expression of Bcl-2,PI3 K,PDK1,Akt,p-Akt protein was significantly higher than yangmingfushi syndrome group( P < 0. 05). Conclusion Soothing liver and regulating the qi method may reduce the apoptosis of Cajal interstitial cells which induced by the yangmingfushi syndrome through the PI3K/PDK1/Akt signal pathway,and effectively improve the gastrointestinal function.
Objective To investigate the effect of soothing liver and regulating the qi method on gastric Cajal interstitial cell apoptosis and its possible mechanism. Methods 120 Wistar rats were divided into three groups: control group,yangmingfushi syndrome group and soothing liver and regulating the qi method group. After the success of the model,the patients in the treatment group were treated with Chaihu Shugan San Decoction,and the rats in the control group and the control group were fed with the same amount of 0. 9% Sodium chloride,continuous treatment for5 d. After 20 days treatment,the survival rate of rats in each group was observed,and the average weight of the surviving rats was measured. Plasma substance P( SP) levels were measured by radioimmunoassay. The Cajal interstitial cells in the stomach of each group rats were separated,and the apoptosis of each group was detected by flow cytometry. The expression of apoptosis related proteins and PI3K/PDK1/Akt signaling pathway related proteins in stomach Cajal cells of was detected by Western-blot. Results After 20 days,the survival rate,the average body weight and SP content of the rats in the yangmingfushi group were significantly lower than those in the control group( P < 0. 05). The survival rate,the average body weight and SP content of the rats treated with soothing liver and regulating qi method were significantly higher than those of the yangmingfushi Group( P < 0. 05). The apoptosis rate and Bax,Cleaved-caspase3,Cleaved-caspase9 protein expression in the soothing liver and regulating the qi method group were significantly higher than those in the control group,and the expression of Bcl-2,PI3 K,PDK1,Akt and p-Akt protein was significantly lower than that in the control group( P < 0. 05); cell apoptosis rate and Bax,Cleaved-caspase3 and Cleaved-caspase9 protein expression in soothing liver and regulating the qi method group were significantly lower than that of yangmingfushi syndrome group,the expression of Bcl-2,PI3 K,PDK1,Akt,p-Akt protein was significantly higher than yangmingfushi syndrome group( P < 0. 05). Conclusion Soothing liver and regulating the qi method may reduce the apoptosis of Cajal interstitial cells which induced by the yangmingfushi syndrome through the PI3K/PDK1/Akt signal pathway,and effectively improve the gastrointestinal function.
引文
[1]Klein S,Seidler B,Kettenberger A,et al.Interstitial cells of Cajal integrate excitatory and inhibitory neurotransmission with intestinal slowwave activity[J].Nat Commun,2013,4(5):1630.
[2]Lee SJ,Hwang CS,Kim A,et al.Gastrointestinal tract spindle cell tumors with interstitial cells of Cajal:Prevalence excluding gastrointestinal stromal tumors[J].Oncol Lett,2016,12(2):1287-1292.
[3]Youm JB,Leem CH,Lee SR,et al.Modeling of stochastic behavior of pacemaker potential in interstitial cells of Cajal[J].Prog Biophys Mol Biol,2014,116(1):56-69.
[4]杨裕忠,王新芳.大承气汤大黄后下时间对阳明腑实证的临床疗效观察[J].世界中医药,2014,(7):904-907.
[5]王琦,秦大莲,熊玉霞.肠源性内毒素血症与阳明腑实证的研究概况[J].北方药学,2014,11(4):76-77.
[6]徐义勇,艾志福,朱丽娟.疏肝理气润肠法治疗便秘型肠易激综合征的疗效及其调节胃肠激素的研究[J].时珍国医国药,2014,25(9):2192-2194.
[7]荀翀,陈海龙.阳明腑实证大鼠模型的制备及其病理机制研究[J].大连:大连医科大学,2009.
[8]Pelaseyed T,Bergstr9m J H,Gustafsson J K,et al.The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system[J].Immunol Rev,2014,260(1):8-20.
[9]白焕焕,周长丽,刘立坤,等.粪便微生物移植与胃肠外相关疾病的研究进展[J].中国老年学杂志,2016,36(4):1005-1008.
[10]Farrugia G,Szurszewski JH.Carbon monoxide,hydrogen sulfide,and nitric oxide as signaling molecules in the gastrointestinal tract[J].Gastroenterology,2014,147(2):303-313.
[11]Li H,Chen Y,Liu S,et al.Long-pulse gastric electrical stimulation protects interstitial cells of Cajal in diabetic rats via IGF-1 signaling pathway[J].World J Gastroenterol,2016,22(23):5353.
[12]Padhi S,Sarangi R,Mallick S.Pancreatic extragastrointestinal stromal tumors,interstitial Cajal like cells,and telocytes[J].JOP,2013,14(1):1-14.
[13]Kant V,Gopal A,Kumar D,et al.Topically applied substance P enhanced healing of open excision wound in rats[J].Eur J Pharmacol,2013,715(1):345-353.
[14]仇莎,杨秀丽,司增顺,等.胃肠ICC网络中P物质的研究进展及中药干预现状[J].世界中西医结合杂志,2013,8(8):861-864.
[15]Lu H,Wang C,Hao L,et al.The expression and significance of programmed cell death 5 and Bcelllymphoma/lewkmia-2 in sinonasal squamous cell carcinoma[J].Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi,2014,28(17):1301-1304.
[16]雷艳,雷雨.结肠癌患者血清IL-8与抗凋亡基因Bcl-2、促凋亡基因Bax的相关性研究[J].临床和实验医学杂志,2014,13(22):1851-1853.
[17]Groe L,Wurm CA,Brüser C,et al.Bax assembles into large ringlike structures remodeling the mitochondrial outer membrane in apoptosis[J].EMBO J,2016,35(4):402-413.
[18]Sikdar S,Mukherjee A,Ghosh S,et al.Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cellproliferation in lung cancer,in vitro and in vivo[J].Environ Toxicol Pharmacol,2014,37(1):300-314.
[19]Han J,Hou W,Goldstein LA,et al.A Complex between Atg7 and Caspase-9 A NOVEL MECHANISM OF CROSS-REGULATION BETWEEN AUTOPHAGY AND APOPTOSIS[J].J Biol Chem,2014,289(10):6485-6497.
[20]Yu W,Ma S,Wang L,et al.Upregulation of GPR34 expression affects the progression and prognosis of human gastric adenocarcinoma by PI3K/PDK1/AKT pathway[J].Histol Histopathol,2013,28(12):1629-1638.
[21]Ohtsubo K,Yamada T,Zhao L,et al.Expression of Akt kinase-interacting protein 1,a scaffold protein of the PI3K/PDK1/Akt pathway,in pancreatic cancer[J].Pancreas,2014,43(7):1093-1100.
[22]Jung Y,Yi YS,Yoo DS,et al.8-(Tosylamino)quinoline inhibits tumour progression through targeting phosphoinositide-3-kinase/Akt pathway[J].Pharmazie,2013,68(2):146-152.
[2]Lee SJ,Hwang CS,Kim A,et al.Gastrointestinal tract spindle cell tumors with interstitial cells of Cajal:Prevalence excluding gastrointestinal stromal tumors[J].Oncol Lett,2016,12(2):1287-1292.
[3]Youm JB,Leem CH,Lee SR,et al.Modeling of stochastic behavior of pacemaker potential in interstitial cells of Cajal[J].Prog Biophys Mol Biol,2014,116(1):56-69.
[4]杨裕忠,王新芳.大承气汤大黄后下时间对阳明腑实证的临床疗效观察[J].世界中医药,2014,(7):904-907.
[5]王琦,秦大莲,熊玉霞.肠源性内毒素血症与阳明腑实证的研究概况[J].北方药学,2014,11(4):76-77.
[6]徐义勇,艾志福,朱丽娟.疏肝理气润肠法治疗便秘型肠易激综合征的疗效及其调节胃肠激素的研究[J].时珍国医国药,2014,25(9):2192-2194.
[7]荀翀,陈海龙.阳明腑实证大鼠模型的制备及其病理机制研究[J].大连:大连医科大学,2009.
[8]Pelaseyed T,Bergstr9m J H,Gustafsson J K,et al.The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system[J].Immunol Rev,2014,260(1):8-20.
[9]白焕焕,周长丽,刘立坤,等.粪便微生物移植与胃肠外相关疾病的研究进展[J].中国老年学杂志,2016,36(4):1005-1008.
[10]Farrugia G,Szurszewski JH.Carbon monoxide,hydrogen sulfide,and nitric oxide as signaling molecules in the gastrointestinal tract[J].Gastroenterology,2014,147(2):303-313.
[11]Li H,Chen Y,Liu S,et al.Long-pulse gastric electrical stimulation protects interstitial cells of Cajal in diabetic rats via IGF-1 signaling pathway[J].World J Gastroenterol,2016,22(23):5353.
[12]Padhi S,Sarangi R,Mallick S.Pancreatic extragastrointestinal stromal tumors,interstitial Cajal like cells,and telocytes[J].JOP,2013,14(1):1-14.
[13]Kant V,Gopal A,Kumar D,et al.Topically applied substance P enhanced healing of open excision wound in rats[J].Eur J Pharmacol,2013,715(1):345-353.
[14]仇莎,杨秀丽,司增顺,等.胃肠ICC网络中P物质的研究进展及中药干预现状[J].世界中西医结合杂志,2013,8(8):861-864.
[15]Lu H,Wang C,Hao L,et al.The expression and significance of programmed cell death 5 and Bcelllymphoma/lewkmia-2 in sinonasal squamous cell carcinoma[J].Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi,2014,28(17):1301-1304.
[16]雷艳,雷雨.结肠癌患者血清IL-8与抗凋亡基因Bcl-2、促凋亡基因Bax的相关性研究[J].临床和实验医学杂志,2014,13(22):1851-1853.
[17]Groe L,Wurm CA,Brüser C,et al.Bax assembles into large ringlike structures remodeling the mitochondrial outer membrane in apoptosis[J].EMBO J,2016,35(4):402-413.
[18]Sikdar S,Mukherjee A,Ghosh S,et al.Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cellproliferation in lung cancer,in vitro and in vivo[J].Environ Toxicol Pharmacol,2014,37(1):300-314.
[19]Han J,Hou W,Goldstein LA,et al.A Complex between Atg7 and Caspase-9 A NOVEL MECHANISM OF CROSS-REGULATION BETWEEN AUTOPHAGY AND APOPTOSIS[J].J Biol Chem,2014,289(10):6485-6497.
[20]Yu W,Ma S,Wang L,et al.Upregulation of GPR34 expression affects the progression and prognosis of human gastric adenocarcinoma by PI3K/PDK1/AKT pathway[J].Histol Histopathol,2013,28(12):1629-1638.
[21]Ohtsubo K,Yamada T,Zhao L,et al.Expression of Akt kinase-interacting protein 1,a scaffold protein of the PI3K/PDK1/Akt pathway,in pancreatic cancer[J].Pancreas,2014,43(7):1093-1100.
[22]Jung Y,Yi YS,Yoo DS,et al.8-(Tosylamino)quinoline inhibits tumour progression through targeting phosphoinositide-3-kinase/Akt pathway[J].Pharmazie,2013,68(2):146-152.