【作者】 陈芳；

【导师】 黄秀深；

【作者基本信息】 成都中医药大学 ， 方剂学， 2018， 硕士

【摘要】 实验目的:通过对模型组大鼠尿液生成调节激素,钠钾泵活性,肝脏各类水转运蛋白表达、含量、mRNA的含量、磷酸化水平的系统研究探究湿阻中焦证对肝脏Na~+-K~+-ATP酶活性、尿液生成调节激素、水转运蛋白的影响及平胃散干预关系,探讨平胃散是否通过影响Na~+-K~+-ATP酶活性,调整尿液生成调节激素失衡状态,使肝脏水转运蛋白含量、表达、活性发生变化,维持水盐平衡,揭示尿液调节激素与水转运蛋白在湿困脾胃后的作用机制,及平胃散对肝细胞跨膜物质转运功能的调控作机理。实验方法:湿阻中焦证造模后比色法测定肝脏钠钾泵活性,过氧化物酶法检测肝脏中葡萄糖含量,HPLC检测肝脏ATP含量,ELISA测定血清中各类激素和肝脏组织中SGLT1、GLUT2、NKCC1含量、PCR法测定肝脏组织SGLT1、GLUT2、NKCC1中mRNA的含量,WB法测定肝脏组织中SGLT1、GLUT2、NKCC1磷酸化水平的影响。实验结果:1湿阻中焦证模型的评估:(1)对大鼠进行一般情况观察,湿困脾胃证大鼠形体消瘦,食欲较差,饮水明显减少,嗜睡倦卧。(2)湿邪侵袭后大鼠的胃肠运动和糖吸收异常,肝脏葡萄糖不能转化利用,ATP生成减少,钠钾泵活力降低。(3)湿困脾胃后各类水转运蛋白在肝组织免疫表达异常。(4)服药治疗后,模型组大鼠宏观体征、客观指标、病理损伤、免疫功能、代谢能力都有所改善。2尿液生成激素的含量造模后,与空白组对照,湿困脾胃证大鼠肾素、血管升压素、内皮素、肾上腺素、血管紧张素Ⅱ的含量出现降低,醛固酮和利钾尿肽的含量升高;与湿困脾胃证大鼠比较,机体进行自我修复后或服用平胃散后肾素、血管紧张素Ⅱ、心房钠尿肽的含量均高于湿困脾胃证大鼠,内皮素、醛固酮、利钾尿肽、抗利尿激素、肾上腺素的含量均低于模型组;与自然恢复组比较,给予不同剂量中药治疗组中肾素、血管紧张素Ⅱ、醛固酮、心房钠尿肽的含量高于自然恢复组,给予不同剂量的中药后,肾上腺素和血管升压素的含量低于自然恢复组大鼠;服用高剂量和中剂量中药组内皮素的含量高于恢复组,服用中药中剂量组利钾尿肽的含量高于自然恢复组,服用中药低剂量组内皮素的含量低于自然恢复组,用平胃散高剂量,和低剂量灌胃后的大鼠利钾尿肽的含量明显比自然恢复组低。3肝脏水转运蛋白的表达、含量、mRNA的含量、磷酸化活性。(1)水糖协同转运蛋白免疫表达、含量、mRNA的含量、磷酸化活性变化湿困脾胃后,与正常组进行对照,湿困脾胃证大鼠肝脏中SGLT1的蛋白免疫表达出现上调,SGLT1的含量表现为降低,mRNA的含量降低,磷酸化活性降低;经过恢复后,与湿困脾胃证大鼠进行对照,恢复组肝脏中SGLT1的蛋白表达表现为上调,SGLT1的含量会降低,mRNA的含量出现降低,磷酸化活性升高;与自然恢复组比较,给予不同剂量的中药组肝脏中SGLT1的蛋白表达上调,SGLT1的含量升高,mRNA的含量升高,SGLT1磷酸化活性降低。湿困脾胃后,与空白组进行对照,湿困脾胃证大鼠肝脏中GLUT2的蛋白表达表现为下调趋势,GLUT2的含量出现降低,mRNA的含量降低,磷酸化活性降低;与湿困脾胃证大鼠进行对照,自然恢复后肝脏中GLUT2的蛋白表达出现下调趋势GLUT2的含量升高,mRNA的含量升高,磷酸化活性升高;与恢复组比较,给予不同剂量的平胃散中药后肝脏中GLUT2的蛋白表达出现下调的趋势,GLUT2的含量高,mRNA的含量升高,平胃散低剂量组肝脏中GLUT2磷酸化活性升高,平胃散高、中剂量有降低趋势。(2)水盐协同转运蛋白免疫表达含量、mRNA的含量、磷酸化活性变化造模后,与空白组进行对照,湿困脾胃证大鼠肝脏中NKCC1的蛋白表达上调,NKCC1的含量升高,mRNA的含量降低,NKCC1磷酸化活性降低。与湿困脾胃证大鼠比较,经恢复后肝脏中NKCC1的蛋白表达出现下调,NKCC1的含量会降低,mRNA的含量升高,NKCC1磷酸化活性升高,与自然恢复组比较,平胃散高剂量组肝脏中NKCC1的蛋白表达有上调趋势,平胃散中、低剂量组肝脏中NKCC1的蛋白表达有下调趋势,不同剂量中药组NKCC1的含量升高,mRNA的含量升高,平胃散高、中、剂量组肝脏中NKCC1磷酸化活性有降低趋势,低剂量中药组肝脏中NKCC1磷酸化活性有升高趋势。(3)水通道蛋白免疫表达含量、mRNA的含量、磷酸化活性变化前期实验发现,湿阻中焦后,肝脏中水通道蛋白1和水通道蛋白4的免疫表达增强,含量,mRNA的含量及磷酸化活性都降低,服用不同剂量的中药平胃散后,水通道蛋白1和水通道蛋白4的免疫表达降低,含量,mRNA的含量及磷酸化活性都升高。实验结论:湿阻中焦证不仅会影响大鼠肾素、血管紧张素、醛固酮、内皮素、血管升压素、心房钠尿肽和利钾尿肽等水液代谢激素;还会导致水转运蛋白肝脏表现异常,SGLT1、NKCC1、AQP1、AQP4免疫表达上调,GLUT2免疫表达下调;SGLT1、GLUT2、AQP1、AQP4含量会降低,NKCC1含量升高;肝脏SGLT1、GLUT2、NKCC1、AQP1、AQP4中mRNA的含量减少;SGLT1、GLUT2、NKCC1、AQP1、AQP4磷酸化水平降低;葡萄糖升高,ATP减少,钠钾泵的活力降低。这可能会是湿阻中焦发生的重要机制。平胃散能通过增强代谢率,进而增强钠钾泵活力。通过调节血清中水液代谢调节激素的平衡,调节尿量使细胞内外电解质达到平衡。同时通过调节肝脏水转运蛋白表达、含量和磷酸化活性,平衡细胞内外的离子,从而使人体的整个水液代谢趋于平衡。更多还原

【Abstract】 Objective: A systematic study on the levels of urinary regulatory hormones,hepatic glucose ATP and Na-K-ATPase activities,the expression of various water transporters in the liver,the content of water transporter mRNA,and the level of phosphorylation in rats with dampness block and middle jiao syndrome.To explore the effect of syndrome of dampness in middle Jiao on the activity of Na-K-ATPase in liver,the effect of urine production regulating hormone,water transport protein and the intervention of Pingwei Powder,and to explore whether Pingwei Powder can regulate the imbalance of urine production regulation hormone by affecting Na-K-ATPase activity.Further affect the liver water-sugar co-transporter to regulate the state of water and liquid metabolism,and reveal some of the molecular mechanism of cell water and liquid metabolism imbalance caused by syndrome of dampness in middle Jiao and the role of Pingwei Powder in regulating cell transmembrane water transport.Method: Spleen and stomach dampness obstruction after making model,through the activity of Na K ATPase in liver was determined by chemical colorimetry after modeling in middle Jiao,glucose content in liver was detected by peroxidase method,and ATP content in liver was detected by high performance liquid chromatography(HPLC).Determination of SGLT1,GLUT2,NKCC1 in liver tissue by enzyme-linked immunosorbent assay(ELISA).The content of mRNA in liver tissue was determined by Polymerase Chain Reaction(PCR).The phosphorylation level of SGLT1,GLUT2,NKCC1 in liver tissue was determined by western blot(WB)method.Results:1 Evaluation of the Syndrome of dampness in middle Jiao model:(1)Observation through macroscopic signs,rats with dampness obstruction and middle Jiao syndrome rats significantly reduce the weight,food intake and drinking water,and poor mental state.(2)The objective index detection,gastric emptying disorder,content of serum D-xylose absorption,hepatic glucose accumulation and ATP production decreased Na-K-ATPase activity.(3)The immune expression ofvarious water transporters in the liver tissue of the spleen and stomach was abnormal.(4)After taking Chinese medicine,feeding,drinking water,weight,gastric residual rate,the intestinal propulsion rate,Speculated that Immune function,serum D-xylose absorption,liver glucose utilization,Reduction of ATP formation and reduction of sodium pump activity.It was speculated that the syndrome of dampness in middle Jiao model rats building success.2 rine production and regulation of hormone content:After animal model building,rats with dampness obstruction and middle Jiao syndrome rats compared with no module,the contents of renin,angiotensin,antidiuretic hormone,endothelin and epinephrine decreased,aldosterone and potassium natriuretic peptide increased,and atrial natriuretic peptide did not change in Rats with dampness obstruction and middle Jiao syndrome.The levels of renin,angiotensin and atrial natriuretic peptide in the rat self-repair group were higher than those in the Rats with dampness obstruction and middle Jiao syndrome,aldosterone,potassium diuretic peptide,antidiuretic hormone,endothelin and adrenaline were lower than those in Rats with dampness obstruction and middle Jiao syndrome.The contents of renin,angiotensin,aldosterone and atrial natriuretic peptide in the high,middle and low dose groups were higher than those in the rat self-repair group,and the levels of antidiuretic hormone and adrenaline in the medium and low dose groups were lower than those in the Rat self-repair group.The content of endothelin in medium dose group of Pingwei Powder was higher than that of Rat self-repair group,the content of potassium diuretic peptide in medium dose group of Pingwei Powder was higher than that of self repairing group,the content of endothelin in low dose group of Pingwei Powder was lower than that of self repairing group,and that of Pingwei Powder was high.The content of potassium diuretic peptide in low dose group was lower than that in self repairing group.3 Expression of liver water transport protein,content of mRNA and phosphorylation activity:(1)Changes of mRNA content and phosphorylation activity of water-sugar co-transporterCompared with the normal group,the expression of SGLT1 protein was up regulated in the liver of rats with damp-trapped spleen and stomach,the content of SGLT1 decreased,and the phosphorylation activity of SGLT1 decreased.After the recovery,the expression of SGLT1 protein in the liver of the recovery group was up-regulated and the content of SGLT1 decreased,and the phosphorylation activity of SGLT1 decreased.Compared with the natural recovery group,the expression of SGLT1 in the liver of the traditional Chinese medicine group was up-regulated,the content of SGLT1 increased,the content of mRNA was increased,the phosphorylation activity of SGLT1 increased and decreased.After dampness of spleen and stomach,compared with blank group,the expression of GLUT2 protein in the liver of rats with dampness trapped spleen and stomach showed that the content of GLUT2 decreased and the activity of phosphorylation decreased,while that of rats with dampness trapped spleen and stomach was compared with that of rats with dampness trapped spleen and stomach.After natural recovery,the expression of GLUT2 protein in the liver decreased,the content of mRNA was increased,the phosphorylation activity increased,and compared with the recovery group,the protein expression of GLUT2 in liver is down-regulated after different doses of Pingwei Powder.The content of GLUT2 is high and the content of GLUT2 is increasing.The activity of GLUT2 phosphorylation in liver of low dose group of Pingwei San was increased,and the activity of phosphorylation of GLUT2 in liver of low dose group was higher than that of medium dose group.(2)The content of immune expression,the content of mRNA and the change of phosphorylation activity of water salt cotransporterAfter modeling,compared with the control group,the expression of NKCC1 protein in the liver of rats with dampness trapped spleen and stomach was up-regulated,the content of NKCC1 was increased,the content of nkCC1 was increased and the phosphorylation activity of NKCC1 was decreased.Compared with the rats with dampness trapped spleen and stomach syndrome,the down-regulation of NKCC1 protein expression in the liver after recovery could decrease the content ofNKCC1 and increase the phosphorylation activity of NKCC1,as compared with the natural recovery group.The protein expression of NKCC1 was up-regulated in high dose group of Pingwei Powder,and down-regulated in low dose group and medium dose group of Pingwei Powder.The content of NKCC1 was increased in different dosage groups,and the content of NKCC1 was higher in Pingwei San group.The activity of NKCC1 phosphorylation in liver of middle dose group was decreased,while that of NKCC1 in low dose group was higher than that in low dose group.(3)The content of aquaporin immuno-expression and the change of mRNA content and phosphorylation activity of aquaporin.The results showed that after dampness trapped spleen and stomach,the expression of aquaporin1 and aquaporin4 in liver was increased,and the content of aquaporin1 and aquaporin4 in liver were decreased,and the activity of phosphorylation was decreased after taking different doses of Pingwei Powder.The immunoexpression of aquaporin1 and aquaporin4 decreased,and the content and phosphorylation activity of aquaporin1 and aquaporin4 increased.The results showed that after dampness obstruction and middle Jiao syndrome,the expression of aquaporin1 and aquaporin4 in liver was increased,and the content of aquaporin1 and aquaporin4 in liver were decreased,and the activity of phosphorylation was decreased after taking different doses of Pingwei Powder.The immunoexpression of aquaporin1 and aquaporin4 decreased,and the content and phosphorylation activity of aquaporin1 and aquaporin 4 increased.Conclusion:Syndrome of dampness in middle Jiao model not only affect the metabolism hormones of renin,angiotensin,aldosterone,endothelin,antidiuretic hormone,atrial natriuretic peptide and potassium uridine peptide,on the other hand,it leads to abnormal expression of liver water transport protein..The expression of liver water-sugar co-transporter SGLT1 and water-salt co-transporter NKCC1 was up-regulated,the expression of liver water-sugar co-transporter GLUT2 was down-regulated,and the contents of liver water-sugar co-transporter SGLT1 and GLUT2 were decreased.The content of mRNA in liver SGLT1 and GLUT2、NKCC1was decreased and reduced phosphorylation levels of GLUT2、NKCC1 and SGLT1 in the liver.Increased hepatic glucose content decreased ATP generation,Na-K-ATP enzyme activity decreased.Pingweisan can improve energy metabolism,enhanced Na-K-ATP enzyme activity in the liver,regulating serum fluid metabolism in regulating hormone balance,regulate the amount of urine cell and electrolyte balance,while the expression by regulating the liver water transport protein content and phosphorylation activity,ion balance inside and outside cells,so that the body of the whole water metabolism balance.更多还原