Molecular Mechanism Research On The Regulation Of Glomerular Podocytes And Mesangial Cells By Jianpi Qushi Heluo Decoction

Chapter One Reference reviewJianpi Qushi Heluo Decoction can alleviate Chronic Glomerulo Nephritis proteinuria and hematuresis remarkable on the clinical.Meanwhile,it can also relieve renal damage and protect renal function.Currently in effect,the result of study related to physiopathology indicated that albuminuria and hematuresis will be caused by the podocytes and mesangial cells ingury.That is duo to podocytes play an important role in building Glomerular filtration barrier which prevents plasma protein passing by.MCs will overproliferation under the pathogenic stimuli.Then oppression of the capillaries will influence the normal structure of the glomerular.Albuminuria and hematuresis will follow the structural changes.In recent years,the related researches suggested that many traditional Chinese medician and Chinese herbal compound prescription can reduce the damage of podocytes and MCs.So they can relieve kidney disease by their abilities of repairation on cells.The purpose of this thesis is to explore the Jianpi Qushi Heluo Decoction's mechanism of action on renal cells.To establish the experimental basis of clinical curative effect.Chapter Two Experiment on effect of Jianpi Qushi Heluo Decoction on injury podocyteResearch Objectives:Experiment aims to induce podocyte injury model by PAN in vitro.Cells model will be cultivated by the prepared medicated serum of Jianpi Qushi Heluo Decoction.Then podocyte marker protein and proteins related to mTOR wil be detection.Result will show the effect of Jianpi Qushi Heluo Decoction on injury podocyte.And Jianpi Qushi Heluo Decoction's clinical significance will be probed.Research Methods:(1)Medicated serum of Jianpi Qushi Heluo Decoction and control group(Telmisartan and Methylprednisolone Acetate)is made by giving the experimental rats gavage administration.(2)Podocyte in vitro is cultured by 10%fetal calf serum+DMEM(cell culture fluid)in Cell incubator.(3)Podocyte injury model in vitro is induced by PAN.(4)Detect poisonous effect of every groups's medicine serum to the podocyte.Then we will detect the cell activity by CCK-8 and Microplate reader after using the drug-containing serum on culturing the podocyte damage model.This experiment will explore the influence of Jianpi Qushi Heluo Decoction for podocyte model.(5)The secretion amount of marker proteins of podocyte which is named nephrin and podocalyxin will be text by method of western blot,mTOR related proteins,too.According to the experiment data,the effection mechanism of drug-containing serum to podocyte model will be analyzed.Research Results:(1)Every groups of medicated serum do not have toxicity to podocyte.(2)PAN can cause podocyte damage.When the concentration of PAN is 100?g/ml,the injury cell of model group is 50%of normal group.It is ideal to build cell injury model at this drug concentration.(3)After cultivating ingury podocyte by the medicated serum,there is no statistically significant between nomal group,low and middle dose group(P>0.05).Compared to model group,high dose group can reduce the damage of podocyte(P<0.05).But there is no statistically significant between high dose group and control group(P>0.05).(4)Fangji is a member of Jianpi Qushi Heluo Decoction,TET can be extract from Fangji.When the concentration of TET is under 6.25 ?g/ml,no toxic effect is found for podocyte.But if the concentration of TET surpass 6.25?g/ml,toxicity will increase significantly(P<0.05).(5)Jianpi Qushi Heluo Decoction can improve the activity of podocyte,promote the expression of nephrin and podocalyxin.And the decoction is non-toxic.(6)The activity of mTOR related pathways in podocyte are induced by PAN obviously.The expression level of mTOR downstream protein is increased significantly,like P-P70S6K and P-4EBP1.Meanwhile,the model group expression level of LC3-II is reduced obviously.LC3-II levels are positively correlated with autophagy in podocyte.This data suggests that the podocyte injury which is induced by PAN is related to the higher mTOR activation and lower level of autophagy.Jianpi Qushi Heluo Decoction high dose group of medicated serum can obviously increase the LC3-II expression level of cell model,reduce the synthesis of mTOR and its downstream factors,improve the restrained level of autophagy in the cell.The western medicine control group and high dose drug-containing serum group of Jianpi Qushi Heluo Decoction have no statistically significant difference between them(P>0.05).Research Conclusion:Jianpi Qushi Heluo Decoction can promote the repair of the podocyte damage induced by PAN.Chapter Three Research on effect of Jianpi Qushi Heluo Decoction on proliferous GMCsResearch Objectives:Experiment aims to study the effect of Jianpi Qushi Heluo Decoction on the expression cententment of laminin fibronectin and Transforming growth factor-?1 which are collected from the supernatant fluid of the proliferation mesangial cell model induced by lipopolysaccharide in vitro.Then we will study the cellular mechanisms of Jianpi Qushi Heluo Decoction in treating Chronic Glomerulo Nephritis.Research Methods:Different concentration medicated serum of Jianpi Qushi Heluo Decoction and control group(Fosinopril Sodium Tablets)is made by giving the experimental rats gavage administration.Mesangial cell proliferation model induced by lipopolysaccharide is cultured by 10%fetal calf serum+DMEM(cell culture fluid)in vitro.Then we will detect the expression cententment of laminin?fibronectin and Transforming growth factor-?1 by ELISA and microplate reader after using the drug-containing serum on culturing the mesangial cell proliferation model.Research Results:(1)The proliferation of GMCs can secrete large amounts of ECM components,which is induced by lipopolysaccharide.Lipopolysaccharide effect the cells longer,the more secretion.(2)Middle and high dose medicated serum of Jianpi Qushi Heluo Decoction can retrain the expression of laminin?fibronectin and Transforming growth factor-?1.No mater action time is 24h?48h or 72h.The difference has statistical significance(P<0.05).And the more drug-containing serum concentration and action time,the more significant effect.Compared to the normal group,the difference between it and the low dose medicated serum do not have statistical significant(P>0.05).Research Conclusion:Jianpi Qushi Heluo Decoction can reduce the excessive secretion of laminin fibronectin and Transforming growth factor-?1 significantly,which are produced by the proliferation of GMCs induced by lipopolysaccharide at pathological state.Jianpi Qushi Heluo Decoction can relieve blood in the urine,protect renal function and prevent renal fibrosis clinically.Clinical application of the decoction and its appropriate dosage is safe?effective and reasonable.Chapter Four Problems and Prospect...