| Porpuse: Allergic purpura nephritis(APN)is seriously harmful disease to children’s health,Inflammation plays a crucial role in the progress of mesangial proliferative glomerulonephritis.However,there is a lack of effective therapeutic medicine in clinical practice.Large number of hormones was used to treat APN in western medicine clinic,which bring a lot of side effects to the growth of children.Traditional Chinese Medicine therapy treated both symptoms and causes of disease,so the curative effect is good to complicated illness.Professor Zhang Jun,a veteran traditional Chinese medicine doctor in the affiliated hospital of our school,has established "Qi Ji Shenkang" for more than 30 years,and the cure rate of apn in children is 78.5 %.Enven thogh,the prescription has good therapeutic effect on APN,it is difficult for children to take it clinically,so it is urgent to optimize the prescription by reducing the medicine ingredients,making the presctiption is beneficial to the use of medicine for children.Based on the fact that the chemical composition is the pharnacodynamic material basis of the prescription of traditional Chinese Medicine,and the principle that "only the chemical components contained in the preparation can be the effective components of the prescription",the meidicine of the prescription is refined,and the pharmacodynamics of the original prescription is studied in parallel to evaluate and to verify the rationality of prescription optimization.All result of this research could provide a basis for the development and utilization of the prescription.Method:(1)Prescription optimization: on the basis of CKNI,Pubmed,Science finder and other databases,the reference chemical component of single medicine in the "Qi Ji Shenkang" prescription were colleted,and the database of "Qi Ji Shenkang" was established using the format of Waters mass spectrometry Unify database.In the positive and negative ion mode,to establish a UPLC-Q-TOF-MS/MS analytical method,comprehensively reflect and fastly explain chemical compositions of Qi Ji Shenkang granules and to accurately classify and identify the main chemical components peaks.The tesed MS and MS/MS spectrum data were inputed into the database established by ourself and the database provided by Waters Company to identify the structure of chemical constituents in Qi Ji Shenkang granules.The pseudo-molecular ions and mass fragmentation were took together to identify the structure of constituents in Qi Ji Shenkang granules and the mass fragmentation rule of different chemical sturcure types was investigated.By comparison with the compnents of single medicine in Qi JiShenkang granules,the main medicin of screpertion were identified.The prescription was optimized according to the result of UPLC-Q-TOF-MS/MS analysis and the doctor’s clinical experience.(2)APN and Ig A nephropathy had same pathogenesis,such as Ig A protein precipitated in glomeruli,proliferation of glomerular Mesangial cells,glomerular fibrosis and hypercoagulable state of renal blood,etc.,so Ig A nephropathy was used as an animal model for pharmacodynamic study.The Ig A nephropathy model of SD rats was established by oral administration of bovine serum albumin(BSA),at a dose of 400 mg/kg by intragastric administration every other day for 8 weeks,followed by subcutaneous injection of castor oil 0.50 ml + CCl4 0.10 m L once a week for 9 weeks,combined with lipopolysaccharide LPS(0.05 mg/ was injected through tail vein at the 6th and 8th week respectively).With reference to the gold standard of clinical renal injury,hematuria,albuminuria,creatinine,urea nitrogen and sections were used as indexes to investigate the effect of Qi Ji Shenkang prescription on renal injury in model rats.At the same time,cell experiments were carried out with Mesangial cell line and vascular endothelial cell line,which are the effector cells of renal injury.Mesangial cell proliferation model was prepared with angiotensin II(Ang II)and vascular endothelial cell injury model was established with hydrogen peroxide(H2O2).MTT,ELISA and Western blot techniques were used to reveal the action mechanism of Qi Ji Shenkang from the point of view of inhibiting the release of cellular factors IL-6,TNF-α,TGF-β,MCP-1,NF-κ B,PAI-1 and related proteins.To evaluate the rationality of prescri Ption optimization by compare the effects of Qi Ji Shenkang before and after optimization.Result:(1)A total of 282 components,including 118 monomer components and 164 isomers,were identified from Qiji Shenkang granules.According to the source of these ingredients in single medicin in the prescription,it is suggested that seven medicines in the original prescription,such as Radix Astragali,small thistle,Salvia miltiorrhiza,Cortex moutan,Herba Astragali,Herba Crane,Glycyrrhiza uralensis,etc.,are the main medicine,and the new prescription is formed by adjusting the dose ratio of main medicine and adding Sabia japonica.The daily dose of the new prescription is 40% lower than that of the original prescription,and can be made into granules with a single dose of 5 g,which can meet the requirements for children.(2)The results of whole animal experiment showed that: compared with the model group,the count of red blood cells and the content of protein in urine(P<0.05)was significantlyreduced in by original and optimized Qi Ji Shenkang,also the circulating immune complex in serum of rats with Ig A nephropathy was reduced(P<0.05),the contents of blood urea nitrogen and creatinine in rats was decreased(P < 0.05),and there was no significant difference between original and optimized Qi Ji Shenkang groups.Under light microscope,the kidney tissue of histological section of the blank group showed that the glomerular volume was normal,the Mesangial area had no obvious proliferation,the Mesangial matrix did not increase,and the capillary loop opened well.Histological section of the model group showed that the glomerular volume increased and the Mesangial area widened obviously,Mesangial cells and Mesangial matrix proliferated moderately and severely,and crescent formation and obvious sclerosing damage were rarely seen;some tubular epithelial turbid swelling,focal epithelial cell exfoliation,renal interstitial lymphocyte invasion and fibrosis.Histological section of original and the optimizated Qi Ji Shenkang granule prescription gourps showed that he glomerular volume of rats in each group increased slightly,the width of Mesangial area was not obvious,Mesangial cells and Mesangial matrix were mainly mild hyperplasia,no crescent formation and sclerosing,a few had tubule epithelial turbid swelling,a few renal interstitium had lymphocyte infiltration and fibrosis.The result suggested that before and after optimized Qi Ji Shenkang granule had the effect of inhibiting kidney injury.The experiment of Mesangial cells showed that after HBZU-1 was treated with Ang Ⅱ at the concentration of 10-5～10-6mol/L,the cells in the model group proliferated significantly(P< 0.05).Compared with the model group,Qi Ji Shenkang group before and after optimization could significantly inhibit the proliferation of Mesangial cells,reduce the release of IL-6,TNF-α,TGF-β,MCP-1 and NF-κ B by Mesangial cells,and inhibit the protein expression of NF-κ B and the phosphorylation of P65(Ser 276)site(P < 0.05),but there was no significant difference between the two groups(P > 0.05).The experiment of vascular endothelial cells showed that H2O2 at the concentration of10-7mol/L could damage the endothelial cells,and the number of endothelial cells decreased significantly(P < 0.01).Compared with the model group,Qi Ji Shenkang group could significantly Protect vascular endothelial cells from damage and reduce the release of PAI-1before and after optimization(P<0.05),but there was no significant difference between the two groups(P > 0.05).Conclusion:(1)Based on the method of LC-MS trace back compound preparations,can quickly identify compound chemical composition,and can take advantage of the compound chemical composition in the effective ingredients in reverse tracking effectively.(2)Results of animal experiment and cell experiment suggested that Qi Ji Shenkang could inhibit the injury of APN,inhibit the proliferation of Mesangial cells and the release of factors,and protect vascular endothelial cells.Thus Qi Ji Shenkang had the effect of inhibiting glomerulosclerosis,regulating the disorder of blood coagulation mechanism of APN,and promoting blood circulation,removing blood stasis,cooling blood and detoxification.The efficacy of the optimized prescription is similar to that of the original prescription,which suggests that the optimization of the prescription guided by the chemical composition of the prescription is reasonable and feasible. |
PDF Full Text Request