| Purpose:IgAN nephropathy,the most common pathological type of kidney disease in China,is taken as the research object.Introducing the theory of "collateral disease" of traditional Chinese medicine of "tonifying kidney collaterals,clearing kidney collaterals and eliminating kidney collaterals".To explore the therapeutic effect of Qiji Shenkang decoction,which is "nourishing,clearing and eliminating kidney collaterals",and its relationship with ERK pathway,MCP-1 and NF-kB cytokines.A new spontaneous IgAN mouse model is constructed and evaluated by transgenic methods,and in vivo experiments and in vitro experiments of human glomerular mesangial cells are carried out.The effects of Qiji Shenkang Decoction with different drug concentrations on IgAN are observed from molecular and cell levels.To clarify the modern biological mechanism of IgAN lesions;To reveal the scientific connotation of IgAN’s "kidney collateral disease" theory.Material and method:1.Modeling method and evaluation of transgenic IgAN animal models: Cooperate with the Institute of Medical Experimental Animals,Chinese Academy of Medical Sciences to complete the work of gene customization,mutation production,vector construction,vector purification,microinjection,embryo transfer,first-time mouse genotype identification,etc.To construct and evaluate transgenic IgAN mice with IGHA1 mutation,marked as IGHA1-MUT group,and the background strain of transgenic mice is C57BL/6J.2.breeding,breeding and identification of transgenic mice:(1)breeding: after the introduction of IGHA1-mutF0 generation of mice,the mice were bred in SPF laboratory of laboratory animal center of Liaoning university of traditional Chinese medicine,with clean internal environment,wind pressure,temperature of 20-25℃,relative humidity of 30%-50%,noise <<60dB,ultraviolet lamp irradiation sterilization for 2 hours every day in the breeding room,and regular cleaning and disinfection.The feed is 60Coγ-ray sterilized sterile full-value nutritional granules.Pregnant and productive female mice are fed with a small amount of egg yolk or sunflower seeds.Irregular fillers ensure sufficient feed.Drinking water is purified water,bottled,autoclaved,and changed 2-3 times a week,with continuous drinking water;Aseptic mixed sawdust or corncob is used as padding;Autoclaved mice were raised in cages.Squirrel cage and padding shall be replaced at any time to keep clean and sterile.Closely observe the diet and activities of mice,record the environmental data and the status of mice.(2)breeding: the age for the initial mating of mice is 60-90 days.F0 generation IGHA1-mut transgenic mice were mated with wild type C57BL/6J mice respectively,female IGHA1-mut mice mated with wild type male mice(1:1),male IGHA1-mut mice mated with wild type female mice(1:2),and were delivered in a single cage,suckled and weaned.F1 generation pups developed well,suckled for 23 days,then separated male and female.At the same time make records.(3)Screening of positive mice: The tail tips of F1 offspring mice were cut for tissue DNA extraction and genotype identification on 23 days,and the mice with positive genotype were screened out from F1 offspring.3.Evaluation method of transgenic IgAN animal model: F1 mice of IGHA1-mut genotype group collected urine samples at 8 weeks old,12 weeks old and 24 weeks old respectively to observe the characteristics,and kidney tissues were taken for IgA immunofluorescence staining and H-E staining,hexamethylene silver staining,MASSON staining and PAS staining to evaluate abnormal immune complex deposition and renal histological changes.Blood was collected from eyeball to separate serum and IgA level was detected.Wild mice of the same age and strain were set as control group at each time point(n=6).4.The therapeutic effect evaluation and dose-effect relationship of Qiji Shenkang Decoction on IgAN mice in treating glomerulosclerosis: C57BL/6J wild-type mice were the blank group;IGHA1-mut genotype transgenic IgAN mice were divided into blank group,model group,western medicine group(telmisartan group)and high,medium and low dose groups of Qiji Shenkang decoction.The general state of mice was observed at 14 days,21 days and 28 days after intragastric administration in each group.Observe urine character;Histomorphology was used to observe the histological changes of kidney in mice.PCR,Western-blot and ELISA were used to detect the expression of ERK1/2,pERK1/2,MCP-1,NF-kB genes and proteins.By comparing the data of each group,the therapeutic effect and dose-effect relationship of Qiji Shenkang Decoction on IgAN mice were evaluated,and its theoretical basis was discussed.5.Preparation of pharmacological serum of Qiji Shenkang Decoction: The decoction method of Qiji Shenkang Decoction refers to the relevant regulations of state administration of traditional chinese medicine and is prepared by the decoction room of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine.The crude drug amounts of high,medium and low dosage groups are 139.32 g/(kg d)respectively in the high dosage group of traditional Chinese medicine.46.44 g/(kg d)in the middle dosage group of traditional Chinese medicine;15.48 g/(kg d)in low dose group of traditional Chinese medicine.After grinding,telmisartan tablets was dissolved in distilled water to 1mg/ml of liquid medicine.The rats were divided into five groups(n=8),namely western medicine group(telmisartan group),high,medium and low dose groups of traditional Chinese medicine and normal serum group.The rats were fed adaptively for 3 days at the first time,and the rats were fed with each dose group of traditional Chinese medicine and the western medicine group according to the 2ml/100 g intragastric dose.The normal rat serum was prepared into normal saline for intragastric administration once a day for 5 days.Aseptic operation was carried out 1 h after the last administration.After anesthesia,blood was collected from abdominal aorta and serum was separated.The blood was allowed to stand at room temperature for 2h,centrifuged at 4℃ for 3,000 r/min for 20 min,and the serum was taken.The drug-containing serum was placed in a 56℃ water bath box and inactivated in a water bath for 30 min.Inactivated serum is filtered,sterilized and subpackaged,and placed in a refrigerator at-20 deg.c for later use6.Culture and grouping of human glomerular mesangial cells: Human glomerular mesangial cells(HGMC)are inoculated in a 75cm2 culture flask,RPMI-1640 culture solution containing 10% calf serum is added,and the cells are placed in an incubator with a constant temperature of 37oC and a carbon dioxide content of 5% to adhere to the wall for growth,the solution is changed every other day,and the orifice plate is inoculated when the adherence of the cells reaches 80%-90% fusion.Inoculating a 24-well culture plate for 24 hours,the blank group was still given RPMI-1640 culture solution containing 10% calf serum,the model group was given RPMI-1640 culture solution containing 10% calf serum containing AngⅡ,and each drug intervention group was replaced with RPMI-1640 culture solution containing 10% pharmacological serum.After continuous culture for 48 hours and 72 hours,cell suspensions were collected respectively at different time points,and rinsed twice with PBS buffer solution first.Add 1.5 mL pancreatin for digestion for 4 ~ 5 min.When the edge of cells is reduced and basically separated from the bottom of the bottle under the microscope,add 12 mL of complete culture solution to stop digestion,repeatedly blow the bottle wall cells,collect the cell suspension in a 15 mL centrifuge tube,centrifuge for 10 min at 2000r/min,suck out the supernatant,and add the complete culture solution to prepare a cell suspension with cell density of 1.0×105mL-1 for later use.7.Effect of Qiji Shenkang Decoction on the Molecular Mechanism of HGMC by Regulating ERK Signal Transduction Pathway: HGMC is cultured in vitro and AngII is applied to induce HGMC proliferation,which is divided into blank group,model group(AngII group),Qiji Shenkang Decoction high and low dose group and western medicine group(telmisartan).MTT assay was carried out at 24 h,48h and 72 h after pharmacological serum was applied to observe cell proliferation.Gene and protein expression was detected at 48 h and 72 h after pharmacological serum was applied according to MTT results: ERK1/2,pERK1/2,MCP-1 and NF-kB protein secretion levels were detected by ELISA.RT-PCR was used to determine the mRNA expression of MCP-1 and NF-kB genes.The protein expression of MCP-1 and NF-kB genes was detected by western-blot.By comparing the data of each group,the mechanism and target of Qiji Shenkang Decoction in treating IgAN were discussed.Results:1.The tail tips of IGHA 1-mut transgenic F1 mice were cut 23 days after birth for tissue DNA extraction,and genotype positive mice were screened from F1 generation.The birth rate,survival rate and sex ratio of F1 generation mice are normal,and the PCR positive rate is between 40% and 60%,which conforms to the genetic law.No macroscopic hematuria or foam was found in urine at 8 weeks,12 weeks and 24 weeks of age.HE staining of kidney tissue showed that slight mesangial cell proliferation was observed at 8 weeks of age in IGHA1-mut group,while mesangial cells and mesangial matrix increased significantly at 12 weeks of age and 24 weeks of age in IGHA1-mut group.Immunofluorescence staining showed that IgA deposition was obvious in the mesangial region of positive mice in IGHA1-mut transgenic group at 12 weeks old and 24 weeks old,which was consistent with IgAN pathological changes.MASSON,PAS and silver hexamethylene staining all confirmed from different angles that renal pathological changes were not obvious at 8 weeks of age,but obvious at 12 weeks of age.Immunocomplex deposition and extracellular matrix increase were observed in mesangial region.At 24 weeks of age,pathological changes were further aggravated with basement membrane thickening and gap widening,but renal interstitium was not accumulated.Western-blot method was used to detect the expression of IgA protein.It was found that the expression of IgA protein increased abnormally at 8 weeks old.The expression level of IgA protein was highest at 12 weeks old and maintained at a high level at 24 weeks old.Referring to the evaluation results of the model,12 weeks old mice were selected for further in vivo experiments of IGHA1-mut transgenic mice.2.F1 mice with positive IGHA1-mut genotype were selected as animal models for in vivo experiments.According to the results of experiment 1,mice were given drugs at 12 weeks old.Samples were collected on 14 days,21 days and 28 days respectively.The changes of kidney tissue,cytokine genes and proteins and serum cytokine secretion were observed from different angles by IgA immunofluorescence staining,HE staining,MASSON staining,PAS staining,hexamethylene silver staining,PCR,western-blot and serum ELISA detection methods.IgA immunofluorescence results showed that the fluorescence intensity of normal group was negative and that of model group was strongly positive,suggesting obvious IgA deposition,which was the main diagnostic index of IgAN.The results of special staining at each time point showed that the glomerulus in the normal group was normal in structure,the glomerulus in the model group was swollen and enlarged,diffuse mesangial hyperplasia,inflammatory cell infiltration and mesangial matrix widening were visible.MASSON staining showed red dot-sheet immune complex deposition.PAS showed immune complex deposition.However,after intervention by western medicine group and high,medium and low dosage groups of Qiji Shenkang decoction,each group has improved to varying degrees.It can be seen that Qiji Shenkang decoction has a very good effect on improving glomerular mesangial cell proliferation.PCR results showed that Qiji Shenkang Decoction could significantly inhibit the expression of inflammatory factors MCP-1 and NF-kB mRNA,which was superior to western medicine group.Western-blot results showed that it had significant inhibitory effect on the expression of IgA protein.ELISA results show that Qiji Shenkang Decoction can increase the secretion of ERK1/2,inhibit the activation of pERK1/2,and reduce the secretion of pERK1/2 in each dosage group,thus achieving the effect of regulating ERK pathway to prevent and treat glomerulosclerosis,and there is a dose-effect relationship in each dosage group of traditional Chinese medicine.3.HGMC was cultured in vitro.AngII was used as a stimulating factor to make HGMC proliferate.Qiji Shenkang Decoction pharmacological serum was used as an intervention factor to explore the effect of Qiji Shenkang Decoction on cell proliferation of each group by MTT,ELISA,PCR and western-blot detection methods.MTT results showed that after pharmacological serum stimulation for 24 h,48 h and 72 h,the OD value of the model group at each time point was significantly higher than that of the normal group,suggesting cell proliferation.After pharmacological serum intervention,the OD value of each drug intervention group was significantly lower than that of the model group,and the inhibitory effect on cell proliferation at 48 h and 72 h was better than that at 24h;Two time points of 48 h and 72 h were selected to observe ELISA test results: compared with the normal group,the expression of pERK1/2 was significantly increased,the expression of ERK1/2 was significantly decreased,and the expressions of NF-κB and MCP-1 were significantly increased in AngII group,indicating that pERK1/2 was activated while ERK1/2 expression was decreased,inflammatory factors MCP-1 and NF-κB were activated.after intervention of qijishenkang decoction,the expressions of pERK1/2,NF-κB and MCP-1 were all decreased,and ERK1/2 expression was increased,with significant differences at all time points.PCR and western-blot results showed that the expression of NF-κB,MCP-1 mRNA and protein in HGMC cultured in vitro were significantly increased after adding AngⅡ stimulation.The drug-containing serum of each group can inhibit the expression of HGMC NF-κB,MCP-1 mRNA and protein after AngⅡ stimulation.Conclusion:1.According to the currently recognized pathogenesis of IgAN,we have successfully constructed and evaluated a new spontaneous IgAN mouse model,IgHA 1-MUT mouse,using transgenic methods to simulate the role of structural changes of human IgA1 molecular hinge region in the pathogenesis of IgAN.IGHA1-mut mice began to show mild cell proliferation at 8 weeks of age,and obvious mesangial IgA deposition,mesangial matrix proliferation and serum IgA level increase were observed at 12 weeks of age,indicating clear pathological changes in kidney of IGHA1-mut mice model.IGHA1-mut transgenic mice model showed obvious pathological changes in kidney at 12 weeks of age and continued abnormal changes until 24 weeks of age.Therefore,12-week-old IGHA1-mut mice were considered as the best observation period.2.To clarify one of the modern biological mechanisms of IgAN lesions through in vivo experiments,which is related to ERK phosphorylation,excessive secretion of MCP-1 and NF-kB cytokines,and induction of mesangial cell proliferation and inflammatory response;Qiji Shenkang Decoction can effectively reduce IgA protein expression in renal tissue and serum of IgAN mice,regulate ERK pathway,inhibit pERK activation,increase ERK content,and inhibit expression of downstream inflammatory factors MCP-1 and NF-kB genes and proteins,thus improving renal pathological changes,reducing deposition in glomerular mesangial region,preventing further development of immune-induced glomerular diseases,and protecting kidney.Qiji Shenkang Decoction can delay glomerular sclerosis by regulating ERK pathway and related factors,and the therapeutic effect of traditional Chinese medicine intervention on IgAN mice is more stable and lasting than that of western medicine,which provides theoretical basis for a course of treatment theory with 4 weeks of clinical application of traditional Chinese medicine for nephropathy.Qiji Shenkang Decoction,which embodies the guiding theory of "tonifying,clearing and eliminating kidney collaterals",can effectively prevent the further development of glomerulosclerosis and protect the kidney.3.In vitro experiments prove that Qiji Shenkang Decoction can effectively inhibit the overexpression of FN,an extracellular matrix of HGMC induced by AngⅡ,inhibit the proliferation of mesangial cells,and delay kidney damage.Qiji Shenkang Decoction can effectively inhibit the activation of ERK signal transduction pathway,balance the normal transduction of ERK pathway by inhibiting PEK1/2 and increasing ERK expression,inhibit the activation of inflammatory factors NF-κB and MCP-1,reduce cellular inflammatory reaction,reduce excessive accumulation of extracellular matrix,reduce glomerular damage,play a protective role on kidney,and reveal the functional approach of Qiji Shenkang Decoction in delaying glomerulosclerosis. |
PDF Full Text Request