Effect And Mechanism Of Fangji Huangqi Decoction On HPBL And HMC In IgAn Patients In Vitro Based On Healthy Serum Containing Drugs

IgA nephropathy is an autoimmune disease characterized by the deposition of IgA immune complexes in the glomerular mesangium.It is the most common primary chronic glomerular disease and end-stage renal disease in China and the world.Despite available treatment options,the prognosis of IgA nephropathy is not optimistic,and about 50% of the patients still progress to end-stage renal disease within 30 years.Fangji Huangqi Decoction has the function of benefiting Qi and firming the surface,dispelling wind,removing dampness and diffusing water.It is mainly used to treat edema due to deficiency of defence and retention of water and dampness.It has a remarkable effect in improving clinical symptoms and delaying the progress of IgA nephropathy in our department.Previous in vitro experiments also confirmed that tetrandrine,one of the effective components of Fangji Huangqi Decoction,could effectively inhibit the proliferation of peripheral blood lymphocyte in healthy volunteers at the concentration of uM,and synergistically increase the immunosuppressive effect of glucocorticoid.Therefore,on the basis of previous studies,this study explored the mechanism of Fangji Huangqi Decoction in the treatment of IgA nephropathy from inflammatory factors secreted by lymphocyte subsets.Objective: To investigate the effect of Fangji Huangqi Decoction on the secretion of inflammatory factors by peripheral blood lymphocytes from patients with IgA nephropathy.Then we studied the changes of p38 MAPK signaling pathway in normal human glomerular mesangial cells induced by IgA1 extracted from patients with IgA nephropathy and the co-intervention of inflammatory factors secreted by peripheral blood lymphocytes and human glomerular mesangial cells in patients with IgA nephropathy,and further explored the effect of Fangji Huangqi Decoction on reducing gonorrhea by blocking p38 MAPK.Some inflammatory factors secreted by the basal cell subsets can reduce the proliferation of mesangial cells and the secretion of extracellular matrix,achieve the goal of treating IgA nephropathy and delaying the progress of the disease,and provide scientific basis for the treatment of IgA nephropathy with the method of "invigorating Qi and strengthening the surface,dispelling wind and removing dampness".Method:?.Experiments in vivo1.Preparation of drug-containing serum.Ten volunteers,five males and five females,were selected.Before the intervention,20 ml of brachial vein blood was collected on an empty stomach in the morning,and the serum was separated and used as the blank control group.Then all the volunteers took Fangji Huangqi Tang granules for 6 consecutive days.The serum was separated and used as drug-containing serum.2.Detection of active ingredients in the serum of healthy volunteers of Fangji Huangqi Decoction based on high performance liquid chromatography(HPLC).Agilent 883975-902 SB-C18 Analytical HPLC Col 4.6 x 150 1 SB-C18 column was used for chromatographic column.The mobile phase was 0.1% formic acid aqueous solution(A)-acetonitrile(B)gradient elution.The gradient elution procedure was 0-20 min,A:90%40%;20-40 min,A:40%25%.Detection wavelength: 205,277 nm;flow rate: 0.8 mL/min;column temperature: 30 C.II.Experiments in vitroExperiment one1.Preparation of human peripheral blood lymphocyte suspension.According to the inclusion and exclusion criteria of IgA nephropathy diagnostic criteria,10 patients with IgA nephropathy were collected from a cohort study of chronic nephropathy,and 15 ml fresh whole blood was collected for the isolation of IgA1 and lymphocyte.Human peripheral blood lymphocyte(HPBL)was isolated from whole blood by gradient density centrifugation of lymphocyte separation solution.2.CCK-8 method was used to detect the equivalence between healthy human serum and 10% fetal bovine serum,and to determine the concentration of HPBL cells cultured in healthy volunteer serum.3.Cell grouping and intervention.The HPBL of the previously isolated healthy person and IgA nephropathy patients were divided into the control group,the normal group,the model group,and the treatment group for corresponding intervention.The cells after the intervention were further cultured for 48 hours,and the subsequent indicators were detected.4.Indicator detection.After 48 hours,the cytokine concentration was measured by centrifugation,and the cytokine levels of Th1,Th2 and Th17(IL-4,IL-6,IL-17,TNF-?,TGF-?1,IFN-?)were measured by ELISA.Experiment two1.The equivalence of healthy human serum and 10% fetal bovine serum was detected by CCK-8 method,and the concentration of human mesangial cells(HMC)cultured in healthy volunteer serum was determined.The toxicity of serum containing drugs on HMC cells was detected by LDH method.2.Preparation of plasma IgA1.Jacalin agglutinin affinity column was used to extract and polymerize IgA1 from plasma collected from healthy people and patients with IgA nephropathy.3.Cell grouping and intervention.The normal human mesangial cells were induced by IgA1 extracted from healthy people and IgA nephropathy,and then divided into three groups: normal group,model group and treatment group for intervention.The corresponding indicators were detected after 48 hours of cell culture in each group.4.Indicator detection.CCK-8 method was used to detect the proliferation rate of human mesangial cells.The levels of Th1,Th2 and Th17 cytokines(IL-4,IL-6,IL-17,TNF-?,TGF-?1 and IFN-?)were measured by ELISA.The levels of extracellular matrix fibronectin(FN),procollagen III(PC III),collagen IV(COL4)secreted by mesangial cells were measured by ELISA.Experiment three1.Experimental grouping and intervention.HMC was induced by inflammatory factors secreted by HPBL from healthy people and IgA nephropathy patients for 24 hours.Cells were divided into five groups: healthy blank group,healthy drugadding group,IgA + blank serum,IgA + drug-containing serum group and IgA + blocker group.After the intervention,cells were cultured for 48 hours,and subsequent indicators were collected for detection.2.Indicator detection.RT-PCR was used to detect the expression of TGF-?1,IL-6 and p38 MAPK,Western Blot was used to detect the expression of TGF-?1,IL-6,p38 and pp38 proteins,and immunofluorescence was used to detect the expression of IL-6,TGF-?1 and p38 in HMC cells.Result:I.Experiments in vivo1.The structure of tetrandrine and atractylode I prototype compounds in the serum of healthy volunteers Fangji Huangqi Decoction was determined by comparing the retention time with that of the control.No astragaloside was detected.The contents of tetrandrine and atractylode I in the serum of healthy volunteers with Fangji Huangqi Decoction were 0.21?g/mL and 0.97?g/mL calculated by linear regression equation.II.Experiments in vitroExperiment one1.The results of CCK-8 test showed that there was a significant difference in cell viability between 5%,15% and 20% healthy human serum and 10% FBS(P<0.05).Compared with 10% FBS,the survival rate of HPBL in 10% healthy serum group was similar,but there was no significant difference(P>0.05).Therefore,the follow-up experiment selected 10% healthy human serum as the final concentration of the experiment.2.The results showed that the levels of inflammatory factors IL-6,TGF-?1,TNF-?,IL-4 and IL-17 secreted by HPBL in the control group and the model group were higher than those in the normal group,and the levels of IFN-gamma secreted by HPBL in the control group and the model group were lower than those in the normal group(P < 0.05).The levels of IL-6,TGF-? 1,IL-4,IL-17 and IFN-? in the treatment group were lower than those in the model group,while the levels of IFN-?were increased(P < 0.05).Although the levels of TNF-? in the treatment group were also decreased,there was no significant difference(P > 0.05).Experiment two1.The results of CCK-8 test showed that there was a significant difference in cell viability between 20%,25% and 30% healthy human serum and 10% FBS(P<0.05).Compared with 10% FBS,the survival rate of HMC in 10% and 15% healthy serum group was similar,but there was no significant difference(P>0.05).The cell survival rate of 10% healthy human serum group was higher than 15% and was closer to that of 10% FBS group.Therefore,10% healthy human serum was chosen as the final concentration in the follow-up experiment.Compared with 10% FBS group,the LDH activity of HMC in 10% healthy blank serum and 10% Fangji Huangqi Decoction containing serum had no significant difference(P>0.05),which indicated that the blank serum and Fangji Huangqi Decoction containing serum had no toxic effect on HMC cells and could be used in subsequent experiments.2.The proliferation rate of HMC in the model group induced by IgA1 extracted from serum of patients with IgA nephropathy was higher than that in the normal group(P<0.05),while the proliferation rate of HMC in the treatment group treated with Fangji Huangqi decoction containing serum was lower than that in the model group(P<0.05).The levels of FN,PCIII and COL4 secreted by the model group were higher than those of the normal group(P<0.05).The levels of FN,PCIII and COL4 in the treatment group were lower than those in the model group(P<0.05).The levels of IL-6,IFN-?,TGF-?1,TNF-?,IL-4 and IL-17 secreted by HMC in model group were significantly higher than those in normal group(P<0.05).The levels of IL-6,IFN-?,TGF-?1,TNF-?,IL-4 and IL-17 secreted by HMC in the treatment group were lower than those in the model group.Except for the decrease of TNF-?,there was no significant difference(P>0.05),the other five inflammatory factors had significant difference(P<0.05).Experiment three1.The expression of IL-6,TGF-?1 and p38 MAPK in HMC cells of IgA + blank serum group was high.The expression of IL-6,TGF-?1 and p38 MAPK in IgA+blank serum group was significantly higher than that in healthy blank group(P<0.05).The expression of IL-6,TGF-?1 and p38 MAPK in IgA+drug-containing serum group was lower than that in IgA+blank serum group(P<0.05).There was no significant difference in the expression of IL-6,TGF-?1 and p38 MAPK between IgA+blocker group and healthy drug-adding group(P>0.05).The expression of IL-6,TGF-?1 and p38 MAPK in IgA+blocker group was lower than that in IgA+blo+ The expression of IL-6,TGF-?1 and p38 MAPK in blank serum group was decreased(P<0.05).2.The expression of IL-6,TGF-?1,p38 and p-p38 protein in HMC cells in IgA + blank serum group was also high,and the expression level was the highest compared with the other four groups.The levels of IL-6,TGF-?1,p38 and p-p38 protein in healthy blank group were lower than those in IgA+blank serum group(P<0.05).The expressions of IL-6,TGF-?1,p38 and p-p38 protein in IgA+blank serum group were lower than those in IgA+blank serum group(P<0.05).There was no significant difference in the expressions of IL-6,TGF-?1,p38 and p-p38 protein between IgA+blocker group and healthy drug-adding group(P>0.05).The expressions of IL-6,TGF-?1,p38 and p-p38 protein in IgA+blocker group Compared with IgA + blank serum group,the expression of IL-6,TGF-?1,p38 and p-p38 proteins decreased(P<0.05).3.Immunofluorescence results showed that the secretion of IL-6,TGF-?1 and p38 in IgA + blank serum group was higher than that in healthy blank serum group.The same effect of IgA + drug-containing serum group and IgA + blocker group could reduce the levels of IL-6,TGF-?1 and p38.Conclusion:1.The active ingredients in the serum of healthy volunteers are tetrandrine and atractylode I,which provide a reference for further pharmacological mechanism study of Fangji Huangqi Decoction.2.HPBL in patients with IgA nephropathy secretes more inflammatory factors such as IL-6,TGF-?1,TNF-?,IL-4 and IL-17,but less IFN-?.IgA1 in patients with IgA nephropathy can induce the proliferation of HMC,increase the secretion of extracellular matrix FN,PCIII and COL 4,and increase the secretion of inflammatory factors such as IL-6,IFN-?,TGF-?1,TNF-?,IL-4 and IL-17.3.Fangji Huangqi Decoction can regulate the secretion of inflammatory factors IL-6,IFN-?,TGF-?1,TNF-?,IL-4 and IL-17 in peripheral blood lymphocytes and mesangial cells,and reduce the proliferation of mesangial cells and the secretion of extracellular matrix FN,PC III and COL 4.Its mechanism may be related to the inhibition of p38 MAPK activation,thereby reducing the inflammatory response of mesangial cells,and then reducing the production of TGFbeta 1 and IL-6,reducing the proliferation of mesangial cells and the deposition of extramesangial matrix,thus delaying the progress of IgA nephropathy.