| ObjectJveM-type phospholipase A2 receptor(PLA2R)as the target autoantigen of adult IMN has been discovered recently.Podocyte’ s injury relate closely to the histology pathogenesis of IMN.However,there is no specific medication for IMN.Our previous studies suggested that the onset of IMN is due to syndrome of Spleen and Kidney Yang deficiency in Chinese medicine theory and Warming Yang and Causing diuresis has a good effect on the disease.This project mainly characterized by investigating the relationship between PLA2R and syndrome of Spleen and Kidney Yang deficiency of IMN and the mechanism of Warming Yang and Causing diuresis.Using data from clinical cases,the study detected the correlation between PLA2R and syndrome of Spleen and Kidney Yang deficiency.In experimental research,conditionally immortalized mouse podocyte cell line was used.By molecular biological methods,we investigated the mechanism of Patient’ s serum extracted from syndrome of Spleen and Kidney Yang deficiency of IMN affecting the structure and apoptosis of podocytes,together with machanism of Warming Yang and Causing diuresis.These lab evidence would support more target based therapy and possible Chinese medicine approaches for IMN prevention and treatment.MethodsIn clinical study,we enrolled 40 patients with renal-biopsy proven IMN from January 2014 to September 2015.These patients were hospitalized for nephrotic syndrome(NS)in the department of Nephrology in the First Affiliated Hospital of Guangzhou University of Chinese Medicine.Among them,there were 31 patients with syndrome of Spleen and Kidney Yang deficiency and 9 patients with non-syndrome of Spleen and Kidney Yang deficiency.ELISA was used to detect serum antibody to PLA2R(anti-PLA2R).Comparison was made between syndrome of Spleen and Kidney Yang deficiency group and non-syndrome of Spleen and Kidney Yang deficiency group in the general data,laboratory examination indexes(ALB,GLO,Scr,UA,TG,CHOL,HDL-C,LDL-C,PT,APTT,UTP and RBC(HPF)),pathological indexes(mesangial proliferation,crescent formation,glomerulosclerosis,tubular atrophy/fibrosis,renal interstitial inflammatory cell infiltration,renal interstitial vascular disease and immunofluorescence deposition).Correlation analysis was employed for the relationship between anti-PLA2R levels and indexes that has significant difference between two groups.In experiment research,conditionally immortalized mouse podocyte cell line was used.To evaluate podocyte proliferation affected by patient’ s serum extracted from syndrome of Spleen and Kidney Yang deficiency of IMN,CCK-8 kit was applied.Based on the results of CCK-8 examination,cellular model of podocyte injured by patient’ s serum extracted from syndrome of Spleen and Kidney Yang deficiency of IMN was developed.5 groups including normal control,model,2mg/ml decoction,4mg/ml decoction and 8mg/ml decoction were divided.Normal control group was builded by ten percent healthy serum and RPMI-1640 medium.Model group was builded by ten percent serum of IMN patients with syndrome of Spleen and Kidney Yang deficiency and RPMI-1640 medium.On the basis of model group,three decoction groups were builded by added 2mg/ml,4mg/ml and 8mg/ml decoction of Warming Yang and Causing diuresis,respectivelly.After 24h and 48h incubation,indexes were detected to explore the protective mechanisms of decoction of Warming Yang and Causing diuresis.Podocyte cytoskeleton of F-actin was observed by immunofluorescence staining.Podocyte apoptosis rates were analysised by flow cytometry.Relative expression of p53 and Bcl-2 protein was detected by Western Blot and mRNA was by real-time quantitative reverse transcription-PCR(qRT-PCR).Results1.The results of clinical studyIn group of syndrome of Spleen and Kidney Yang deficiency group,there were 31 patients,of which 16 males and 15 females.In the other group,there was 9 patients,of which 8 males and 1 female.70.97 percent of patients in syndrome of Spleen and Kidney Yang deficiency group showed positive anti-PLA2R and 29.03 percent of patients in non-syndrome of Spleen and Kidney Yang deficiency group showed positive.There was no significant difference in the positive rate of anti-PLA2R between these two groups.Patients in group of syndrome of Spleen and Kidney Yang deficiency were higher in age,the optical density of serum anti-PLA2R as well as GLO(P<0.05).These two groups showed no significant difference in gender,course,MAP,UTP,ALB,Scr,UA,TG,CHOL,HDL-C,LDL-C,PT,APTT,UTP,RBC(HPF).In pathological parameters,only renal interstitial inflammatory cell infiltration showed significantly different between two groups(P<0.05).Spearman correlation analysis showed that the optical density of anti-PLA2R only had a positive correlation with serum globulin in patients with syndrome of Spleen and Kidney Yang deficiency(P<0.05).2.The results of CCK-8 examinationTen percent serum of IMN patients with syndrome of Spleen and Kidney Yang deficiency had a negative effect on podocyte proliferation.2mg/ml,4mg/ml and 8mg/ml decoction of Warming Yang and Causing diuresis had no effect on podocyte proliferation.3.Podocyte cytoskeleton of F-actin was observed by immunofluorescence stainingAfter 24h incubation,enough F-actin filaments distributed as stress fiber-like bundles along the axis of the cells was detected in normal control group.However,the expression of F-actin filaments was down-regulation and the filaments was redistributed without along the axis of the cells in model group.Compared to model group,the injury and redistribution of F-actin filaments was relatively smooth in decoction group.After 48h incubation,less enough F-actin filaments was expressed but also distributed as stress fiber-like bundles along the axis of the cells was detected in normal control group.However,the expression of F-actin filaments was down-regulation severity and the filaments was redistributed heavily in model group.Compared to model group,the injury and redistribution of F-actin filaments was relatively smooth in decoction group.4.Podocyte apoptosis rates were analysised by flow cytometryAfter 24h incubation,podocyte apoptosis rates were higher in model group than normal control group.There was a significant difference between normal control group and model group(P<0.05).Compared to model group,apoptosis rates were lower in three decoction groups.Among three decoction groups,there was a negative relationship between concentration of decoction and apoptosis rates but without a significant difference.After 48h incubation,podocyte apoptosis rates were low in normal control but higher in model group.There was a significant difference between normal control group and model group(P<0,05).Compared to model group,apoptosis rates were lower in three decoction groups.There was a significant difference between model group and 4mg/ml as well as 8mg/ml decoction group(P<0.05).Among three decoction groups,there was a negative relationship between concentration of decoction and apoptosis rates.Significant difference was determined among them(P<0.05).5.Relative expression of p53 and Bcl-2 protein was detected by Western BlotAfter 24h incubation,the relative expression of p53 protein was higher and Bcl-2 was lower in model group than normal control.However,there was no significant difference between them.Neither the relative expression of p53 protein nor Bcl-2 protein showed a significant difference between model group and decoction group.Among three decoction groups,there was no significant difference on the relative expression of p53 protein and Bcl-2 protein either.After 48h incubation,the relative expression of p53 protein was higher and Bcl-2 was lower in model group than normal control.There was a significant difference between these two groups on the relative expression of Bcl-2 protein(P<0.05).In comparison to model group,both of 4mg/ml and 8mg/ml group had a lower level of p53 protein expression and a higher Bcl-2 protein expression.There was a significant difference among these groups on the relative expression of Bcl-2 protein(P<0.05).Among three decoction groups,the relative expression of p53 protein was gradually down-regulation while Bcl-2 was gradually up-regulation with the increase of decoction concentration.Besides,there was a significant difference on Bcl-2 expression between 2mg/ml and 4mg/ml as well as 8mg/ml decoction group(P<0.05).6.Relative expression of p53 and Bcl-2 mRNA was detected by qRT-PCRAfter 24h incubation,neither the relative mRNA expression of p53 nor Bcl-2 showed a significant difference between normal control group and model group.Compared to model group,both of the relative mRNA expression of p53 and Bcl-2 reduced.However,there was no significant difference among them.There was also no significant difference among three decoction groups.After 48h incubation,the relative mRNA expression of p53 was higher and Bcl-2 was lower in model group than normal control group.Both of p53 and Bcl-2 mRNA relative expression showed a significant difference between these two groups(P<0.05).In comparison to model group,the relative mRNA expression of p53 was lower in 2mg/ml and 4mg/ml decoction group.In comparison to model group,the relative mRNA expression of Bcl-2 was higher in 4mg/ml and 8mg/ml decoction group.Moreover,there was a significant difference between model group and 4mg/ml as well as 8mg/ml decoction group(P<0.05).Among three decoction groups,the relative expression of p53 mRNA showed no significant difference.However,the relative expression of Bcl-2 mRNA was higher in 4mg/ml and 8mg/ml decoction group and both showed a significant difference compared to 2mg/ml decoction group(P<0.05).ConclusionThere may be a correlation between syndrome of Spleen and Kidney Yang deficiency and PLA2R in IMN.The mechanism of decoction of Warming Yang and Causing diuresis in the treatment of IMN patients with syndrome of Spleen and Kidney Yang deficiency,may be related to its attenuation of podocyte apoptosis through prevent p53 expression and up-regulate Bcl-2 expression and so as to stablize structure of F-actin cytoskeleton. |
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