| BackgroundPrimary nephrotic syndrome(PNS)in children is caused by increased permeability of the glomerular basement membrane induced by a variety of reasons and the loss of a large amount of proteins in the blood from the urine,which leads to a series of pathological changes and physiological syndromes.PNS ranks first in hospitalized children with urological diseases in China.The etiology and pathogenesis of PNS in children are not fully understood.It has been thought that the occurrence of PNS in children may be related to a variety of factors such as flora disorders,immune disorders,inflammatory mediators,genetic factors,and lipid metabolism disorders.Changes in the intestinal microbial flora of PNS may cause systemic immune inflammation by disrupting the intestinal epithelial barrier and producing toxic byproducts,and changing the intestinal mucosal immune system.This study aims to investigate the correlations between PNS and gut microbiota dysbiosis and Th17/Treg immune imbalance in children,explore the possible pathogenesis,and provide theoretical and experimental basis for finding new biological target for the diagnosis and treatment of children’s PNS.Part Ⅰ Study on the correlations between gut microbiota dysbiosis and inflammation and Th17/Treg immune imbalance in children with PNSObjectiveTo investigate the gut microbiota dysbiosis and Th17/Treg immune imbalance in children’s PNS,and analyze its correlations and possible pathogenesis in order to find new biological target for the diagnosis and treatment of children’s PNS.Methods1.This study took 29 children diagnosed with PNS admitted to the Children’s Hospital of Soochow University from December 2017 to December 2018 as the research objects,and divided them into the before treatment group and the after treatment group.In addition,there were 15 healthy children in the hospital during the same period enrolled as a study control group.2.The fecal specimens naturally excreted from 29 children with PNS before and after treatment and from 15 healthy children were collected.Based on 16S rRNA technology,the changes of intestinal lactobacilli,bifidobacteria,and E.coli were detected by quantitative real-time PCR(qRT-PCR),and calculate the Bifidobacteria/E.Coli(B/E)ratio.3.Fasting peripheral blood was collected from 29 PNS children before and after treatment and from 15 healthy children.Mononuclear cells were extracted from peripheral blood,and the proportions of Th17 and Treg cells in mononuclear cells were detected by flow cytometry analysis.4.Serum IL-17,IL-10,IL-6 and TNF-α levels were detected by ELISA.5.Western blot was used to detect the level of NF-κB in peripheral blood mononuclear cells.6.One-way analysis of variance was used to analyze the differences among the three groups.7.Pearson correlation was used to analyze the correlations between the corresponding items.Results1.The changes of intestinal flora:Compared with the control group,the counts of Lactobacillus,bifidobacteria and E.coli in children with PNS decreased before treatment(P<0.05),and partly recovered after treatment,but did not reach the normal level of the control group(P<0.05);Compared with the control group,children with PNS had lower B/E value before treatment(P<0.05),increased after treatment,and returned to normal level of the control group(P>0.05).2.Percentages of Th17 and Treg cells in peripheral blood:Compared with the control group,the proportion of Th17 cells increased and the proportion of Treg cells decreased in children with PNS before treatment(P<0.05).After treatment,the proportion of Th17 cells decreased,but was still higher than the control group(P<0.05),and the proportion of Treg cells increased,although it was lower than the control group,there was no significant difference between the two groups(P>0.05);Compared with the control group,the ratio of Th17/Treg cells before treatment in children with PNS increased(P<0.05),and the ratio of Th17/Treg cells returned to normal level of the control group after treatment(P>0.05).3.Serum IL-17,IL-10,IL-6 and TNF-α levels:Compared with the control group,serum levels of IL-17,IL-6 and TNF-α increased before treatment in children with PNS,and decreased after treatment,but still higher than the control group(P<0.05).Compared with the control group,the serum IL-10 level in children with PNS decreased before treatment and partly recovered after treatment,but did not reach the normal level of the control group(P<0.05).4.NF-κB protein level in peripheral blood:Western blot result showed that compared with the control group,the level of NF-κB protein in peripheral blood of children with PNS were significantly increased before treatment and partly recovered after treatment,but did not reach the normal level in the control group(P<0.05).5.Correlation analysis between the levels of IL-17,IL-10 and the proportions of Th17 and Treg cells:Pearson correlation analysis showed that serum IL-17 level in children with PNS had a similar trend of change with the proportion of Th17 cells,and had a positive correlation with the proportion of Th17 cells(r=0.66,P<0.05);the serum IL-10 level in children with PNS had a similar trend to Treg cells,and had a positive correlation with the proportion of Treg cells(r=0.52,P<0.05).6.Correlation analysis between the counts of intestinal flora and the ratio of Th17/Treg cells:Pearson correlation analysis showed that there were no correlations between the counts of lactobacillus,bifidobacteria,E.coli and the ratio of Th17/Treg cells(P>0.05)There was negative correlation between B/E and Th17/Treg cell ratio(r＝-0.32,P<0.05).7.Correlation analysis between the number of intestinal flora and serum IL-17,IL-10,IL-6 and TNF-α levels:Pearson correlation analysis showed that the count of lactobacillus was positively correlated with the level of IL-10(r=0.34,P<0.05)and was negatively correlated with TNF-α level(r=-0.40,P<0.05);The count of bifidobacteria was positively correlated with the level of IL-10(r=0.36,P<0.05),and was negatively correlated with the levels of IL-17 and IL-6(r=-0.35,P<0.05;r=-0.46,P<0.05);There was no correlation between the count of E.coli and the levels of IL-17,IL-10,IL-6,and TNF-α(P>0.05);B/E value was negatively correlated with IL-17 and IL-6 levels(r=-0.48,P<0.05;r=-0.32,P<0.05),and B/E value was positively correlated with IL-10(r=0.50,P<0.05).Conclusions:Children with PNS showed gut microbiota dysbiosis,peripheral blood Th17/Treg cell imbalance,and increased inflammatory mediators,which improved after treatment.Gut microbiota dysbiosis may be involved in the pathogenesis of children with PNS,and are related to Th17/Treg cell imbalance and immune inflammation.PartⅡ Study on the therapeutic effect and mechanism of fecal microbiota transplantation on young rats with adriamycin-induced nephropathyObjective:To explore the therapeutic effect of fecal microbiota transplantation(FMT)on young rats with adriamycin-induced nephropathy and its possible related mechanisms,and to provide experimental and theoretical basis for the study of adjusting thegut microbiota dysbiosis as a target for PNS treatment in children.Methods:1.Male SD young rats were purchased and divided into control group,adriamycin-induced nephropathy(AIN group and FMT group according to the digital random table method.2.Young rats of AIN group and FMT group were injected with adriamycin 6.5 mg/kg by the tail vein.Young rats of control group were injected with the same amount of normal saline by the tail vein.One week later,urine was collected for 24 h to test urine protein.3.FMT bacterial solution was prepared from the feces in the proximal large intestine of healthy young rats,and 0.2 ml of the prepared bacterial solution was aspirated with an intragastric needle and injected into the stomachs of young rats in the FMT group.In the control group and the AIN group,0.2 ml of sterile PBS(containing 20%glycerol)was given to each young rat per day.The young rats were treated continuously for 7 days.4.After treatment for 7 days,urine was collected from the young rats in the three groups for 24 h to detect urine protein.5.After collecting the urine for 24 h,the young rats of each group were anesthetized intraperitoneally with compound anesthesia,and the heart blood was collected to extract the mononuclear cells,which were used to detect the numbers of Th17 and Treg cells by flow cytometry.Serum IL-17,IL-10,IL-6 and TNF-α levels were detected by ELISA.6.After blood sample has been collected,the proximal large intestine is obtained.Collect fecal specimens from the proximal large intestine,detect the changes in intestinal Lactobacillus,Bifidobacteria,and E.coli by qRT-PCR based on 16S rRNA technology,and calculate Bifidobacteria/E.Coli(B/E)ratio.7.After the proximal large intestine was repeatedly rinsed with sterile normal saline,the levels of Jaggedl,Hesl,ROR-yt,and Foxp3 proteins in the intestine were measured by Western blot.8.The kidneys in longitudinal dissection were taken and fixed in formalin for HE staining.9.One-way analysis of variance was used to analyze the differences among the three groups.Pearson correlation was used to analyze the correlations between the corresponding items.Results:1.General situation of young rats:After 7 days of one-time tail vein injection of adriamycin,young rats in the AIN group and FMT group reduced appetite,lost weight,had hair loss,poor coat color,reduced activity,and obvious edema of scrotum and quadrupeds.Quantitative analysis of 24 h urine protein showed that the 24 h urine proteins in AIN group and FMT group were 35.75±6.02 mg/24 h and 37.34±3.39 mg/24 h,which was significantly higher than that in control group(P<0.05).The rats of control group had no abnormalities.2.Quantitative analysis of 24 h urine protein in young rats:After 7 days of FMT treatment,the urine protein of young rats in the AIN group and FMT group increased,which were 62.16±5.62 mg/24 h,44.73±9.91 mg/24 h,but the 24 h urine protein of young rats in the FMT group was significantly lower than that in the AIN group,and the difference between the groups was statistically significant(P<0.05).3.Pathological changes in the kidneys of yong rats:Normal glomeruli could be seen in the kidneys of the control group,and renal pathological damage such asinflammatory cell infiltration,renal tubular dilatation or basement membrane thickening,and proteinuria could be observed in the kidneys of the AIN and FMT groups.The pathological changes of kidney in young rats in FMT group were less than those in AIN group.4.Percentages of Th17 and Treg cells in blood:Compared with the control group,the proportion of Th17 cells in AIN group and FMT group increased,but the proportion of Th 17 cells in young rats of FMT group was significantly lower than that in AIN group(P<0.05);Compared with the control group,the proportion of Treg cells in the AIN group and FMT group was reduced,but the proportion of Treg cells in the young rats of the FMT group was significantly higher than that in the AIN group(P<0.05);Compared with the control group,the ratio of Th17/Treg cells in young rats of AIN group and FMT group increased,but the ratio of Th17/Treg cells in young rats of FMT group was significantly lower than that in AIN group(P<0.05).5.Levels of IL-17,IL-10,IL-6 and TNF-α in serum:Compared with the control group,the serum levels of IL-17,IL-6 and TNF-α in young rats in the AIN and FMT groups increased,but the serum levels of IL-17,IL-6 and TNF-α in the young rats of the FMT group were significantly lower than those in the AIN group(P<0.05).Compared with the control group,serum IL-10 level decreased,but the serum IL-10 level in the FMT group was significantly higher than that in the AIN group(P<0.05).6.Correlation analysis between the levels of IL-17,IL-10 and proportions of Th17,Treg cells:Pearson correlation analysis showed that the serum IL-17 level of young rats with AIN had a similar tendency to the change of the proportion of Th17 cells,and was positively correlated with the the proportion of Th17 cells(r=0.73,P<0.05);Serum IL-10 level in rats with AIN showed similar trends to the change of Treg cells,and was positively correlated with the proportion of Treg cells(r=0.87,P<0.05).7.The changes of intestinal flora in young rats:The count of intestinal Lactobacillus in the AIN group was significantly lower than that in the control group(P<0.05),and the count of intestinal Lactobacillus in FMT group was slightly lower than that in the control group,but the difference was no statistical significance(P>0.05).The count of intestinal Lactobacillus in FMT group was significantly higher than that in AIN group(P<0.05).Compared with the control group,the count of the bifidobacteria in AIN group and FMT group was decreased(P<0.05),but the count of intestinal bifidobacteria in FMT group was significantly higher than that in AIN group(P<0.05);The count of intestinal E.coli in AIN group was significantly higher in the FMT group(P<0.05),the count of intestinal E.coli in FMT group was slightly higher than that in the control group,but the difference was not statistically significant(P>0.05);Compared with the control group,the B/E value in the AIN group and the FMT group was significantly reduced,but the B/E value of the FMT group was significantly higher than that of the AIN group(P<0.05).8.Correlation analysis between the counts of intestinal flora and the ratio of Th17/Treg cells:Pearson correlation analysis showed that there was a positive correlation between the count of E.coli and the ratio of Th17/Treg cells(r=0.60,P<0.05)in rats with AIN.The counts of lactobacillus and bifidobacteria was negatively correlated with the ratio of Th17/Treg cells(r=-0.58,P<0.05;r＝-0.41,P<0.05)in rats with AIN.There was a negative correlation between B/E value and Th17/Treg cell ratio(r＝-0.60,P<0.05).9.Correlation analysis between the counts of intestinal flora and levels of serum IL-17,IL-10,IL-6,and TNF-α:Pearson correlation analysis showed that the count of lactobacillus was negatively correlated to the levels of IL-17,IL-6,and TNF-α(r=-0.70,P<0.05;r=-0.64,P<0.05;r=-0.52,P<0.05),and was positively correlated with the level of IL-10(r=0.62,P<0.05).The count of bifidobacteria was positively correlated with the level of IL-10(r=0.53,P<0.05).The count of E.coli was positively correlated with the levels of IL-17,IL-6,and TNF-α(r=0.55,P<0.05;r＝0.76,P<0.05;r=0.51,P<0.05),and the count of E.coli was negatively correlated related to IL-10 level(r=-0.61,P<0.05).The B/E value was negatively correlated with the levels of IL-17,IL-6,and TNF-α(r=-0.55,P<0.05;r=-0.65,P<0.05;r=-0.47,P<0.05).B/E value was positively correlated with IL-10 level(r=0.67,P<0.05).10.Jaggedl,Hesl,ROR-yt,and Foxp3 protein levels in the large intestine of young rats:Compared with the control group,the relative expressions of Jaggedl,Hesl,ROR-yt proteins in the large intestine of AIN and FMT groups was increased.However,the relative expressions of Jaggedl,Hesl,ROR-yt protein in the large intestine of young rats in the FMT group was significantly lower than those in the AIN group(P<0.05).Compared with the control group,the relative expression of Foxp3 protein in the large intestine of young rats in the AIN group and the FMT group decreased,but the relative expression of Foxp3 protein in the large intestine of the young rats in the FMT group was significantly higher than that in theAIN group(P<0.05).Conclusions:FMT treatment for SD yong rats with AIN can improve proteinuria and reduce renal pathological changes.This is related to the reduction of gut microbiota dysbiosis,Th17/Treg cell imbalance,and increase of inflammatory mediators after FMT treatment.The mechanism of FMT in treating SD yong rats with AIN may be related to Notch signal.FMT may be a new biological approach for PNS treatment. |
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