| Objectives:To investigate the role and effect of CD4~+CD25~+regulatory T(Treg)cells in type 1 diabetic nephropathy(DN).Control experiments were carried out in a mouse model to dynamically observe the changes in the number and function of Treg cells in each group of mice.Fasting blood glucose,body weight,24h urine volume,water intake,urine protein,islet function and renal pathology were regularly monitored.The levels of Th1,Th2,Th17 and Treg cells were analyzed to study the effect of CD4~+CD25~+Treg cells on the prevention of diabetic nephropathy,and to further clarify the underlying mechanisms of CD4~+CD25~+Treg cells in the treatment of DN,which may provide some important evidence for immune cell therapy of DN.Methods:1.Thirty 8-week-old C57BL/6 male mice were randomly divided into normal control group(n=6)and T1DM model group(n=24).The T1DM model group was fed with normal diet combined with multiple low-dose STZ injection to establish the model,and the normal control group was fed with water and normal diet.The changes of symptoms and signs before and after the disease were observed.2.CD4~+CD25~+Tregs from the spleen of spare normal mice were obtained and cultured in vitro.The expanded Tregs were used to treat T1DM model mice.According to different treatment regimens,the T1DM model group was subdivided into T1DM untreated group(n=6)and Treg low-,medium-,and high-dose groups,with 6 mice in each dose group.The corresponding mice were treated with three concentrations(1×10~6/kg,5×10~6/kg,and 10×10~6/kg),respectively.Fasting blood glucose,body weight,24h urine volume,water intake,urine protein,and islet function were measured at the prescribed time points.The changes of these indexes after treatment were observed to determine the efficacy and safe and effective dose of CD4~+CD25~+Treg in the treatment of DN.3.The kidney sections of the 5 groups of mice were obtained at the proposed time points for HE staining and PDGF immunohistochemical detection to observe the pathological changes of the kidney and evaluate the renal inflammation score,and to explore whether Treg cell treatment has a protective effect on the kidney of DN mice.4.The spleens of the five groups of mice were obtained at the proposed time points,and the levels of Th1,Th2,Th17 and Treg cells in the spleen were analyzed to further explore the molecular mechanism of Treg cells in the treatment of DN mice.5.The differences between two groups were compared using two-sample t-tests,and one-way ANOVA for three or more groups.P<0.05 was considered to be statistically significant.Results:1.Comparison of fasting blood glucose(mmol/L)among groups:Compared with the normal control group(5.72±0.24),the fasting blood glucose of T1DM group(19.12±0.52),Treg low dose group(17.68±0.09),Treg medium dose group(16.30±0.50),Treg high dose group(15.85±0.63)increased significantly on Day14.(P<0.001).The hypoglycemic effect of Treg high-dose group was better than that of Treg medium-dose group,and the difference was statistically significant compared with T1DM group(P<0.01).The hypoglycemic effect of Treg medium dose group was better than that of Treg low dose group,and the difference was statistically significant compared with T1DM group(P<0.05).There was no significant difference between Treg low dose group and T1DM group(P>0.05).Compared with the normal control group(5.33±0.23),T1DM group(21.03±1.20),Treg low dose group(18.10±1.35),Treg middle dose group(16.50±0.38),Treg high dose group(15.60±0.62),the difference was statistically significant(P<0.001).Compared with the T1DM group,the blood glucose in the Treg high-dose group(P<0.01)and Treg medium-dose group(P<0.05)had significant differences,and the blood glucose in the medium-and high-dose groups increased less than that in the low-dose group.These results suggest that Treg can improve fasting blood glucose in DN mice,and the higher the concentration of Treg,the more effective the effect is.2.Comparison of body weight(g)between groups:Compared with the normal control group(21.78±0.310),the body weight of T1DM group(16.25±0.20),Treg low dose group(16.72±0.49),Treg middle dose group(17.71±0.23),Treg high dose group(17.23±0.41)decreased significantly on Day14.(P<0.001).Compared with the T1DM group,the Treg high-dose group had a lighter weight loss(P<0.05),and there was no significant difference between the Treg medium-low dose group and the T1DM group(P>0.05).Compared with the normal control group(21.63±0.46),T1DM group(15.43±0.19),Treg low dose group(16.50±0.35),Treg middle dose group(16.63±0.38),Treg high dose group(16.67±0.26)were statistically significant(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).These results suggest that Treg can improve the symptoms of weight loss in DN mice,and the effect is significant on Day14,and the higher the concentration of Treg,the more effective the effect is.3.Comparison of 24h urine volume(ml)among groups:On Day14,compared with the normal control group(0.09±0.01),the urine output levels of T1DM group(1.66±0.17),Treg low-dose group(1.13±0.12),Treg medium-dose group(1.13±0.07),Treg high-dose group(0.97±0.04)were significantly increased.(P<0.001).Compared with the T1DM group,the 24h urine volume in the Treg medium dose group(P<0.05)and the high dose group(P<0.01)were significantly improved,but there was no significant difference between the Treg low dose group and the T1DM group(P>0.05).Compared with normal control group(0.08±0.02)at Day34,T1DM group(1.25±0.15),Treg low dose group(1.22±0.06),Treg middle dose group(1.02±0.09),Treg high dose group(0.95±0.03),the difference was statistically significant(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).These results suggest that Treg can improve the symptoms of polyuria in DN mice,and the effect is significant on Day14,and the higher the concentration of Treg,the more obvious the effect is.4.Comparison of 24h drinking water(ml)among groups:On Day14,compared with the normal control group(5.33±0.33),T1DM group(27.17±2.02),Treg low dose group(23.17±1.64),Treg medium dose group(23.33±2.20),Treg high dose group(20.67±1.38)24h water intake increased significantly.(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).Compared with normal control group(5.00±0.58)at Day34,T1DM group(26.00±2.52),Treg low dose group(22.00±1.15),Treg middle dose group(18.00±1.53),Treg high dose group(15.67±0.88),the difference was statistically significant(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).The average water intake of mice in the middle and high dose groups on Day34 was lower than that on Day14,suggesting that Treg may improve the symptoms of polydipsia in DN mice.5.Comparison of 24h urinary protein(mg)among groups:On Day14,compared with the normal control group(0.04±0.01),the levels of urinary protein in T1DM group(0.65±0.04),Treg low-dose group(0.58±0.05),Treg medium-dose group(0.50±0.03)and Treg high-dose group(0.44±0.02)were significantly increased.(P<0.001).Compared with T1DM group,the urine protein in Treg medium dose group(P<0.05)and high dose group(P<0.01)decreased significantly,but there was no significant difference between Treg low dose group and T1DM group(P>0.05).Compared with the normal control group(0.03±0.01),T1DM group(0.60±0.03),Treg low dose group(0.56±0.02),Treg middle dose group(0.48±0.05),Treg high dose group(0.43±0.04),the difference was statistically significant(P<0.001).There was significant difference between the high dose group and the T1DM group(P<0.05).These results suggest that Treg can improve the symptoms of proteinuria in DN mice,and the higher the concentration of Treg,the more effective the effect is.6.Comparison of insulin concentrations(n IU/ml)among groups:Compared with the normal control group(62.73±4.47),the insulin levels of T1DM group(19.47±1.18),Treg low dose group(20.05±1.84),Treg middle dose group(21.78±1.59),Treg high dose group(28.94±1.95)were significantly decreased on Day14.(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).Compared with the normal control group(63.28±4.01),T1DM group(22.06±1.50),Treg low dose group(25.40±1.99),Treg middle dose group(29.08±1.11),Treg high dose group(36.21±1.52),the difference was statistically significant(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).The average insulin level of the medium-and high-dose groups on Day34 was higher than that on Day14,suggesting that Treg may improve insulin levels in DN mice.7.Comparison of C-peptide concentration(ng/ml)among groups:Compared with the normal control group(13.25±0.71),the C-peptide levels of T1DM group(6.27±0.44),Treg low dose group(6.42±0.48),Treg middle dose group(7.80±0.36),Treg high dose group(8.50±0.15)were significantly decreased on Day14.(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).Compared with the normal control group(13.38±0.23),T1DM group(6.46±0.06),Treg low dose group(7.37±0.39),Treg middle dose group(8.94±0.37),Treg high dose group(10.56±0.70),the difference was statistically significant(P<0.001).Treg low dose group,middle dose group,high dose group and T1DM group had no significant difference(P>0.05).The average C-peptide level in the middle and high dose groups at Day34 was higher than that at Day14,suggesting that Treg may improve C peptide levels in DN mice.8.Comparison of kidney HE staining pathological sections between groups:the morphology and structure of kidney tissue in the control group were normal,and there was no obvious lesion.In T1DM group,the renal tubules showed dilatation,vacuolar degeneration of renal tubular epithelial cells,renal interstitial edema,and moderate granular degeneration of renal tubules accompanied by glomerular basement membrane thickening.With the increase of modeling time,the pathological changes of renal tissue became more serious.After Treg treatment,the renal tissue lesions were relatively alleviated:Treg low-dose group was lighter than T1DM group,there was a small amount of renal tubular granular degeneration,the middle dose group was lighter than the low dose group,the high dose group was better than the other two treatment groups,there was only a trace of renal tubular granular degeneration,the pathological changes were similar to the control group.These results suggest that Treg can improve the degree of renal inflammation in DN mice,and the higher the concentration of Treg,the more effective the effect is.9.Comparison of renal PDGF expression levels between groups:in the control group,there were only a very small amount of brown particles in the renal tissue,and the color was very light;Compared with the control group,the expression of PDGF in renal tissues of T1DM mice was significantly increased,and a large number of brown particles were deposited in renal tissues.With the increase of modeling time,the expression of PDGF in renal tissues increased.Compared with T1DM group,the expression of PDGF in renal tissue of Treg treatment group was reduced,and the degree of expression was:low dose group>middle dose group>high dose group.These results suggest that Treg can reduce the expression of PDGF in kidney of DN mice,and the higher the concentration of Treg,the more obvious the effect.10.Comparison of renal pathological scores among groups:Compared with the normal control group(0.00±0.00),the inflammatory scores of T1DM group(3.00±1.00),Treg low dose group(2.67±0.58),Treg medium dose group(2.33±0.58),Treg high dose group(1.33±0.58)increased on Day14.The differences were statistically significant,including T1DM group(t=5.133,P<0.01),Treg low-dose group(t=4.399,P<0.01),Treg medium-dose group(t=4.033,P<0.05),each group showed different degrees of renal inflammatory lesions,combined with the results of section,The scores were T1DM group>Treg low dose group>Treg middle dose group>Treg high dose group.However,there was no significant difference in Treg low dose group,middle dose group,high dose group and T1DM group(P>0.05).Compared with normal control group(0.00±0.00),T1DM group(3.33±0.58),Treg low dose group(3.00±1.00),Treg medium dose group(2.33±0.58),Treg high dose group(1.67±0.58),T1DM group(t=5.105,P<0.05).P<0.001),Treg low dose group(t=4.741,P<0.01),Treg medium dose group(t=4.376,P<0.05),Treg low dose group and Treg medium dose group had reduced renal lesions,Treg high dose group and control group had no significant difference.These results suggest that Treg can alleviate renal pathological changes in DN mice,and the higher the concentration of Treg,the more obvious the therapeutic effect.11.Comparison of spleen immune cells among each group:on the day of successful modeling,there was no significant difference in the levels of Th1,Th2,Th17 and Treg in spleen cells among the five groups.After Treg treatment,the levels of Th1,Th2 and Th17 in T1DM mice on Day14 were down-regulated,and the level of Treg was increased.Compared with the normal control group,Th1(P<0.001)and Th2(P<0.05)had significant differences,but Th17 and Treg had no significant differences compared with the normal control group.On Day14,the low-dose Treg group had the best effect on increasing the level of Th2 cells,and the medium-dose Treg group had the best effect on increasing the level of Treg cells.However,the effects of the three treatment groups on Th1 cells were not regular,and the effects on Th17 cells were not significantly different among the three treatment groups.On Day34,there was no significant difference in the levels of Th1,Th2,Th17 and Treg in spleen cells among the five groups.Conclusions:1.CD4~+CD25~+Treg cells may improve fasting blood glucose,body weight,24h urine volume,water intake,urine protein,insulin and C-peptide levels in DN mice.2.CD4~+CD25~+Treg cells can improve renal inflammatory lesions in DN,and the effect of high-dose group is obvious,which may be related to increasing the number of Treg cells in kidney tissue.3.Th1,Th2,Th17,and Treg levels were unbalanced in T1DM.After treatment with different concentrations of CD4~+CD25~+Treg cells,the medium-dose group had the best effect on increasing Treg level on Day14,and the low-dose group had the best effect on increasing Th2 cell level.However,Treg had an irregular effect on Th1 level,and there was no significant difference in Th17 level among the groups. |
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