Effect Of Esculentoside A On Nephritis In MRL/lpr Mice And Expression Of CD19~+IL-35~+breg Cells And IL-35

Objective:By observing the effect of esculentoside A（EsA）on the expression of CD19⁺IL-35⁺Breg cells,IL-35,and other cytokines in MRL/lpr mice to explore the possible therapeutic mechanism of EsA on lupus nephritis.Methods:Select 24 MRL/lpr mice of SPF rate,（female mice have onset）,divide randomly into EsA group,EsA+anti-IL-12p35 group and model control group.Administer the drug daily by intraperitoneal injection for 4 weeks in each group.4 weeks later,blood samples were collected and mice were sacrificed.Kidney and spleen tissues were collected.The spleen index was calculated by weighing the mice and spleen weight.HE staining and Masson+PASM staining were used to observe the pathological changes of the kidney and score the acute index（AI）.The expression levels of serum IL-10,IL-17 and IL-35 were detected by enzyme-linked immunosorbent assay（ELISA）.The proportion of CD19⁺IL-35⁺Breg cells in spleen was tested by flow cytometry.Use SPSS 21.0 for statistical analysis of the data.P≤0.05 was considered statistically significant.Results:1.General conditions Before the treatment,MRL/LPR mice in each experimental group ate normally,moved normally and also had normal hair.As the experiment progressed,the mice in the control group and EsA+anti-IL-12p35 group gradually performed mental fatigue,decreased feeding and activity,shortness of breath,hair loss,alopecia maculae skin injury and scab formation.No significant change in feeding and activity in the EsA group,except that the hair was thinner and the gloss was lower than before.2.Spleen index The spleen index of MRL/LPR mice in the model control group,EsA+anti-IL-12p35 group,and EsA group was respectively 228.12±11.8mg/g,191.69±10.75mg/g,and 134.47±7.64mg/g,with statistically significant differences（P<0.05）.3.Acute index The pathological manifestations of MRL/LPR mice in the model control group were:severe diffuse proliferation of mesangial cells and endothelial cells,deposition of a large number of immune complexes in the glomerulus,formation of cell fiber crescent and ring body;Diffuse lymphocytes and monocytes are seen in the renal stroma.The pathological manifestations of mouse kidney tissue in the EsA+anti-IL-12p35 group were:mesangial cells and endothelial cells of the glomerulus showed moderate to severe hyperplasia,a large number of immune complexes were deposited in the glomerulus,and rare crescent bodies and ring bodies were formed.Diffuse infiltration of lymphocytes and monocytes in the renal stroma.The pathological manifestations of the kidney in EsA group were:slight hyperplasia of mesangial cells and endothelial cells,deposition of immune complex in the glomerulus,formation of rare crescent.The renal interstitium was infiltrated with focal lymphocytes and monocytes.The Austin scoring system was used to score the kidney pathology of mice in each group.The AI indexes of MRL/LPR mice in the control group,EsA+anti-IL-12p35 group,and EsA group were 10.25±1.22,8.38±0.79,and 4.81±1.07,respectively.The differences between the three groups were statistically significant（P<0.05）.4.Proportion of CD19⁺IL-35⁺Breg cells in spleen of MRL/lpr mice The proportion of CD19⁺IL-35⁺Breg cells in spleen of MRL/lpr mice in in the model control group,EsA+anti-IL-12p35 group,and EsA group were 0.13±0.05%,0.35±0.16%,1.19±0.30%,respectively.The differences between the three groups were statistically significant（P<0.05）.5.Serum levels of IL-10,IL-35,and IL-17 in MRL/lpr mice Serum IL-10 levels of MRL/lpr mice in the model control group,EsA+anti-IL-12p35 group,and EsA group were63.30±12.83pg/m,101.34±21.67pg/ml,137.40±22.86pg/ml（P<0.05）.Serum IL-35 levels of MRL/lpr mice in model control group,EsA+anti-IL-12p35 group and EsA group were116.04±13.86pg/ml,129.56±12.22pg/ml and 141.96±5.83pg/ml（P<0.05）.Serum IL-17levels of MRL/lpr mice in the model control group,EsA+anti-IL-12p35 group,and EsA group were 17.90±1.89pg/ml,14.70±1.81pg/ml,12.54±0.94pg/ml（P<0.05）.6.Pearson correlation analysis and linear regression analysis Pearson correlation analysis showed that serum IL-35 level in MRL/lpr mice was positively correlated with IL-10 level（r=0.906,P<0.05）.The serum IL-35 level in MRL/lpr mice was positively correlated with the proportion of CD19⁺IL-35⁺Breg cells（r=0.667,P<0.05）.The serum levels of IL-35 and IL-17 in MRL/lpr mice were negatively correlated（r=-0.954,P<0.05）.Linear regression analysis of serum IL-35 and IL-10,IL-17,CD19⁺IL-35⁺Breg in MRL/lpr mice showed that the regression equation between peripheral blood IL-35（X）and IL-10（Y₁）in MRL/lpr mice was Y₁=-177.75+2.2X,with statistical significance（P<0.05）,the regression equation between IL-35（X）and IL-17（Y₂）in peripheral blood of mice was Y₂=37.14-0.17X,with statistical significance（P<0.05）,the regression equation between the expression level of IL-35 and the proportion of CD19+IL-35+Breg cells in peripheral blood of mice was Y₃=-2.3+0.02X,with statistical significance（P<0.05）.Conclusion:1.EsA can reduce the AI index and spleen index of mice in the model of spontaneous systemic lupus erythematosus,and has a certain therapeutic effect on MRL/lpr mice.2.The therapeutic effect of EsA on MRL/lpr mice may be related to the down-regulation of IL-17 by promoting the proliferation of CD19⁺IL-35⁺Breg cells to produce IL-35.