Regulatory Effect Of Compound Shengdi Mixture On TLR-NF-?B Signaling Pathway And Thl7/Treg Cell Balance In MRL/lpr Mice

Objective:To investigate the regulation of compound Shengdi Mixture on TLR-NF-?B signaling pathway and Thl7/Treg cell balance in MRL/lpr mice.Methods:MRL/lpr mice were randomly divided into model group,Chinese medicine group and western medicine group.C57BL/6 mice were used as normal control group.Ten mice in each group were female mice of 12 weeks old.The Chinese medicine group was given the compound Shengdi mixture for intragastric administration,and the western medicine group was given the treatment with prednisone.The control group and the model group were replaced with physiological saline.After 8 weeks,the peripheral blood and spleen were taken and treated.Tested separately from the following two aspects:1.Effect of Compound Shengdi Mixture on Thl7/Treg Cell Balance in MRL/lpr Mice:The expression of cytokines TGF-?and IL-17 in peripheral blood was detected by enzyme-linked immunosorbent assay(ELISA).The levels of Th17 cells and Treg cells were detected by FCM.The expression of ROR?tmRNA?Foxp3mRNA in spleen was determined by quantitative Polymerase Chain Reaction.2.The effect of compound Shengdi mixture on the TLR-NF-?B signaling pathway in MRL/lpr lupus mice:The expression of IFN-?and TNF-?in peripheral blood was detected by ELISA.The expression of TLR7mRNA?TLR9mRNA?My D88mRNA and NF-?BmRNA in spleen was determined by quantitative Polymerase Chain Reaction.Expression of related proteins in TLR7,My D88,and NF-?B in the spleen was determined by Western blot analysis.Result:1.The effect of Compound Shengdi mixture on Th17/Treg cells MRL/lpr in mice:(1)The percentage of Th17 cells/CD4~+T cells in the blank group,western medicine group and Chinese medicine group was lower than that in the model group(P<0.01,P<0.01,P<0.05).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(2)The proportion of Treg cells/CD4~+T cells in the blank group,Chinese medicine group and western medicine group was higher than that in the model group(P<0.01,P<0.05,P<0.05).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(3)The ratio of Th17 cells/Treg cells in the blank group,western medicine group and Chinese medicine group was significantly lower than that in the model group(P<0.01).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).2.The effect of compound Shengdi mixture on cytokine levels of TLR-NF-?B signaling pathway and Th17/Treg in MRL/lpr mice:(1)The levels of IL-17A in the Chinese medicine group,western medicine group and blank group were lower than those in the model group(P<0.05,P<0.01,P<0.01).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(2)The levels of TGF-?1 in the blank group,western medicine group and Chinese medicine group were higher than those in the model group(P<0.01,P<0.05,P<0.05).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(3)The levels of TNF-?in the blank group and the Chinese medicine group were lower than those in the model group(P<0.01,P<0.05).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(4)The IFN-?levels in the blank group,the Chinese medicine group and the western medicine group were lower than those in the model group(P<0.05).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).3.The effect of compound Shengdi mixture on gene expression of TLR-NF-?B signaling pathway and Th17/Treg in MRL/lpr mice:(1)The expression levels of ROR?t mRNA in spleen of Chinese medicine group,western medicine group and blank group were lower than those in model group(P<0.05,P<0.05,P<0.01).There was no significant difference between Chinese medicine group and western medicine group(P>0.05).(2)The expression level of Foxp3 mRNA in spleen tissue of blank group and western medicine group was higher than that of model group(P<0.01,P<0.05).There was no significant difference between Chinese medicine group and western medicine group(P>0.05).(3)The expression levels of TLR7 mRNA in spleen of blank group,Chinese medicine group and western medicine group were lower than those in model group(P<0.01,P<0.05,P<0.01).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(4)The expression levels of TLR9mRNA in spleen of Chinese medicine group,western medicine group and blank group were lower than those in model group((P<0.05,P<0.01,P<0.01).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(5)The expression level of My D88 mRNA in spleen of Chinese medicine group,western medicine group and blank group were lower than that of model group(P<0.05,P<0.01,P<0.01).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).(6)The expression levels of NF-?B mRNA in spleen of Chinese medicine group,western medicine group and blank group were lower than those in model group((P<0.05,P<0.01,P<0.01).There was no significant difference between the Chinese medicine group and the western medicine group(P>0.05).4.The effect of compound Shengdi mixture on protein expression of TLR-NF-?B signaling pathway in MRL/lpr mice:(1)The TLR7/?-catin levels in blank group,traditional Chinese medicine group and Western medicine group were lower than those in model group(P<0.01);there was no significant difference between traditional Chinese medicine group and Western medicine group(P>0.05).(2)The My D88/?-catin levels in blank group,traditional Chinese medicine group and Western medicine group was lower than that in model group(P<0.01,P<0.01,P<0.05).Compared with western medicine group,My D88/beta-catin in western medicine group was higher(P<0.01).(3)The NF-?B p65/?-catin levels in blank group,traditional Chinese medicine group and Western medicine group were lower than those in model group(P<0.01);there was no significant difference between traditional Chinese medicine group and Western medicine group(P>0.05).Conclusion:1.Compound Shengdi Mixture can improve Th17 cells,decrease ROR?t mRNA and serum IL-17A levels in MRL/lpr mice,increase Treg cells and increase serum TGF-?1 levels,decrease Th17 cell/Treg cell ratio,and improve Th17/Treg Cell balance.2.Compound Shengdi Mixture can reduce TLR7?My D88 on TLR-NF-?B pathway in MRL/lpr mice,reduce the activation of NF-?B and the production of serum TNF-?and IFN-?.3.Compound Shengdi Mixture may reduce the activation of Th17cells by down-regulating the TLR-NF-?B signaling pathway in MRL/lpr mice,thereby improving the Th17/Treg cell balance in MRL/lpr mice.