| Objective The aim of this study was to investigate the variation of intestinal microbiota,calcium/calmodulin-dependent protein kinase type 4(CaMK4)and spleen tyrosine kinase(Syk)in MRL/lpr mice.Observe the regulatory effect of Jieduquyuziyin prescription(JP)on intestinal microbiota of systemic lupus erythematosus(SLE)mice and investigate the possible mechanism of the CaMK4 and Syk on the balance of Th17/Treg,suggesting a novel therapy for treatment of SLE.Methods 1.Thirty 8-week female MRL/lpr mice were randomly divided into three groups(n=10):the SLE model group,the JP-treated group and the prednisone(PN)-treated group.Ten C57BL/6J mice are treated as the healthy mice.Fecal samples were obtained from the four groups and then bacteria(Escherichia coli,Enterococcus faecalis,Baeteroides,Lactobacillus and Bifidobacteria)DNA were extracted from those samples,RT-PCR was used to analyze the amount of the bacteria;Urinary samples were collected from the four groups and determined the content of proteinuria;HE staining was used to detect the pathological changes of the kidneys.2.Forty 8-week female MRL/lpr mice were randomly divided into four groups(n=10):the SLE model group,the JP-treated group,the KN-93(an inhibitor of CaMK4)-treated group and the Prednisone(PN)-treated group.Ten C57BL/6J mice are treated as the healthy mice.After the treatment of 10 weeks all mice were drawn blood from eyeball and were sacrificed.The coomassie blue staining was employed to detect the concentration of urinary protein,the lymph node and spleen removed and weighed values were expressed as a percentage of body.The expression of CaMK4 was studied by RT-PCR and western blot.The effect of JP on the balance of Th17 cells and Treg cells in spleen lymphocyte was tested by the technology of flow cytometry.ELISA was used to measure the level of Th17-related cytokines and Treg-related cytokines.3.Ten C57BL/6J mice are treated as the healthy mice.Forty 8-week female MRL/lpr mice were randomly divided into the SLE model group,the JP-treated group,the R-406(an inhibitor of Syk)-treated group and the Prednisone(PN)-treated group.10 weeks later,mice were sacrificed after taking blood sample.The lymph node and spleen removed and weighed values were expressed as a percentage of body,ELISA was used to measure the level of ds-DNA.The preventative or therapeutic effects of JP in MRL/lpr mice were investigated.The expression of Syk was studied by RT-PCR and western blot.The effect of JP on the balance of Th17 cells and Treg cells in spleen lymphocyte was taken by the technology of flow cytometry.ELISA was used to measure the level of Th17-related cytokines and Treg-related cytokines.Results 1.The amounts of Escherichia coli,Enterococcus faecalis,Baeteroides,Lactobacillus and Bifidobacteria were respectively(9.35±0.32,9.83±0.44),(7.28±0.75,7.89±0.87),(9.01±0.38,8.63±0.29),(8.24±0.12,7.90±0.41),(6.81±0.25,7.39±0.52)in the fecal samples of the normal control group and the model group.Statistical analysis showed that the amount of bacteria were significantly different with those of control group(P<0.01);Urine protein in model group was obviously higher than control group(P<0.01);In each treating group,proteinuria and pathological injuries were apparently decreased compared with control group.The prednisone had no prominent regulation effect on intestinal microbiota while Jieduquyuziyin prescription showed promotive effect on intestinal microbiota(P<0.01).2.The expression levels of CaMK4 was significantly increased in the model SLE group compare to the healthy mice(P<0.01).Treatment with JP,KN-93 or PN remarkably suppressed expression of CaMK4(P<0.05).Analysis results of FACS showed that the Th17 cells in the MRL/lpr mice were significantly higher compared with the healthy mice(P<0.05).When treatment groups were compared to the model group,the level of Th17 cells was considerably reduced and the difference was statistically significant(P<0.05).The level of Treg cells in the model group was significantly lower compared with the healthy mice(P<0.05).When the treatment groups were compared to the MRL/lpr mice,the level of Treg cells was considerably increased and the difference was statistically significant(P<0.05).The Th17/Treg restored immune balance.3.The results showed that SLE T cells express abnormally high levels of Syk,while using JP or R406 resulted in decreasing in the expression of Syk,and an imbalance between Th17 cells and Treg cells was involved in the occurrence and development of SLE.Treatment with JP,R406 or R406 remarkably suppressed expression of Syk(P<0.05).The frequencies of Th17 cells were further significantly reduced in each treatment groups compared to the model group(P<0.05).The frequencies of Treg cells were markedly increased in each treatment groups compared to the model group(P<0.05).The Th17/Treg restored immune balance.Conclusion 1.Experimental evidence of pharmacological showed that JP contributes to reducing the skin disease and spleen and lymph node enlargement in the MRL/lpr mice;JP treatment strongly decreased autoantibody and proteinuria production,ameliorated lethal renal injury,improved the histopathologic changes of glomerulonephritis,and thus prolonged the lifespan of MRL/lpr mice.We investigate the mechanism involved in the therapeutic efficacy of JP.2.We showed that JP can suppress the expression of mRNA and protein of CAMK4 in T cells from MRL/lpr mice.We also demonstrated that JP has a central role in the balance of Th17 cell and Treg cell.Taken together,JP might be used as a potential therapeutic strategy to regulate the the balance of Th17 cell and Treg cell by inhibiting the express of CaMK4.3.Our data support that JP has a profound inhibitory effect on SLE development as well as its severity.Mechanistically,our results support the concept that Syk is a crucial aspect in the pathogenesis of SLE.The inhibitory effect appeared to be mediated through reciprocal regulation of Th17 and Treg cells by suppressive effect on Syk of JP.Therefore,JP may be a novel therapeutic agent for the treatment of SLE. |
PDF Full Text Request