Acknowledgements
This work is supported by National Key Research and Development Program of China (2019YFE0111700) and National Natural Science Foundation of China (81971517). XY was funded by the Science and Technology Development Fund, Macau SAR (File no. 0075/2019/AMJ).
References
Figure legends
Fig. 1 Silybin ameliorated lupus-like features and improved the efficacy of MSCs in R848-induced mice. (A ) The survival rate for 12 weeks old acetone-induced B6 mice, R848-induced B6 mice, and R848-induced mice treated with silybin, MSCs, and silybin plus MSCs (n =6 in B6 group, n =9 in the rest four groups). (B ) Spleen size of mice among five groups. (C-E ) Serum IgG, antinuclear antibody and anti-ds-DNA antibody levels among five groups. (F-I ) Urine protein, urine creatinine, serum nitrogen and serum creatinine levels among five groups. (J ) Representative renal histology and immunofluorescence among five groups (Magnification: ×400). *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001, ns: not significant.
Fig. 2 Changes in T/B lymphocyte subsets in vivo after silybin and MSCs treatment. (A ) Representative flow cytometry plots of Th1 cell (CD4+INF-γ+), Th2 cell (CD4+IL-4+) and Th17 cell (CD4+IL-17+) percentages in splenocytes among five groups. (B ) Representative flow cytometry plots of Tfh cell (CD4+CXCR5+PD-1+) and Treg cell (CD4+CD25+Foxp3+) percentages in splenocytes among five groups. (C ) Representative flow cytometry plots of memory B cell (B220+IgD+CD38+), GC B cell (B220+CD95+GL7+) and plasmablast (B220+CD138+) percentages in splenocytes among five groups. (D ) Statistical results of flow cytometry data for each group. n = 6 in B6 and R848 groups, n = 9 in silybin or MSCs treated groups, and n = 8 in silybin plus MSCs group after removal of dead mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001, ns: not significant.
Fig. 3 Effects of silybin on lymphocyte subsets in vitro . (A ) The cell subset proportions were determined by flow cytometry using splenocytes from two groups (n = 6 for Th1, Th2, Th17, Treg and Tfh cells, n= 5 for GC B cells and plasmablast). (B ) The percentages of Tfh cells of R848-induced mice treated with different concentrations of silybin in vitro (n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001, ns: not significant.
Fig. 4 Regulatory effects of silybin and MSCs on Tfh cell proliferation, apoptosis and differentiation-related genes. (A ) The percentages of apoptosis (n=5) and proliferation (n=6) of Tfh cells in R848-induced mice treated with silybin and MSCs in vitro . (B ) Expression of Tfh-related genes in acetone-induced B6 mice and R848-induced B6 mice (n=6). (C ) Expression of Tfh-related genes in R848-induced B6 mice and R848-induced mice treated with silybin and/or MSCs (n=6). (D ) Western blot analysis of p-STAT3, STAT3 and β-actin in splenocytes of B6, R848-induced and silybin treated R848-induced mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001, ns: not significant.
Fig. 5 Impact of IL-6 and its downstream pathways on silybin regulated Tfh cells. (A ) IL-6 counteracted the effect of silybin on Tfh cell expansion in splenocytes of R848-induced mice (n=6). (B ) The mean fluorescence intensity of pAKT and pS6 in Tfh cells of R848-induced mice increased after IL-6 treatment (n=6). (C ) The mean fluorescence intensity of pSTAT3 in Tfh cells of R848-induced mice increased after IL-6 treatment (n=6). (D-E ) Both rapamycin and stattic partially inhibited the effect of IL-6 on Tfh cell expansion in normal B6 mice splenocytes (n=6). (F ) Both colivelin and MHY1485 counteracted the effect of silybin on Tfh cell expansion in splenocytes of R848-induced mice (n=6). *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001.