The effects of Jieduquyuzishen prescription-treated rat serum on the BAFF/BAFF-R signal pathway

Abstract

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease mainly characterized by B cell hyperactivity. Glucocorticoid (GC) is widely used in SLE for its potent anti-inflammatory and immunosuppressive effects. Despite its important clinical efficacy, high-dose or long-term use of GC can cause severe side effects, such as osteoporosis, osteonecrosis, cataracts, hyperglycemia, coronary heart disease and cognitive impairment. Our early clinical studies have shown that Jieduquyuzishen prescription (JP) can effectively reduce the adverse effects and improve the curative effect of GC in the treatment of SLE. The BAFF/BAFF-R signaling pathway plays an important role in the development of SLE and has been regarded as a potential target for the therapy of SLE. In this study, we attempt to investigate the effect of JP on the BAFF/BAFF-R signaling pathway to explore the mechanism of JP in reducing the toxicity and enhancing the efficacy of GC. YAC-1 cells, isolated rat peripheral blood lymphocytes, polymorphonuclear neutrophils and spleen lymphocytes were treated with drug-containing serum. The results of RT-PCR, Western blot and dual-luciferase reporter gene assays indicate that either JP or GC can inhibit the mBAFF-induced up-regulation of BAFF, BAFF-R, Bcl-2, IL-10 and NF-κB in YAC-1 cells and WEHI-231 cells. Furthermore, MTS, flow cytometry and CFSE results reveal that the proliferation and survival of lymphocytes activated by mBAFF are suppressed by JP, GC and their combination. Contrary to GC, JP can reduce the apoptosis and raise the survival of polymorphonuclear neutrophils and can't increase the apoptosis of the peripheral blood lymphocytes and spleen lymphocytes. Therefore, it is possible that JP can down-regulate the BAFF/BAFF-R signaling pathway as effectively as GC, which may result in the dosage reduction of GC, thus decreasing the toxicity and improving the efficacy of GC-based treatment of SLE.

Fig 1. Effect of JP on the mRNA sxpression of BAFF, BAFF-R, NF-κB, Bcl-2 and IL-10 in YAC-1 Cells.

The mRNA expression was determined by RT-PCR, the mRNA level of β-actin was used as an internal control, and gene-specific mRNA expression was normalized against β-actin expression (a and b). The protein expression of BAFF-R, NF-κB and phospho-NF-κB in YAC-1 Cells was determined by Western blot (c and d). A GAPDH protein antibody was used as an internal control. Target protein expression was normalized against GAPDH protein expression. C represents the 10% blank-control serum group, R represents the 10% blank-control serum plus mBAFF group, G represents the 10% blank-control serum plus mBAFF and GC group and J represents the 10% moderate-dose JP-treated rat serum plus mBAFF group. Results are shown as means ± SD. * p<0.05 compared with the C group, ▲p<0.05 compared with the R group.

Fig 2. mRNA expression of BAFF-R, NF-κB, Bcl-2 and IL-10 in WEHI-231 cells treated with JP at different doses (a and b) and different times (c and d).

The mRNA level of β-actin was used as an internal control, and gene-specific mRNA expression was normalized against β-actin expression. C represents the 10% blank-control serum group, R represents the 10% blank-control serum plus mBAFF group, L represents the 10% low-dose JP-treated rat serum plus mBAFF group, M represents 10% moderate-dose JP-treated rat serum plus mBAFF group, H represents 10% high-dose JP-treated rat serum plus mBAFF group. 3h, 6h, 12h and 24h means that cells were treated with moderate-dose JP-treated rat serum for 3 hours, 6 hours, 12 hours and 24 hours respectively. Results are shown as means ± SD. * p<0.05 compared with the C group, ▲ p<0.05 compared with the R group, # p<0.05 compared with the L group, △ p<0.05 compared with the 3h group.

Fig 3. Effect of JP on the apoptosis of rat peripheral blood lymphocytes (a), polymorphonuclear neutrophils (b), spleen lymphocytes (c) and on the transcriptional activity of NF-κB in YAC-1 cells (d).

The percentage of apoptotic cells was assessed by flow cytometry as described. The blue characters in the lower right part of each figure represent different treatment groups. The transcriptional activity of NF-κB was detected by dual-luciferase reporter gene assay and expressed as the ratio of firefly to Renilla luciferase abundance (Fluc/Rluc). Data is shown as means ± SD. *p<0.05 compared with the C group, ▲p<0.05 compared with the R group. C represents the 10% blank-control serum group, R represents the 10% blank-control serum plus mBAFF group, G represents the 10% blank-control serum plus mBAFF and GC group, J represents the 10% moderate-dose JP-treated rat serum plus mBAFF group.

Fig 4. Effect of JP on the proliferation of cells.

(a) The proliferation of YAC-1 cells, rat peripheral blood lymphocytes and polymorphonuclear neutrophils determined by MTS. The absorbance was subsequently measured at 490 nm using an automatic microplate reader. (b) The proliferation of rat spleen lymphocytes determined by CFSE method. The proliferation dynamics of CFSE labeled cells were analyzed by flow cytometry within 1 hour. The B region of the cells was showed as group of proliferation cells. * p<0.05 compared with the C group, ▲ p<0.05 compared with the R group.