Jieduquyuziyin Prescription-Treated Rat Serum Suppresses Activation of Peritoneal Macrophages in MRL/Lpr Lupus Mice by Inhibiting IRAK1 Signaling Pathway

Abstract

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, and Jieduquyuziyin prescription (JP) is a traditional Chinese medicine (TCM) formula that has been testified to be effective for SLE treatment as an approved hospital prescription for many years in China. However, its mechanism of action in the treatment of this disease is largely unknown. The purpose of this study was to determine whether JP-treated rat serum can inhibit the activation of peritoneal macrophages in MRL/lpr mice by downregulating the IRAK1 signaling pathway, thereby achieving the effect of improving SLE. The JP-treated rat serum was prepared, and the peritoneal macrophages of MRL/lpr lupus mice were isolated in vitro, and the effect of JP on cell viability was detected by the CCK8 method. After LPS induction and shRNA lentiviral transfection, the effect of JP on the expression of IRAK1 in cells was detected by immunofluorescence staining. The content of TNF-α and IL-6 in the cell supernatant was determined by ELISA. The expression of IRAK1, NF-κB, TNF-α, and IL-6 mRNA was detected by RT-PCR, and the expression levels of IRAK1, p-IRAK1, TRAF6, IKBα, p-IKBα, IKK + IKK, NF-κB, and p-NF-κB proteins was detected by western blot method. We investigated the role of JP in peritoneal macrophages of the MRL/lpr mouse and identified the possible mechanisms of action. The results showed that JP could reduce the phosphorylation of IRAK1 and its downstream proteins induced by LPS and inhibit the expression of inflammatory cytokines, including TNF-α and IL-6. In addition, after the transfection of cells with shRNA lentiviral, the results of JP tended to be consistent. In conclusion, JP may inhibit the activation of peritoneal macrophages in MRL/lpr mice by downregulating the IRAK1-NF-κB signaling pathway, and IRAK1 may be a potential target for JP treatment of SLE.

Figure 1

The effects of JP-treated rat serum on cell proliferation. Peritoneal macrophages of MRL/lpr lupus mice were seeded onto 96-wells plate and treated with various concentrations of JP-treated rat serum (0, 2.5, 5, 10, 15, or 20%) for 24 h Cell proliferation was assessed using CCK-8 assay. The values were expressed as the mean ± SD of three independent experiments. ^∗P < 0.05, and ^∗∗P < 0.01 versus blank cells.

Figure 2

JP suppresses IRAK1 activation in LPS-stimulated peritoneal macrophages of MRL/lpr lupus mice. Cells were treated with LPS either alone or with JP (2.5%) for 24 h later, the cells were subjected by immunofluorescent assays using DAPI (blue) and FITC (green). Data are representative images (magnification, 40× for immunofluorescent staining) of individual groups from three separate experiments. A control group; B LPS group; C LPS plus JP group.

Figure 3

JP downregulates LPS-induced signaling pathway in peritoneal macrophages of MRL/lpr mice. (a) The expression alterations in IRAK1 and NF-κB mRNA. (b) The expression alterations in phosphorylation and nonphosphorylation of IRAK1 and NF-κB protein. (c) The protein expression alterations in TRAF6, IKBα, p-IKBα, and IKKα + IKKβ. (d) The related protein bands. Values are expressed as mean ± SD of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01 versus control cells; ^#P < 0.05 and ^##P < 0.01 versus LPS-treated cells.

Figure 4

JP inhibits the secretion of inflammatory cytokines in peritoneal macrophages of MRL/lpr lupus mice. (a) The expression alterations in TNF-α and IL-6 mRNA. (b) Concentrations of TNF-α and IL-6 in the supernatant of peritoneal macrophages of MRL/lpr mice. (c) Transfection protein bands and transfection efficiency of IRAK1 shRNA in peritoneal macrophages of MRL/lpr mice. (d) After transfection and JP treatment, the alterations of inflammatory cytokine production and TNF-α and IL-6 mRNA in cells. Values are expressed as mean ± SD of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01 versus vehicle control cells; ^#P < 0.05 and ^##P < 0.01 versus LPS or shRNA-treated cells.

Figure 5

JP inhibits the activation of peritoneal macrophages in MRL/lpr lupus mice by downregulating IRAK1 and NF-κB signaling pathway. (a) After transfection and JP treatment, the expression alterations in IRAK1 and NF-κB mRNA. (b) First of all, JP could inhibit the expression of IRAK1 protein. (c) Next, after the JP intervention, the level of TRAF6 protein was inhibited. (d) Protein bands of each group of TRAF6. (e) Finally, the expression alterations in IKBα, p-IKBα, IKKα + IKKβ, NF-κB, and p-NF-κB protein after transfection and JP treatment. (f) Protein bands of each group of the related antibodies. Values are expressed as mean ± SD of three independent experiments. ^∗P < 0.05 and ^∗∗P < 0.01 versus vehicle control cells; ^#P < 0.05 and ^##P < 0.01 versus shRNA-treated cells.