Yi Shen Juan Bi Pill Regulates the Bone Immune Microenvironment via the JAK2/STAT3 Signaling Pathway in Vitro

Abstract

Rheumatoid arthritis (RA) is characterized by an impaired articular bone immune microenvironment, which is associated with regulatory T cells (Tregs) hypofunction and osteoclasts (OCs) hyperfunction and leads to articular bone erosion and systemic bone loss. Studies have shown that Tregs slow bone loss in RA by regulating the bone resorption function of OCs and the JAK/STAT signaling pathway can regulate the immunosuppressive function of Tregs and reduce the bone erosion function of OCs. Yi Shen Juan Bi Pill (YSJB) is a classic Chinese herbal compound for the treatment of RA. However, whether YSJB regulates bone immune microenvironment homeostasis through JAK/STAT signaling pathway remains unclear. Based on in vitro OC single culture, Treg single culture and OC-Treg coculture systems, treatments were performed using drug-containing serum, AG490 and JAK2 siRNA to explore whether YSJB-containing serum regulates the homeostasis of the bone immune microenvironment through the JAK/STAT signaling pathway. In vitro, YSJB treatment decreased the number of TRAP⁺ cells and the areas of bone resorption and inhibited the expression of RANK, NFATc1, c-fos, JAK2, and STAT3 in both the OC single culture system and the OC-Treg coculture system. Tregs further reduced the number of TRAP⁺ cells and the areas of bone resorption in the coculture system. YSJB promoted the secretion of IL-10 while inhibiting the expression of JAK2 and STAT3 in Tregs. Moreover, inhibiting the expression of JAK2 with the JAK2 inhibitor AG490 and JAK2 siRNA improved the immunosuppressive functions of Treg, inhibited OC differentiation and bone resorption. Our study demonstrates that YSJB can regulate OC-mediated bone resorption and Treg-mediated bone immunity through the JAK2/STAT3 signaling pathway. This study provides a new strategy for regulating the bone immune microenvironment in RA with traditional Chinese medicine.

Keywords: JAK2/STAT3 signaling pathway; Yi Shen Juan Bi Pill; osteoclast; regulatory T cell; rheumatoid arthritis.

FIGURE 1

Effect of AG490 on cells viability and osteoclastogenesis in the OC single culture system. (A) Cells (OPCs and OCs) viability was assessed by CCK-8 assays after treatment with AG490 (0, 2.5, 5, 10, 20, or 50 μM). (B,C) TRAP staining in OCs after treatment with AG490 (0, 2.5, 5 or 10 μM). Scale bars = 100 µm. The data are shown as the mean ± SEM (n ≥ 2). ^# p < 0.05, ^## p < 0.01 compared with the control group.

FIGURE 2

Effect of YSJB and JAK2 siRNA on the JAK2/STAT3 signaling pathway. (A) Confirmation of JAK2 knockdown in OCs. (B) The relative mRNA levels of JAK2 in OCs. (C) The relative mRNA levels of STAT3 in OCs. (D) Representative bands of JAK2, p-JAK2, STAT3, and p-STAT3 protein detected by western blot assay in OCs. (E) Protein expression levels of p-JAK2/JAK2 and p-STAT3/STAT3 in OCs. (F) The relative mRNA levels of JAK2 in Tregs. (G) The relative mRNA levels of STAT3 in Tregs. (H) Representative bands of JAK2, p-JAK2, STAT3, and p-STAT3 protein detected by western blot assay in Tregs. (I) Protein expression levels of p-JAK2/JAK2 and p-STAT3/STAT3 in Tregs. Cells were treated with drug-containing serum and JAK2 siRNA. ^# p < 0.05, ^## p < 0.01 compared with the control group; *p < 0.05, **p < 0.01 compared with the CIA group.

FIGURE 3

Effect of YSJB, AG490 and JAK2 siRNA on osteoclastogenesis in the OC single culture system. (A) Images of TRAP staining in OPCs treated with drug-containing serum (Control, CIA, CIA + MTX, CIA + YSJB), AG490 and JAK2 siRNA. Scale bar = 100 μm. (B,C) The number of TRAP⁺ cells. The data are shown as the mean ± SEM (n ≥ 2). ^# p < 0.05, ^## p < 0.01 compared with the control group; *p < 0.05; **p < 0.01 compared with the CIA group.

FIGURE 4

Effect of YSJB, AG490 and JAK2 siRNA on osteoclastogenesis in the OC-Treg coculture system (25:1). (A) Images of TRAP staining in the coculture system after treatment with drug-containing serum (Control, CIA, CIA + MTX, CIA + YSJB), AG490 and JAK2 siRNA. Scale bar = 100 μm. (B,C) The number of TRAP⁺ cells in the coculture system treated as described in (A). (D–G) The number of TRAP⁺ OCs after the addition of Tregs. The data are shown as the mean ± SEM (n ≥ 2). ^# p < 0.05, ^## p < 0.01 compared with the control group; *p < 0.05, **p < 0.01 compared with the CIA group.

FIGURE 5

Effect of YSJB, AG490 and JAK2 siRNA on bone resorption in the OC single culture system. (A) Images of bone resorption pits. Scale bar = 100 μm. (B,C) The areas of the bone resorption pits were measured by the image analysis program. The data are shown as the mean ± SEM (n ≥ 2). ^# p < 0.05, ^## p < 0.01 compared with the control group; *p < 0.05, **p < 0.01 compared with the CIA group.

FIGURE 6

Effect of YSJB, AG490 and JAK2 siRNA on bone resorption in the coculture system. (A) Images of bone resorption pits. Scale bar = 100 μm. (B,C) The areas of the bone resorption pits were measured by the image analysis program. (D–G) The areas of the bone resorption pits after the addition of Tregs. The data are shown as the mean ± SEM (n ≥ 2). ^# p < 0.05, ^## p < 0.01 compared with the control group; *p < 0.05, **p < 0.01 compared with the CIA group; ^p < 0.05, ^ ^p < 0.01 compared with the CIA+MTX group.

FIGURE 7

YSJB, AG490 and JAK2 siRNA decreased the mRNA levels of RANK, NFATc1, and c-fos, which were associated with the differentiation and maturation of OCs. (A–F) The mRNA levels of RANK, NFATc1, and c-fos in the OC single culture system. (G–L) The mRNA levels of RANK, NFATc1, and c-fos in the coculture system. Cells were treated with drug-containing serum, AG490 or JAK2 siRNA. ^# p < 0.05, ^## p < 0.01 compared with the control group; *p < 0.05, **p < 0.01 compared with the CIA group; ^p < 0.05, ^ ^p < 0.01 compared with the CIA+MTX group.

FIGURE 8

YSJB, AG490 and JAK2 siRNA promoted the secretion of IL-10 by Tregs. The mRNA levels of IL-10 and TGF-β1 were measured by qRT-PCR (A–D) and ELISA (E–H) in Tregs treated with drug-containing serum, AG490 or JAK2 siRNA. ^# p < 0.05, ^## p < 0.01 compared with the control group; *p < 0.05, **p < 0.01 compared with the CIA group.

FIGURE 9

Mechanism of YSJB regulates the bone immune microenvironment. YSJB directly regulates the JAK2/STAT3 signaling pathway in OCs, thereby inhibiting OC differentiation and bone loss. Moreover, YSJB regulates the JAK2/STAT3 signaling pathway in Tregs and upregulates the expression of IL-10. YSJB enhances the immunosuppressive function of Tregs and indirectly inhibits OC differentiation and bone loss.