Anti-Inflammatory Effect of Geniposide on Regulating the Functions of Rheumatoid Arthritis Synovial Fibroblasts via Inhibiting Sphingosine-1-Phosphate Receptors1/3 Coupling Gαi/Gαs Conversion

Abstract

The activated Gα protein subunit (Gαs) and the inhibitory Gα protein subunit (Gαi) are involved in the signal transduction of G protein coupled receptors (GPCRs). Moreover, the conversion of Gαi/Gαs can couple with sphingosine-1-phosphate receptors (S1PRs) and have a critical role in rheumatoid arthritis (RA). Through binding to S1PRs, sphingosine-1-phosphate (S1P) leads to activation of the pro-inflammatory signaling in rheumatoid arthritis synovial fibroblasts (RASFs). Geniposide (GE) can alleviate RASFs dysfunctions to against RA. However, its underlying mechanism of action in RA has not been elucidated so far. This study aimed to investigate whether GE could regulate the biological functions of MH7A cells by inhibiting S1PR1/3 coupling Gαi/Gαs conversion. We use RASFs cell line, namely MH7A cells, which were obtained from the patient with RA and considered to be the main effector cells in RA. The cells were stimulated with S1P (5 μmol/L) and then were treated with or without different inhibitors: Gαi inhibitor pertussis toxin (0.1 μg/mL), S1PR1/3 inhibitor VPC 23019 (5 μmol/L), Gαs activator cholera toxin (1 μg/mL) and GE (25, 50, and 100 μmol/L) for 24 h. The results showed that GE may inhibit the abnormal proliferation, migration and invasion by inhibiting the S1P-S1PR1/3 signaling pathway and activating Gαs or inhibiting Gαi protein in MH7A cells. Additionally, GE could inhibit the release of inflammatory factors and suppress the expression of cAMP, which is the key factor of the conversion of Gαi and Gαs. GE could also restore the dynamic balance of Gαi and Gαs by suppressing S1PR1/3 and inhibiting Gαi/Gαs conversion, in a manner, we demonstrated that GE inhibited the activation of Gα downstream ERK protein as well. Taken together, our results indicated that down-regulation of S1PR1/3-Gαi/Gαs conversion may play a critical role in the effects of GE on RA and GE could be an effective therapeutic agent for RA.

Keywords: fibroblast-like synoviocytes; geniposide; gαi-gαs conversion; rheumatoid arthritis; sphingosine-1-phosphate; sphingosine-1-phosphate receptor 1/3.

FIGURE 1

Chemical structure of geniposide.

FIGURE 2

Effects of GE on proliferation, migration and invasion of MH7A cells induced by S1P. MH7A cells were treated with S1P (5 μmol/L) with or without different inhibitors Gαs activator CTX (1 μg/mL), Gαi inhibitor PTX (0.1 μg/mL), S1PR1/3 inhibitor VPC 23019 (5 μmol/L) and GE (25, 50 and 100 μmol/L) for 24 h. (A) Effect of S1P on the proliferation activity in MH7A was determined by CCK-8 kit (n = 6). (B) Effects of GE on the proliferation activity of MH7A induced by S1P was determined by CCK-8 kit (n = 6). (C) The proliferation protein level of Ki 67 was determined by Western blot (n = 3). (D) Effects of GE on the migration activity of MH7A was determined by Transwell (n = 5). (E) Effects of GE on the insion activity of MH7A was determined by Transwell (n = 5). Data are represented as mean ± SEM. ^* p < 0.05, ^** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus S1P (5 μmol/L) group.

FIGURE 3

Effects of GE on the expression of S1PR1/3 and Gαi/Gαs. MH7A cells were treated with S1P (5 μmol/L) with or without different inhibitors Gαs activator CTX (1 μg/mL), Gαi inhibitor PTX (0.1 μg/mL), S1PR1/3 inhibitor VPC 23019 (5 μmol/L) and GE (25, 50 and 100 μmol/L) for 24 h. (A) Effects of GE on the expression of S1PR1 and S1PR3 mRNA in MH7A induced by S1P (n = 3). (B) mRNA expression of Gαi1/2/3 and Gαs was detected by qRT-PCR (n = 3). (C) The protein levels of S1PR1/3 and Gαi/Gαs were determined by Western blot analyses using specific antibodies for each protein (n = 3). Data are represented as mean ± SEM. ^* p < 0.05, ^** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus S1P (5 μmol/L) group.

FIGURE 4

Effects of GE on the expression of cAMP in MH7A induce by S1P. MH7A cells were treated with S1P (5 μmol/L) with or without different inhibitors Gαs activator CTX (1 μg/mL), Gαi inhibitor PTX (0.1 μg/mL), S1PR1/3 inhibitor VPC 23019 (5 μmol/L) and GE (25, 50 and 100 μmol/L) for 24 h. (A) cAMP expression in MH7A was determined by ELISA analysis (n = 3). (B) mRNA expression of cAMP was detected by qRT-PCR (n = 3). Data are represented as mean ± SEM. ^* p < 0.05, ^** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus S1P (5 μmol/L) group.

FIGURE 5

Effect of GE on S1PR1/3 co-expressing and coupling with Gαi/Gαs in MH7A induced by S1P. MH7A cells were treated with S1P (5 μmol/L) with or without different inhibitors Gαs activator CTX (1 μg/mL), Gαi inhibitor PTX (0.1 μg/mL), S1PR1/3 inhibitor VPC 23019 (5 μmol/L) and GE (25, 50 and 100 μmol/L) for 24 h. (A) Confocal imaging of S1PR1 and the Gαi in MH7A (n = 5). (B) Confocal imaging of S1PR1 and the Gαs in MH7A (n = 5). (C) Confocal imaging of S1PR3 and the Gαi in MH7A (n = 5). (D) Confocal imaging of S1PR3 and the Gαs in MH7A (n = 5). Data are represented as mean ± SEM. ^* p < 0.05, ^** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus S1P (5 μmol/L) group.

FIGURE 6

Effect of GE on modulate cytokine secretion in MH7A induced by S1P. MH7A cells were treated with S1P (5 μmol/L) with or without different inhibitors Gαs activator CTX (1 μg/mL), Gαi inhibitor PTX (0.1 μg/mL), S1PR1/3 inhibitor VPC 23019 (5 μmol/L) and GE (25, 50 and 100 μmol/L) for 24 h. Expression of (A) IL-1β, (B) IL-6, (C) IL-8, (D) PGE2 and (E) TGF-β1 in MH7A was determined by ELISA analysis (n = 3). Data are represented as mean ± SEM. ^* p < 0.05, ^** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus S1P (5 μmol/L) group.

FIGURE 7

Effect of GE on ERK1/2 in S1P-stimulated MH7A. MH7A cells were treated with S1P (5 μmol/L) with or without different inhibitors Gαs activator CTX (1 μg/mL), Gαi inhibitor PTX (0.1 μg/mL), S1PR1/3 inhibitor VPC 23019 (5 μmol/L) and GE (25, 50 and 100 μmol/L) for 24 h. Western blot analysis of ERK1/2 and p-ERK1/2 in MH7A cells (n = 3). Data are represented as mean ± SEM. ^* p < 0.05, ^** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.01 versus S1P (5 μmol/L) group.

FIGURE 8

The potential mechanism of cells dysfunctions that S1P may stimulus S1PR1/3 coupled Gαi/Gαs conversion and the regulation of GE. S1P binds to the ligand S1PR1/3, resulting in an imbalance in the conversion of Gαi and Gαs and inducing an inflammatory response. Meanwhile, it mediates the activation of the downstream signal pathway AC/cAMP and protein ERK1/2, which leads to the dysfunctions of FLSs. GE exerts regulation by blocking S1PR1/3 coupling Gαi/Gαs conversion, restoring the dynamic balance of Gαi and Gαs protein and anti-/pro-inflammatory factors.