Objective: To observe the effects of paeoniflorin on 3T3 fibroblast activation, proliferation and collagen production through IL-13/STAT6 signaling pathway.
Methods: 3T3 cell strain was cultured with serum-free medium for 12 h, then stimulated by paeoniflorin (200, 400, 600, 800, and 1000 mg/L) or rIL-13 (6.25, 12.5, 50, 100, and 200 microg/L) for another 24 h. At the same time the blank control group for paeoniflorin or rIL-13 was observed. 3T3 cell proliferation was assayed by Cell Counting Kit-8 (CCK-8), and an appropriate concentration (100 microg/L) of rIL-13 was chosen according to the result of cell proliferation. Subsequently, 3T3 cell cultured with serum-free medium for 12 h was stimulated by 100 microg/L rIL-13 for 12 h, and then was treated with different concentrations of paeoniflorin (200, 400, 600, 800, and 1000 mg/L) for another 24 h. Untreated 3T3 cell served as blank control Cell proliferation was measured by CCK-8. Hydroxyproline content in cell supernatant was determined by alkaline lysis method. IL-13Ralpha1, alpha-SMA and STAT6 protein expression were detected by Western blotting. Col-I, Col-III, IL-13Ralpha1 and STAT6 mRNA expression were analyzed by RT-PCR.
Results: Paeoniflorin inhibited 3T3 cell proliferation in a concentration-dependent manner (r = -0.980, P < 0.01), and there was a statistically significant difference among all groups (F = 198.599, P < 0.01). rIL-13 caused a remarkably concentration-dependent increase in proliferation of 3T3 cells (r = 0.538, P < 0.05). Paeoniflorin (200, 400, 600, 800, and 1000 mg/L) inhibited proliferation of 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (1.780 +/- 0.177, 1.636 +/- 0.073, 0.965 +/- 0.066, 0.623 +/- 0.037, 0337 +/- 0.022, r = -0.971, P < 0.01), and among all groups there existed a significant difference (F = 198.537, P < 0.01). Moreover, paeoniflorin also suppressed secretion of hydroxyproline from 3T3 cell stimulated by rIL-13 in a concentration-dependent manner (3.030 +/- 0.094, 2.976 +/- 0.047, 2.814 +/- 0.047, 2.652 +/- 0.124, 2.408 +/- 0.124, r = -0.916, P < 0.01) with a statistical significance among all groups (F = 13.642, P < 0.01). Further investigations showed that paeoniflorin decreased both protein expression of alpha-SMA, IL-13Ralpha1, and STAT6, and mRNA expression of Col-I, Col-III, IL-13Ralpha1, and STAT6 in 3T3 cell stimulated by rIL-13.
Conclusion: Paeoniflorin inhibits activation, proliferation of fibroblasts and production of collagen from fibroblasts through IL-13/STAT6 signaling pathway, which might be one of mechanisms of anti-hepatic fibrosis of paeoniflorin in schistosomiasis japonica.