Oridonin promotes CD4+/CD25+ Treg differentiation, modulates Th1/Th2 balance and induces HO-1 in rat splenic lymphocytes

Abstract.

Objective and design:

Oridonin is an ent-kaurene diterpenoid extracted from Isodon Serra, and we have previously demonstrated its immunosuppressive effect. Our goal was to study how Oridonin impacts CD4⁺/CD25⁺ regulatory T cells (Tregs) and Th1/Th2 balance, as well as its effect on the anti-inflammatory target HO-1.

Material:

Splenic lymphocytes were prepared from male 6–8-week-old SD rats.

Treatment:

Cells were cultured in four groups as Oridonin-L (Oridonin 12.5 μmol/l), Oridonin-H (Oridonin 25 μmol/l), Cobalt protoporphyrin (Copp 50 μmol/l) and control (DMSO) with stimulation of ConA (5 μg/ml) for 48 h or with no stimulation for 12 h.

Method:

We set up a model of Th1 polarization in vitro using ConA stimulation; ratios of CD4⁺/CD25⁺ Tregs (confirmed by the expression of Foxp3) were measured by flow cytometry, and levels of IL-2, IFN-γ, TGF-β and IL-10 were measured by ELISA. In addition, HO-1 expression was measured without stimulation with ConA by RT-PCR and Western blotting, and HO-1 level in vitro was then measured by enzyme activity assay. p<0.05 (t-test) was taken as the level of statistical significance.

Result:

Oridonin promoted differentiation towards CD4⁺/CD25⁺ Tregs, inhibited IL-2 and IFN-γ but induced TGF-β and IL-10, thus rectifed the Th1 polarization. Moreover, Oridonin induced the expression of HO-1 mRNA and protein, and HO-1 activity in vitro was enhanced accordingly.

Conclusion:

The results suggest that Oridonin has a distinct effect on promoting CD4⁺/CD25⁺ Treg differentiation and modulating Th1/Th2 balance, and this effect may be achieved via inducing the anti-inflammatory target HO-1.