Paeoniflorin inhibits pulmonary artery smooth muscle cells proliferation via upregulating A2B adenosine receptor in rat

Abstract

Paeoniflorin (PF), which is the main active ingredient in the root of Paeonia Radix, has many pharmacological effects. Here, we investigated the effect of PF on rat pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions and explored the mechanisms of the effects. The anti-proliferative effect of PF increased in a dose dependent manner. At the highest dose (20 μmol/L), the anti-proliferative effect of PF peaked at 24 h after administration. However, the selective A2B adenosine receptor (A2BAR) antagonist MRS1754 abolished it. PF increased A2BAR mRNA levels from 0.0763±0.0067 of β-actin mRNA levels (hypoxia group) to 0.1190±0.0139 (P<0.05) measured by Real Time Reverse Transcription-Polymerase Chain Reaction. A2BAR protein expression measured by Western Blot was also increased. PF inhibited the proliferation of PASMCs by blocking cell cycle progression in the S phase. These data indicated that activation of A2BAR might be involved in the anti-proliferative effect of PF on PASMCs under hypoxic conditions. The results suggested that a new mechanism of PF could be relevant to the management of clinical hypoxic pulmonary hypertension.

Figure 1. Chemical structure of paeoniflorin.

Figure 2. PASMCs morphology

. A) Typical “hill and valley” appearance of PASMCs under phase contrast microscope. B) Immunofluorescent identification of α-smooth muscle actin was positive.

Figure 3. Cell viability was tested with CCK-8

. * P<0.05 vs. the N group; # P<0.05 vs. the H group; Δ P<0.05 vs. the H+PF20 group. Mean ± SD, n = 6.

Figure 4. Paeoniflorin decreased the proportion of S phase in hypoxic PASMCs

. PASMCs were incubated under normoxic (N), hypoxic (H) condition, or pretreated with PF (20 μmol/l) when under hypoxic condition. * P<0.05 vs. the N group; # P<0.05 vs. the H group; n = 3.

Figure 5. Effects of hypoxia and paeoniflorin on the expression of adenosine receptors.

The PASMCs were incubated under normoxic (N), hypoxic (H) condition, or pretreated with PF (20 μmol/l) when under hypoxic condition. ※ P>0.05 vs. the N group; * P<0.05 vs. the N group; # P<0.05 vs. the H group; ## P<0.05 vs. the H+PF20 group; Δ P<0.05 vs. the H group n = 3.

Figure 6. Paeoniflorin upregulate A_2BAR protein expression in hypoxic PASMCs.

Treat PASMCs were assayed for A_2BAR protein. * P<0.05 vs. the N group; # P<0.05 vs. the H group; ## P<0.05 vs. the H+PF20 group; Δ P<0.05 vs. the H group. n = 3.