The traditional Japanese formula keishibukuryogan inhibits the production of inflammatory cytokines by dermal endothelial cells

Abstract

Keishibukuryogan (KBG) is one of the traditional herbal formulations widely administered to patients with blood stagnation for improving blood circulation; currently, it is the most frequently prescribed medicine in Japan. KBG has been reported to improve conjunctional microcirculation. The aim of this study was to evaluate the role of KBG and paeoniflorin, a bioactive compound of KBG, in inhibiting the production of inflammatory cytokines using human dermal microvessel endothelial cells (HDMECs). The authors observed that lipopolysaccharide (LPS; 1 μg/mL) stimulated the secretion of proinflammatory cytokines in HDMECs. KBG treatment (10 mg/mL) significantly suppressed the mRNA levels of migration inhibitory factor (MIF), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in LPS-stimulated cultured HDMECs. Similarly, paeoniflorin significantly suppressed the mRNA levels of these cytokines in LPS-stimulated cultured HDMECs. ELISA showed that KBG and paeoniflorin suppressed the production of MIF, IL-6, IL-8, and TNF-α in LPS-stimulated HDMECs. Moreover, KBG and paeoniflorin decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) in these cells. These results suggest that KBG may be useful for improving microvascular inflammation in patients with skin diseases.

Figure 1

Effect of KBG on LPS-stimulated induction of cytokines in HDMECs. (a) HDMEC cells were treated with 1 μg/mL LPS and/or KBG (10 mg/mL) for 6 or 24 h and were subjected to the MTT assay. (b) HDMEC cells were treated with 1 μg/mL LPS and/or 10 mg/mL of KBG for 24 h. The MIF, IL-6, IL-8, and TNF-α content of cultured supernatants was analyzed by ELISA. Data are presented as the means ± S.D (n = 5). *P < .001, **P < .005, ***P < .05. (c) The cells were treated with 1 μg/mL LPS and/or 10 mg/mL of KBG for 6 h. Total RNA was isolated, and the mRNA expression levels of MIF, IL-6, IL-8, and TNF-α were detected by RT-PCR. (These experiments were repeated three times with similar results).

Figure 2

Effect of paeoniflorin on LPS-stimulated induction of cytokines in HDMECs. (a) HDMEC cells that were treated with LPS (1 μg/mL) and/or paeoniflorin (100 μg/mL) for 6 or 24 h and were subjected to the MTT assay. (b) HDMEC cells were treated with 1 μg/mL LPS and/or 100 μg/mL of paeoniflorin for 24 h. The MIF, IL-6, IL-8, and TNF-α protein content of cultured supernatants was analyzed by ELISA. Data are presented as the means ± S.D (n = 5). *P < .0005. (c) The cells were treated with 1 μg/mL LPS and/or 100 μg/mL of paeoniflorin for 6 h. Total RNA was isolated, and the mRNA expression levels of MIF, IL-6, IL-8, and TNF-α were detected by RT-PCR. (These experiments were repeated three times with similar results).

Figure 3

Effect of paeoniflorin COX-2 and iNOS protein expression. HDMEC cells were treated with 1 μg/mL LPS and/or 100 μg/mL of paeoniflorin for 24 h. COX-2 and iNOS protein expression in the cells was detected by a Western blot analysis, using β-actin as the internal control. The results of densitometric analysis, normalized with respect to β-actin using a bioimaging analyzer, are presented. Data are presented as the means ± S.D (n = 5). *P < .0001. (This experiment was repeated three times with similar results).