Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance.

Rheumatoid arthritis is characterized by the imbalance of T cells, which leads to increased pro-inflammatory and reduced anti-inflammatory cytokines. Modulating the balance among T cells is crucial for the treatment of RA. Kirenol is a major diterpenoid components of Herba Siegesbeckiae, which has been applied for arthritic therapy for centuries. Since prior research showed Kirenol exhibited anti-inflammatory effect in rats, in this study we have evaluated the effect and mechanism of bioactive Kirenol in a rat model of collagen-induced arthritis (CIA) on modulation of T cells. After immunization with bovine type II collagen (CII), Wistar rats were orally administered saline (CIA group), 2 mg/kg Kirenol or 2 mg/kg prednisolone daily for 30 days. The severity of arthritis was clinically and histologically assessed. The numbers of CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs) and IFNγ⁺CD4⁺ and IL4⁺CD4⁺ T cells were determined by flow cytometry, the mRNA expression level of Foxp3 was quantified by RT-PCR, cytokine levels were measured by ELISA and CII-induced cell proliferation was quantified in vitro. Kirenol significantly delayed the occurrence and reduced the disease severity of CIA. Histological analysis confirmed Kirenol suppressed joint inflammation and inhibited cartilage and bone destruction, compared to the CIA group. Kirenol also upregulated the mRNA expression of Foxp3, increased the numbers of CD4⁺CD25⁺Foxp3⁺ and IL4⁺CD4⁺ T cells, and reduced the number of IFNγ⁺CD4⁺ T cells. Kirenol reduced the levels of TNF-α, IL-17A and IL-6 in synovial fluid and TNF-α, IL-17A and IFN-γ in serum, and increased the serum levels of IL-4, IL-10 and TGF-ß1. In addition, Kirenol inhibited the ability of CII to induce splenocyte, PBMC and lymph node cell proliferation in vitro, compared to cells from CIA rats. In conclusion, these results suggest that Kirenol may be a potential immunosuppressant for the treatment for rheumatoid arthritis.

[Comparative study on anti-inflammatory and antipyretic effects between Dao-di herb and non Dao-di herb of Huangqin].

OBJECTIVE: To do some comparative study on anti-inflammatory and antipyretic effects between the Dao-di herb and non Dao-di herb of Huangqin (the roots of Scutellaria baicalensis) and provide thinking and evidence for study on geoherbalism and clinical usage of Huangqin. METHOD: The anti-inflammatory action was assessed by auricular swelling induced by dimethylbenzene in mice and the antipyretic action was monitored by dried yeast-induced mice fever. RESULT: All samples of both Dao-di and non Dao-di herbs of Huangqin showed antipyretic effect. The Dao-di Huangqin samples showed antipyretic effect between 61% to 53% , whereas the non Dao-di Huangqin samples between 53% to 43%. Six Dao-di Huangqin samples showed better antipyretic effect than four non Dao-di Huangqin samples. All samples of both Dao-di and non Dao-di Huangqin showed anti-inflammatory effect. Dao-di Huangqin showed anti-inflammatory effect between 73% to 54%, whereas non dao-di Huangqin between 53% to 34%. Six Dao-di Huangqin showed better anti-inflammatory effect than four non Dao-di Huangqin. In totality, results from analysis of geoherbalism showed that geoherbal production areas of Huangqin had better effect than that of the non geoherbal production areas in anti-inflammatory and antipyretic effects. CONCLUSION: Both the Dao-di and non Dao-di Huangqin have effects of anti-inflammatory and antipyretic to a certain extent, but the efficacy of the Dao-di Huangqin surpass the non Dao-di Huangqin.

Kirenol upregulates nuclear annexin-1 which interacts with NF-κB to attenuate synovial inflammation of collagen-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Kirenol is a diterpenoid compound purified from the Chinese Herba Siegesbeckiae. Siegesbeckiae has been employed for the treatment of arthritis for centuries, its safety and efficacy are documented through a long history of human use. AIM OF THE STUDY: To investigate the effects on collagen-induced arthritis (CIA) and anti-inflammatory mechanism of kirenol. MATERIALS AND METHODS: Kirenol was administrated intragastrically in rats after the onset of CIA. Pathological changes were evaluated by paw swelling and histopathology. Concentration of IL-1ß in synovial fluid and adrenal corticotropin (ACTH) in plasma were determined by Elisa. Western blot was performed to detect the expression of annexin-1 and glucocorticoid receptor alpha (GRα) in synovium. NF-κB DNA binding activity was assessed by electrophoretic mobility shift assays (EMSA). RESULTS: Kirenol (1, 2, and 4 mg/kg) and prednisolone depressed paw swelling and reduced IL-1ß of synovial fluid in the CIA rats (p<0.05 or p<0.01). Kirenol and prednisolone upregulated nuclear annexin-1 and inhibited NF-κB activity in synovium of CIA. The inhibitory effect of kirenol and prednisolone on NF-κB activity was enhanced by anti-annexin-1 Ab. Prednisolone, but not kirenol, downregulated plasma ACTH and GRα expression significantly (p<0.01). CONCLUSION: Kirenol and prednisolone can upregulate nuclear annexin-1 which interacts with NF-κB to inhibit NF-κB activity, reduce cytokines expression and thereby attenuate inflammation of CIA joints. Kirenol does not lead to ACTH or GR downregulation, which is in contrast to classic glucocorticoid prednisolone. Kirenol shares with GCs similar anti-inflammatory mechanism but bypass the considerable limitation of GCs treatment.

A rapid and simple RP-HPLC method for quantification of kirenol in rat plasma after oral administration and its application to pharmacokinetic study.

A rapid and simple reverse-phase high-performance liquid chromatography (RP-HPLC) was developed and validated for the quantification of kirenol in rat plasma after oral administration. Kirenol and darutoside (internal standard, IS) were extracted from rat plasma using Cleanert™ C(18) solid-phase extraction (SPE) cartridge. Analysis of the extraction was performed on a Thermo ODS-2 Hypersil C(18) reversed-phase column with a gradient eluent composed of acetonitrile and 0.1% phosphoric acid. The flow rate was 1.0 mL/min and the detection wavelength was set at 215 nm. The calibration curve was linear over the range of 9.756-133.333 µg/mL (r(2) = 0.9991) in rat plasma. The lower limits of detection and quantification were 2.857 and 9.756 µg/mL, respectively. The intra- and inter-day precisions (relative standard deviation, RSD) were between 2.24 and 4.46%, with accuracies ranging from 91.80 to 102.74%. The extraction recovery ranged from 98.16 to 107.62% with RSD less than 4.81%. Stability studies showed that kirenol was stable in preparation and analytical process. The present method was successfully applied to the pharmacokinetic study of kirenol in male Sprague-Dawley rats after oral administration at a dose of 50 mg/kg.