Identification of anti-inflammatory constituents in Hypericum species and unveiling the underlying mechanism in LPS-stimulated mouse macrophages and H1N1 influenza virus infected BALB/c mouse

Hypericum species are a large family of plants with potential medicinal value. To date, only H. perforatum has been thoroughly studied for its bioactivities due to its popularity among depression patients. Other than its anti-depression and anti-viral activities, H. perforatum also has anti-inflammatory activity, which is not well characterized. Previous studies by Hammer et al. (2007) evaluated the inhibitory effect of different H. perforatum extracts on lipopolysaccharide (LPS)-induced macrophage prostaglandin E2 (PGE2) production. The subsequent study also identified 4 synergistic anti-inflammatory constituents in a fraction of the H. perforatum extract, namely pseudohypericin, quercetin, amentoflavone, and chlorogenic acid (referred to as the 4 compounds). Lastly, the janus kinase - signal transducer and activator of transcription (JAK-STAT) pathway were proposed as molecular targets for the 4 compounds\u27 anti-inflammatory activity. The current study set out to test the central hypothesis that the 4 compounds contributed significantly to the anti-inflammatory activity of the H. perforatum extract by inducing suppressor of cytokine signaling 3 (SOCS3) both in vitro and in vivo. The first part of this study was to compare the chemical profiles and anti-inflammatory potential of extracts of H. perforatum, H. gentianoides, H. beanii, H. densiflorum, H. balearicum, H. forrestii, H.bellum, and H. patulum. At a concentration of 20 yg/mL, all nine extracts included had significant inhibitory effect on LPS-induced PGE2 and nitric oxide (NO) production in RAW 264.7 mouse macrophages. The extracts made from H. perforatum and H. gentianoides had distinctive chromatograms in LC-MS analysis and relatively stronger PGE2 and NO reducing efficacy. The 4 compounds accounted for a portion of the H. perforatum extract\u27s PGE2 inhibition and the majority of its NO and interleukin (IL)-1b reducing effects. LPS-stimulated tumor necrosis factor (TNF)-a production was only suppressed by the 4 compounds but not by the extract, suggesting the presence of counteractive constituents. Uliginosin A, one of the acylphloroglucinols found in the H. gentianoides extract, inhibited PGE2 and NO by more than 70% at 2 yM. Then, the importance of SOCS3 activation in the anti-inflammatory potential of the H. perforatum extract and the 4 compounds was investigated using SOCS3 knockdown RAW 264.7 macrophages. The results indicated that pseudohypericin was the major PGE2 and NO inhibiting constituent among the 4 compounds and required SOCS3 activation to exert the effect. At the same time, amentoflavone and quercetin accounted for the inhibition of pro-inflammatory cytokines TNF-a, IL-6, and IL-1b in a SOCS3 independent manner. Interestingly, although the 4 compounds\u27 PGE2 and NO inhibitory activities were compromised with SOCS3 knockdown, H. perforatum extract\u27s efficacy was not affected, suggested that components other than the 4 compounds inhibited these inflammatory mediators without activating SOCS3. Because a cell culture model cannot comprehensively reflect the complex nature of inflammation, influenza A infected-BALB/c mice were used to assess the in vivo immune-regulatory impact of H. perforatum. When the mice were infected with a high dose of H1N1 virus, H. perforatum oral treatment at 110 mg/kg body weight significantly increased viral titer in the lung 5 days post-infection. H. perforatum treatment also resulted in elevation of 18 pro-inflammatory cytokine and chemokine levels and increased the number of pro-inflammatory cells in the bronchoalveolar lavage, as compared to the 5% ethanol vehicle treatment. SOCS3 transcription in the lung was elevated after viral infection, and further potentiated by the H. perforatum extract. These results suggested that influenza might be a contraindication for H. perforatum, because SOCS3 elevation could impair the immune response against influenza virus infection, possibly through blocking type I interferon signaling. H. perforatum was applied to mice only during the later phase of influenza infection, in the hope that inflammatory tissue damage can be alleviated. But no significant improvement was found. Overall, the current study showed that the 4 compounds in H. perforatum partially depend on SOCS3 activation to exert their in vitro anti-inflammatory activity. However, the elevated SOCS3 by H. perforatum during influenza infection can be detrimental due to the impaired immune response