Effect of the Miaoyao Fanggan sachet-derived isorhamnetin on TLR2/4 and NKp46 expression in mice

ETHNOPHARMACOLOGICAL RELEVANCE: Miaoyao Fanggan Sachets (MFS) has long been used as a folk medicine for the prevention of influenza in Southeast of Guizhou Province, China. However, the precise immunological mechanisms by which MFS confers protection have not been defined. STUDY AIM: To explore the effects of MFS on innate immune system responses using a cold stress-induced immune impairment model as a means of examining MFS-mediated influenza prevention. We investigated the effects of MFS on toll-like receptor 2 and 4 (TLR2/4) gene and protein expression levels and on the percentage of NKp46⁺ cells present in serum. No overt toxicity was observed following continuous administration of MFS at high doses. METHODS: Kunming male mice (n=40) were randomly divided into 4 groups consisting of the continuous inhalation Sachet group, Yu-Ping-Feng powder (YPF-P) gavage positive control group, discontinuous inhalation MFS group and untreated controls. After 4 weeks, mice were sacrificed and lungs harvested. The expression of toll-like receptors 2 and 4 (TLR2/4) gene and protein levels was assessed using real-time polymerase chain reaction (RT-PCR) and Western blot analyses, respectively. An additional 60 Kunming mice were randomly divided into 6 groups comprised of a blank control group, continuous MFS inhalation group, an immune-compromised continuous MFS inhalation group, an immuno-compromised group, an immune-compromised MFS discontinuous inhalation group and an immune-compromised positive control group. Immune suppression was induced by cold stress (4°C/4h daily for 3 days) and mice were treated with MFS or YPF-P before cold stress treatments. Immuno-compromised mice were treated with MFS continuously or intermittently, or treated with YPF-P. Blood samples were collected and examined for natural killer cells based on positive NKp46 staining. The isorhamnetin associated with MFS-induced immune modulation was obtained from ‘wo ga le’ which is considered to be a major component of MFS, and analyzed by HPLC. RESULTS: Mice continuously inhaling MFS showed a moderate increase in TLR2/4 mRNA and protein levels compared to mice in the control and discontinuous inhalation groups. MFS significantly increased the TLR2/4 expression in a dose-dependent manner. Furthermore, there was also a slightly significant increase in the number of NKp46⁺ cells in the continuous inhalation group compared to controls and discontinuous inhalation group. Pretreatment with MFS partially prevented cold stress-induced inhibition of NKp46⁺ cells. HPLC analysis of the ‘wo ga le’ associated with immune function identified the major component to be isorhamnetin. CONCLUSIONS: Taken together, these data suggested that MFS significantly enhanced TLR2/4 expression levels and the number of NKp46⁺ cells in mice and moderately affected innate immune responses associated with protection against influenza, suggesting that isorhamnetin in the MFS enhanced innate immune potency. The use of MFS for the prevention of various respiratory tract infections can be attributed to its antimicrobial properties. In a pilot study, a large quantity (40g) was administered over a prolonged period did not produce apparent toxicity.