Hispolon is a potent anticancer, anti-inflammatory, antioxidant, and antidiabetic agent isolated from Phellinus linteus, an oriental medicinal mushroom. However, the immunomodulatory mechanisms by which hispolon affects macrophages and lymphocytes remain poorly characterized. We investigated the immunomodulatory effects of hispolon on oxidative stress, inflammatory responses, and lymphocyte proliferation using lipopolysaccharide (LPS)-treated RAW264.7 macrophages or mitogen/alloantigen-treated mouse splenocytes. Hispolon inhibited LPS-induced reactive oxygen and nitrogen species (ROS/RNS) generation and decreased total sulfhydryl (SH) levels in a cell-free system and RAW264.7 cells. Hispolon exerted significant anti-inflammatory effects by inhibiting production of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) and activation of nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) in LPS-treated RAW264.7 cells. Hispolon also modulated NF-κB and STAT3 activation by suppressing the NF-κB p65 interaction with phospho-IκBα and the STAT3 interaction with JAK1, as determined via coimmunoprecipitation analysis. Additionally, hispolon significantly decreased lymphocyte proliferation, T cell responses and T helper type 1 (Th1)/type 2 (Th2) cytokines production in mitogen/alloantigen-treated splenocytes. We conclude that hispolon exerts immunomodulatory effects on LPS-treated macrophages or mitogen/alloantigen-treated splenocytes through antioxidant, anti-inflammatory, and antiproliferative activities. Thus, hispolon may be a therapeutic agent for treating immune-mediated inflammatory diseases.

The purpose of this study was to determine the antiallergic effects of AF-343, a mixture of natural plant extracts from Cassia tora L., Ulmus pumila L., and Taraxacum officinale, on rat basophilic leukemia (RBL-2H3) cells. The inhibitory effects on cell degranulation, proinflammatory cytokine secretion, and reactive oxygen species (ROS) production were studied in compound 48/80-treated RBL-2H3 cells. The bioactive compounds in AF-343 were also identified by HPLC–UV. AF-343 was found to effectively suppress compound 48/80-induced β-hexosaminidase release, and interleukin (IL)-4 and tumor necrosis factor-α (TNF-α) production in RBL-2H3 cells. In addition, AF-343 exhibited DPPH free radical scavenging effects in vitro (half-maximal inhibitory concentration (IC₅₀) = 105 μg/mL) and potently inhibited compound 48/80-induced cellular ROS generation in a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay. Specifically, treatment with AF-343 exerted stronger antioxidant effects in vitro and antiallergic effects in cells than treatment with three single natural plant extracts. Furthermore, AF-343 was observed to contain bioactive compounds, including catechin, aurantio-obtusin, and chicoric acid, which have been reported to elicit antiallergic responses. This study reveals that AF-343 attenuates allergic responses via suppression of β-hexosaminidase release, IL-4 and TNF-α secretion, and ROS generation, perhaps through mechanisms related to catechin, aurantio-obtusin, and chicoric acid. The results indicate that AF-343 can be considered a treatment for various allergic diseases.

MicroRNAs (miRNAs) are key biomarkers for the diagnosis, prevention, and treatment of various diseases. Most miRNAs interact with one another to influence disease occurrence, making multiplex detection essential for miRNA analysis. In this study, we developed a quantitative analysis method for nine miRNAs using a bead-based flow cytometry system. The method was validated using calibration curves, working range, precision, accuracy, specificity, and carry-over assessments in accordance with bioanalysis guidelines. We analyzed the expression of eight inflammation-related miRNAs, including miR-21, miR-24, miR-29a, miR-34a, miR-122, miR-125a, miR-146a, and miR-155, using cel-miR-39 as an internal control. To demonstrate the applicability of the proposed method, we analyzed nine miRNAs in the livers of male and female rats administered CpG ODN 1826 and LPS and compared the results obtained using the RT–qPCR. The results revealed that the calibration standards for the nine miRNAs included nine points, covering a quantification range of 0.20 to 51.20 pg/μL, with a five-parameter logistic (5-PL) curve fit within ±20%. The quality control (QC) samples for all nine miRNAs met the accuracy and precision criteria, with a %RE within ±20% (25% at the LLOQ and ULOQ) and a %CV of ≤20% (25% at the LLOQ and ULOQ). The total error at each QC level was within the acceptance criteria of ≤30% (40% at the LLOQ and ULOQ). Specificity and carry-over evaluations indicated that specific analytes in the multiplex assay were successfully quantified without interference from other analytes, with no carry-over effects observed. Finally, a total of nine miRNAs were quantitatively analyzed in the livers of rats with inflammation induced by CpG ODN 1826 and LPS, compared with the control group. Of these, the expression of miR-21, miR-34a, miR-146a, and miR-155 tended to increase during liver inflammation in both male and female rats. These results were consistent with the RT–qPCR data. In conclusion, this analytical method, validated in accordance with FDA guidelines, enables accurate and quantitative multiplex detection of inflammation-related miRNA biomarkers in liver tissue. It demonstrates strong reliability in current tissue analyses and provides a foundation for developing multiplex miRNA assays capable of analyzing body fluids, with potential applications in future clinical studies.