Broad-Spectrum Antiviral Agents against SARS-CoV-2 Variants Inhibit the Conserved Viral Protein Nsp1-RNA Interaction.

The ongoing global threats posed by COVID-19 pandemic, catalyzed by SARS-CoV-2, underscores the pressing need for effective antiviral strategies. The viral non-structural protein 1 (Nsp1) significantly influences pathogenicity by impeding host protein expression and enhancing viral RNA translation through its interaction with the stem-loop 1 (SL1) in the 5' untranslated region (UTR). We have developed a novel dual-luciferase reporter assay, designed to investigate the critical Nsp1-SL1 interaction, and identified P23E02 as a potential inhibitor. Our investigation, combining molecular docking studies and alanine mutagenesis, has unveiled that P23E02 disrupts Nsp1-40S ribosomal subunit interaction, liberating translational inhibition and empowering host antiviral responses. P23E02 exhibits antiviral efficacy against various sarbecoviruses, making it a promising candidate for combatting COVID-19 and related diseases. This study underscores the therapeutic potential of targeting the Nsp1/SL1 axis and lays the foundation for innovative antiviral interventions, ultimately fortifying global preparedness against future viral threats.

What Are the Challenges of School Nurses in South Korea in Managing Obese Children From Low-Income Households?

Given that the obesity rate among school-age children is increasing, school nurses can play a vital role in managing obesity and encouraging healthy living in school settings. Obese children from low-income backgrounds are more vulnerable than other students and require more careful attention and intervention. This qualitative study aimed to explore and understand the barriers recognized by school nurses in managing obesity in low-income household children. A focus group interview was conducted with 17 school nurses working at an elementary school. Children, home, school, political and structural, and social areas were revealed as intricate factors in obesity management. This study can help understand school nurses' obstacles in managing obese children from low-income families and can help them prepare practical measures to overcome these obstacles.

Targeting the Receptor-Binding Motif of SARS-CoV-2 with D-Peptides Mimicking the ACE2 Binding Helix: Lessons for Inhibiting Omicron and Future Variants of Concern.

The COVID-19 pandemic continues to spread around the world, with several new variants emerging, particularly those of concern (VOCs). Omicron (B.1.1.529), a recent VOC with many mutations in the spike protein's receptor-binding domain (RBD), has attracted a great deal of scientific and public interest. We previously developed two D-peptide inhibitors for the infection of the original SARS-CoV-2 and its VOCs, alpha and beta, in vitro. Here, we demonstrated that Covid3 and Covid_extended_1 maintained their high-affinity binding (29.4-31.3 nM) to the omicron RBD. Both D-peptides blocked the omicron variant in vitro infection with IC50s of 3.13 and 5.56 µM, respectively. We predicted that Covid3 shares a larger overlapping binding region with the ACE2 binding motif than different classes of neutralizing monoclonal antibodies. We envisioned the design of D-peptide inhibitors targeting the receptor-binding motif as the most promising approach for inhibiting current and future VOCs of SARS-CoV-2, given that the ACE2 binding interface is more limited to tolerate mutations than most of the RBD's surface.

Synthesis and structure-activity relationship study of saponin-based membrane fusion inhibitors against SARS-CoV-2.

We previously discovered that triterpenoid saponin platycodin D inhibits the SARS-CoV-2 entry to the host cell. Herein, we synthesized various saponin derivatives and established a structure-activity relationship of saponin-based antiviral agents against SARS-CoV-2. We discovered that the C3-glucose, the C28-oligosaccharide moiety that consist of (→3)-ß-d-Xyl-(1 â†’ 4)-α-l-Rham-(1 â†’ 2)-ß-d-Ara-(1 â†’ ) as the last three sugar units, and the C16-hydroxyl group were critical components of saponin-based coronavirus cell entry inhibitors. These findings enabled us to develop minimal saponin-based antiviral agents that are equipotent to the originally discovered platycodin D. We found that our saponin-based antiviral agents inhibited both the endosomal and transmembrane protease serine 2-mediated cell surface viral entries. Cell fusion assay experiment revealed that our newly developed compounds inhibit the SARS-CoV-2 entry by blocking the fusion between the viral and host cell membranes. The effectiveness of the newly developed antiviral agents over various SARS-CoV-2 variants hints at the broad-spectrum antiviral efficacy of saponin-based therapeutics against future coronavirus variants.

Anatomy-mimetic design preserves natural kinematics of knee joint in patient-specific mobile-bearing unicompartmental knee arthroplasty.

PURPOSE: This study aims to evaluate whether different tibial-femoral conformities for patient-specific mobile-bearing unicompartmental knee arthroplasties (UKAs) preserve natural knee kinematics, using computational simulations. METHODS: Different designs for patient-specific mobile-bearing UKAs were evaluated using finite element analysis. Three designs for the identical femoral component were considered: flat (non-conforming design), anatomy-mimetic, and conforming for the tibial insert. RESULTS: The conforming design for the patient-specific mobile-bearing UKAs exhibited a 1.2 mm and 0.7° decrease in the translation and rotation, respectively, in the swing phase compared with those of the natural knee. In addition, the femoral rollback and internal rotation were 2.6 mm and 1.2° lower, respectively, than those of the natural knee, for the conforming design under the deep-knee-bend condition. The flat design for the patient-specific mobile-bearing UKAs exhibited a 2.2 mm and 1.4° increase in the femoral rollback and rotation compared with the natural knee under the deep-knee-bend condition. The anatomy-mimetic patient-specific mobile-bearing UKAs best preserved the natural knee kinematics under the gait and deep-knee-bend loading conditions. CONCLUSIONS: The kinematics of the loading conditions in patient-specific mobile-bearing UKAs was determined to closely resemble those of a native knee. In additional, by replacing the anatomy-mimetic design with a mobile-bearing, natural knee kinematics during gait and deep-knee-bend motions is preserved. These results confirm the importance of tibiofemoral conformity in preserving native knee kinematics in patient-specific mobile-bearing UKA.

Computational analysis of customized cruciate retaining total knee arthroplasty restoration of native knee joint biomechanics.

The purpose of this study was to verify if customized prosthesis better preserves the native knee joint kinematics and provides lower contact stress on the polyethylene (PE) insert owing to the wider bone preservation than that of standard off-the-shelf prosthesis in posterior cruciate-retaining type total knee arthroplasty (TKA). Validated finite element (FE) models for were developed to evaluate the knee joint kinematics and contact stress on the PE insert after TKA with customized and standard off-the-shelf (OTS) prostheses as well as in normal healthy knee through FE analysis under dynamic loading conditions. The contact stresses on the customized prosthesis decreased by 18% and 8% under gait cycle loading conditions, and 24% and 9% under deep-knee-bend loading conditions, in the medial and lateral sides of the PE insert, respectively, compared with the standard OTS prosthesis. The anterior-posterior translation and internal-external (IE) rotation in customized TKA were more similar to native knee joint behaviors compared with standard OTS TKA under gait loading conditions. The difference from normal knee kinematics was lower for femoral rollback and IE rotation in customized TKA than in standard OTS TKA in the deep-knee-bend condition. In general, customized prostheses achieve kinematics that are close to those of the native healthy knee joint and have better contact stresses than standard OTS prostheses in gait and deep-knee-bend loading conditions.

Acquisition of reproducible transmission near-infrared (NIR) spectra of solid samples with inconsistent shapes by irradiation with isotropically diffused radiation using polytetrafluoroethylene (PTFE) beads.

A bead-incorporated transmission scheme (BITS) has been demonstrated for collecting reproducible transmission near-infrared (NIR) spectra of samples with inconsistent shapes. Isotropically diffused NIR radiation was applied around a sample and the surrounding radiation was allowed to interact homogeneously with the sample for transmission measurement. Samples were packed in 1.40 mm polytetrafluoroethylene (PTFE) beads, ideal diffusers without NIR absorption, and then transmission spectra were collected by illuminating the sample-containing beads using NIR radiation. When collimated radiation was directly applied, a small portion of the non-fully diffused radiation (NFDR) propagated through the void space of the packing and eventually degraded the reproducibility. Pre-diffused radiation was introduced by placing an additional PTFE disk in front of the packing to diminish NFDR, which produced more reproducible spectral features. The proposed scheme was evaluated by analyzing two different solid samples: density determination for individual polyethylene (PE) pellets and identification of mining locality for tourmalines. Because spectral collection was reproducible, the use of the spectrum acquired from one PE pellet was sufficient to accurately determine the density of nine other pellets with different shapes. The differentiation of tourmalines, which are even more dissimilar in appearance, according to their mining locality was also feasible with the help of the scheme.

Chronic Social Defeat Stress Gives Rise to Social Avoidance Through Fear Learning.

Chronic social defeat stress (CSDS), a widely used rodent model of stress, reliably leads to decreased social interaction in stress susceptible animals. Here, we investigate a role for fear learning in this response using 129Sv/Ev mice, a strain that is more vulnerable to CSDS than the commonly used C57BL/6 strain. We first demonstrate that defeated 129Sv/Ev mice avoid a CD-1 mouse, but not a conspecific, indicating that motivation to socialize is intact in this strain. CD-1 avoidance is characterized by approach behavior that results in running in the opposite direction, activity that is consistent with a threat response. We next test whether CD-1 avoidance is subject to the same behavioral changes found in traditional models of Pavlovian fear conditioning. We find that associative learning occurs across 10 days CSDS, with defeated mice learning to associate the color of the CD-1 coat with threat. This leads to the gradual acquisition of avoidance behavior, a conditioned response that can be extinguished with 7 days of repeated social interaction testing (5 tests/day). Pairing a CD-1 with a tone leads to second-order conditioning, resulting in avoidance of an enclosure without a social target. Finally, we show that social interaction with a conspecific is a highly variable response in defeated mice that may reflect individual differences in generalization of fear to other social targets. Our data indicate that fear conditioning to a social target is a key component of CSDS, implicating the involvement of fear circuits in social avoidance.

Discovery of (-)-epigallocatechin gallate, a novel olfactory receptor 2AT4 agonist that regulates proliferation and apoptosis in leukemia cells.

Olfactory receptors (ORs), the largest family of G protein-coupled receptors (GPCRs), are ectopically expressed in cancer cells and are involved in cellular physiological processes, but their function as anticancer targets is still potential. OR2AT4 is expressed in leukemia cells, influencing the proliferation and apoptosis, yet the limited number of known OR2AT4 agonists makes it challenging to fully generalize the receptor's function. In this study, we aimed to identify new ligands for OR2AT4 and to investigate their functions and mechanisms in K562 leukemia cells. After producing the recombinant OR2AT4 protein, immobilizing it on a surface plasmon resonance chip, and conducting screening to confirm binding activity using 258 chemicals, five novel OR2AT4 ligands were discovered. As a result of examining changes in intracellular calcium by five ligands in OR2AT4-expressing cells and K562 cells, (-)-epigallocatechin gallate (EGCG) was identified as an OR2AT4 agonist in both cells. EGCG reduced the viability of K562 cells and induced apoptosis in K562 cells. EGCG increased the expression of cleaved caspase 3/8 and had no effect on the expression of Bax and Bcl-2, indicating that it induced apoptosis through the extrinsic pathway. Additionally, the initiation of the extrinsic apoptosis pathway in EGCG-induced K562 cells was due to the activation of OR2AT4, using an OR2AT4 antagonist. This study highlights the potential of EGCG as an anti-cancer agent against leukemia and OR2AT4 as a target, making it a new anti-cancer drug.

Engineering of Cell Derived-Nanovesicle as an Alternative to Exosome Therapy.

BACKGROUND: Exosomes, nano-sized vesicles ranging between 30 and 150 nm secreted by human cells, play a pivotal role in long-range intercellular communication and have attracted significant attention in the field of regenerative medicine. Nevertheless, their limited productivity and cost-effectiveness pose challenges for clinical applications. These issues have recently been addressed by cell-derived nanovesicles (CDNs), which are physically synthesized exosome-mimetic nanovesicles from parent cells, as a promising alternative to exosomes. CDNs exhibit structural, physical, and biological properties similar to exosomes, containing intracellular protein and genetic components encapsulated by the cell plasma membrane. These characteristics allow CDNs to be used as regenerative medicine and therapeutics on their own, or as a drug delivery system. METHODS: The paper reviews diverse methods for CDN synthesis, current analysis techniques, and presents engineering strategies to improve lesion targeting efficiency and/or therapeutic efficacy. RESULTS: CDNs, with their properties similar to those of exosomes, offer a cost-effective and highly productive alternative due to their non-living biomaterial nature, nano-size, and readiness for use, allowing them to overcome several limitations of conventional cell therapy methods. CONCLUSION: Ongoing research and enhancement of CDNs engineering, along with comprehensive safety assessments and stability analysis, exhibit vast potential to advance regenerative medicine by enabling the development of efficient therapeutic interventions.

Lindera obtusiloba Blume Alleviates Non-Alcoholic Fatty Liver Disease Promoted by Nε-(carboxymethyl)lysine.

Non-alcoholic fatty liver disease (NAFLD) is a major issue because it is closely associated with metabolic diseases. Advanced glycation end products (AGEs) are implicated as risk factors for steatosis during NAFLD progression. AGEs influence NAFLD progression through a receptor-independent pathway involving AGE cross-link formation and a receptor-dependent pathway that binds to receptors like receptors for advanced glycation end products (RAGE). The objectives of this study are to examine the effect of Lindera obtusiloba Blume (LO) on NAFLD promoted by Nε-(carboxymethyl)lysine (CML), one of the most common dietary AGEs. The anti-glycation effects of LO were evaluated by inhibiting the AGEs formation and AGEs-collagen cross-links breaking. The efficacy of LO against NAFLD promoted by CML was assessed using both in vitro and in vivo models. NAFLD was induced in mice by feeding a high-fat diet and orally administering CML over a period of 12 weeks, and the effects of LO on lipid metabolism and its regulatory mechanisms were investigated. LO showed the effect of inhibited AGEs formation and breakage, and collagen cross-linking. Fed a high-fat diet with administered CML by gavage, LO administration resulted in a reduction in body weight, fat mass, serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels. LO reduced hepatic CML accumulation and RAGE expression in mice fed a high-fat diet and orally administered CML. LO alleviated hepatic steatosis accompanied by lipid accumulation and histological damage by suppressing the expression of sterol regulatory element-binding protein 1c, carbohydrate response element binding protein, fatty acid synthase, stearoyl-CoA desaturase1, tumor necrosis factor-α, and interleukin-1ß. LO alleviated the MAPK/NF-κB expression by attenuating CML and RAGE expression. Taken together, our results demonstrate that LO alleviates the progression of NAFLD by lowering the levels of AGEs by downregulating CML/RAGE expression.

An ancestral SARS-CoV-2 vaccine induces anti-Omicron variants antibodies by hypermutation.

The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyze blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyze their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) is achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counterprotective mechanism against virus variant immune escape.

A herb mixture to ameliorate non-alcoholic fatty liver in rats fed a high-fat diet.

This study was performed to investigate the effects of an herb extract mixture (HM) in ameliorating non-alcoholic fatty liver disease (NAFLD). The HM contained equal amounts of 70% ethanol extracts from Zingiber officinale, Centella asiatica, and Boehmeria nivea. In vitro, the HM significantly inhibited lipid accumulation in oleic acid-stimulated HepG2 cells. We further evaluated the anti-NAFLD activities of the HM in vivo in an animal model. Rats were fed two different amounts of the HM (50 and 200 mg/kg body weight) along with a high-fat diet for 6 weeks. HM supplementation reduced liver weight; epididymal, peri-renal, and intra-abdominal fat content; and serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels as well as increased high-density lipoprotein cholesterol levels in a dose-dependent manner. Histological evaluation of liver specimens further demonstrated that administration of HM significantly prevented hepatic lipid accumulation and subsequent development of hepatic steatosis. These findings suggest that HM can be used as an alternative nutraceutical for ameliorating NAFLD.

Serotonergic Modulation of the BNST-CeA Circuit Promotes Sex Differences in Fear Learning.

Post-traumatic stress disorder (PTSD) is characterized by intense fear memory formation and is diagnosed more often in women than men. Here, we show that serotonin differentially affects fear learning and communication in the extended amygdala of male and female mice. Females showed higher sensitivity to the effects of pharmacologically increasing serotonin during auditory fear conditioning, which enhanced fear memory recall in both sexes. Optogenetic stimulation of dorsal raphe terminals in the anterior dorsal bed nucleus of the stria terminalis (adBNST) during fear conditioning increased c-Fos expression in the BNST and central nucleus of the amygdala (CeA), and enhanced fear memory recall via activation of adBNST 5-HT2C receptors in females only. Likewise, in females only, serotonin stimulation during learning enhanced adBNST-CeA high gamma (90-140Hz) synchrony and adBNST-to-CeA communication in high gamma during fear memory recall. We conclude that sex differences in the raphe-BNST-CeA circuit may increase risk of PTSD in women.

Effect of surface matching mismatch of focal knee articular prosthetic on tibiofemoral contact stress using finite element analysis.

Aims: Focal knee arthroplasty is an attractive alternative to knee arthroplasty for young patients because it allows preservation of a large amount of bone for potential revisions. However, the mechanical behaviour of cartilage has not yet been investigated because it is challenging to evaluate in vivo contact areas, pressure, and deformations from metal implants. Therefore, this study aimed to determine the contact pressure in the tibiofemoral joint with a focal knee arthroplasty using a finite element model. Methods: The mechanical behaviour of the cartilage surrounding a metal implant was evaluated using finite element analysis. We modelled focal knee arthroplasty with placement flush, 0.5 mm deep, or protruding 0.5 mm with regard to the level of the surrounding cartilage. We compared contact stress and pressure for bone, implant, and cartilage under static loading conditions. Results: Contact stress on medial and lateral femoral and tibial cartilages increased and decreased, respectively, the most and the least in the protruding model compared to the intact model. The deep model exhibited the closest tibiofemoral contact stress to the intact model. In addition, the deep model demonstrated load sharing between the bone and the implant, while the protruding and flush model showed stress shielding. The data revealed that resurfacing with a focal knee arthroplasty does not cause increased contact pressure with deep implantation. However, protruding implantation leads to increased contact pressure, decreased bone stress, and biomechanical disadvantage in an in vivo application. Conclusion: These results show that it is preferable to leave an edge slightly deep rather than flush and protruding.

Deubiquitinase USP1 enhances CCAAT/enhancer-binding protein beta (C/EBPß) stability and accelerates adipogenesis and lipid accumulation.

Dysregulation of the ubiquitin-proteasome system has been implicated in the pathogenesis of several metabolic disorders, including obesity, diabetes, and non-alcoholic fatty liver disease; however, the mechanisms controlling pathogenic metabolic disorders remain unclear. Transcription factor CCAAT/enhancer binding protein beta (C/EBPß) regulates adipogenic genes. The study showed that the expression level of C/EBPß is post-translationally regulated by the deubiquitinase ubiquitin-specific protease 1 (USP1) and that USP1 expression is remarkably upregulated during adipocyte differentiation and in the adipose tissue of mice fed a high-fat diet (HFD). We found that USP1 directly interacts with C/EBPß. Knock-down of USP1 decreased C/EBPß protein stability and increased its ubiquitination. Overexpression of USP1 regulates its protein stability and ubiquitination, whereas catalytic mutant of USP1 had no effect on them. It suggests that USP1 directly deubiquitinases C/EBPß and increases the protein expression, leading to adipogenesis and lipid accumulation. Notably, the USP1-specific inhibitor ML323-originally developed to sensitize cancer cells to DNA-damaging agents-decreased adipocyte differentiation and lipid accumulation in 3T3-L1 cells without cytotoxicity. Oral gavage of ML323 was administered to HFD-fed mice, which showed weight loss and improvement in insulin and glucose sensitivity. Both fat mass and adipocyte size in white adipose tissues were significantly reduced by ML323 treatment, which also reduced the expression of genes involved in adipogenesis and inflammatory responses. ML323 also reduced lipid accumulation, hepatic triglycerides, free fatty acids, and macrophage infiltration in the livers of HFD-fed mice. Taken together, we suggest that USP1 plays an important role in adipogenesis by regulating C/EBPß ubiquitination, and USP1-specific inhibitor ML323 is a potential treatment option and further study by ML323 is needed for clinical application for metabolic disorders.

Glutathione dynamics is a potential predictive and therapeutic trait for neoadjuvant chemotherapy response in bladder cancer.

Radical cystectomy with preoperative cisplatin-based neoadjuvant chemotherapy (NAC) is the standard care for muscle-invasive bladder cancers (MIBCs). However, the complete response rate to this modality remains relatively low, and current clinicopathologic and molecular classifications are inadequate to predict NAC response in patients with MIBC. Here, we demonstrate that dysregulation of the glutathione (GSH) pathway is fundamental for MIBC NAC resistance. Comprehensive analysis of the multicohort transcriptomes reveals that GSH metabolism and immune-response genes are enriched in NAC-resistant and NAC-sensitive MIBCs, respectively. A machine-learning-based tumor/stroma classifier is applied for high-throughput digitalized immunohistochemistry analysis, finding that GSH dynamics proteins, including glutaminase-1, are associated with NAC resistance. GSH dynamics is activated in cisplatin-resistant MIBC cells, and combination treatment with a GSH dynamics modulator and cisplatin significantly suppresses tumor growth in an orthotopic xenograft animal model. Collectively, these findings demonstrate the predictive and therapeutic values of GSH dynamics in determining the NAC response in MIBCs.

Hepatoprotective and Antioxidative Activities of Cornus officinalis against Acetaminophen-Induced Hepatotoxicity in Mice.

The fruit of Cornus officinalis Sieb. et Zucc. is commonly prescribed in Asian countries as a tonic formula. In this study, the hepatoprotective effect of ethanolic extracts of the fruit of C. officinalis (ECO) was investigated in a mouse model of acetaminophen- (APAP-) induced liver injury. Pretreatment of mice with ECO (100, 250, and 500 mg/kg for 7 days) significantly prevented the APAP (200 mg/kg) induced hepatic damage as indicated by the serum marker enzymes (AST, ALT, and LDH). Parallel to these changes, ECO treatment also prevented APAP-induced oxidative stress in the mice liver by inhibiting lipid peroxidation (MDA) and restoring the levels of antioxidant enzymes (SOD, CAT, and HO-1) and glutathione. Liver injury and collagen accumulation were assessed using histological studies by hematoxylin and eosin staining. Our results indicate that ECO can prevent hepatic injuries associated with APAP-induced hepatotoxicity by preventing or alleviating oxidative stress.

Asiasari sieboldii suppresses inflammatory mediators through the induction of hemeoxygenase-1 expression in RAW264.7 cells.

OBJECTIVE: Asiasari sieboldii is widely used in Korean traditional medicine. In this study, we examined the anti-inflammatory effects of A. sieboldii ethanolic extract (ASEE) in a lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cell, and then sought to understand the mechanism(s) underlying the observed effects. MATERIALS AND METHODS: The production levels of nitrite oxide (NO), prostaglandin E2 (PGE2) and cytokines were measured using the Griess reagent and enzyme-linked immunosorbent assays (ELISA), while the cell protein expression levels were monitored by Western blot analysis. RESULTS: Our results revealed that ASEE had prominent inhibitory effects on NO, PGE2, interleukin (IL)-6 and tumor necrosis factor (TNF)-α production, as well as the expression of inducible nitrite oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in LPS-induced RAW264.7 cells. Mechanistically, ASEE upregulated the expression of hemeoxygenase-1 (HO-1), and inhibited the nuclear translocation of nuclear factor (NF)-κB by preventing degradation of inhibitor κB-α (IκB-α). CONCLUSION: These results indicate that the anti-inflammatory activity of ASEE occurs at least partially through the induction of HO-1 and subsequent suppression of the NF-κB pathway.

Spirodela polyrhiza (L.) Sch. ethanolic extract inhibits LPS-induced inflammation in RAW264.7 cells.

OBJECTIVE: Spirodela polyrhiza (L.) Sch. is widely used in Korean traditional medicine. No previous work has investigated in detail the anti-inflammatory activities of S. polyrhiza or assessed in vitro their potential underlying mechanism(s). We assessed the effects of S. polyrhiza ethanolic extract (SPEE) on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and investigated some potential underlying mechanisms. Additionally, we performed simultaneous determination of seven flavonoids in S. polyrhiza by high-performance liquid chromatography (HPLC)-photodiode array (PDA). MATERIALS AND METHODS: RAW264.7 cells were subjected to 5, 10, 20, and 50 µg/mL of SPEE for 1 h then treated with LPS for 24 h. Production of namely nitric oxide (NO), prostaglandin E(2) and cytokine levels were measured by the Griess reagent and ELISA, respectively. To investigate the underlying mechanisms of the anti-inflammatory activities of SPEE, expression of NO synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor-kappa B (NF-κB) proteins were evaluated by western blot analysis. HPLC analysis was performed using a Gemini C(18) column at 40°C and PDA detection at 340 nm. RESULTS: SPEE treatment significantly inhibited the LPS-induced production of NO, prostaglandin E(2), interleukin-6, and tumor necrosis factor-α and inhibited the expression of iNOS and COX-2 via attenuation of NF-κB p65 expression. The contents of the seven flavonoids in S. polyrhiza range from 0.25 to 8.77 mg/g. CONCLUSIONS: These results indicate that the anti-inflammatory activity of SPEE may be NF-κB p65 signaling. Also, the method will help to improve quality control of S. polyrhiza.