Identification of plasma and urinary inflammatory markers in severe knee osteoarthritis: Relations with synovial fluid markers.

BACKGROUND: This study aimed to identify plasma and urinary cytokines as potential biomarkers for severe knee osteoarthritis (OA). It also investigated associations between these cytokines and cartilage markers, as well as their connections with synovial fluid (SF) markers. METHODS: Samples of plasma, urine, and SF were obtained from patients (n = 40) undergoing total knee arthroplasty (TKA) or unicompartmental knee arthroplasty (UKA) due to severe knee OA. Control samples of plasma and urine were collected from non-OA individuals (n = 15). We used a Luminex immunoassay for the simultaneous measurement of 19 cytokines, MMP-1, and MMP-3 levels. COMP, CTX-II, and hyaluronan (HA) levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Receiver operating characteristic (ROC) curves were utilized to analyze each biomarker's performance. Correlations among these biomarkers were evaluated via Spearman's correlation. RESULTS: The levels of plasma (p)CCL11, pCXCL16, pIL-8, pIL-15, pHA, urinary (u)CCL2, uCCL11, uCCL19, uCXCL16, uIL-1ß, uIL-6, uIL-8, uIL-12p70, uIL-15, uIL-33, uMMP-3, uHA, uCTX-II, and uCOMP were significantly elevated in individuals with severe knee OA. Notably, specific correlations were observed between the plasma/urine biomarkers and SF biomarkers: pCCL11 with sfHA (r = 0.56) and sfTNF-α (r = 0.58), pIL-15 with sfCCL19 (r = 0.43) and sfCCL20 (r = 0.44), and uCCL19 with sfCCL11 (r = 0.45) and sfIL-33 (r = 0.51). Positive correlations were also observed between uCCL11 and its corresponding sfCCL11(r = 0.49), as well as between sfCCL11 and other cytokines, namely sfCCL4, sfCCL19, sfCCL20, sfIL-33, and sfTNF-α (r = 0.46-0.63). CONCLUSION: This study provides an extensive profile of systemic inflammatory mediators in plasma of knee OA and identified four inflammatory markers (pCCL11, pIL-15, uCCL11, and uCCL19) reflecting joint inflammation.

Evaluation of Human Platelet Lysate as an Alternative to Fetal Bovine Serum for Potential Clinical Applications of Stem Cells from Human Exfoliated Deciduous Teeth.

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H2O2. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

Anti-Metastatic Effects of Standardized Polysaccharide Fraction from Diospyros kaki Leaves via GSK3ß/ß-Catenin and JNK Inactivation in Human Colon Cancer Cells.

A polysaccharide fraction from Diospyros kaki (PLE0) leaves was previously reported to possess immunostimulatory, anti-osteoporotic, and TGF-ß1-induced epithelial-mesenchymal transition inhibitory activities. Although a few beneficial effects against colon cancer metastasis have been reported, we aimed to investigate the anti-metastatic activity of PLE0 and its underlying molecular mechanisms in HT-29 and HCT-116 human colon cancer cells. We conducted a wound-healing assay, invasion assay, qRT-PCR analysis, western blot analysis, gelatin zymography, luciferase assay, and small interfering RNA gene silencing in colon cancer cells. PLE0 concentration-dependently inhibited metastasis by suppressing cell migration and invasion. The suppression of N-cadherin and vimentin expression as well as upregulation of E-cadherin through the reduction of p-GSK3ß and ß-catenin levels resulted in the outcome of this effect. PLE0 also suppressed the expression and enzymatic activity of matrix metalloproteinases (MMP)-2 and MMP-9, while simultaneously increasing the protein and mRNA levels of the tissue inhibitor of metalloproteinases (TIMP-1). Furthermore, signaling data disclosed that PLE0 suppressed the transcriptional activity and phosphorylation of p65 (a subunit of NF-κB), as well as the phosphorylation of c-Jun and c-Fos (subunits of AP-1) pathway. PLE0 markedly suppressed JNK phosphorylation, and JNK knockdown significantly restored PLE0-regulated MMP-2/-9 and TIMP-1 expression. Collectively, our data indicate that PLE0 exerts an anti-metastatic effect in human colon cancer cells by inhibiting epithelial-mesenchymal transition and MMP-2/9 via downregulation of GSK3ß/ß-catenin and JNK signaling.

Implant Removal After Medial Opening Wedge High Tibial Osteotomy Provides Implant-Related Pain Relief and Functional Improvement.

PURPOSE: To (1) investigate the incidence of implant-related pain after medial opening wedge high tibial osteotomy (MOWHTO) using a locking plate, (2) determine whether implant removal provides pain relief and functional improvement, and (3) evaluate bone healing and loss of correction after implant removal. METHODS: Between March 2014 and September 2017, MOWHTO was performed without bone graft. The inclusion criteria were patients who underwent implant removal after MOWHTO and were followed up for a minimum of 2 years. Patients were evaluated for implant removal 1 and 2 years after surgery. Clinical and functional evaluations were conducted to investigate implant-related pain using the visual analog scale, Lysholm score, and Tegner score. The radiographic indices measured were the gap-filling rate, weightbearing line (WBL) ratio, hip-knee-ankle angle (HKAA), medial proximal tibial angle (MPTA), and posterior tibial slope angle (PTSA). RESULTS: A total of 55 patients were enrolled. Fifty-one (92.7%) patients experienced implant-related pain prior to implant removal, with 43 and 8 patients reporting mild pain and moderate pain, respectively. At 1 and 2 years after implant removal, mild pain occurred in 6 (10.9%) and 5 (9.1%) patients, respectively. The remaining patients reported no implant-related pain. Prior to implant removal and 1 year after implant removal, the Lysholm score improved from 77.0 ± 5.6 to 86.8 ± 5.7 (P < .001), and the Tegner score improved from 3.3 ± 1.2 to 3.9 ± 1.3 (P < .001). The mean gap-filling rate was 84.4% ± 9.6% at implant removal, and it significantly increased to 93.7% ± 5.4% and 97.4% ± 2.6% at 1 and 2 years after implant removal, respectively (P < .001). For the WBL ratio, HKAA, MPTA, and PTSA, no statistically significant differences were found after implant removal. CONCLUSIONS: The incidence of implant-related pain after MOWHTO using the medial proximal tibial locking plate was high. Implant removal provides pain relief and functional improvement (met minimal clinically important differences). Even after implant removal, bone healing progressed gradually without a loss of correction in all patients. LEVEL OF EVIDENCE: Level IV, case series.

The Potential Application of Human Gingival Fibroblast-Conditioned Media in Pulp Regeneration: An In Vitro Study.

Regenerative endodontic treatment based on tissue engineering has recently gained interest in contemporary restorative dentistry. However, low survival rates and poor potential differentiation of stem cells could undermine the success rate of pulp regenerative therapy. Human gingival fibroblast-conditioned medium (hGF-CM) has been considered a potential therapy for tissue regeneration due to its stability in maintaining multiple factors essential for tissue regeneration compared to live cell transplantation. This study aimed to investigate the potency of hGF-CM on stem cells from human dental pulp (DPSC) in pulp regeneration. A series of experiments confirmed that hGF-CM contributes to a significant increase in proliferation, migration capability, and cell viability of DPSC after H2O2 exposure. Moreover, it has been proved to facilitate the odontogenic differentiation of DPSC via qRT-PCR, ALP (alkaline phosphatase), and ARS (Alizarin Red S) staining. It has been discovered that such highly upregulated odontogenesis is related to certain types of ECM proteins (collagen and laminin) from hGF-CM via proteomics. In addition, it is found that the ERK pathway is a key mechanism via inhibition assay based on RNA-seq result. These findings demonstrate that hGF-CM could be beneficial biomolecules for pulp regeneration.

Mosloflavone-Resveratrol Hybrid TMS-HDMF-5z Exhibits Potent In Vitro and In Vivo Anti-Inflammatory Effects Through NF-κB, AP-1, and JAK/STAT Inactivation.

TMS-HDMF-5z is a hybrid of the natural products mosloflavone and resveratrol. It was discovered to show potent inhibitory effects against lipopolysaccharide (LPS)-induced production of inflammatory mediators in RAW 264.7 macrophages. However, its mechanism of action is unknown. Hence this study aimed to demonstrate and explore in vitro and in vivo anti-inflammatory effects of TMS-HDMF-5z and its mechanism of action employing RAW 264.7 macrophages and carrageenan-induced hind paw edema. This work revealed that TMS-HDMF-5z suppressed the LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein, mRNA, and promoter binding levels and tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6, and interferon-ß (IFN-ß) at the mRNA expression in RAW 264.7 macrophages. The results showed that TMS-HDMF-5z reduced the transcription and DNA binding activities of nuclear factor-κB (NF-κB) through inhibiting nuclear translocation of p65 and phosphorylation of κB inhibitor α (IκBα), IκB kinase (IKK), and TGF-ß activated kinase 1 (TAK1). Additionally, TMS-HDMF-5z attenuated the LPS-induced transcriptional and DNA binding activities of activator protein-1 (AP-1) by suppressing nuclear translocation of phosphorylated c-Fos, c-Jun, and activating transcription factor 2 (ATF2). TMS-HDMF-5z also reduced the LPS-induced phosphorylation of Janus kinase 1/2 (JAK1/2), signal transducers and activators of transcription 1/3 (STAT1/3), p38 mitogen-activated protein kinase (MAPK), and MAPK-activated protein kinase 2 (MK2). In rats, TMS-HDMF-5z alleviated carrageenan-induced hind paw edema through the suppressing iNOS and COX-2 via NF-κB, AP-1, and STAT1/3 inactivation. Collectively, the TMS-HDMF-5z-mediated inhibition of NF-κB, AP-1, and STAT1/3 offer an opportunity for the development of a potential treatment for inflammatory diseases.

Anti-inflammatory potential of Patrineolignan B isolated from Patrinia scabra in LPS-stimulated macrophages via inhibition of NF-κB, AP-1, and JAK/STAT pathways.

Patrineolignan B (PB), a lignan compound isolated from the radix and rhizomes of Patrinia scabra, was previously reported to possess a strong tumor-specific cytotoxic activity and beneficial effects on nitric oxide (NO) levels in macrophages induced by lipopolysaccharide (LPS). In this study, we assessed the effects of PB on LPS-induced inflammation in RAW 264.7 cells and clarified its molecular mechanisms. PB reversed LPS-induced increase in NO levels and prostaglandin E2 (PGE2) production, as well as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA levels in macrophages. Besides, PB prevented the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in a concentration-dependent manner. The regulatory effects of PB on LPS-induced inflammatory mediators and overproduction of pro-inflammatory cytokines were shown to depend partly on the suppression of nuclear factor kappa B (NF-κB)-mediated transcription and AP-1 activation regulated by a c-Jun amino-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK). Its anti-inflammatory activity was also mediated by regulating the phosphorylation of Janus kinase (JAK)/signal transducers and activators of transcription 1/3 (STAT1/3) signaling pathway. Taken together, our results suggest that PB exhibits anti-inflammatory potency through interfering with the NF-κB, AP-1, and JAK/STAT signaling pathway in LPS-stimulated macrophages.

Patriscabrin F from the roots of Patrinia scabra attenuates LPS-induced inflammation by downregulating NF-κB, AP-1, IRF3, and STAT1/3 activation in RAW 264.7 macrophages.

BACKGROUND: The roots of Partrinia scabra have been used as a medicinal herb in Asia. We previously reported that the inhibitory effect of patriscabrin F on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was the most potent than that of other isolated iridoids from the roots of P. scabra. PURPOSE: We investigated the anti-inflammatory activity of patriscabrin F as an active compound of P. scabra and related signaling cascade in LPS-activated macrophages. METHOD: The anti-inflammatory activities of patriscabrin F were determined according to its inhibitory effects on NO, prostaglandin E2 (PGE2), and pro-inflammatory cytokines. The molecular mechanisms were revealed by analyzing nuclear factor-κB (NF-κB), activator protein-1 (AP-1), interferon regulatory factor 3 (IRF3), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. RESULTS: Patriscabrin F inhibited the LPS-induced production of NO, PGE2, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in both bone-marrow derived macrophages (BMDMs) and RAW 264.7 macrophages. Patriscabrin F downregulated LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), TNF-α, IL-1ß, and IL-6 at the transcriptional level. Patriscabrin F suppressed LPS-induced NF-κB activation by decreasing p65 nuclear translocation, inhibitory κBα (IκBα) phosphorylation, and IκB kinase (IKK)α/ß phosphorylation. Patriscabrin F attenuated LPS-induced AP-1 activity by inhibiting c-Fos phosphorylation. Patriscabrin F suppressed the LPS-induced phosphorylation of IRF3, JAK1/JAK2, and STAT1/STAT3. CONCLUSION: Taken together, our findings suggest patriscabrin F may exhibit anti-inflammatory properties via the inhibition of NF-κB, AP-1, IRF3, and JAK-STAT activation in LPS-induced macrophages.

Inhibitory effects of triarylpyrazole derivatives on LPS-induced nitric oxide and PGE2 productions in murine RAW 264.7 macrophages.

In this article, a series of 22 triarylpyrazole derivatives were evaluated for in vitro antiinflammatory activity as inhibitors of nitric oxide (NO) and prostaglandin E2 (PGE2) release induced by lipopolysaccharide (LPS) in murine RAW 264.7 macrophages. The synthesized compounds 1a-h, 2a-f and 3a-h were first examined for their cytotoxicity for determination of the non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE2 production were not caused by non-specific cytotoxicity. Compounds 1h and 2f were the most active PGE2 inhibitors with IC50 values of 2.94 µM and 4.21 µM, respectively. Western blotting and cell-free COX-2 screening revealed that their effects were due to inhibition of COX-2 protein expression. Moreover, compound 1h exerted strong inhibitory effect on the expression of COX-2 mRNA in LPS-induced murine RAW 264.7 macrophages.

The Anti-Proliferative Activity of the Hybrid TMS-TMF-4f Compound Against Human Cervical Cancer Involves Apoptosis Mediated by STAT3 Inactivation.

We previously reported the potential anti-proliferative activity of 3-(5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl)-N-(3,4,5-trimethoxyphenyl) benzamide (TMS-TMF-4f) against human cancer cells; however, the underlying molecular mechanisms have not been investigated. In the present study, TMS-TMF-4f showed the highest cytotoxicity in human cervical cancer cells (HeLa and CaSki) and low cytotoxicity in normal ovarian epithelial cells. Annexin V-FITC and propidium iodide (PI) double staining revealed that TMS-TMF-4f-induced cytotoxicity was caused by the induction of apoptosis in both HeLa and CaSki cervical cancer cells. The compound TMS-TMF-4f enhanced the activation of caspase-3, caspase-8, and caspase-9 and regulated Bcl-2 family proteins, which led to mitochondrial membrane potential (MMP) loss and resulted in the release of cytochrome c and Smac/DIABLO into the cytosol. Also, TMS-TMF-4f suppressed both constitutive and IL-6-inducible levels of phosphorylated STAT3 (p-STAT3) and associated proteins such as Mcl-1, cyclin D1, survivin, and c-Myc in both cervical cancer cells. STAT-3 overexpression completely ameliorated TMS-TMF-4f-induced apoptotic cell death and PARP cleavage. Docking analysis revealed that TMS-TMF-4f could bind to unphosphorylated STAT3 and inhibit its interconversion to the activated form. Notably, intraperitoneal administration of TMS-TMF-4f (5, 10, or 20 mg/kg) decreased tumor growth in a xenograft cervical cancer mouse model, demonstrated by the increase in TUNEL staining and PARP cleavage and the reduction in p-STAT3, Mcl-1, cyclin D1, survivin, and c-Myc expression levels in tumor tissues. Taken together, our results suggest that TMS-TMF-4f may potentially inhibit human cervical tumor growth through the induction of apoptosis via STAT3 suppression.

Chemical Constituents from the Aerial Parts of Agastache rugosa and Their Inhibitory Activities on Prostaglandin E2 Production in Lipopolysaccharide-Treated RAW 264.7 Macrophages.

A new flavone glucoside, acacetin-7-O-(3″-O-acetyl-6″-O-malonyl)-ß-d-glucopyranoside (1), two new phenolic glucosides, (3R,7R)-tuberonic acid-12-O-[6'-O-(E)-feruloyl]-ß-d-glucopyranoside (14) and salicylic acid-2-O-[6'-O-(E)-feruloyl]-ß-d-glucopyranoside (15), and two new phenylpropanoid glucosides, chavicol-1-O-(6'-O-methylmalonyl)-ß-d-glucopyranoside (17) and chavicol-1-O-(6'-O-acetyl)-ß-d-glucopyranoside(18), as well as 26 known compounds, 2-13, 16, and 19-31, were isolated from the aerial parts of Agastache rugose. The structures of the new compounds were established by spectroscopic/spectrometric methods such as HRESIMS, NMR, and ECD. The anti-inflammatory effect of the isolated compounds was evaluated by measuring their inhibitory activities on prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. New compounds 1, 15, 17, and 18 inhibited LPS-induced PGE2 production with IC50 values of 16.8 ± 0.8, 33.9 ± 4.8, 14.3 ± 2.1, and 48.8 ± 4.4 µM, respectively. Compounds 5, 7, 9-11, 13, 19, 20, 22, and 27-30 showed potent inhibitory activities with IC50 values of 1.7-8.4 µM.

Anti-Inflammatory Mechanisms of Koreanaside A, a Lignan Isolated from the Flower of Forsythia koreana, against LPS-Induced Macrophage Activation and DSS-Induced Colitis Mice: The Crucial Role of AP-1, NF-κB, and JAK/STAT Signaling.

The current treatment options for inflammatory bowel disease (IBD) are unsatisfactory. Therefore, novel and safer therapies are needed. We previously reported that koreanaside A (KA) showed high radical scavenging activity and suppressed vascular cell adhesion molecule 1 (VCAM-1) expression in vascular smooth muscle cells. However, the molecular mechanisms involved in its anti-inflammatory effect have not been reported. KA inhibited pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nitric oxide (NO), and prostaglandin E2 (PGE2). KA inhibited the production and mRNA expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) induced by LPS. KA downregulated the myeloid differentiation primary response 88 (MyD88)-dependent inflammatory gene expressions in the MyD88-overexpressed cells. KA suppressed the LPS-induced transcriptional and DNA-binding activities of activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB). KA was found to inhibit the phosphorylation of Janus kinase 1/2 (JAK1/2) and signal transducers and activators of transcription 1/3 (STAT1/3). In DSS-induced colitis mice, KA relieved the symptoms of colitis by suppressing inflammatory cell infiltration, restoring tight junction (TJ)- and epithelial-mesenchymal transition (EMT)-related protein expression, and inactivating AP-1, NF-κB, and STAT1/3. Therefore, KA reduced inflammatory responses by downregulating AP-1, NF-κB, and JAK/STAT signaling in LPS-induced macrophages and DSS-induced colitis mice.

Hydrangenol Isolated from the Leaves of Hydrangea serrata Attenuates Wrinkle Formation and Repairs Skin Moisture in UVB-Irradiated Hairless Mice.

Our previous study showed that hydrangenol isolated from Hydrangea serrata leaves exerts antiphotoaging activity in vitro. In this study, we determined its antiphotoaging effect in UVB-irradiated HR-1 hairless mice. We evaluated wrinkle formation, skin thickness, histological characteristics, and mRNA and protein expression using qRT-PCR and Western blot analysis in dorsal skins. Hydrangenol mitigated wrinkle formation, dorsal thickness, dehydration, and collagen degradation. Hydrangenol increased the expression of involucrin, filaggrin, and aquaporin-3 (AQP3) as well as hyaluronic acid (HA) production via hyaluronidase (HYAL)-1/-2 downregulation. Consistent with the recovery of collagen composition, the expression of Pro-COL1A1 was increased by hydrangenol. Matrix metalloproteinase (MMP)-1/-3, cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) expression was reduced by hydrangenol. Hydrangenol attenuated the phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK and p38, activator protein 1 (AP-1) subunit, and signal transduction and activation of transcription 1 (STAT1). Hydrangenol upregulated the expression of nuclear factor-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO-1), glutamate cysteine ligase modifier subunit (GCLM), and glutamate cysteine ligase catalysis subunit (GCLC). Taken together, our data suggest that hydrangenol can prevent wrinkle formation by reducing MMP and inflammatory cytokine levels and increasing the expression of moisturizing factors and antioxidant genes.

Repurposing mosloflavone/5,6,7-trimethoxyflavone-resveratrol hybrids: Discovery of novel p38-α MAPK inhibitors as potent interceptors of macrophage-dependent production of proinflammatory mediators.

Herein, we address repurposing hybrids of mosloflavone or 5,6,7-trimethoxyflavone with amide analogs of resveratrol from anticancer leads to novel potent anti-inflammatory chemical entities. To unveil the potent anti-inflammatory molecules, biological evaluations were initiated in LPS-induced RAW 264.7 macrophages at 1 µM concentration. Promising compounds were further evaluated at various concentrations. Multiple proinflammatory mediators were assessed including NO, PGE2, IL-6, TNF-α and IL-1ß. Compound 5z inhibited the induced production of NO, PGE2, IL-6, TNF-α and IL-1ß at the low 1 µM concentration by 44.76, 35.71, 53.48, 29.39 and 41.02%, respectively. Compound 5z elicited IC50 values as low as 2.11 and 0.98 µM against NO and PGE2 production respectively. Compounds 5q and 5g showed potent submicromolar IC50 values of 0.31 and 0.59 µM respectively against PGE2 production. Reverse docking of compound 5z suggested p38-α MAPK, which is a key signaling molecule within the pathways controlling the transcription of proinflammatory mediators, as the molecular target. Biochemical testing confirmed these compounds as p38-α MAPK inhibitors explaining its potent inhibition of proinflammatory mediators' production. Collectively, the results presented 5z as a promising compound for further development of anti-inflammatory agents for treatment of macrophages-and/or immune mediated inflammatory diseases.

Caffeoyloxy-5,6-dihydro-4-methyl-(2H)-pyran-2-one isolated from the leaves of Olinia usambarensis attenuates LPS-induced inflammatory mediators by inactivating AP-1 and NF-κB.

We have previously reported the isolation of four compounds, caffeoyloxy-5,6-dihydro-4-methyl-(2H)-pyran-2-one (CDMP), olinioside, caffeic acid and 3-hydroxylup-12-en-28-oic acid, from the leaves of Olinia usambarensis. Here, we evaluated the inhibitory effects of these compounds on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages, and found that CDMP is the most potent of these two pro-inflammatory mediators (IC50; 12.12 µM and 10.78 µM, respectively). Consistent with these results, CDMP also down-regulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interleukin 6 (IL-6) at the protein and mRNA levels in LPS-treated RAW 264.7 macrophages. Furthermore, CDMP suppressed LPS-induced nuclear factor κB (NF-κB) activation by decreasing p65 nuclear translocation through the phosphorylation and degradation of the inhibitory κBα (IκBα). CDMP also attenuated LPS-induced transcriptional and DNA-binding activities of activator protein 1 (AP-1) by suppressing the phosphorylation and expression of c-Fos and c-Jun. Finally, CDMP considerably suppressed the LPS-induced phosphorylation of c-Jun N-terminal kinase (JNK), but did not affect the phosphorylation of p38 or extracellular signal-regulated kinase (ERK). Taken together, our data suggest that CDMP down-regulates genes encoding pro-inflammatory mediators and cytokines, such as iNOS, COX-2, TNF-α, IL-1ß, and IL-6 via NF-κB and JNK/AP-1 inactivation in LPS-induced RAW 264.7 macrophages.

EGFR inhibitors from cancer to inflammation: Discovery of 4-fluoro-N-(4-(3-(trifluoromethyl)phenoxy)pyrimidin-5-yl)benzamide as a novel anti-inflammatory EGFR inhibitor.

EGFR inhibitors are well-known as anticancer agents. Quite differently, we report our effort to develop EGFR inhibitors as anti-inflammatory agents. Pyrimidinamide EGFR inhibitors eliciting low micromolar IC50 and the structurally close non-EGFR inhibitor urea analog were synthesized. Comparing their nitric oxide (NO) production inhibitory activity in peritoneal macrophages and RAW 246.7 macrophages indicated that their anti-inflammatory activity in peritoneal macrophages might be a sequence of EGFR inhibition. Further evaluations proved that compound 4d significantly and dose-dependently inhibits LPS-induced iNOS expression and IL-1ß, IL-6, and TNF-α production via NF-κB inactivation in peritoneal macrophages. Compound 4d might serve as a lead compound for development of a novel class of anti-inflammatory EGFR inhibitors.

Anti-Colitic Effects of Ethanol Extract of Persea americana Mill. through Suppression of Pro-Inflammatory Mediators via NF-κB/STAT3 Inactivation in Dextran Sulfate Sodium-Induced Colitis Mice.

Persea americana Mill, cv. Hass, also known as avocado, has been reported to possess hypolipidemic, anti-diabetic, anti-oxidant, cardioprotective, and photoprotective potencies. However, few studies have reported its anti-colitic effects. In this study, we investigated anti-colitic effects of ethanol extract of P. americana (EEP) in dextran sulfate sodium (DSS)-induced colitic mice and the involved molecular mechanisms. EEP effectively improved clinical signs and histological characteristics of DSS-induced colitis mice. In DSS-exposed colonic tissues, EEP reduced expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α. Moreover, EEP suppressed DSS-induced activation of nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3). Consistent with in vivo results, EEP also suppressed protein and mRNA expression levels of iNOS, COX-2, and pro-inflammatory cytokines via NF-κB and STAT3 inactivation in LPS-induced RAW 264.7 macrophages. Taken together, our data indicate that ethanol extract of avocado may be used as a promising therapeutic against inflammatory bowel diseases by suppressing the NF-κB and STAT3 signaling pathway.

Saikosaponin B2 Suppresses Inflammatory Responses Through IKK/IκBα/NF-κB Signaling Inactivation in LPS-Induced RAW 264.7 Macrophages.

Bupleurum falcatum (Umbelliferae) have been widely used to treat inflammatory diseases as traditional medicines in East Asian region. Although saikosaponins are main bioactive molecules of B. falcatum, there is little information on bioactivity of saikosaponin B2 (SSB2). This study was conducted to assess the anti-inflammatory activities and the involved mechanisms of SSB2 in LPS-induced RAW 264.7 macrophages. SSB2 suppressed the releases of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukins (IL)-6, and IL-1ß by suppressing mRNA levels of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1ß, and IL-6 in LPS-induced macrophages. SSB2 blocked LPS-induced DNA binding and nuclear factor kappa B (NF-κB) transcriptional activity by inhibiting nuclear translocation p65 and p50, inhibitory κBα (IκBα) degradation, and IκB kinase ß (IKKß) phosphorylation and activity. In IKKß-overexpressing cells, SSB2 significantly suppressed IKKß-dependent NF-κB transcriptional activity. Moreover, SSB2 reduced phosphorylation of p38 and extracellular signal-regulated kinase1/2 (ERK1/2). SSB2 effectively inhibits LPS-induced pro-inflammatory mediator releases by interfering with IKKß and IκBα activation, thus preventing NF-κB activation. Our data indicates that SSB2 could be a potential therapeutic application for inflammation-associated diseases.

Natural products hybrids: 3,5,4'-Trimethoxystilbene-5,6,7-trimethoxyflavone chimeric analogs as potential cytotoxic agents against diverse human cancer cells.

Cancer still represents a major global health problem. All currently available anticancer agents have disadvantages like resistance or side effects. Therefore, introduction of novel anticancer agents is needed. Intrigued by the high success rate for natural products-based drug discovery, we designed and synthesized antiproliferative chemical entities as hybrids of two natural products; 3,5,4'-trimethoxystilbene and 5,6,7-trimethoxyflavone. To probe the spectrum of the synthesized compounds, in vitro evaluation was conducted against nine panels representing major cancer diseases. The results revealed the hybrid analogs 4f, 4h, 4k and 4q as promising broad-spectrum anticancer lead compounds eliciting high growth inhibition of several cell lines representing multiple cancers diseases. Evaluation of the promising lead compounds against normal human cell lines suggested a selective cytotoxic effect on cancer cells. Mechanistic investigation of the cytotoxic activity of compound 4f in human cervical cancer HeLa cells showed that it triggers cell death through induction of apoptosis. As a whole, this study presents the natural products hybrid analogs 4f, 4h, 4k and 4q as potential lead compounds for further development of novel anticancer therapeutics.

Synthesis of New Triarylpyrazole Derivatives Possessing Terminal Sulfonamide Moiety and Their Inhibitory Effects on PGE2 and Nitric Oxide Productions in Lipopolysaccharide-Induced RAW 264.7 Macrophages.

This article describes the design, synthesis, and in vitro anti-inflammatory screening of new triarylpyrazole derivatives. A total of 34 new compounds were synthesized containing a terminal arylsulfonamide moiety and a different linker between the sulfonamide and pyridine ring at position 4 of the pyrazole ring. All the target compounds were tested for both cytotoxicity and nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Compounds 1b, 1d, 1g, 2a, and 2c showed the highest NO inhibition percentages and the lowest cytotoxic effect. The most potent derivatives were tested for their ability to inhibit prostaglandin E2 (PGE2) in LPS-induced RAW 264.7 macrophages. The IC50 for nitric oxide inhibition, PGE2 inhibition, and cell viability were determined. In addition, 1b, 1d, 1g, 2a, and 2c were tested for their inhibitory effect on LPS-induced inducible nitric oxide synthase (iNOS) and Cyclooxygenase 2 (COX-2) protein expression as well as iNOS enzymatic activity.