JAK/STAT3 Signaling Activation Related to Distinct Clinicopathologic Features in Systemic ALK - Anaplastic Large Cell Lymphomas : New Insights into Their Heterogeneity.

Systemic anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a group of heterogenous CD30 + T-cell non-Hodgkin lymphomas. Previous studies have highlighted the importance of JAK/STAT3 signaling activation in the molecular pathogenesis of ALK - ALCLs. In the present study, we aimed to establish a potential relationship between JAK/STAT3 signaling activation and clinicopathologic features in ALK - ALCLs, and further recognize the heterogenous nature of these neoplasms. Immunohistochemistry staining of the phosphorylated-STAT3 (p-STAT3) and dual-specificity protein phosphatase 22 ( DUSP22 ) gene rearrangement analysis were performed. Forty-five cases of ALK - ALCL were divided into 3 groups, including 9 DUSP22 -rearranged ALCLs, 21 p-STAT3 + double-negative (DN) ALCLs (both ALK and DUSP22 rearrangement negative), and 15 p-STAT3 - DN-ALCLs. Morphologically, p-STAT3 + DN-ALCLs exhibited sheet-like neoplastic cells and sometimes showed large pleomorphic cells scattered in a lymphocyte-rich background more frequently than those in other ALK - ALCLs subtypes. Phenotypically, the p-STAT3 + DN-ALCLs frequently expressed cytotoxic molecules, epithelial membrane antigen, and programmed death-ligand 1, whereas CD3 and CD5 expression was not observed. Clinically, patients with p-STAT3 + DN-ALCLs had a better prognosis than those with p-STAT3 - DN-ALCLs. These observations suggest that p-STAT3 + DN-ALCLs represent a distinct subtype of ALK - ALCLs. Identifying ALK - ALCL subtypes by using p-STAT3 staining and DUSP22 rearrangement is a promising approach that may contribute to risk stratification and better treatment decisions in the future clinical practice.

Molecular profiling of Chinese systemic anaplastic large cell lymphoma patients: novel evidence of genetic heterogeneity.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is a rare non-Hodgkin lymphoma. A comprehensive understanding of the genetic and clinical heterogeneity of ALCL may help to improve the clinical management of patients with ALCL. However, due to the rarity of the disease, the genetic heterogeneity of ALCL has not been well elucidated. This study aimed to comprehensively elucidate the mutational landscape of tumor tissue samples from patients with systemic ALCL. METHODS: Thirty-six patients with systemic ALCL were enrolled in this retrospective study. Immunohistochemistry (IHC) was performed on tumor tissues at baseline to identify anaplastic lymphoma kinase (ALK) fusions. Capture-based targeted next-generation sequencing (NGS) with a panel spanning 112 lymphoma-related genes, including ALK rearrangements, was also performed on tumor tissue samples. RESULTS: A total of 102 mutations were identified in the entire cohort. Among the 36 patients included in this analysis, 14 (38.8%) were ALK positive, as determined by IHC, while NGS showed 12 patients (33.3%) to harbor ALK rearrangements. Younger patients were more likely to have ALK-positive ALCL (P=0.011). Patients with wild-type (WT) ALK were more likely to have single-nucleotide variants (SNVs) and insertions or deletions (INDELs) than patients with ALK rearrangements (P=0.027). Among the 22 patients with WT ALK, the most commonly mutated genes were TP53 (n=6, 27.3%), followed by NOTCH1 (n=5, 22.7%), KMT2D (n=3, 13.6%), KRAS (n=3, 13.6%), TET2 (n=3, 13.6%), and JAK1 (n=2, 9.1%). Mutations in PRDM1, a commonly mutated gene in ALK-negative patients, were not detected in our ALK-negative cohort. Start-loss of beta-2-microglobulin (B2M) was detected in another patient; this patient had a favorable prognosis, with an overall survival exceeding 19 months. CONCLUSIONS: Our study revealed the unique genomic profiles of Chinese ALCL patients and represents an incremental step in deepening the understanding of the genetic heterogeneity of ALCL patients.

Comprehensive Flow-Cytometry-Based Immunophenotyping Analysis for Accurate Diagnosis and Management of Extranodal NK/T Cell Lymphoma, Nasal Type.

BACKGROUND: Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTL-N) is an aggressive lymphoma typically diagnosed by examining small biopsy specimens. Flow cytometry is very valuable for the diagnosis and classification of several kinds of hematolymphoid neoplasms but has not been widely used for diagnosing ENKTL-N. METHODS: We systematically investigated the flow cytometry characteristics of 26 solid tissue biopsy specimens of ENKTL-N at initial diagnosis and compared the results with those from reactive NK-cells in the nasal/nasopharyngeal region and peripheral blood. RESULTS: Our study revealed seven flow cytometry (FCM)-based characteristics for distinguishing between the neoplastic cells and reactive NK-cells, including (1) the proportion of NK-cells among total lymphocytes >10%; (2) forward scatter >105 ; (3) mean fluorescence intensity of CD56 > 5,000; (4) aberrant antigen expression or loss; (5) skewed killer cell immunoglobulin-like receptor repertoire; (6) homogenously positive for CD38; and (7) positive for CD30 or CD336. FCM-based immunophenotyping is a potentially feasible and convenient approach for discriminating cellular lineages, evaluating the activation status of NK-cells, and selecting potential therapy targets of ENKTL-N. CONCLUSIONS: Flow cytometry is very valuable for facilitating routine diagnosis, confirming clonality, predicting the cellular lineage, and guiding individual treatment for ENKTL-N. © 2019 International Clinical Cytometry Society.

RIP3 promotes colitis-associated colorectal cancer by controlling tumor cell proliferation and CXCL1-induced immune suppression.

Rationale: Necroptosis is a programmed form of non-apoptotic cell death that requires receptor-interacting protein 3 (RIP3). RIP3 has been shown to be relevant in multiple tumor types and has differential impact on tumor progression. We investigated whether RIP3 is involved in the progression of colitis-associated cancer (CAC) in mice. Methods: Tissues from colorectal cancer patients were examined for RIP3 expression. CAC was induced using azoxymethane (AOM) injection followed by dextran sodium sulfate (DSS) treatment in RIP3-deficient or wild-type mice. Colon tissues were collected and analyzed by Western blotting and gene expression profile analyses. Immune cell infiltration and CXCL1 expression were examined by flow cytometry and Real-time PCR, respectively. Results: RIP3 expression was upregulated in mouse CAC and human colon cancer. RIP3-deficient mice showed significantly attenuated colitis-associated tumorigenesis. Bone marrow transplantation experiments suggested that RIP3's function in hematopoietic cells primarily contributes to the phenotype. RIP3 supported epithelial proliferation and tumor growth via JNK signaling but had no effect on apoptosis. RIP3 deletion increased T cell accumulation and reduced infiltration by immunosuppressive subsets of myeloid cells during acute colitis and CAC. The immune-suppressive tumor microenvironment was dependent on RIP3-induced expression of the chemokine attractant CXCL1, and administration of recombinant CXCL1 during CAC restored tumorigenesis in Rip3-/- mice. Conclusion: Our results reveal an unexpected function of RIP3 in enhancing the proliferation of premalignant intestinal epithelial cells (IECs) and promoting myeloid cell-induced adaptive immune suppression. These two distinct mechanisms of RIP3-induced JNK and CXCL1 signalling contribute to CAC progression.

Receptor-Interacting Protein Kinase 3 Deficiency Recruits Myeloid-Derived Suppressor Cells to Hepatocellular Carcinoma Through the Chemokine (C-X-C Motif) Ligand 1-Chemokine (C-X-C Motif) Receptor 2 Axis.

Receptor-interacting protein kinase 3 (RIP3) is the core regulator that switches cell death from apoptosis to necrosis. However, its role in tumor immunity is unknown. In this study, decreased RIP3 expression was observed in patients with hepatocellular carcinoma (HCC), which correlates with myeloid-derived suppressor cell (MDSC) accumulation. Moreover, RIP3 is a prognosis factor for patients with HCC. We further found that RIP3 knockdown results in an increase of MDSCs and a decrease of interferon gamma-positive (IFN-γ+ ) cluster of differentiation 8-positive (CD8+ ) tumor-infiltrating lymphocytes (IFN-γ+ CD8+ T cells) in hepatoma tissues, thus promoting immune escape and HCC growth in immunocompetent mice. By phosphorylating P65Ser536 and promoting phosphorylated P65Ser536 nuclear translocation, RIP3 knockdown increases the expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in HCC cells. RIP3 knockdown induces MDSC recruitment through the CXCL1-chemokine (C-X-C motif) receptor 2 (CXCR2) axis. Furthermore, a CXCR2 antagonist substantially suppresses MDSC chemotaxis and HCC growth in RIP3 knockout mice. Conclusion: RIP3 deficiency is an essential factor directing MDSC homing to HCC and promoting CXCL1/CXCR2-induced MDSC chemotaxis to facilitate HCC immune escape and HCC progression; blocking the CXCL1-CXCR2 chemokine axis may provide an immunological therapeutic approach to suppress progression of RIP3 deficiency HCC.

Chrysophanol Suppresses Hypoxia-Induced Epithelial-Mesenchymal Transition in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common human malignancy, accounting for 600,000 death cases annually worldwide. Chrysophanol is a naturally occurring anthraquinone compound and exhibits anti-neoplastic activities. This study aims to explore the biological effects of chrysophanol on CRC metastasis and the relevant underlying mechanism. Cell proliferation assay, wound scratch assay, and Transwell invasion assay were used to examine the effect of chrysophanol on proliferation, migration, and invasion of CRC cells. Hypoxia-inducible factor-1α (HIF-1α) shRNA was utilized to transfect CRC cells to examine the role of HIF-1α in chrysophanol suppression of hypoxia-induced epithelial to mesenchymal transition (EMT). The suppression effect of chrysophanol on hypoxia-induced EMT in vivo was also validated in xenograft tumor models. In the present study, our findings indicated that chrysophanol has the capability to suppress hypoxia-induced EMT in CRC in vitro and in vivo, and the possible mechanism involved is the inhibition of HIF-1α via modulating PI3k/Akt signaling pathway. Collectively, the results indicated that chrysophanol can be used as an EMT and cancer metastasis inhibitor in the treatment of CRC. Anat Rec, 302:1561-1570, 2019. © 2019 American Association for Anatomy.

Alterations of the Immunologic Co-Stimulator B7 and TNFR Families Correlate with Hepatocellular Carcinoma Prognosis and Metastasis by Inactivating STAT3.

Blockade of the immunosuppressive checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or programmed death 1 (PD-1) and its cognate ligand, programmed death 1 ligand (PD-L1), has altered the landscape of anti-tumor immunotherapy. B7 family and tumor necrosis factor receptor (TNFR) superfamily play a crucial role in T cell activation, tolerance, and anergy through co-stimulatory and inhibitory signal transduction. Investigating the immune molecular landscapes of the B7 and TNFR families is critical in defining the promising responsive candidates. Herein, we performed comprehensive alteration analysis of the B7 and TNFR family genes across six hepatocellular carcinoma (HCC) datasets with over 1000 patients using cBioPortal TCGA data. About 16% of patients had both B7 and TNFR gene alterations. TNFR gene amplifications were relatively more common (1.73⁻8.82%) than B7 gene amplifications (1.61⁻2.94%). Analysis of 371 sequenced samples revealed that all genes were upregulated: B7 and TNFR mRNA were upregulated in 23% of cases (86/371) and 28% of cases (105/371), respectively. Promoter methylation analysis indicated an epigenetic basis for B7 and TNFR gene regulation. The mRNA levels of B7 and TNFR genes were inversely correlated with promoter methylation status. B7-H6 expression was significantly associated with worse overall survival, and B7-H6 mRNA was increased gradually in cases with gene copy number alterations. B7-H6 overexpression was associated with aggressive clinicopathologic features and poor prognosis in HCC. Downregulation of B7-H6 in HCC cells significantly inhibited cell adhesion, proliferation, migration, and invasion. Knockdown of B7-H6 in HCC cells inhibited tumor growth and metastasis in vivo. B7-H6 promoted HCC metastasis via induction of MMP-9 expression and STAT3 activation. B7-H6 and STAT3 performed functional overlapping roles on enhancing the MMP-9 promoter activity in HCC cells. These results suggest that alterations of the immunologic co-stimulator B7 and TNFR families correlate with HCC metastasis and prognosis, and especially B7-H6 plays a critical role in promoting metastasis of HCC.

Prevalence And Clinical Significance Of Oncogenic CD79B And MYD88 Mutations In Primary Testicular Diffuse Large B-Cell Lymphoma: A Retrospective Study In China.

PURPOSE: In this study, we investigated the prevalence of CD79B and MYD88 mutations and their relation to clinical characteristics in a cohort of Chinese patients with primary testicular diffuse large B cell lymphoma (PT-DLBCL). PATIENTS AND METHODS: We examined the mutational status of CD79B and MYD88 by Sanger sequencing, and the gene amplification and protein expression of MYD88 in tissue samples from 30 cases of PT-DLBCL by quantitative polymerase chain reaction and immunohistochemistry, respectively. Western blotting was used to analyze phosphorylated STAT3 (p-STAT3) and phosphorylated p65 (p-p65) protein expression in cell lines harboring retroviral constructs for WT MYD88 or MYD88 mutant. RESULTS: Immunophenotypically, MYD88 protein staining was positive in 26/30 (86.67%) cases, and 23/30 (76.7%) cases tested positive for p65 in the nucleus. Genetically, CD79B mutation was found in 13/30 (43.3%) cases, whereas the MYD88 L265P mutation was found in 18/30 (60.0%) cases. Interestingly, CD79B and MYD88 mutations were more prevalent in the non-germinal center B cell (GCB) subtype (83.3% and 76.9%, respectively) and were relatively rare in the GCB subtype (16.7% and 23.1%, respectively). Furthermore, although MYD88 was significantly amplified in PT-DLBCL, the amplification status showed no correlation with its mutational status and protein expression. Clinicopathological comparison between the mutant and wild-type group showed that both CD79B mutation and MYD88 L265P were not significantly correlated with age, anatomical site, Ann Arbor stage, non-GCB/GCB subtype, p65 protein expression, BCL-2 protein expression, or BCL-2/c-MYC double expression (P>0.05). Survival analyses showed that high IPI and advanced stage (stage III-IV) associated with worse outcome (P<0.05). The expression of p-STAT3 and p-p65 protein was upregulated in the mutant group, indicating that MYD88 mutant activated NF-κB and JAK-STAT3 signaling. CONCLUSION: Our results suggest that MYD88 and CD79B mutations are important drivers of immune-privileged site-associated DLBCL and highlight potential therapeutic targets for personalized treatment.

Surface treatment with Fenton for separation of acrylonitrile-butadiene-styrene and polyvinylchloride waste plastics by flotation.

Surface treatment with Fenton was applied to flotation separation of acrylonitrile-butadienestyrene (ABS) and polyvinylchloride (PVC). After treatment, the floatability of ABS has a dramatic decrease, while the floatability of PVC is not affected. Fourier transform infrared spectroscopy (FT-IR) spectra and X-ray photoelectron spectroscopy (XPS) spectra were recorded to ascertain the mechanism of Fenton treatment. FT-IR and XPS analysis confirms that the introduction of oxygen-containing group occurs on the surface of ABS. The optimum conditions are molar ration (H2O2:Fe2+) 10000, H2O2 concentration 0.4M/L, pH 5.8, treatment time 2min and temperature 25°C, frother concentration 15mg/L and flotation time 3min. Particle sizes and mixing ratios were also investigated. Plastic mixtures of ABS and PVC with different particle sizes and mixing ratios can be effectively separated. The purity of ABS and PVC are up to 100% and 99.78%, respectively; the recovery of ABS and PVC are up to 99.89% and 100%, respectively. A practical, environmentally friendly and effective reagent, namely Fenton, was originally applied to surface treatment of ABS and PVC waste plastics for flotation separation of their mixtures.

Bufalin inhibits pancreatic cancer by inducing cell cycle arrest via the c-Myc/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Bufalin, a cardiotonic steroid isolated from toad venom (bufo gargarizans Cantor or B. melanotictus Schneider), has widely demonstrated antitumor effects and exhibits potential antitumor activity in various human cancer cells lines. AIMS OF THE STUDY: The main characteristic of cancers including pancreatic cancer is the ability of uncontrolled proliferation. The aim of this study is to clarify the underlying mechanism by which bufalin inhibits pancreatic cancer cell proliferation. MATERIALS AND METHODS: The effect of bufalin on the suppression of tumor growth in vivo was studied in a bioluminescent mouse model generated using the pancreatic cancer cell line BxPC3-luc2 and the cytotoxicity was evaluated in BcPc3 and Sw1990 cells with MTT. Flow cytometry and western blotting analyses were utilized to detect the effect of bufalin on the cell cycle and to detect the cell cycle-related proteins, respectively. Then, a luciferase reporter assay was applied to screen the activity of potent transcription factors following bufalin exposure and their expression was detected by western blotting. RESULTS: Bufalin suppressed tumor growth in a bioluminescence mouse model generated using BxPC3-luc2 cells and inhibited cell proliferation in vitro through inducing cell cycle arrest at S phase. Bufalin treatment inhibited cyclin D1 and cyclin E1 expression and therefore increased expression of p27, a regulatory molecular that controls cell cycle transition from S to G2 phase. Furthermore, luciferase reporter screening studies revealed that bufalin inhibited the expression and activity of the transcription factors c-Myc and NF-κB, which might cause cell cycle arrest at S phase and the inhibition of cell proliferation. CONCLUSIONS: Taken together, our results indicate that bufalin can inhibit pancreatic cancer by targeting c-Myc, thus suggesting that the mechanism of c-Myc regulation by bufalin might be worthy of further study regarding its potential as a therapeutic target for pancreatic cancer treatment.

Short term effects of small incision lenticule extraction surgery on corneal endothelium.

AIM: To assess the effects of small incision lenticule extraction (SMILE) surgery on the corneal endothelium at 1d to 1mo postoperatively. METHODS: A retrospective, observational study was conducted on 47 patients (47 eyes) who received SMILE surgery. Patients were grouped according to contact lens wear condition. The corneal endothelium was examined preoperatively and at 1d, 1wk and 1mo postoperatively. The corneal endothelium was analyzed for endothelial cell density (ECD), percentage of hexagonal cells, and coefficient of variation (CV) of cell size. RESULTS: There were no significant decrease in the ECD, percentage of hexagonal cells or increase in CV at 1d, 1wk and 1mo postoperatively (P>0.05). However, there was a small increase of ECD by 2.88% in contact lens wearers (78.26±113.62 cell/mm(2), P<0.05). CONCLUSION: SMILE has no significant adverse effects on the corneal ECD and morphology during 1mo follow-up time.

Clinicopathologic Characterization of Aggressive Natural Killer Cell Leukemia Involving Different Tissue Sites.

Aggressive natural killer cell leukemia (ANKL) is a rare disease with an extremely aggressive clinical course. The etiology of ANKL is unclear with few genetic/epigenetic aberrations described to date. Moreover, misdiagnosis of ANKL is a frequent problem. Clinicopathologic characteristics of 35 retrospective cases of ANKL were investigated with the aim of improving diagnosis and to find the genetic/epigenetic aberrations associated with ANKL etiology. Because of the relatively low number of leukemic cells in the peripheral blood and bone marrow, diagnosis of ANKL can be missed; therefore, it is important to perform biopsy on solid tissues, if necessary. We describe the pathology of ANKL in the lymph nodes, bone marrow, spleen, liver, and skin, with focus on diagnosis and differentiated diagnosis. We observed young male predominance in our cohort, and the clinical course was more aggressive than reported previously. Low lactate dehydrogenase (<712 IU/L), chemotherapy or L-asparaginase administration were found to be associated with more favorable outcomes. SH2 domains of STAT5B and STAT3 also were screened for the presence of activating mutations. Moreover, CpG island methylation status of HACE1, a candidate tumor-suppressor gene, was determined in ANKL samples. We observed activating STAT5B mutations (1/5) and hypermethylation of HACE1 (3/4) in ANKL cases, suggesting that these aberrations may contribute to ANKL pathogenesis.

CD147 reinforces [Ca2+]i oscillations and promotes oncogenic progression in hepatocellular carcinoma.

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC.

Two new ionone glycosides from the roots of Rehmannia glutinosa Libosch.

Two new ionone glycosides, named frehmaglutoside G (1) and frehmaglutoside H (2), together with six known compounds, rehmapicroside (3), sec-hydroxyaeginetic acid (4), dihydroxy-ß-ionone (5), trihydroxy-ß-ionone (6), rehmaionoside A (7) and rehmaionoside C (8), were isolated from the 95% EtOH extract of the dried roots of Rehmannia glutinosa Libosch. Their structures were determined on the basis of extensive spectroscopic analyses, including HR-ESI-MS, UV, IR, 1D and 2D NMR ((1)H-(1)H COSY, HSQC, HMBC and NOESY) methods. The absolute configurations were confirmed via the circular dichroism spectra.

[Comparison of efficacies for two surgical methods in the treatment of medial tibial-femoral osteoarthritis].

OBJECTIVE: To compare the clinical efficacies of two surgical methods in the treatment of medial tibial-femoral osteoarthritis. METHODS: Between October 2007 and June 2010, a total of 22 cases (25 knees) with severe osteoarthritis in medial tibial-femoral compartment underwent minimally invasive unicompartmental knee arthroplasty (UKA) with Sled prosthesis after arthroscopic procedure. And its clinical efficacy was compared with that of 22 cases (25 knees) undergoing total knee arthroplasty (TKA) with Gemini MKII prosthesis almost simultaneously. RESULTS: There were no significant difference in general data between 2 groups (P > 0.05). Compared with the TKA group, the UKA group had a smaller blood loss ((148 ± 26) vs (278 ± 36) ml), a shorter operative duration ((68 ± 12) vs (86 ± 12) min), a faster progress of resuming 90° flexion ((3.18 ± 1.8) vs (9.1 ± 2.2) d) and an earlier off-bed time (P < 0.05). All patients were followed up for 6 - 34 months. There was no significant difference in KSS (Knee Society Score), function score or WOMAC (Western Ontario and McMaster Universities) score between 2 groups at the last follow-up (P > 0.05). CONCLUSION: The treatment of medial tibial-femoral osteoarthritis with minimally invasive UKA is superior to that with TKA in that it is less invasive, there is a faster recovery of joint functions and no significant difference exists in the mid-term clinical efficacies between them.

Inhibition of inflammatory mediators and related signaling pathways by macrophage-stimulating protein in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To evaluate the mechanism of macrophage-stimulating protein (MSP)-mediated inhibition of inflammatory cytokine and chemokine production in rheumatoid arthritis synovial fibroblasts (RASF). MATERIALS AND METHODS: RASF were treated with different concentrations (0, 0.5, 1, 5 and 10 ng/ml) of MSP with or without 1 µg/mL lipopolysaccharide (LPS). The protein expressions of IL-1ß, TNF-α, IL-18, MIP-1, MCP-1, RANTES and PGE(2) were analyzed by enzyme-linked immunosorbent assays (ELISA). The total nitric oxide (NO) concentration was determined using the Griess reaction. The protein expressions of iNOS, COX-2, NF-κB(p-p65), IKB-α, IKB-ß, p-P38, p-Erk1/2 (P-P42/44) and p-AKT were detected by Western blotting. RESULTS: MSP markedly inhibited expression of inflammatory cytokines (IL-1ß, TNF-α and IL-18), chemokines (MIP-1, MCP-1 and RANTES) and iNOS, NO, COX-2 and PGE(2) in RASF stimulated by LPS. MSP treatment decreased expressions of p-IκBα, p-IKBß and p-P65 in RASF in a concentration-dependent manner. Expressions of p-AKT, p-p38 and p-Erk1/2 were also inhibited markedly in RASF stimulated by LPS after treatment with MSP in a concentration-dependent manner. CONCLUSION: MSP could inhibit the inflammatory cycle by suppressing inflammatory mediators and activation of NF-κB as well. The inhibitory effect of MSP on LPS-stimulated RASF may act through suppression of multiple signals such as the PI3K/AKT and/or MAPK pathways.