HAb18G/CD147 functions in invasion and metastasis of hepatocellular carcinoma

CD147 molecule is reported to be correlated with the malignancy of some cancers; however, it remains unclear whether it is involved in the progression of hepatocellular carcinoma (HCC). Here, we investigated the function of HAb18G/CD147, a member of CD147 family, and its antibodies, HAb18 and LICARTIN, in HCC invasion and metastasis. We observed that HAb18G/CD147 gene silence in HCC cells significantly decreased the secretion of matrix metalloproteinase (MMP) and the invasive potential of HCC cells (P < 0.001). MMP silence in HCC cells also significantly suppressed the invasion of the cells when cocultured with fibroblasts; however, its inhibitory effect was significantly weaker than that of both HAb18G/CD147 silence in HCC cells and that of MMP silence in fibroblasts (P < 0.001). Blocking theHAb18G/CD147 molecule on HCC cells with HAb18 monoclonal antibody resulted in a similar suppressive effect on MMP secretion and cell invasion, but with no significant effects on the cell growth. (131)I-labeled HAb18 F(ab')(2) (LICARTIN), however, significantly inhibited the in vitro growth of HCC cells (P < 0.001). In an orthotopic model of HCC in nude mice, HAb18 and LICARTIN treatment effectively reduced the tumor growth and metastasis as well as the expression of three major factors in the HCC microenviroment (MMPs, vascular endothelial growth factor, and fibroblast surface protein) in the paracancer tissues. Overall, these results suggest that HAb18G/CD147 plays an important role in HCC invasion and metastasis mainly via modulating fibroblasts, as well as HCC cells themselves to disrupt the HCC microenviroment. LICARTIN can be used as a drug targeting to HAb18G/CD147 in antimetastasis and recurrence therapy of HCC.     

Dimerization is essential for HAb18G/CD147 promoting tumor invasion via MAPK pathway

HAb18G/CD147 is a transmembrane glycoprotein of the immunoglobulin superfamily (IgSF) and is reported to be correlated with invasion and metastasis of many cancers. The crystal structure of HAb18G/CD147 ectodomain has shown that it can form homodimers in crystal. However, the functional significance of HAb18G/CD147 dimerization remains unclear. In the present study, guided by the crystal structure, we performed extensive mutational and functional studies to identify residues critical for dimerization and molecular function of HAb18G/CD147. Fourteen mutants were purified and evaluated for their ability to form dimers in solution and in living cells. Subsequent functional validation revealed that K63E and S193A mutants, which disrupted CD147 dimerization both in solution and in living cells, showed clearly dominant-negative effects on MAPK activation, MMP2 induction and invasiveness in tumor cells. Taken together, the present study provides mutational and functional evidences demonstrating for the first time the functional importance of CD147 dimerization and its direct correlation with invasion and metastasis of tumor cells.     

Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

HAb18G/CD147, a glycoprotein of the immunoglobulin super‐family (IgSF), is a T cell activation‐associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4⁺ and CD8⁺ T cells was up‐regulated. In vitro cross‐linking of T cells with an anti‐HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co‐stimulation inhibited T cell proliferation by down‐regulating the expression of CD25 and interleukin‐2 (IL‐2), decreased production of IL‐4 but not interferon‐γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti‐HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody–antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N‐terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co‐stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.     

The glycosylation characteristic of hepatoma-associated antigen HAb18G/CD147 in human hepatoma cells

HAb18G/CD147 is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. Our previous studies have demonstrated that overexpressing HAb18G/CD147 enhances the metastatic potentials of human hepatoma cells. In the present study, to investigate the glycosylation characteristic of HAb18G/CD147 in human hepatoma cells, HAb18G/CD147 was first purified from human FHCC-98 hepatoma cells by immunoaffinity chromatography, and then introduced into human fibroblasts culture system for matrix metalloproteinases induction. As a result, the elevated levels of matrix metalloproteinases secreted by fibroblasts were detected by gelatin zymography. The lysates of human hepatoma FHCC-98 cell revealed two major forms of HAb18G/CD147 (43-66 and 35 kDa) by western blot assay. To elucidate whether the variation of molecule size were caused by different glycosylation, two different approaches were employed to accomplish this goal: deglycosylation with N-glycosylation inhibitor tunicamycin or endoglycosidases. A single deglycosylated core protein with molecular weight approximately 27 kDa was obtained from both methods. Furthermore, the results of endoglycosidases treatment also showed that two forms of HAb18G/CD147 contain different types of oligosaccharide chains, thus sensitive to different endoglycosidase. In conclusion, the present study demonstrated that purified native HAb18G/CD147 has the bioactivity of stimulating human fibroblasts to produce elevated levels of matrix metalloproteinases, and that the two different forms of HAb18G/CD147 are derived from the single core protein but differ in their degree and types of glycosylation.     

BetaIg-h3 is involved in the HAb18G/CD147-mediated metastasis process in human hepatoma cells

HAb18G/CD147, a new hepatoma-associated antigen cloned and screened from human hepatocellular carcinoma cDNA library, is closely correlated with metastasis process in human hepatoma cells. In the present study we aimed to identify the pivotal molecules of the HAb18G/CD147 signal transduction pathway. The investigation showed that betaig-h3, a secretory extracellular matrix (ECM) protein, was upregulated in HAb18G/CD147-expressing human hepatoma T7721 cells and was downregulated by depressing HAb18G/CD147 expression. The expression of betaig-h3, upregulated in human hepatoma cells, was positively relative to the expression of HAb18G/CD147 in different human hepatoma cell lines. By overexpressing betaig-h3 in human SMMC-7721 hepatoma cells, we discovered that betaig-h3 promoted cell adhesion, invasion, and matrix metalloproteinase (MMP) secretion potential. HAb18G/CD147-induced invasion and metastasis potential of human hepatoma cells can be attenuated by antibodies specific for betaig-h3, and no significant differences on inhibitory effects were observed among T7721 cells incubated with antibodies for betaig-h3 or HAb18G/CD147 or both types together. Taken together, our study suggests that betaig-h3, regulated by the expression of HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates the HAb18G/CD147-induced invasion and metastasis process of human hepatoma cells.     

HAb18G/CD147 Regulates Vinculin-Mediated Focal Adhesion and Cytoskeleton Organization in Cultured Human Hepatocellular Carcinoma Cells

Focal adhesions (FAs), integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147) is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC) cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001), length/width ratios (P<0.0001), and mean intensities (P<0.0001). CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t _1/2) recovery times (22.7±3.3 s) of vinculin-mediated focal adhesions (P<0.0001). In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2). Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.     

The involvement of HAb18G/CD147 in regulation of store-operated calcium entry and metastasis of human hepatoma cells

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca(2+) mobilization and metastatic process of human hepatoma cells. HAb18G/CD147 cDNA was transfected into human 7721 hepatoma cells to obtain a cell line stably expressing HAb18G/CD147, T7721, as demonstrated by Northern blot and immunocytochemical studies. 8-Bromo-cGMP (cGMP) inhibited the thapsigargin-induced Ca(2+) entry in a concentration-dependent manner in 7721 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 microm). However, expression of HAb18G/CD147 in T7721 cells decreased the inhibitory response to cGMP. A similar concentration-dependent inhibitory effect on the Ca(2+) entry was observed in 7721 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was significantly reduced in HAb18G/CD147-expressing T7721 cells, indicating a role for HAb18G/CD147 in NO/cGMP-regulated Ca(2+) entry. Experiments investigating metastatic potentials demonstrated that HAb18G/CD147-expressing T7721 cells attached to the Matrigel-coated culture plates and invaded through Matrigel-coated permeable filters at the rate significantly greater than that observed in 7721 cells. Both the attachment and invasion rates could be suppressed by SNAP, and the inhibitory effect of SNAP could be reversed by NO inhibitor, N(G)-nitro-l-arginine methyl ester. The sensitivity of the attachment and invasion rates to cGMP was significantly reduced in T7721 cells as compared with 7721 cells when cells were pretreated with thapsigargin. The difference in the sensitivity between the two cells could be abolished by a Ca(2+) channel blocker, Ni(2+) (3 mm). These results suggest that HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca(2+) entry by NO/cGMP.     

HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner

Background  

Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory.     

HAb18G/CD147 enhances the secretion of matrix metalloproteinases (MMP) via cGMP/NO-sensitive capacitative calcium entry (CCE) and accordingly attenuates adhesion ability of fibroblasts

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca2+ mobilization to induce matrix metalloproteinases (MMP) production and attenuate adhesion ability of mouse fibroblast NIH/3T3 cells. HAb18G/CD147 cDNA was transfected into fibroblast 3T3 cells to obtain a cell line stably expressing HAb18G/CD147, t3T3, as demonstrated by immunofluorescence staining and flow cytometry assays. 8-Bromo-cGMP inhibited the thapsigargin-induced Ca2+ entry in 3T3 cells, whereas an inhibitor of protein kinase G, KT5823 (1 microM), led to an increase in Ca2+ entry. Expression of HAb18G/CD147 in t3T3 cells decreased the inhibitory response to cGMP. A similar effect on the Ca2+ entry was observed in 3T3 cells in response to an NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca2+ entry was also reduced in HAb18G/CD147-expressing t3T3 cells, indicating a role for HAb18G/CD 147 in NO/cGMP-regulated Ca2+ entry. Results of gelatin zymography assays showed that addition of extracellular Ca2+ induced MMP (MMP-2, MMP-9) release and activation in a dose-dependent manner, and expression of HAb18G/CD147 enhanced the secretion of MMP-2 and MMP-9 in 3T3 cells. 8-Bromo-cGMP and SNAP reduced the production of MMP in 3T3 cells but not in t3T3 with HAb18G/CD147 expression. RT-PCR experiments substantiated that the expression of MMP-2 and MMP-9 mRNA in HAb18G/CD 147-expressing t3T3 cell was significantly greater than that in 3T3 cells. Experiments investigating adhesion potentials demonstrated that HAb18G/CD147-expressing t3T3 cells pretreated with Ca2+ attached to Matrigel-coated culture plates significantly less efficiently than 3T3 cells. The proportion of attached cells could be increased by treatment with 8-bromo-cGMP and SNAP in 3T3 cells, but not in t3T3. These results suggest that HAb18G/CD147 attenuates adhesion potentials in fibroblasts by enhancing the secretion of MMP through NO/cGMP-sensitive capacitative Ca2+ entry.     

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.     

Crystal structure of CD14 and its implications for lipopolysaccharide signaling

Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 angstroms resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.     

Crystal structure of the M-fragment of alpha-catenin: implications for modulation of cell adhesion.

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.     

Crystal structure of an aminoimidazole riboside kinase from Salmonella enterica: implications for the evolution of the ribokinase superfamily

The crystal structures of a Salmonella enterica aminoimidazole riboside (AIRs) kinase, its complex with the substrate AIRs, and its complex with AIRs and an ATP analog were determined at 2.6 angstroms, 2.9 angstroms, and 2.7 angstroms, respectively. The product of the Salmonella-specific gene stm4066, AIRs kinase, is a homodimer with one active site per monomer. The core structure, consisting of an eight-stranded beta sheet flanked by eight alpha helices, indicates that AIRs kinase is a member of the ribokinase superfamily. Unlike ribokinase and adenosine kinase in this superfamily, AIRs kinase does not show significant conformational changes upon substrate binding. The active site is covered by a lid formed by residues 16-28 and 86-100. A comparison of the structure of AIRs kinase with other ribokinase superfamily members suggests that the active site lid and conformational changes that occur upon substrate binding may be advanced features in the evolution of the ribokinase superfamily.     

Crystal structure of Rnd3/RhoE: functional implications

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up-regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that ms1 may be important in striated muscle biology and the development of pressure-induced left ventricular hypertrophy.     

Constitutive and stimulus-induced phosphorylation of CD11/CD18 leukocyte adhesion molecules

The leukocyte CD11/CD18 adhesion molecules (beta 2 integrins) are a family of three heterodimeric glycoproteins each with a distinct alpha subunit (CD11a, b, or c) and a common beta subunit (CD18). CD11/CD18 mediate crucial leukocyte adhesion functions such as chemotaxis, phagocytosis, adhesion to endothelium, aggregation, and cell-mediated cytotoxicity. The enhanced cell adhesion observed upon activation of leukocytes is associated with increased surface membrane expression of CD11/CD18, as well as a qualitative upregulation of CD11/CD18 functions. To elucidate the nature of the qualitative modifications that occur, we examined the phosphorylation status of these molecules in resting human leukocytes and upon activation with PMA or with the chemotactic peptide F-met-leu-phe (FMLP). In unstimulated cells, all three CD11 subunits were found to be constitutively phosphorylated. In contrast, phosphorylation of the common CD18 subunit was minimal. PMA induced rapid and sustained phosphorylation of CD18 that occurred at high stoichiometry, but had only minimal effects on phosphorylation of the associated CD11 subunits. FMLP also induced rapid phosphorylation of CD18, but the effect was of short duration. FMLP-induced phosphorylation of CD18 was not related to its Ca++-mobilizing effect, as CD18 phosphorylation was not observed upon treatment of leukocytes with the Ca++ ionophore, ionomycin. Phosphoamino acid analysis of CD11/CD18 in PMA- or FMLP-treated monocytes revealed a predominance of phosphoserine residues in all CD11/CD18 subunits. A small component of phosphothreonine was present in CD11c and CD18 and a minor component of phosphotyrosine was also detected in CD18 upon leukocyte activation may regulate the adhesion functions mediated by the CD11/CD18 family of molecules.     

Crystal structure of cell adhesion molecule nectin-2/CD112 and its binding to immune receptor DNAM-1/CD226

The nectin and nectin-like molecule (Necl) family includes important cell adhesion molecules (CAMs) characterized by their Ig-like nature. Such CAMs regulate a broad spectrum of cell-cell interactions, including the interaction between NK cells and cytotoxic T lymphocytes (CTLs) and their target cells. CAM members nectin-2 (CD112) and Necl-5 (CD155) are believed to form homodimers (for nectin-2) or heterodimers in their functions for cell adhesion, as well as to interact with immune costimulatory receptor DNAX accessory molecule 1 (DNAM-1) (CD226) to regulate functions of both NK and CTL cells. However, the structural basis of the interactive mode of DNAM-1 with nectin-2 or Necl-5 is not yet understood. In this study, a soluble nectin-2 Ig-like V-set domain (nectin-2v) was successfully prepared and demonstrated to bind to both soluble ectodomain and cell surface-expressed full-length DNAM-1. The 1.85-? crystal structure of nectin-2v displays a perpendicular homodimer arrangement, revealing the homodimer characteristics of the nectin and Necls. Further mutational analysis indicated that disruption of the homodimeric interface of nectin-2v led to a failure of the homodimer formation, as confirmed by crystal structure and biochemical properties of the mutant protein of nectin-2v. Interestingly, the monomer mutant also loses DNAM-1 binding, as evidenced by cell staining with tetramers and surface plasmon resonance assays. The data indicate that interaction with DNAM-1 requires either the homodimerization or engagement of the homodimeric interface of nectin-2v. These results have implications for immune intervention of tumors or autoimmune diseases in the DNAM-1/nectin-2-dependent pathway.     

Crystal structure of human thioesterase superfamily member 2

Hotdog-fold has been identified in more than 1000 proteins, yet many of which in eukaryotes are less studied. No structural or functional studies of human thioesterase superfamily member 2 (hTHEM2) have been reported before. Since hTHEM2 exhibits about 20% sequence identity to Escherichia coli PaaI protein, it was proposed to be a thioesterase with a hotdog-fold. Here, we report the crystallographic structure of recombinant hTHEM2, determined by the single-wavelength anomalous dispersion method at 2.3A resolution. This structure demonstrates that hTHEM2 indeed contains a hotdog-fold and forms a back-to-back tetramer as other hotdog proteins. Based on structural and sequence conservation, the thioesterase active site in hTHEM2 is predicted. The structure and substrate specificity are most similar to those of the bacterial phenylacetyl-CoA hydrolase. Asp65, located on the central alpha-helix of subunit B, was shown by site-directed mutagenesis to be essential to catalysis.     

CD2F-10: a new member of the CD2 subset of the immunoglobulin superfamily

The CD2 subset of the immunoglobulin superfamily consists of a rapidly expanding family of leukocyte cell surface receptors, at least five of which (CD2, CD48, CD58, CD150, and CD244) are involved in lymphocyte activation as either receptors or ligands. Completion of the draft sequence of the human genome offers the possibility of systematically identifying the full set of proteins and interactions of this important family. Here we describe the identification and characterization of the first new member of the subset, CD2F-10, found exclusively by genome searching.     

Expression of HAb18G in non-small lung cancer and characterization of activation, migration, proliferation, and apoptosis in A549 cells following siRNA-induced downregulation of HAb18G

VSL#3 probiotics can be effective on induction and maintenance of the remission of clinical ulcerative colitis. However, the mechanisms are not fully understood. The aim of this study was to examine the effects of VSL#3 probiotics on dextran sulfate sodium (DSS)-induced colitis in rats. Acute colitis was induced by administration of DSS 3.5 % for 7 days in rats. Rats in two groups were treated with either 15 mg VSL#3 or placebo via gastric tube once daily after induction of colitis; rats in other two groups were treated with either the wortmannin (1 mg/kg) via intraperitoneal injection or the wortmannin + VSL#3 after induction of colitis. Anti-inflammatory activity was assessed by myeloperoxidase (MPO) activity. Expression of inflammatory related mediators (iNOS, COX-2, NF-κB, Akt, and p-Akt) and cytokines (TNF-α, IL-6, and IL-10) in colonic tissue were assessed. TNF-α, IL-6, and IL-10 serum levels were also measured. Our results demonstrated that VSL#3 and wortmannin have anti-inflammatory properties by the reduced disease activity index and MPO activity. In addition, administration of VSL#3 and wortmannin for 7 days resulted in a decrease of iNOS, COX-2, NF-κB, TNF-α, IL-6, and p-Akt and an increase of IL-10 expression in colonic tissue. At the same time, administration of VSL#3 and wortmannin resulted in a decrease of TNF-α and IL-6 and an increase of IL-10 serum levels. VSL#3 probiotics therapy exerts the anti-inflammatory activity in rat model of DSS-induced colitis by inhibiting PI3K/Akt and NF-κB pathway.