Anti-inflammatory and anti-infectious effects of Evodia rutaecarpa (Wuzhuyu) and its major bioactive components

This article reviews the anti-inflammatory relative and anti-infectious effects of Evodia rutaecarpa and its major bioactive components and the involvement of the nitric oxide synthases, cyclooxygenase, NADPH oxidase, nuclear factor kappa B, hypoxia-inducible factor 1 alpha, reactive oxygen species, prostaglandins, tumor necrosis factor, LIGHT, amyloid protein and orexigenic neuropeptides. Their potential applications for the treatment of endotoxaemia, obesity, diabetes, Alzheimer's disease and their uses as cardiovascular and gastrointestinal protective agents, analgesics, anti-oxidant, anti-atherosclerosis agents, dermatological agents and anti-infectious agents are highlighted. Stimulation of calcitonin gene-related peptide release may partially explain the analgesic, cardiovascular and gastrointestinal protective, anti-obese activities of Evodia rutaecarpa and its major bioactive components

Activation of Akt by advanced glycation end products (AGEs): involvement of IGF-1 receptor and caveolin-1.

Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs

Promotion of adipogenesis of 3T3-L1 cells by AGEs-treatment.

<p>(<b>A</b>) 3T3-L1 cells were treated with and without 100 µg/ml AGEs during differentiation.The adipogenic induction medium contains 0.5 mM isobutylmethylxanthine, 1 mM dexamethasone, and 1.7 mM insulin. At day 12, cells were subjected to the Oil Red O staining. The oil droplet contents were quantified by measuring the OD510 nm. (*, <i>P</i><0.05 <i>versus</i> control) (<b>B</b>) 3T3-L1 cells were treated with and without 100 µg/ml AGEs during differentiation. At day 5, cell lysates were subjected to Western blot analysis using the PPARγ, C/EBPα, aP2 and the loading control actin antibodies. (<b>C</b>) Cells from 3T3-L1 and 100 µg/ml AGEs-treated cells were harvested on day 8 after adipogenic induction and were subjected to GPDH activity assay by reading the absorbance of NADH at 340 nm. The results are the means ± SEM of three independent experiments. (*, <i>P</i><0.05 <i>vs.</i> control) (<b>D</b>) 3T3-L1 cells were treated with and without 100 µg/ml AGEs in the presence of and absence of 10 µM AG1024, 15 µM LY294002, or 5 µM Akt inhibitor during differentiation. Four days after adipogenic induction, cells were subjected to the Oil Red O staining and the oil droplet contents were quantified by measuring the OD510 nm. (E) 3T3-L1 cells were treated with and without 100 µg/ml AGEs in the presence of and absence of 10 µM AG1024, 15 µM LY294002, or 5 µM Akt inhibitor during differentiation. The concentration of DMSO is 0.05% for control and inhibitor-treated groups. At day 5, cell lysates were subjected to Western blot analysis using PPARγ, aP2 and actin antibodies. (*, <i>P</i><0.05 DMSO <i>versus</i> inhibitors; **, <i>P</i><0.05 DMSO vs. DMSO+AGEs, ***, P<0.05 DMSO+AGEs vs. inhibitors+AGEs).</p

The effect of NAC, Tiron, DPI and apocynin on AGEs-stimulated Akt activation in 3T3-L1 cells.

<p>(<b>A</b>) Cells were exposed to 100 µg/ml AGEs for 15 min in the serum-free medium. After treatment, cells were incubated with 20 µM CM-H2DCFDA for 30 min at 37°C. The ROS production was determined by a fluorescence reader (excitation/emission: 485/520 nm). The data represent mean ± the standard error (SE) of results from three independent experiments. Increases in the AGEs-induced ROS were statistically significant at this time point. (*: <i>P</i><0.05) (B) After 18 hr serum starvation, 3T3-L1 cells were treated with 2 mM NAC, 2 mM Tiron, 50 µM DPI, 25 µM apocynin, or RAGE antibodies (1 µg/ml) for 60 min, and then ROS production was determined by a fluorescence reader (excitation/emission: 485/520 nm). After 18 hr serum starvation, 3T3-L1 cells were treated with 2 mM NAC (<b>C</b>), 2 mM Tiron (<b>D</b>), 50 µ M DPI (<b>E</b>), or 25 µM apocynin (<b>F</b>) for 60 min, and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were immunoblotted with antibodies specific for phospho-Akt (Akt^ser473), Akt or actin. Data are representative of three independent experiments yielding similar results.</p

Transactivation of IGF-1R by AGEs is mediated by Src in 3T3-L1 cells.

<p>(<b>A</b>) Serum-depleted 3T3-L1 cells were pretreated with and without 10 µM PP2 for 30 min and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were immunoprecipitated with IGF-1Rβantibody followed by immunoblotting with phospho-tyrosine, IGF-1Rβ or phospho-IGF-1Rβ (IGF-1Rβ^pY1135/1136) antibodies. (<b>B</b>) Total lysates from cells treated with and without 2 µM AG1024 and then challenged with 100 µg/ml AGEs for 15 min were subjected to Western blotting with antibodies specific for phospho-Src (Src^Tyr416), Src or actin antibodies. (<b>C</b>) Total lysates from cells treated with and without 100 µg/ml AGEs were immunoprecipitated with the IGF-1Rβantibody followed by immunoblotting with IGF-1Rβ, Src and p-Src antibodies. (<b>D</b>) Serum-starved 3T3-L1 cells were pretreated with and without 2 mM NAC for 60 min and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were immunoprecipitated with IGF-1Rβantibody followed by immunoblotting with phospho-tyrosine or phospho-IGF-1Rβ (IGF-1Rβ^pY1135/1136) antibodies. The data represent mean ± the standard error (SE) of results from three independent experiments. The densitometrical data were shown as the means ± SEM of three independent experiments. *P<0.05 compared with control group and **P<0.05 compared with AGEs group.</p

Time- and dose-dependent activation of Akt by AGEs in 3T3-L1 cells.

<p>(<b>A</b>) Serum-starved quiescent 3T3-L1 cells were exposed to AGEs (100 µg/ml) for the indicated times. (<b>B</b>) Serum-starved quiescent 3T3-L1 cells were exposed to various concentrations of AGEs for 15 min. (<b>C</b>) Serum-starved quiescent 3T3-L1 cells were pretreated with 15 µM LY294002 for 30 min, and then exposed to 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies recognizing phospho-Akt (Akt^ser473), Akt or actin. (<b>D</b>) Serum-starved quiescent 3T3-L1 cells were pretreated with 15 µM LY294002 for 30 min, and then exposed to 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies recognizing phospho-Akt (Akt^Thr308), Akt or actin. (<b>E</b>) Serum-starved quiescent 3T3-L1 cells were pretreated with 15 µM LY294002 for 30 min, and then exposed to 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies recognizing phospho-PDK1 (PDK1^Ser241), PDK1 or actin. Data are representative of three independent experiments yielding similar results. *, statistically significant differences (*, <i>P <</i>0.05 <i>versus</i> control).</p

Involvement of phospho-Cav-1 in AGEs-mediated Akt activation in 3T3-L1 cells.

<p>(<b>A</b>) Serum-depleted 3T3-L1 cells were pretreated with and without 50 µM β-MCD for 60 min and then challenged with 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies specific for phospho-Akt (Akt^ser473), Akt or actin. Data are representative of three independent experiments yielding similar results. (<b>B</b>) Serum-depleted 3T3-L1 cells were pretreated with and without 20 µM β-MCD for 60 min and then challenged with 100 µg/ml AGEs for 15 min. Cell lysates were subjected to Western blotting with antibodies specific for phospho-Src (Src^Tyr416), total Src (Src) or actin antibodies. (<b>C</b>) Serum-depleted 3T3-L1 cells were pretreated with and without 50 µM β-MCD for 60 min and then challenged with 100 µg/ml AGEs for 15 min. After treatment, cells were incubated with 20 µM CM-H2DCFDA for 30 min at 37°C. The ROS production was determined by a fluorescence reader (excitation/emission: 485/520 nm). The data represent mean ± the standard error (SE) of results from three independent experiments. (<b>D</b>) Serum-depleted 3T3-L1 cells were stimulated with 100 µg/ml AGEs for 15 min, and cell lysates were immunoprecipitated with IGF-1Rβ antibody followed by immunoblotting with phospho-Cav-1 (Cav-1 ^tyr14), Cav-1 or IGF-1Rβ. (<b>E</b>) Cell lysates from vector control and cells expressing Cav-1 Y14F were subjected to Western blotting with phospho-Cav-1 (Cav-1 ^tyr14), Cav-1, phospho-Akt or Akt. (<b>F</b>) Cell lysates from vector control and cells expressing Cav-1 Y14F were subjected to Western blotting with phospho-Src (Src^Tyr416), Src or actin antibodies. Cell lysates were also immunoprecipitated with IGF-1Rβantibody followed by immunoblotting with phospho-IGF-1Rβ (IGF-1Rβ^pY1135/1136) or IGF-1Rβ. (<b>G</b>) Serum-depleted cells were pretreated with and without 10 µM PP2 for 30 min and then challenged with 100 µg/ml AGEs for 15 min. Total cell lysates were immunoblotted with antibodies specific for phospho-caveolin-1 (Cav-1 ^tyr14), Cav-1 or actin. Data are representative of three independent experiments yielding similar results. *, statistically significant differences (* and **, P<0.05 <i>versus</i> control).</p

Mirtazapine Inhibits Tumor Growth via Immune Response and Serotonergic System

<div><p>To study the tumor inhibition effect of mirtazapine, a drug for patients with depression, CT26/<em>luc</em> colon carcinoma-bearing animal model was used. BALB/c mice were randomly divided into six groups: two groups without tumors, i.e. <em>wild-type</em> (no drug) and <em>drug</em> (mirtazapine), and four groups with tumors, i.e. <em>never</em> (no drug), <em>always</em> (pre-drug, i.e. drug treatment before tumor inoculation and throughout the experiment), <em>concurrent</em> (simultaneously tumor inoculation and drug treatment throughout the experiment), and <em>after</em> (post-drug, i.e. drug treatment after tumor inoculation and throughout the experiment). The “psychiatric” conditions of mice were observed from the immobility time with tail suspension and spontaneous motor activity post tumor inoculation. Significant increase of serum interlukin-12 (sIL-12) and the inhibition of tumor growth were found in mirtazapine-treated mice (<em>always</em>, <em>concurrent</em>, and <em>after</em>) as compared with that of <em>never</em>. In addition, interferon-γ level and immunocompetent infiltrating CD4+/CD8+ T cells in the tumors of mirtazapine-treated, tumor-bearing mice were significantly higher as compared with that of <em>never.</em> Tumor necrosis factor-α (TNF-α) expressions, on the contrary, are decreased in the mirtazapine-treated, tumor-bearing mice as compared with that of <em>never</em>. Ex vivo autoradiography with [¹²³I]ADAM, a radiopharmaceutical for serotonin transporter, also confirms the similar results. Notably, better survival rates and intervals were also found in mirtazapine-treated mice. These findings, however, were not observed in the immunodeficient mice. Our results suggest that tumor growth inhibition by mirtazapine in CT26/<em>luc</em> colon carcinoma-bearing mice may be due to the alteration of the tumor microenvironment, which involves the activation of the immune response and the recovery of serotonin level.</p> </div