Effects of berberine and red yeast on proinflammatory cytokines IL-6 and TNF-alpha in peripheral blood mononuclear cells (PBMCs) of human subjects

Background and Aims: Obesity is a condition associated with chronic or acute inflammatory response characterized by an increase of proinflammatory cytokine levels. Peripheral blood mononuclear cells (PBMCs) migrate in adipose tissue inducing synthesis and secretion of adipocytokines as IL-6 and TNF-α. The aim of this study was to investigate the effect of berberine (a natural alkaloid) and red yeast (a natural antioxidant) on IL-6 and TNF-α cytokines release and gene expression, in circulating lipopolisaccarides (LPS) stimulated PBMCs. Methods and Results: PBMCs isolated from whole blood of healthy donors were stimulated with LPS to induce cytokines production; simultaneously cells were treated with increasing doses of berberine and red yeast. The substances were administered alone or in association. IL-6 and TNF-α protein levels in the culture medium and their mRNA levels were assessed by ELISA and real time PCR, respectively. Berberine and red yeast treatment prevented the LPS induction of IL-6 release in the culture medium of PBMCs. In addition, berberine plus red yeast treatment showed a synergic inhibitory effect on IL-6 release at low concentration. Berberine and red yeast showed an inhibitory effect also on LPS induction of TNF-α release exerting a synergic effect mainly at high concentrations. On the contrary, berberine and red yeast did not significantly affect IL-6 and TNF-α mRNA levels induced by LPS. In this case, only concomitant treatment of PBMCs with high doses of berberine and red yeast inhibits LPS induced IL-6 or TNF-α mRNA levels. Conclusions: The results of our study show that both berberine and red yeast were able to carry out anti-inflammatory action through an inhibition of proinflammatory IL-6 and TNF-α protein release. Moreover, when given in combination these substances were able to inhibit IL-6 and TNF-α gene expression in PBMCs activated by LPS. Therefore, these substances could represent a useful pharmacological treatment to reduce the proinflammatory status accompanied with obesity

The critical role of didodecyldimethylammonium bromide on physico-chemical, technological and biological properties of NLC

Exploiting the experimental factorial design and the potentiality of Turbiscan AG Station, we developed and characterized unmodified and DDAB-coated NLC prepared by a low energy organic solvent free phase inversion temperature technique. A 22 full factorial experimental design was developed in order to study the effects of two independent variables (DDAB and ferulic acid) and their interaction on mean particle size and zeta potential values. The factorial planning was validated by ANOVA analysis; the correspondence between the predicted values of size and zeta and those measured experimentally confirmed the validity of the design and the equation applied for its resolution. The DDAB-coated NLC were significantly affected in their physico-chemical properties by the presence of DDAB, as showed by the results of the experimental design. The coated NLC showed higher physical stability with no particles aggregation compared to the unmodified NLC, as demonstrated by Turbiscan (R) AGS measurements. X-ray diffraction, Raman spectroscopy and Cryo-TEM images allowed us to assert that DDAB plays a critical role in increasing the lipids structural order with a consequent enhancement of the NLC physical stability. Furthermore, the results of the in vitro biological studies allow the revisiting of the role of DDAB to the benefit of glioblastoma treatment, due to its efficacy in increasing the NLC uptake and reducing the viability of human glioblastoma cancer cells (U87MG)

Activation of 5-HT7 serotonin receptors reverses metabotropic glutamate receptor-mediated synaptic plasticity in wild-type and Fmr1 knockout mice, a model of Fragile X Syndrome

Background: Fragile X syndrome (FXS) is a genetic cause of intellectual disability and autism. Fmr1 knockout (Fmr1KO) mice, an animal model of FXS, exhibit spatial memory impairment and synapse malfunctioning in the hippocampus, with abnormal enhancement of long-term depression mediated by metabotropic glutamate receptors (mGluR-LTD). The neurotransmitter serotonin (5-HT) modulates hippocampal-dependent cognitive functions through 5-HT1A and 5-HT7 receptors, respectively impairing and improving learning; the underlying mechanisms are unknown. Methods: we used electrophysiology to test the effects of 5-HT on mGluR-LTD in wild-type and Fmr1KO mice, and immunocytochemistry and biotinylation assay to study related changes of GluR2 AMPA receptor subunit surface expression. Results: application of 5-HT or 8-OH-DPAT (a mixed 5-HT1A/5-HT7 agonist) reversed mGluR-LTD induced by DHPG, a group-I mGluR agonist, on CA1 pyramidal neurons in hippocampal slices. Reversal of mGluR-LTD by 8-OH-DPAT persisted in the presence of the 5-HT1A receptor antagonist WAY- 100635, was abolished by SB-269970 (5-HT7 receptor antagonist) and was mimicked by LP-211, a novel selective 5-HT7 receptor agonist. Consistently, 8-OH-DPAT decreased DHPG-mediated reduction of GluR2 surface expression in hippocampal slices and in cultured hippocampal neurons, an effect mimicked by LP-211 and blocked by SB-269970. In Fmr1KO mice, mGluR-LTD was abnormally enhanced; similarly to wild-type, 8-OH-DPAT reversed mGluR-LTD and decreased DHPG-induced reduction of surface AMPA receptors, an effect antagonized by SB-269970. Conclusions: 5-HT7 receptor activation reverses mGluR-induced AMPA receptor internalization and LTD both in wild-type and in Fmr1KO mice, correcting excessive mGluR-LTD. Therefore, selective activation of 5-HT7 receptors may represent a novel strategy in the therapy of FXS

Heat shock response relieves ER stress

Accumulation of misfolded protein in the endoplasmic reticulum (ER) causes stress. The unfolded protein response (UPR), a transcriptional induction pathway, is activated to relieve ER stress. Although UPR is not essential for viability, UPR-deficient cells are more sensitive to ER stress; ire1Δ cells cannot grow when challenged with tunicamycin or by overexpression of misfolded CPY*. In these cells, multiple functions are defective, including translocation, ER-associated degradation (ERAD), and ER-to-Golgi transport. We tested whether heat shock response (HSR) can relieve ER stress. Using a constitutively active Hsf1 transcription factor to induce HSR without temperature shift, we find that HSR rescues growth of stressed ire1Δ cells, and partially relieves defects in translocation and ERAD. Cargo-specific effects of constitutively active Hsf1 on ER-to-Golgi transport are correlated with enhanced protein levels of the respective cargo receptors. In vivo, HSR is activated by ER stress, albeit to a lower level than that caused by heat. Genomic analysis of HSR targets reveals that >25% have function in common with UPR targets. We propose that HSR can relieve stress in UPR-deficient cells by affecting multiple ER activities

CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1-3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4-6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8