St. John's Wort and hyperforin inhibit multiple phosphorylation steps of cytokine signalling in beta cells and modulate functional, inflammatory and apoptotic gene expression

Background and aims:Cytokines released by mononuclear cells infiltratingthe islets of Langerhans during an autoimmune attack are considered responsible for the β-cell destruction leading to type 1 diabetes, through STAT-1 and NF-kB activation and consequent expression of deleterious target genes. We have previously shown that the extract of Hypericum perforatum (St. John’s wort, SJW) and its phloroglucinol component hyperforin (HPF), are potent inhibitors of cytokine-induced STAT-1 and NF-kB activation in β-cells and prevent dysfunction and apoptosis in INS-1E β-cell line, as well as in rat and human islets. Aims of this study were: a) to further clarify the mechanism of the regulatory activity of SJW and HPF on cytokine signaling pathways in INS-1E cells; b) to assess their ability to counteract the cytokine-driven changes in the expression of STAT-1 and NF-kB target genes involved in β-cell function, inflammatory response and apoptosis regulation.Materials and methods:INS-1E cells, exposed to mixtures of IFN-γ, IL-1β and TNF-α for various time periods with/without SJW extract (1-2 μg/ml) or HPF (1-2 μmol/l), were used for RT-qPCR gene expression analysis and assessment of the phosphorylation state of various components of STAT-1, NF-kB and MAPK pathways by western blotting. STAT-1 and NF-kB activation was also evaluated by EMSA on nuclear extracts.Results:Cytokine-induced STAT-1 phosphorylation in both tyrosine and serine residues (2-3-fold increase vs. controls) was significantly hindered by SJW or HPF in a dose-dependent manner. These compounds prevented NF-kB activation by suppressing phosphorylation of the p65 subunit and theinhibitory subunit IkB activating kinase (IKK). Furthermore, MAPK pathway was also modulated by the vegetal compounds through dose-dependentpartial or total restriction of ERK1/2, p38 MAPK and JNK cytokine-inducedphosphorylations. Inhibition of DNA binding of STAT-1 and NF-kB in thepresence of SJW or HPF was confirmed by EMSA. Expressions of a number of β-cell functional genes, such as PDX-1, GLUT-2 and FOXO1, weredown-regulated by 60-80% (p<0.05 vs. controls) upon cytokine treatmentand restored in the presence of SJW and HPF, while expressions of insulin,glucokinase, MAFA, PAX-6 genes were less affected. A remarkable induction (>10-fold vs. controls) of iNOS and other pro-inflammatory genes (CXCL9, CXCL10, COX-2, ICAM-1, MHC-2 trans-activator) was elicited by cytokines in INS-1E cells and significantly reduced or totally abolished by vegetal compounds in a dose-dependent fashion. SJW and HPF were able to partially correct the cytokine-induced unbalance between anti- and pro-apoptotic factors, mainly by preventing down-regulation of anti-apoptotic members of BCL-2 family.Conclusion:We provide evidence that SJW extract and hyperforin exert their protective effects against cytokine-induced β-cell damage by inhibitingmultiple phosphorylation steps along the STAT-1, NF-kB and MAPK signaling pathways and modulating target gene expression. Thus, SJW components represent novel promising pharmacological tools for prevention or limitation of β-cell loss in type 1 diabetes.Supported by: Telethon-Italy/JDRF (grant n. GJT08016)