Total coumarins from Urtica dentata Hand prevent murine autoimmune diabetes via suppression of the TLR4-signaling pathways

Abstract

Ethnopharmacological relevanceUrtica dentata Hand (UDH), the root of Laportea bulbifera (Sieb. et. Zucc.) Wedd, has been traditionally used in traditional Chinese medicine as an anti-inflammatory and immuno-regulatory agent for rheumatoid arthritis and some other autoimmune diseases treatment. And the coumarins are the major components of UDH. Aim of the studyTo investigate the effect of total coumarins (TC) isolated from UDH on the development of autoimmune diabetes. Materials and methodsEight-week-old non-obese diabetic (NOD) mice were randomly divided into four groups: control group, low-dose (37.5mg/kg), middle-dose (75mg/kg), and high-dose (150mg/kg) TC-treatment groups. NOD mice were then given with a suspension of TC or saline by intragastric (i.g.) administration every other day. After 4 weeks of treatment, 8 mice at 12-weeks of age per group were randomly selected to be sacrificed to perform intraperitoneal glucose tolerance test, examine histopathological insulitis, spleen T lymphocyte proliferation, the percentage of CD4+CD25+Foxp3+ T regulatory cell (Treg), dendritic cell (DC) surface molecules, toll-like receptor (TLR)4 expression and signal pathways involved. The remaining 10 mice per group were kept until 26 weeks of age to assess the incidence of diabetes. We also studied the direct effect of TC on DC and CD4+CD25+ Tregs in vitro. ResultsTreatment with TC for 4 weeks significantly inhibited insulitis, increased pancreatic islet number, delayed the onset and decreased the development of diabetes by 26 weeks of age in NOD mice, compared with the untreated control mice. TC suppressed spleen T lymphocyte proliferation, induced Th2-biased cytokine response, the generation of CD4+CD25+Foxp3+ Tregs and Foxp3 mRNA expression. And TC-treated DCs were characterized as low expression of MHC class II and CD86 molecules. TLR4 gene and protein expressions in the spleen, thymus and pancreas were down-regulated in TC-treated groups. The key molecules in the downstream signaling cascades of TLR4, including myeloid differentiation factor (MyD)88, nuclear factor (NF)-κB, IL-1β, Toll–IL-1 receptor domain-containing adaptor inducing interferon-β(TRIF), TRIF-related adaptor molecule (TRAM), interferon regulatory factor (IRF)-3 and IFN-β, all decreased significantly in TC groups, suggesting that TC inhibits both MyD88-dependent and -independent pathways of TLR4. At the cellular level, however, TLR4 protein expression in DCs, but not in Tregs, was downregulated by TC. And TC strengthened the role of DC, not Treg, in negative immune regulation in vitro. In contrast, anti-TLR4 antibody could block the effect of TC on DCs immune function. ConclusionThese results suggest that TC extracted from UDH prevent the development of autoimmune diabetes in NOD mice via suppression of the TLR4-signaling pathways. TC maintain the DCs in an immature tolerogenic state, at least in part, mediated by down-regulating TLR4-signaling pathways in DCs, then enhance Treg differentiation, shift toward Th2 and suppress T lymphocyte proliferation.