Identification and Comparison of Tannins in Gall of Rhus chinensis Mill. and Gall of Quercus infectoria Oliv. by High-Performance Liquid Chromatography-Electrospray Mass Spectrometry.

Gall of Rhus chinensis Mill. (Chinese galls) and gall of Quercus infectoria Oliv. (Turkish galls) have similar applications and chemical compositions, and their extracts have been widely used for industrial production and for medicinal applications. In this study, high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS/MS) methods were established for profiling the components of Chinese galls and Turkish galls. Compounds representing 96.56 and 99.15% of the total peak area of Chinese galls and Turkish galls were identified. The results identified that the ellagic acid, galloyl-HHDP-glucose and pedunculagin act as the identifying markers for the comparison of Chinese galls and Turkish galls in HPLC-ESI-MS/MS. The peak area of tetragalloyl-glucoside, heptagalloyl-glucoside and pentagalloyl-glucoside can be used to distinguish these two phytomedicines. This work provides a reference for the study of the chemical composition of Chinese galls and Turkish galls, which not only introduce a simple and reliable method to prevent the adulteration or misuse of Chinese galls and Turkish galls but also lay the foundations for clarifying the material basis of their similar pharmacological action.

Systematic investigation of the mechanism of Cichorium glandulosum on type 2 diabetes mellitus accompanied with non-alcoholic fatty liver rats.

Cichorium glandulosum(CG) can treat various diseases with multiple targets effectively. It has been widely used in folk medicine to treat nonalcoholic fatty liver disease (NAFLD) as well as type 2 diabetes mellitus (T2DM). However, the active compounds and underlying mechanisms of CG on T2DM accompanied with NAFLD (T2DM-NAFLD) remain unclear. In this study, a systems pharmacology method was used to explain the pharmacology mechanism of CG for treatment of T2DM-NAFLD. Twenty four main compounds were detected by UPLC-Q-TOF-MS, of which 13 showed favorable pharmacokinetic profiles. We demonstrated with target fishing and pathway analysis that CG has protective effects on T2DM-NAFLD, probably through the regulation of 88 targets and 86 pathways. Forty nine targets were related to T2DM, and 39 were related to NAFLD, while 27 targets, primarily involved in insulin resistance and inflammation were common to T2DM and NAFLD related pathways. A NF-κB signaling pathway was chosen to validate the impacts of CG on T2DM-NAFLD because CG can ameliorate T2DM-NAFLD by regulating the NF-κB signaling pathway according to animal experiments. These findings systematically interpreted the active compounds and mechanism of the efficiency of CG for treating T2DM-NAFLD. This study not only laid a basis for understanding the active compounds and action mechanism of CG, but also provides a reference for a study of the mechanism of a herbal medicine for the treatment of multiple diseases.

Adjuvant immunotherapy increases beta cell regenerative factor Reg2 in the pancreas of diabetic mice.

Insulin-producing ß cells can partially regenerate in adult pancreatic tissues, both in human and animal models of type 1 diabetes (T1D). Previous studies have shown that treatment with mycobacterial adjuvants such as CFA and bacillus Calmette-Guérin prevents induction and recurrence of T1D in NOD mice with partial recovery of ß cell mass. In this study, we investigated factors involved in the regeneration of ß cells in the pancreas of NOD mice during diabetes development and after treatment with adjuvants. The Regeneration (Reg) gene family is known to be involved in regeneration of various tissues including ß cells. Reg2 expression was found to be upregulated in pancreatic islets both during diabetes development and as a result of adjuvant treatment in diabetic NOD mice and in C57BL/6 mice made diabetic by streptozotocin treatment. The upregulation of Reg2 by adjuvant treatment was independent of signaling through MyD88 and IL-6 because it was not altered in MyD88 or IL-6 knockout mice. We also observed upregulation of Reg2 in the pancreas of diabetic mice undergoing ß cell regenerative therapy with exendin-4 or with islet neogenesis-associated protein. Reg2 expression following adjuvant treatment correlated with a reduction in insulitis, an increase in insulin secretion, and an increase in the number of small islets in the pancreas of diabetic NOD mice and with improved glucose tolerance tests in streptozotocin-treated diabetic C57BL/6 mice. In conclusion, adjuvant immunotherapy regulates T1D in diabetic mice and induces Reg2-mediated regeneration of ß cells.

A novel mechanism of regulatory T cell-mediated down-regulation of autoimmunity.

We have established a novel CD4 and CD8 double-positive CD25+ T regulatory (Treg) clone, MT-5B, from lymph nodes of type 1 diabetes prone non-obese diabetic (NOD) mice immunized with CFA. CFA has previously been shown to prevent the onset of diabetes by inducing Treg cells. In vitro, clone MT-5B was anergic to a panel of antigen stimulations and exerted an immunosuppressive effect in antigen-non-specific and cell contact-independent manners. In vivo, clone MT-5B blocked the adoptive transfer of diabetes. Proteomics and immunoadsorption studies identified the suppressive proteins secreted by clone MT-5B as granzyme B (GrB) and perforin (PFN). GrB-mediated immune suppression was PFN dependent. Removal of GrB or PFN from the culture supernatant (SN) of MT-5B cells or pre-incubation of MT-5B cells with ethyleneglycol-bis(aminoethylether)-tetraacetic acid which blocks PFN activity reduced the immunosuppressive effect in vitro. Pre-incubation of diabetogenic splenocytes from NOD mice with MT-5B SN impaired their ability to transfer disease by inducing T cell apoptosis, and removal of GrB from MT-5B SN by immunoadsorption decreased the effector function of MT-5B SN on diabetogenic splenocytes. Immunization of NOD mice with CFA increased the expression of GrB+ CD4 T cells, indicating that these cells are present in vivo. In conclusion, we describe a novel mechanism of cell contact-independent immune suppression in which Treg cells maintain immune homeostasis by secreting GrB/PFN.

In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha.

Immunization with mycobacterial preparation such as Bacille Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. We found that the potential of splenocytes from BCG-immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-alpha, IFN-gamma and IL-4 by splenocytes, increased the expression of Fas(high) (Apo-1/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-gamma and TNF-alpha in BCG-immunotherapy was demonstrated by (i) reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anti-cytokine antibodies, (ii) inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNFR1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-alpha and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells. Our results suggest that BCG down-regulates destructive autoimmunity by TNF-alpha/IFN-gamma-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. These studies provide a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.

CD4+CD25+ regulatory T cells generated in response to insulin B:9-23 peptide prevent adoptive transfer of diabetes by diabetogenic T cells.

NOD mice have a relative deficiency of CD4+CD25+ regulatory T cells that could result in an inability to maintain peripheral tolerance. The aim of this study was to induce the generation of CD4+CD25+ regulatory T cells in response to autoantigens to prevent type 1 diabetes (T1D). We found that immunization of NOD mice with insulin B-chain peptide B:9-23 followed by 72 h in vitro culture with B:9-23 peptide induces generation of CD4+CD25+ regulatory T cells. Route of immunization has a critical role in the generation of these cells. Non-autoimmune mice BALB/c, C57BL/6 and NOR did not show up regulation of CD4+CD25+ regulatory T cells. These cells secreted large amounts of TGF-beta and TNF-alpha with little or no IFN-gamma and IL-10. Adoptive transfer of these CD4+CD25+ regulatory T cells into NOD-SCID mice completely prevented the adoptive transfer of disease by diabetogenic T cells. Although, non-self antigenic OVA (323-339) peptide immunization and in vitro culture with OVA (323-339) peptide does result in up regulation of CD4+CD25+ T cells, these cells did not prevent transfer of diabetes. Our study for the first time identified the generation of antigen-specific CD4+CD25+ regulatory T cells specifically in response to immunization with B:9-23 peptide in NOD mice that are capable of blocking adoptive transfer of diabetes. Our results suggest the possibility of using autoantigens to induce antigen-specific regulatory T cells to prevent and regulate autoimmune diabetes.

Type 1 diabetes alters anti-hsp90 autoantibody isotype.

The 90-kDa chaperon family includes heat shock protein (hsp) 90 and glucose-regulated protein (grp) 94. These proteins play an important role in normal cellular architecture, in the etiology of some autoimmune and infectious diseases and in antigen presentation to T cells. Owing to its role in autoimmunity, we explored anti-hsp90 autoantibody (hsp90AA) response in the sera of persons with type 1 diabetes, first-degree relatives (FDR) and in normal subjects. Significant high level of hsp90AA was found in FDR, but there was no significant difference between the normal and diabetic persons. The IgG1 and IgG3 isotypes of hsp90AA were higher in persons with type 1 diabetes and FDR than in normal subjects. We found a good correlation between hsp90AA measured by ELISA and RIA. A positive correlation between serum hsp90AA and glutamic acid decarboxylase (GAD65) autoantibody (GAA) was also observed. Hsp90AA positive sera from diabetic persons immunoblotted recombinant hsp90, GAD65 and corresponding proteins in islet lysates. Our study suggests that hsp90AA are present in normal, FDR and diabetic persons. However, there is a higher level of IgG1 and IgG3 isotypes of hsp90AA in FDR and type 1 diabetic subjects. Thus, autoimmunity leading to type 1 diabetes significantly alters the autoantibody isotype to autoantigens, such as hsp90.