Gu-Jiun Lin

Triptolide exerts anti-tumor effect on oral cancer and KB cells in vitro and in vivo

Triptolide (TPL), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been reported to potentiate the anti-tumor effect in various cancer cells. However, the effect of TPL on oral cancers is not yet evaluated. Herein we first demonstrate that TPL induces prominent growth inhibition and apoptosis in two oral cancer cell lines, SCC25 and OEC-M1 and in KB cells. Our results indicate that TPL induces a dose-dependent apoptosis of these cells at nanomolar concentration. Apoptosis signalings are both activated through time upon TPL treatment detected by elevated caspase-3, 8, 9 activities. In xenograft tumor mouse model, TPL injection successfully inhibits the tumor growth via apoptosis induction which was demonstrated by TUNEL assay. These results demonstrate that TPL exerts anti-tumor effect on oral cancer and KB cells and suggest further the potential of TPL combining with other chemotherapeutic agents or radiotherapy for advanced oral cancer.

Melatonin prolongs islet graft survival in diabetic NOD mice

Abstract:  Islet transplantation has been established as a potential therapy for type 1 diabetes. However, inflammation, allorejection, and on‐going autoimmune damage contribute to early graft loss and failure of islet transplantation. Melatonin is the major secretory product of the pineal gland during the dark period of each day and displays multifunctional properties including the regulation of circadian and seasonal rhythms, antioxidation reactions and immune modulation. Based on the immunosuppressive properties of melatonin, we investigated whether melatonin treatment prolonged the survival of islet grafts in non‐obese diabetic (NOD) mice. The mean islet graft survival time was 7.33 ± 1.51 and 7.75 ± 2.66 days in untreated controls and in the solvent‐treated animals, respectively. Strikingly, the mean survival time of islet grafts in recipients treated with melatonin (200 mg/kg/bw) was 17 ± 7.76 days. Moreover, melatonin treatment reduced the proliferation of splenocytes in NOD mice. Using a T1 and T2 double transgenic mouse model, we found that T helper 1 (Th1) cells in mice treated with melatonin were significantly decreased. The reduction of Th1 cells and T cell proliferation may result from an increase in the immunosuppressive cytokine IL‐10. Our results indicate that melatonin treatment suppresses autoimmune recurrence by inhibiting the proliferation of Th1 cells in NOD mice and thus prolongs the survival of syngeneic islet grafts.

Modulation by Melatonin of the Pathogenesis of Inflammatory Autoimmune Diseases

Melatonin is the major secretory product of the pineal gland during the night and has multiple activities including the regulation of circadian and seasonal rhythms, and antioxidant and anti-inflammatory effects. It also possesses the ability to modulate immune responses by regulation of the T helper 1/2 balance and cytokine production. Autoimmune diseases, which result from the activation of immune cells by autoantigens released from normal tissues, affect around 5% of the population. Activation of autoantigen-specific immune cells leads to subsequent damage of target tissues by these activated cells. Melatonin therapy has been investigated in several animal models of autoimmune disease, where it has a beneficial effect in a number of models excepting rheumatoid arthritis, and has been evaluated in clinical autoimmune diseases including rheumatoid arthritis and ulcerative colitis. This review summarizes and highlights the role and the modulatory effects of melatonin in several inflammatory autoimmune diseases including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes mellitus, and inflammatory bowel disease.

Transgenic expression of haem oxygenase-1 in pancreatic beta cells protects non-obese mice used as a model of diabetes from autoimmune destruction and prolongs graft survival following islet transplantation

Aims/hypothesisHaem oxygenase 1 (HO-1) has strong anti-apoptotic, anti-inflammatory and antioxidative effects that help protect cells against various forms of immune attack. We investigated whether transgenic expression of Ho-1 (also known as Hmox1) in pancreatic beta cells would protect NOD mice from autoimmune damage and prolong graft survival following islet transplantation.MethodsTo evaluate the protective effect of beta cell-specific HO-1 in autoimmune diabetes, we used an insulin promoter-driven murine Ho-1 construct (pIns-mHo-1) to generate a transgenic NOD mouse. Transgene expression, insulitis and the incidence of diabetes in mice were characterised. Lymphocyte composition, the development of T helper (Th)1, Th2 and T regulatory (Treg) cells, T cell proliferation and lymphocyte-mediated disease transfer were analysed. The potential effects of transgenic islets and islet transplantation on apoptosis, inflammation and the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were evaluated.ResultsTransgenic mice showed less severe insulitis and a lower incidence of diabetes than non-transgenic control littermates. Lymphocyte composition and functions were not affected. Islets from transgenic mice expressed lower levels of proinflammatory cytokines/chemokines, proapoptotic gene expression and amounts of ROS/RNS, and were more resistant to TNF-α- and IFN-γ-induced apoptosis. Islet grafts from transgenic mice also survived longer in diabetic recipients than control islets.Conclusions/interpretationTransgenic overexpression of Ho-1 in beta cells protected NOD mice from diabetes and delayed the autoimmune destruction of islet grafts, providing valuable insight into the development of better strategies for clinical islet transplantation in patients with type 1 diabetes.

Melatonin enhances endogenous heme oxygenase-1 and represses immune responses to ameliorate experimental murine membranous nephropathy.

Abstract:  Idiopathic membranous nephropathy (MN), an autoimmune‐mediated glomerulonephritis, is one of the most common causes of nephrotic syndrome in adults. Therapeutic agents for MN remain ill defined. We assessed the efficacy of melatonin therapy for MN. Experimental murine MN was induced with cationic bovine serum albumin, and the mice were immediately administered 20 mg/kg melatonin or phosphate‐buffered saline subcutaneously once a day. Disease severity was verified by examining serum and urine metabolic profiles and renal histopathology. The expression of cytokines and oxidative stress markers, cell apoptosis, and the associated mechanisms were also determined. Mice treated with melatonin displayed a significant reduction in proteinuria and a marked amelioration of glomerular lesions, with attenuated immunocomplex deposition. The subpopulations of T cells were not altered, but the CD19+ B‐cell subpopulation was significantly reduced in the MN mice treated with melatonin. The expression of cytokine mRNAs in splenocytes indicated that melatonin reduced the expression of proinflammatory cytokines and increased the expression of anti‐inflammatory cytokines (interleukin 10). The production of reactive oxygen species and TUNEL‐positive apoptotic cells in the kidney were also significantly reduced in the melatonin‐treated MN mice. Melatonin also upregulated heme oxygenase 1 (HO1) and ameliorated MN. The blockade of HO1 expression with SnPP, a HO1 inhibitor, attenuated HO1 induction by melatonin and thus mitigated its renoprotective effects during MN. Our results suggest that melatonin treatment ameliorates experimental MN via multiple pathways, including by its antioxidative, antiapoptotic, and immunomodulatory effects. Melatonin should be considered a potential therapeutic intervention for MN in the future.

Melatonin enhances interleukin-10 expression and suppresses chemotaxis to inhibit inflammation in situ and reduce the severity of experimental autoimmune encephalomyelitis.

Melatonin is the major product secreted by the pineal gland at night and displays multifunctional properties, including immunomodulatory functions. In this study, we investigated the therapeutic effect of melatonin in experimental autoimmune encephalomyelitis (EAE). We demonstrated that melatonin exhibits a therapeutic role by ameliorating the clinical severity and restricting the infiltration of inflammatory Th17 cells into the CNS of mice with myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Furthermore, melatonin enhances splenic interleukin (IL)-10 expression in regulatory T cells by inducing IL-27 expression in the splenic DC; it also suppresses the expression of IFN-γ, IL-17, IL-6, and CCL20 in the CNS and inhibits antigen-specific T cell proliferation. However, there were no significant differences in the percentage of splenic regulatory T cells. These data provide the first evidence that the therapeutic administration of melatonin is effective in mice with EAE and modulates adaptive immunity centrally and peripherally. Thus, we suggest that melatonin could play an adjunct therapeutic role in treating human CNS autoimmune diseases such as multiple sclerosis. Melatonin merits further studies in animals and humans.

α-Lipoic acid enhances endogenous peroxisome-proliferator-activated receptor-γ to ameliorate experimental autoimmune encephalomyelitis in mice

ALA (α-lipoic acid) is a natural, endogenous antioxidant that acts as a PPAR-γ (peroxisome-proliferator-activated receptor-γ) agonist to counteract oxidative stress. Thus far, the antioxidative and immunomodulatory effects of ALA on EAE (experimental autoimmune encephalomyelitis) are not well understood. In this study, we found that ALA restricts the infiltration of inflammatory cells into the CNS (central nervous system) in MOG (myelin oligodendrocyte glycoprotein)-EAE mice, thus reducing the disease severity. In addition, we revealed that ALA significantly suppresses the number and percentage of encephalitogenic Th1 and Th17 cells and increases splenic Treg-cells (regulatory T-cells). Strikingly, we further demonstrated that ALA induces endogenous PPAR-γ centrally and peripherally but has no effect on HO-1 (haem oxygenase 1). Together, these data suggest that ALA can up-regulate endogenous systemic and central PPAR-γ and enhance systemic Treg-cells to inhibit the inflammatory response and ameliorate MOG-EAE. In conclusion, our data provide the first evidence that ALA can augment the production of PPAR-γ in vivo and modulate adaptive immunity both centrally and peripherally in EAE and may reveal further antioxidative and immunomodulatory mechanisms for the application of ALA in human MS (multiple sclerosis).

Transgenic expression of murine chemokine decoy receptor D6 by islets reveals the role of inflammatory CC chemokines in the development of autoimmune diabetes in NOD mice

Aims/hypothesisAutoimmune diabetes results from a progressive destruction of insulin-producing beta cells in the pancreatic islets by chemokine-attracted lymphocytes. Because islet cells in NOD mice produce chemokines during the development of autoimmune diabetes, we investigated the role of inflammatory CC chemokines in disease progression in these mice.MethodsWe generated a transgenic NOD mouse model that overproduces the inflammatory CC chemokine decoy receptor D6 in pancreatic islets.ResultsThe frequency of diabetes and insulitis scores of transgenic mice were decreased significantly, compared with non-transgenic control littermates. Transgenic expression of D6 (also known as Ccbp2) did not affect systemic lymphocyte development or alter: (1) the T cell subsets such as T helper (Th)1, Th2 and T regulatory cells; or (2) antigen-presenting cells such as dendritic cells or macrophages. The percentages and numbers of T and B lymphocytes were decreased significantly in the pancreas. Activation status, autoantigen-specific proliferation and diabetogenicity of lymphocytes were also markedly reduced.Conclusions/interpretationInflammatory CC chemokines play a critical role in the development of autoimmune diabetes. Transgenic expression of D6 in pancreatic islets of NOD mice reduced this pathogenic process by suppressing activation of autoreactive lymphocytes and by reducing migration of lymphocytes to the pancreas.

Triptolide ameliorates autoimmune diabetes and prolongs islet graft survival in nonobese diabetic mice.

Objectives Triptolide (TPL) possesses profound immunosuppressive effects and has potential in allograft transplantation. We investigated whether TPL treatment prevents autoimmune diabetes in nonobese diabetic (NOD) mice and prolongs the survival of islet grafts against autoimmune attack or allograft rejection. Methods Diabetic incidence was monitored in TPL-treated NOD mice. Nonobese diabetic or BALB/c islets were transplanted into diabetic recipients treated with TPL. Different T-cell subsets in grafts or spleen were analyzed. The proliferation, apoptosis, cytokines, and activities of AKT, NF&kgr;B, and caspases 3, 8, and 9 of T cells were determined. Results Diabetic incidence was reduced and inflammatory cytokines were decreased in islets and spleen under TPL treatment. T-cell proliferation was reduced and the survival of syngeneic or allogeneic grafts was significantly increased in TPL-treated mice. The populations of CD4, CD8, CD4CD69, CD8CD69, and interferon-&ggr;–producing T cells in islet grafts and spleen were reduced. Triptolide treatment increased the apoptosis of T cells in the spleen of recipients. Levels of phosphorylated protein kinase B and phosphorylated inhibitor of kappa B in splenocytes were reduced and caspases 3, 8, and 9 were increased in TPL-treated mice. Conclusions Triptolide treatment not only reduced the diabetic incidence in NOD mice but also prolonged the survival of syngeneic or allogeneic grafts.

Enhanced Anti-Tumor Activity of Triptolide in Combination with Irradiation for the Treatment of Oral Cancer

Advanced oral cancer has a poor prognosis because of the lack of an effective treatment. We explored the efficiency of combined treatment with triptolide and ionizing radiation for treating oral cancer. Human tongue cancer cells were treated with triptolide, ionizing radiation, or triptolide plus ionizing radiation. Cell proliferation, cell cycle arrest, and apoptotic influences were analyzed by FACS and immunohistochemistry. Tumor potency was examined in an in vivo human tongue cancer cells xenograft mouse model. Our results demonstrated that triptolide caused a marked reduction in colony number that was further enhanced with increasing doses of ionizing radiation. Triptolide increased apoptosis and decreased the expression of anti-apoptotic proteins. In vivo, combination treatment synergistically reduced tumor weight and volume possibly via the induction of apoptosis and reduction in anti-apoptotic protein expression. In conclusion, triptolide plus ionizing radiation treatment had synergistic anti-tumor effects, especially in vivo, and may be a promising combined modality therapy for advanced oral cancer.