Effect of SHED-CM on DPN

Aims/Introduction: Transplantation of stem cells promotes axonal regeneration and angiogenesis in a paracrine manner. In the present study, we examined whether the secreted factors in conditioned medium of stem cells from human exfoliated deciduous teeth (SHED‐CM) had beneficial effects on diabetic polyneuropathy in mice. Materials and Methods: Conditioned medium of stem cells from human exfoliated deciduous teeth was collected 48 h after culturing in serum‐free Dulbecco's modified Eagle's medium (DMEM), and separated into four fractions according to molecular weight. Dorsal root ganglion neurons from C57BL/6J mice were cultured with SHED‐CM or DMEM to evaluate the effect on neurite outgrowth. Streptozotocin‐induced diabetic mice were injected with 100 μL of SHED‐CM or DMEM into the unilateral hindlimb muscles twice a week over a period of 4 weeks. Peripheral nerve functions were evaluated by the plantar test, and motor and sensory nerve conduction velocities. Intraepidermal nerve fiber densities, capillary number‐to‐muscle fiber ratio, capillary blood flow and morphometry of sural nerves were also evaluated. Results: Conditioned medium of stem cells from human exfoliated deciduous teeth significantly promoted neurite outgrowth of dorsal root ganglion neurons compared with DMEM. Among four fractions of SHED‐CM, the only fraction of <6 kDa promoted the neurite outgrowth of dorsal root ganglion neurons. In addition, SHED‐CM significantly prevented decline in sensory nerve conduction velocities compared with DMEM in diabetic mice. Although SHED‐CM did not improve intraepidermal nerve fiber densities or morphometry of sural nerves, SHED‐CM ameliorated the capillary number‐to‐muscle fiber ratio and capillary blood flow. Conclusions: These results suggested that SHED‐CM might have a therapeutic effect on diabetic polyneuropathy through promoting neurite outgrowth, and the increase in capillaries might contribute to the improvement of neural function

Omega‐3 polyunsaturated fatty acids exert anti‐oxidant effects through the nuclear factor (erythroid‐derived 2)‐related factor 2 pathway in immortalized mouse Schwann cells

Abstract Aims/Introduction Recent studies advocate that omega‐3 polyunsaturated fatty acids (ω‐3 PUFAs) have direct anti‐oxidative and anti‐inflammatory effects in the vasculature; however, the role of ω‐3 PUFAs in Schwann cells remains undetermined. Materials and methods Immortalized mouse Schwann (IMS32) cells were incubated with the ω‐3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The messenger ribonucleic acid levels of several anti‐oxidant enzymes (heme oxygenase‐1 [Ho‐1], nicotinamide adenine dinucleotide [phosphate] H quinone oxidoreductase 1, catalase, superoxide dismutase and glutathione peroxidase) were identified using real‐time reverse transcription polymerase chain reaction. Ho‐1 and nicotinamide adenine dinucleotide [phosphate] H quinone oxidoreductase 1 protein levels were evaluated using Western blotting. Nuclear factor (erythroid‐derived 2)‐related factor 2 (Nrf2) of the nuclear fraction was also quantified using western blotting. Catalase activity and glutathione content were determined by colorimetric assay kits. Nrf2 promoter‐luciferase activity was evaluated by a dual luciferase assay system. Results Treatment with tert‐butyl hydroperoxide decreased cell viability dose‐dependently. DHA or EPA pretreatment significantly alleviated tert‐butyl hydroperoxide‐induced cytotoxicity. DHA or EPA increased the messenger ribonucleic acid levels of Ho‐1, nicotinamide adenine dinucleotide (phosphate) H quinone oxidoreductase 1 and catalase dose‐dependently. Ho‐1 protein level, catalase activity, Nrf2 promoter‐luciferase activity and intracellular glutathione content were significantly increased by DHA and EPA. Conclusions These findings show that DHA and EPA can induce Ho‐1 and catalase through Nrf2, thus protecting Schwann cells against oxidative stress. ω‐3 PUFAs appear to exert their neuroprotective effect by increasing defense mechanisms against oxidative stress in diabetic neuropathies

Glucagon Prevents Cytotoxicity Induced by Methylglyoxal in a Rat Neuronal Cell Line Model

Although diabetic polyneuropathy (DPN) is a frequent diabetic complication, no effective therapeutic approach has been established. Glucagon is a crucial hormone for glucose homeostasis but has pleiotropic effects, including neuroprotective effects in the central nervous system. However, the importance of glucagon in the peripheral nervous system (PNS) has not been clarified. Here, we hypothesized that glucagon might have a neuroprotective function in the PNS. The immortalized rat dorsal root ganglion (DRG) neuronal cell line 50B11 was treated with methylglyoxal (MG) to mimic an in vitro DPN model. The cells were cultured with or without glucagon or MG. Neurotoxicity, survival, apoptosis, neurite projection, cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA) were examined. Glucagon had no cytotoxicity and rescued the cells from neurotoxicity. Cell survival was increased by glucagon. The ratio of apoptotic cells, which was increased by MG, was reduced by glucagon. Neurite outgrowth was accelerated in glucagon-treated cells. Cyclic AMP and PKA accumulated in the cells after glucagon stimulation. In conclusion, glucagon protected the DRG neuronal cells from MG-induced cellular stress. The cAMP/PKA pathway may have significant roles in those protective effects of glucagon. Glucagon may be a potential target for the treatment of DPN

Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death via the AMPK-Dependent Signaling Pathway in Immortalized Fischer Rat Schwann Cells 1

Autophagy is the process by which intracellular components are degraded by lysosomes. It is also activated by oxidative stress; hence, autophagy is thought to be closely related to oxidative stress, one of the major causes of diabetic neuropathy. We previously reported that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) induced antioxidant enzymes and protected Schwann cells from oxidative stress. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) decreased the cell survival rate, as measured by an MTT assay in immortalized Fischer rat Schwann cells 1 (IFRS1). A DHA pretreatment significantly prevented tBHP-induced cytotoxicity. tBHP increased autophagy, which was revealed by the ratio of the initiation markers, AMP-activated protein kinase, and UNC51-like kinase phosphorylation. Conversely, the DHA pretreatment suppressed excessive tBHP-induced autophagy signaling. Autophagosomes induced by tBHP in IFRS1 cells were decreased to control levels by the DHA pretreatment whereas autolysosomes were only partially decreased. These results suggest that DHA attenuated excessive autophagy induced by oxidative stress in Schwann cells and may be useful to prevent or reduce cell death in vitro. However, its potentiality to treat diabetic neuropathy must be validated in in vivo studies