| Background:One of the most common complications of early-onset diabetes mellitus is peripheral diabetic neuropathy, which is manifested either by loss of nociception or by allodynia and hyperalgesia. Diabetes mellitus is a common metabolic disease in human beings with characteristic symptoms of hyperglycemia, chronic inflammation and insulin resistance. Dietary fatty acids, especially polyunsaturated fatty acids, have been shown anti-inflammatory role in various experimental conditions. The present study investigated the effects of fish oil supplementation on the inflammation in the dorsal root ganglion of streptozotocin-induced diabetes rats. The effects of diabetes and fish oil treatment on the allodynia and hyperalgesia were also evaluated. Dietary fish oil effectively attenuated both allodynia and hyperalgesia induce by STZ injection. Along with the behavioral findings, DRG from fish oil treated diabetic rats displayed a decrease in inflammatory cytokines and the expression of NF-κB compared with untreated diabetic rats. Fish oil supplementation also increased the phosphorylation of AKt in DRG of diabetic rats. These results suggested that dietary fish oil inhibited allodynia and hyperalgesia in diabetic rats may stem from its anti-inflammatory potential by regulate NF-κB and AKt. Fish oil might be useful as an adjuvant therapy for the prevention and treatment of diabetic complications.ObjectiveDiabetes mellitus(DM) is a metabolic disease in human beings with characteristic symptoms of hyperglycemia, chronic inflammation and insulin resistance. Painful diabetic neuropathy(PDN) is a neurological disorder that is a common complication of diabetes. Patients with PDN can suffer from painful disorder characterized by hyperalgesia and allodynia. The mechanisms underlying abnormal nociception in diabetes are unclear.It has been reported that nuclear factor-κ-gene binding(NF-κB) activation enhanced the transcription of tumor necrosis factor α(TNF-α) and interleukin-1β(IL-1β), and both of these cytokines were in turn known to activate NF-κB. The positive feedback of NF-κB may amplify inflammatory signals. Inflammatory cytokines were proved to play an important role in the development of neuropathic pain. It has been reported that the dysfunction of phosphatidylinositol 3-kinase(PI3K)and PI3K-protein kinase B/AKt(PKB/AKt) signaling pathway increased glycogen synthase kinase-3β(GSK-3β) activity, and it participates in the signal regulating of insulin path and NF-κB path.N-3 polyunsaturated fatty acids(PUFA) have been shown anti-inflammtory role in nervous system. In addition, it has been reported that n-3 PUFA had beneficial effects in pain states. In rat DRG neurons, extracellular docosahexaenoic acid(DHA, C22: 6n-3) and eicosapentaenoic acid(EPA,C20:5 n-3) inhibited sodium current and reduced neuron excitability.Although many studies show pain relieve effects of n-3 PUFA, the effects of n-3PUFA on the DRG in diabetes are not fully known. Therefore, the purpose of the current study was to determine whether supplementation with Fish oil(FO), a rich source of n-3 PUFA such as DHA and EPA, could alleviate the allodynia and hyperalgesia in streptozotocin(STZ)-induced diabetic rats.MethodsA total of 100 adult(200-230 g) male Sprague–Dawley rats were housed individually, food and water were available ad libitum. 40 animals were randomly divided into four groups.Control animals received saline injection. Ten days before STZ administration, Control group and diabetic group were provided standard chow,while control + fish oil and diabetic + fish oil group were fed a diet containing 4% fish oil.Diabetes was induced by a 60mg/kg intraperitoneal injection of STZ. Sixty animals were used in the second experiment. In two of diabetic groups,the NF-κB inhibitor PDTC(50 m M, Beyotime) was injected intrathecally(10 μl) and flushed with 10 μl of saline which was started before STZ-injection and once daily thereafter for 35 days. In another group of the diabetic rats, the injection of PDTC was performed on day 14 after STZ-injection and once daily thereafter until day-35. The vehicle groups received same volume of vehicle(saline contained 3% DMSO) injection at same time as above.The fish oil chow was made by uniformly mixing standard chow and fish oil,which contained 30% DHA/EPA.Three days after STZ administration, blood glucose levels were determined with a blood glucometer. Animals were considered diabetic if plasma glucose levels exceeded 16.7 mmol/L. Body weights were assessed weekly.Animals were inspected and tested every three days for 10 days prior to STZ-injection, twice during the first two weeks and every three days for the last 3weeks, for a total of 14 testing sessions.After sacrifice, the bilateral DRGs(L4 and L5)were isolated from the rats, the supernatant was collected for TNF-α, CXCL1, IKKβ,IκBα, p-AKt, AKt, NF-κB, IRβ, IRS-1, p-GSK-3βand GSK-3β assay.Relative TNF-αand CXCL1 expression was determined by quantitative PCR.ResultsMean blood glucose levels in the diabetic groups was significantly higher than the control group(P < 0.01) after STZ injection. Significant weight loss was observed in STZ-induced diabetic rats compared with the control group(P < 0.01). fish oil did not exert a significant effect on weight loss of diabetic rats(P > 0.05).After the injection of STZ rats show mechanical hyperalgesia and allodynia,administration of fish oil significantly attenuated the development in diabetic rats(P<0.01).STZ-injection significantly increased the levels of TNF-α and CXCL1 in DRGs of diabetes rats(P < 0.05). The levels of TNF-α and CXCL1 were both reduced towards normal levels in diabetic animals treated with fish oil(P < 0.05).Diabetes significantly increased IKKβ/NF-κB activation by increasing the phosphorylation of IKKβ(Ser181)(P<0.05), inducing the degradation of IκBα(P<0.05).Higher intensity of NF-κB immunoreactivity was found in nucleus compared to the controls(P < 0.05). Fish oil treatment significantly decreased the p-IKKβ and increased NF-κB immunoreactivity in cytoplasm compared to the diabetic group(P < 0.05).The amount of IR protein was slightly down-regulated in diabetic rats although this trend was not significant(P>0.05). Diabetes markedly decreased the serine phosphorylation of AKt. IRS-1 was significantly higher in diabetic group compared with control, and fish oil partially reversed the change(P<0.05). Fish oil treatment significantly stimulated AKt phosphorylation compared to diabetic group(P < 0.05).On the contrary, higher level of phosphorylated GSK-3β was observed in diabetic group, and administration of fish oil decreased the GSK-3β phosphorylation(P<0.05).Compared with vehicle group, PDTC treatment significantly reduced the mechanical allodynia and thermal hyperalgesia(P < 0.05). Intrathecal injection of PDTC had no effect on the behavioral test in normal rats(P > 0.05). The intrathecal injection of PDTC(50 m M) or vehicle was designed on day 14 after STZ-injection and once daily thereafter for 35 days in another two groups of animals,there were not alleviated in either of groups(P > 0.05).Compared with vehicle group, PDTC treatment on day 1 significantly decreased the levels of TNF-α and CXCL1 in DRGs(P < 0.05), but not on day 14 after STZ-injection. PDTC treatment on both time points significantly decreased IKKβactivation(P < 0.05) and inhibited the nuclear translocation of NF-κB in theDRGs(P < 0.05). Whereas after PDTC treatment, the significant inhibitory effect on the phosphorylation of GSK-3β only was observed in the rats that received the drug injection started on day 1(P < 0.05).Conclusion:Dietary fish oil effectively attenuated both allodynia and hyperalgesia induced by STZ injection. Along with the behavioral findings, DRG from fish oil treated diabetic rats displayed a decrease in inflammatory cytokines and the expression of NF-κB compared with untreated diabetic rats. Fish oil supplementation also increased the phosphorylation of AKt in DRG of diabetic rats. These results suggested that dietary fish oil inhibited allodynia and hyperalgesia in diabetic rats may stem from its anti-inflammatory potential by regulate NF-κB and AKt. |
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