Melatonin modulates neuroinflammation and oxidative stress in experimental diabetic neuropathy: effects on NF-κB and Nrf2 cascades

Abstract: Melatonin exhibits an array of biological activities, including antioxidant and anti‐inflammatory actions. Diabetic neuropathy is one of the complications of diabetes with a prevalence rate of 50–60%. We have previously reported the protective effect of melatonin in experimental diabetic neuropathy. In this study, we investigated the role of nuclear factor‐kappa B (NF‐κB) and nuclear erythroid 2‐related factor 2 (Nrf2) in melatonin‐mediated protection against streptozotocin‐induced diabetic neuropathy. Melatonin at doses of 3 and 10 mg/kg was administered daily in seventh and eighth week after diabetes induction. Motor nerve conduction velocity and nerve blood flow were improved in melatonin‐treated animals. Melatonin also reduced the elevated expression of NF‐κB, IκB‐α, and phosphorylated IκB‐α. Further, melatonin treatment also reduced the elevated levels of proinflammatory cytokines (TNF‐α and IL‐6), iNOS and COX‐2 in sciatic nerves of animals. The capacity of melatonin to modulate Nrf2 pathway was associated with increased heme oxygenase‐1 (HO‐1) expression, which strengthens antioxidant defense. This fact was also established by decreased DNA fragmentation (because inhibition of excessive oxidant‐induced DNA damage) in the sciatic nerve of melatonin‐treated animals. The results of this study suggest that melatonin modulates neuroinflammation by decreasing NF‐κB activation cascade and oxidative stress by increasing Nrf2 expression, which might be responsible at least in part, for its neuroprotective effect in diabetic neuropathy.     

Protective effects of mineralocorticoid receptor blockade against neuropathy in experimental diabetic rats

Aims: Mineralocorticoid receptor (MR) blockade is an effective treatment for hypertension and diabetic nephropathy. There are no data on the effects of MR blockade on diabetic peripheral neuropathy (DPN). The aim of this study was to determine whether MRs are present in the peripheral nerves and to investigate the effectiveness of MR blockade on DPN in streptozotocin (STZ)‐induced diabetic rats. Methods: Expression of MR protein and messenger RNA (mRNA) was examined in the peripheral nerves using Western blot analysis and RT‐PCR. We next studied the effects of the selective MR antagonist eplerenone and the angiotensin II receptor blocker candesartan on motor and sensory nerve conduction velocity (NCV), morphometric changes and cyclooxygenase‐2 (COX‐2) gene and NF‐κB protein expression in the peripheral nerves of STZ‐induced diabetic rats. Results: Expression of MR protein and mRNA in peripheral nerves was equal to that in the kidney. Motor NCV was significantly improved by 8 weeks of treatment with either eplerenone (39.1 ± 1.2 m/s) or candesartan (46.4 ± 6.8 m/s) compared with control diabetic rats (33.7 ± 2.0 m/s) (p < 0.05). Sensory NCV was also improved by treatment with candesartan or eplerenone in diabetic rats. Eplerenone and candesartan caused significant improvement in mean myelin fibre area and mean myelin area compared with control diabetic rats (p < 0.05). COX‐2 mRNA and NF‐κB protein were significantly elevated in the peripheral nerves of diabetic rats compared with control rats, and treatment with eplerenone or candesartan reduced these changes in gene expression (p < 0.05). Conclusion: MR blockade may have neuroprotective effects on DPN.     

Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor‐κB activation

Constitutive activation and gemcitabine induction of nuclear factor‐κB (NF‐κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF‐κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF‐κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF‐κB by suppressing IκBα phosphorylation and decreased the expression of NF‐κB response genes in MiaPaCa‐2, AsPc‐1, Panc‐28 cells and gemcitabine resistance MiaPaCa‐2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor‐independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug‐treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF‐κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF‐κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.     

Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis

Abstract: We explored anti‐inflammatory potential of melatonin against the lipopolysaccharide (LPS)‐induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment with melatonin or vehicle at various time intervals. In a mouse model of meningitis induced by LPS, melatonin (5 mg/kg) or vehicle was intravenously injected at 30 min postinsult. The activity of matrix metalloproteinase‐2 (MMP‐2) and metalloproteinase‐9 (MMP‐9) was determined by gelatin zymography. Nuclear factor‐kappa B (NFκB) translocation and binding activity were determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA). Our results showed that either pretreatment or cotreatment with melatonin at 50–500 μm effectively inhibited the LPS‐induced proMMP‐9 activation in the RAW 264.7 and BV2 cells, respectively (P < 0.05). This melatonin‐induced proMMP‐9 inhibition remained effective when treatment was delayed up to 2 and 6 hr postinsult for RAW 264.7 and BV2 cells, respectively (P < 0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP‐9 activity, respectively (P < 0.05) and reduced the loss of body weight (P < 0.05) in mice with meningitis. Moreover, melatonin (50 μm ) effectively inhibited nuclear factor‐kappa B (NFκB) translocation and binding activity in the LPS‐treated RAW 264.7 and BV2 cells, respectively (P < 0.05). These results demonstrate direct inhibitory actions of melatonin against postinflammatory NFκB translocation and MMP‐9 activation and highlight its ability to inhibit systemic and cerebral MMP‐9 activation following brain inflammation.     

核转录因子κB在糖尿病大鼠坐骨神经中的表达及其与传导速度的关系

目的研究核转录因子出（NF-κB）在糖尿病大鼠坐骨神经中的表达动态变化及其意义。方法建立糖尿病大鼠模型后，分别在实验1个月、3个月、6个月时测定坐骨神经的传导速度和NF-κB的表达量。结果（1）坐骨神经传导速度：与正常对照（NC）组相比，糖尿病模型1个月组大鼠坐骨神经传导速度未见明显下降，3个月、6个月组则明显下降（P〈0．05）；（2）EMSA电泳条带灰度分析：与NC相比，NF-κB表达在糖尿病各组均明显增强（P〈0．05）。结论NF-κB在糖尿病大鼠坐骨神经中持续活化，推断NF-κB在糖尿病周围神经病变的发病机制中起重要作用。     

Anti-oxidant treatment prevents the development of peripheral nerve dysfunction in streptozotocin-diabetic rats

Summary We tested the notion that oxidative stress makes an important contribution to the aetiology of diabetic neuropathy. The effect of treatment with a 1% dietary supplement of the anti-oxidant butylated hydroxytoluene was studied during 2 months of streptozotocin-induced diabetes mellitus. In final experiments, sciatic motor and saphenous sensory conduction velocities were measured in vivo, and resistance to hypoxic conduction failure for sciatic trunk was examined in vitro. There were 20% and 12% decreases in motor and sensory conduction velocity, respectively after 2 months of diabetes (p<0.001). These were completely prevented by butylated hydroxytoluene treatment (p<0.001). Resistance to hypoxic conduction failure, shown by the time taken for sciatic compound action potential amplitude to decline by 80%, was 55% increased by diabetes, and this was limited to 31% (p<0.01) by treatment. There were no significant effects of treatment on the 9–10 fold elevation of sciatic nerve sorbitol and fructose levels with diabetes, or on the non-significant 22% reduction in myoinositol content. Butylated hydroxytoluene treatment also did not affect sciatic nerve capillary density. We conclude that oxidative stress makes an important contribution to the aetiology of early experimental diabetic neuropathy. Amelioration of oxidative stress could potentially be a final common mechanism whereby a number of diverse treatments exert a beneficial effect on diabetic nerve function.     

RhoA‐mediated,tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF‐κB in tumor necrosis factor α (TNFα)–stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα‐induced activation of RhoA and NF‐κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

Methods

Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull‐down assay. NF‐κB DNA binding activity and nuclear translocation of NF‐κB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

Results

TNFα stimulation elicited a robust increase in RhoA activity in a dose‐dependent manner, and SMV mitigated this increase. TNFα also hastened NF‐κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin‐1β (IL‐1β) and IL‐6. SMV prevented the increase in NF‐κB activation and rise in IL‐1β and IL‐6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF‐κB and RhoA. Furthermore, cotransfection with a dominant‐negative mutant of RhoA demonstrated that the TNFα‐induced signaling pathway involved sequential activation of RhoA, leading to NF‐κB activation and, ultimately, to secretion of cytokines.

Conclusion

This study identifies RhoA as the key regulator of TNFα‐induced NF‐κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

     

The effect of the calcium antagonist nifedipine on peripheral nerve function in streptozotocin-diabetic rats

Summary Recent data suggests that reduced nerve blood flow is implicated in the aetiology of experimental diabetic neuropathy, which may be prevented by manipulations that reduce receptor-mediated vasoconstrictor activity. This investigation examines the effects of nifedipine, a voltage-sensitive calcium channel antagonist which has a direct vasodilatory effect on vessels, on nerve conduction, hypoxic resistance and capillary density in streptozotocin-induced diabetic rats. Treated and non-treated non-diabetic and diabetic groups were employed. Diabetes duration was 2 months. Treatment was preventive, groups received a nifedipine dietary supplement (40 mg · kg^–1 · day^–1) for 2 months from the start of the study. Conduction was measured in sciatic motor branches supplying tibialis anterior and gastrocnemius muscles, and sensory saphenous nerve. Diabetes resulted in a 23–28 % reduction in motor conduction velocity (p<0.001), and a 15% deficit for sensory saphenous nerve (p<0.001). In the nifedipine-treated diabetic group, motor and sensory conduction deficits were minimal compared with non-treated diabetes (p<0.001). Nifedipine treatment had no significant effect on conduction velocity in nondiabetic rats. In vitro measurement of sciatic nerve hypoxic resistance revealed a 60 % increase in the time taken for compound action potential amplitude to reach half its initial value with diabetes (p<0.001). This was not significantly affected by nifedipine treatment. Experimental diabetes or nifedipine treatment did not significantly alter sciatic nerve endoneurial capillary density. We conclude that nifedipine, a vasodilator which acts directly on vascular smooth muscle, prevents nerve conduction deficits in experimental diabetes.     

Angiotensin II receptor blockade improves nerve function,modulates nerve blood flow and stimulates endoneurial angiogenesis in streptozotocin-diabetic ratsand nerve function

Summary We examined the effect of the angiotensin II receptor blocker, ZD 8731, on nerve function, capillary density, and blood flow in streptozotocin-diabetic rats. Deficits in sciatic motor and saphenous sensory nerve conduction velocity of 21% and 15%, respectively, were observed after 1 month of diabetes mellitus (p <0.001). These were completely ameliorated by a further month of ZD 8731 treatment (p <0.001). Treatment of non-diabetic rats for 1 month with ZD 8731 had no effect on motor or sensory conduction velocity. Sciatic nerve capillary density was not significantly affected by 1- or 2-month untreated diabetes, however, there was a 15% increase in density with ZD 8731 treatment (p <0.001). Treatment of non-diabetic rats for 1 month had no effect on capillary density. Diabetes prolonged the time taken for 80% conduction failure by 19% (p <0.05) and 49% (p <0.001) for 1 and 2 months of diabetes, respectively, when sciatic nerve was exposed to hypoxia in vitro. ZD 8731 treatment during the second month of diabetes limited the prolongation to 22%, not significantly different from 1 month of untreated diabetes but less than for the 2-month diabetic group (p <0.001). Concentrations of sciatic nerve polyol pathway metabolites were elevated six-fold and myo-inositol was reduced 40% by diabetes; ZD 8731 treatment was without effect. Acute experiments examined the effect of ZD 8731 on sciatic nerve blood flow using laser-Doppler flowmetry. In non-diabetic rats, blood flow changes followed the dose-dependent reductions in systemic arterial pressure and there were no significant variations in sciatic vascular resistance. In marked contrast, nerve blood flow was elevated by 47% (p <0.01), and vascular resistance decreased by 32% (p <0.01) in diabetic rats despite similar changes in blood pressure compared with the non-diabetic group. Thus, the investigation has identified abnormalities in vasa nervorum reactivity which are ameliorated by angiotensin II receptor blockade and may contribute to experimental diabetic neuropathy.     

Protein kinase C effects on nerve function, perfusion, Na+,K+-ATPase activity and glutathione content in diabetic rats

Abstract Aims//hypothesis. Increased protein kinase C activity has been linked to diabetic vascular complications in the retina and kidney, which were attenuated by protein kinase C antagonist treatment. Neuropathy has a vascular component, therefore, the aim was to assess whether treatment with WAY151 003 or chelerythrine, inhibitors of protein kinase C regulatory and catalytic domains respectively, could correct nerve blood flow, conduction velocity, Na⁺,K⁺-ATPase, and glutathione deficits in diabetic rats. Methods. Diabetes was induced by streptozotocin. Sciatic nerve conduction velocity was measured in vivo and sciatic endoneurial perfusion was monitored by microelectrode polarography and hydrogen clearance. Glutathione content and Na⁺,K⁺-ATPase activity were measured in extracts from homogenised sciatic nerves. Results. After 8 weeks of diabetes, sciatic blood flow was 50 % reduced. Two weeks of WAY151 003 (3 or 100 mg/kg) treatment completely corrected this deficit and chelerythrine dose-dependently improved nerve perfusion. The inhibitors dose-dependently corrected a 20 % diabetic motor conduction deficit, however, at high doses ( > 3.0 mg/kg WAY151003; > 0.1 mg/kg chelerythrine) conduction velocity was reduced towards the diabetic level. Sciatic Na⁺,K⁺-ATPase activity, 42 % reduced by diabetes, was partially corrected by low but not high dose WAY151 003. In contrast, only a very high dose of chelerythrine partially restored Na⁺,K⁺-ATPase activity. A 30 % diabetic deficit in sciatic glutathione content was unchanged by protein kinase C inhibition. The benefits of WAY151 003 on blood flow and conduction velocity were blocked by nitric oxide synthase inhibitor co-treatment. Conclusion/interpretation. Protein kinase C contributes to experimental diabetic neuropathy by a neurovascular mechanism rather than through Na⁺,K⁺-ATPase defects. [Diabetologia (1999) 42: 1120–1130] Received: 21 December 1998 and in final revised form: 23 April 1999