2/15-lipoxygenase Inhibition Counteracts Mapk Phosphorylation in Mouse and Cell Culture Models of Diabetic Peripheral Neuropathy

Background: Increased mitogen-activated protein kinase (MAPK) phosphorylation has been detected in peripheral nerve of human subjects and animal models with diabetes as well as high-glucose exposed human Schwann cells, and have been implicated in diabetic peripheral neuropathy. In our recent studies, leukocyte-type 12/15-lipoxygenase inhibition or gene deficiency alleviated large and small nerve fiber dysfunction, but not intraepidermal nerve fiber loss in streptozotocin-diabetic mice. Methods: To address a mechanism we evaluated the potential for pharmacological 12/15-lipoxygenase inhibition to counteract excessive MAPK phos-phorylation in mouse and cell culture models of diabetic neuropathy. C57Bl6/J mice were made diabetic with streptozotocin and maintained with or without the 12/15-lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC). Human Schwann cells were cultured in 5.5 mM or 30 mM glucose with or without CDC. Results: 12(S) HETE concentrations (ELISA), as well as 12/15-lipoxygenase expression and p38 MAPK, ERK, and SAPK/JNK phosphorylation (all by Western blot analysis) were increased in the peripheral nerve and spinal cord of diabetic mice as well as in high glucose-exposed human Schwann cells. CDC counteracted diabetes-induced increase in 12(S)HETE concentrations (a measure of 12/15-lipoxygenase activity), but not 12/15-lipoxygenase overexpression, in sciatic nerve and spinal cord. The inhibitor blunted excessive p38 MAPK and ERK, but not SAPK/ JNK, phosphorylation in sciatic nerve and high glucose exposed human Schwann cells, but did not affect MAPK, ERK, and SAPK/JNK phos-phorylation in spinal cord. Conclusion: 12/15-lipoxygenase inhibition counteracts diabetes related MAPK phosphorylation in mouse and cell culture models of diabetic neuropathy and implies that 12/15-lipoxygenase inhibitors may be an effective treatment for diabetic peripheral neuropathy.

Multiple specific small molecule MAPK inhibitors are now in clinical trials for chronic diseases including several types of cancer, inflammatory and autoimmune diseases, neuropathic pain following nerve trauma, as well as Parkinson's and Alzheimer's diseases [33].Regardless of these efforts and outcomes, the search for alternative approaches to inhibit excessive MAPK phosphorylation in specific pathological conditions including diabetic neuropathy is highly warranted.In our previous experiments [24,25,31], 12/15-lipoxygenase inhibition and gene deficiency improved several diabetic neuropathy associated endpoints in streptozotocin-diabetic mice including nerve conduction deficits and behavioral changes suggesting that preventing 12/15-lipoxygenase activation may be an effective treatment for diabetic neuropathy.

Animals
The experiments were performed in accordance with The Guide for the Care and Handling of Laboratory Animals (NIH Publication No. 85-23) and Pennington Biomedical Research Center Protocol for Animal Studies.Mature male C57Bl6/J mice were purchased from Jackson Laboratories.All the mice were fed standard mouse chow (PMI Nutrition International, Brentwood, MO, USA) and had ad libitum access to water.After a 7-day acclimation in a new environment, the mice were randomly divided into two groups.In one group, diabetes was induced by streptozotocin (STZ) as we described previously [24,25].The mice with blood glucose ≥13.8 mM, three days post streptozotocin were considered diabetic.The control and diabetic mice were kept for 12 weeks without treatment, and then divided into two subgroups that were maintained with or without treatment with cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC), 8 mg kg/d, s.c., for another 4 weeks.CDC, at the aforementioned dose, counteracted multiple manifestations of diabetic neuroapthy and oxidative-nitrosative stress in peripheral nerve and spinal cord in our previous study [24].Non-fasting blood glucose measurements were performed after induction of diabetes and at the end of the study period.

Anesthesia, Euthanasia and Tissue Sampling
The animals were sedated by CO 2 , and immediately sacrificed by cervical dislocation.Sciatic nerves and spinal cords were rapidly dissected and frozen in liquid nitrogen for subsequent assessment of LO as well as total and phosphorylated p38 MAPK, ERK, and SAPK/JNK levels, and 12(S)-HETE concentrations.

Human Schwann Cell Culture
Schwann cells play a key role in the pathology of various inflammatory, metabolic, and hereditary polyneuropathies, including diabetic neuropathy [34,35].Previous studies demonstrated that cultured human Schwann cells (cell line cat.#1700, ScienCell, Carlsbad, CA) manifest increased superoxide production, accumulation of nitrated and poly(ADP-ribosyl)ated proteins and 4hydroxynonenal adducts, inducible nitric oxide synthase overexpression, 12/15-Lipoxygenase overexpression and activation, increased p38 MAPK phosphorylation, downregulation of taurine transporter, as well as impaired insulin signaling early (1 -7 d) after exposure to high glucose [24,[36][37][38].They therefore represent a good model for studying interactions among individual pathobiochemical mechanisms in the peripheral nerve.In the present study, human Schwann cells (passages 7 -10) were cultured in 6-well plates in media containing 5.5 mM D-glucose.At ~70% confluence, the media were replaced with those containing either 5.5 mM D-glucose or 30 mM D-glucose with or without CDC, 10 M (6 -8 plates per condition).After 24 hr, the cells were used for assessment of total and phosphorylated p38 MAPK, ERK, and SAPK/JNK.

Statistical Analysis
The results are expressed as Mean ± SEM.Data were subjected to equality of variance F test, and then to log transformation, if necessary, before one-way analysis of variance.Where overall significance (p < 0.05) was attained, individual between group comparisons for multiple groups were made using the Student-Newman-Keuls multiple range test.When between-group variance differences could not be normalized by log transformation (datasets for body weights and plasma glucose), the data were analyzed by the nonparametric Kruskal-Wallis oneway analysis of variance, followed by the Bonferroni/ Dunn test for multiple comparisons.Individual pair-wise comparisons in experiments 3 and 4 were made using the unpaired two-tailed Student's t-test or Mann-Whitney rank sum test where appropriate.Significance was defined at p < 0.05.

Body Weights and Blood Glucose Concentrations
The initial (prior to streptozotocin administration) body weights were similar in all experimental groups (Table 1).Weight gain during the 16-wk study was lower in both untreated and CDC-treated diabetic mice than in the non-diabetic control group.CDC treatment did not affect weight gain in either control or diabetic mice.Initial (after streptozotocin administration) non-fasting blood glucose concentrations were 2.0-fold and 1.9-fold higher in untreated and CDC-treated diabetic mice than in the control group.Hyperglycemia progressed with the prolongation of diabetes, and the differences between final blood glucose concentrations in both diabetic groups and non-diabetic controls exceeded 3-fold.CDC treatment did not affect non-fasting blood glucose concentrations in either non-diabetic or diabetic mice.1(a) and (b)) and spinal cord (Figures 1(d) and (e)) 12/15-lipoxygenase expression was increased by 40% and 57%, respectively, in diabetic mice compared with controls.CDC treatment did not affect sciatic nerve and spinal cord 12/15-lipoxygenase expression in either control or diabetic mice.Sciatic nerve (Figure 1(c)) and spinal cord (Figure 1(f)) 12(S)HETE concentrations, a measure of 12/15-lipoxygenase activity, were increased by 223% and 48%, respectively, in diabetic mice compared with controls.CDC treatment blunted diabetes-associated sciatic nerve and spinal cord 12(S)HETE accumulation.

DISCUSSION
The findings described herein indicate that pharmacological inhibition of 12/15-lipoxygenase suppresses diabetes-induced excessive p38 MAPK and ERK, but not SAPK/JNK, phosphorylation in mouse sciatic nerve.Furthermore, 12/15-lipoxygenase inhibition blunts high glucose-induced p38 MAPK and ERK, but not SAPK/ JNK, phosphorylation in human Schwann cells, thus suggesting the existence of the similar relationship between 12/15-lipoxygenase and MAPK in diabetic peripheral neuropathy in humans.
Evidence for the important role of the enzymes of arachidonic acid metabolism, cyclooxygenase-2 (COX-2, [23,39]) and 12/15-lipoxygenase [24,25], in functional, morphological, and biochemical abnormalities in diabetic peripheral neuropathy is emerging.Increased activity of COX-2 was implicated in motor nerve conduction velocity and sensory nerve conduction velocity deficits, oxidative stress, and inflammation associated with experimental diabetic peripheral neuropathy [23,39], as well as in diabetic cardiac autonomic neuropathy and left ventricular dysfunction [40].In our previous studies [24,25], increased activity of 12/15-lipoxygenase was identified as an important contributor to diabetes-induced motor and sensory nerve conduction slowing, thermal and mechanical hypoalgesia, axonal atrophy of large myelinated fibers, and oxidative-nitrosative stress in peripheral nerve and spinal cord.Despite its clear role in oxidativenitrosative stress, 12/15-lipoxygenase activation was not involved in diabetes-associated reduction in intraepidermal nerve fiber density [24,25].This makes exploration of the protective and pathobiochemical processes triggered through 12/15-lipoxygenase in tissue-sites for diabetic peripheral neuropathy particularly interesting.
In conclusion, the present pharmacological study dissects the role for12/15-lipoxygenase in diabetes-and high glucose-induced MAPK activation in tissue-sites for diabetic peripheral neuropathy and human Schwann cells.They support interaction between the two mechanisms in the peripheral nerve, but not in spinal cord.The findings are relevant to understanding the mechanisms of diabetic peripheral neuropathy in humans, and suggest, that together with MAPK inhibitors, 12/15-lipoxygenase inhibitors can be used as pharmacological tool for inhibiting excessive MAPK phosphorylation in experimental, and, potentially, future clinical studies.In addition, our findings suggest that the 12/15-lipoxygenase inhibitor CDC can be used for dissection of the pathobiochemical mechanisms triggered by leukocyte-type 12/15-lipoxygenase, because the biochemical effects of CDC closely mimic those of leukocyte-type 12/15-lipoxygenase gene deficiency.

Table 1 .
, Initial and final body weights and blood glucose concentrations in control and diabetic mice maintained with and without CDC inhibitor treatment.