Neuropeptide FF Study In Diabetic Corneal Lesions And Molecular Mechanisms Of Action

Purpose:Diabetes mellitus is a major cause of peripheral nerve damage and can decrease the release of neuropeptides in neurons. Using an mRNA real-time PCR array, we found that NPFF significantly decreased in trigeminal ganglion (TG) tissues of type2diabetic BKS-db/db mice compared with control mice. Then, we investigated the role of NPFF in recovery from peripheral nerve injury in experimental diabetic neuropathy in vitro using cultured TG neurons and in vivo following corneal injury healing responses.Methods:Animal experimentsDiabetic BKS-db/db and nondiabetic control db/+were used in this study. Tear secretion was evaluated with phenol red-impregnated cotton threads.and an esthesiometer was used to determine corneal sensation. Corneal nerve staining was performed with anti-neuron-specific beta III tubulin antibody. NPFF levels in TG tissues of BKS-db/db and nondiabetic control db/+mice were determined by ELISA.The mice were randomly divided into five groups: untreated control db/+mice, untreated BKS-db/db mice, saline-treated BKS-db/db mice, NPFF-treated BKS-db/db mice, NPFF-treated control db/+mice. The in vivo corneal injury model was made. For groups3to5,10μl saline or100μM recombinant mouse NPFF was injected into the subconjunctival site of the right eye on the same day as corneal epithelium injury. The mice were assessed using fluorescein sodium and photographed at0,24,48, and72hours.Cell culturePrimary TG neuronal cells were cultured as described previously. TKE2cell line was maintained in keratinocyte serum-free medium. TG neurons and TKE2cells were maintained in their respective media containing5mM D-glucose (normal glucose [NG]),25mM D-glucose (high glucose [HG]), or5mmol/1D-glucose plus20mmol/1mannitol (high mannitol,[HM]). In addition, TG neurons and TKE2cells were treated with10or100μM NPFF in HG medium. Treatment with NG was used as a control. The viability of these cells was quantified with a Live/Dead Viability-Cytotoxicity Kit according to the manufacturer’s protocol. The blots were probed with the following primary antibodies: AKT1, phospho-AKT, phospho-p44/42MAPK (Erkl/2), and p44/42MAPK (Erkl/2).Results:BKS-db/db mice had significantly less tears than the control db/+mice. The mean overall corneal sensitivity of the BKS-db/db mice and control db/+mice was5.4±0.04cm and5.8±0.07cm, respectively. Corneal sensitivity was significantly reduced in BKS-db/db mice compared to control db/+mice. The expression of NPFF protein was also reduced in TG tissues of the BKS-db/db mice compared with that of non-diabetic control db/+mice (p<0.05). We found NPFF can enhance neurite elongation in isolated TG neurons and overcome the detrimental corneal wound healing effects of hyperglycemia by stimulating corneal nerve regeneration. More importantly, the recovery effect of NPFF on corneal nerve injury is associated with functional recovery of corneal sensation in hyperglycemic conditions. For corneal epithelial wound healing in the presence of high glucose, exogenous expression of NPFF activates the ERK1/2pathway, which is responsible for cell proliferation and division.Conclusions:24-week BKS-db/db mice expressed typical characteristic of diabetic keratopathy, which may be used as a good model to further study this disease. NPFF can enhance neurite elongation in isolated TG neurons and overcome the detrimental corneal wound healing effects of hyperglycemia by stimulating corneal nerve regeneration.These results suggest that NPFF is a potential neuroregenerative factor for peripheral nerve injury in experimental diabetic neuropathy.