The Study To The Ocular Anti-inflammatory Effect Of Alpha-melanocyte Hormone To Animals After Extracapsule Lens Extraction

α-Melanocyte-stimulating hormone (a-MSH) is a basic tricapeptide whose amino acid sequence is identical to the 1-13 (N-terminal) amino acid sequence of adrenocorticotrophic hormone (ACTH). Pro-opio-melanocortin (POMC), found in the pituitary, brain, inflammatory cells, skin, and at other sites, is the precursor of ACTH. MSH (α-, β- and γ-MSH), lipotrophins, and endorphins, which constitute a family of biologically active peptides termed melanocortins. MSH, named for its effect on pigmentation in amphibian skin, is widely known as an anti-inflammatory and antipyretic agent with greater potency than paracetamol. The peptide inhibits chronic and acute inflammation mediated by various cytokines and alleviates allergic reactions. The anti-inflammatory effect of MSH takes place through a central and peripheral mechanisms and is related to its ability to counteract the effects and the production of pro-inflammatory cytokines such as nitric oxide, tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1). In addition, the chemotactic activity of macrophages and neutrophils is inhibited by these peptides. Studies conducted twodecades ago showed that a-MSH mediated changes in the blood-aqueous barrier (BAB) and in intraocular pressure. In these studies crude extracts of the peptide were used in high nonphysiological doses. Recently a-MSH was detected in the normal aqueous of humans, mice, and rabbits and was demonstrated to play a role in activation of regulatory T cells in the aqueous. It also modulates the melanocyte response in the retina-choroid. Despite the fact that a-MSH takes part in anterior segment immune reactions and BAB permeability changes, its role in ocular inflammation has only been studied in one reseach. In this reseach, rabbits received a nonperforating corneo-limbal cut, followed by paracentesis. Treatment involved topical or intramuscular a-MSH, topical steroids or saline. In a-MSH- and steroid-treated eyes the protein and cells in the aqueous was counted 24 hours after surgery. The results demonstrated that a-MSH reduced the clinical signs of ocular inflammation, curtailed blood-aqueous barrier (BAB) disruption, and reduced aqueous inflammatory cell number. The efficacy of a-MSH applied systemically or topically was similar to that of steroids in reducing clinical signs of trauma, but the efficacy of a-MSH in maintaining BAB integrity surpassed that of steroids. The authors suggested that the melanocortins might be a useful anti-inflammatory agents in ocular trauma. However, this reseach was limited, because the trauma was slight compared with cataract surgery or severe ocular trauma;the whole course was only 24 hours while the anti-inflammation course after surgery or severe trauma was several weeks or months;the potential mechanism including celluar and molecular level has not been studied in this reseach. So, in the first part of our study the long term anti-inflammatory activity of a-MSH in extracapsule lens extraction induced-ocular inflammation was investigated using physiological doses administered topically and their effect was compared to that of topical dexamethasone and diclofenac sodium. And in the second part of the study, the experiments in molecular level were carried out to investigate the potentialmechanism of the ocular anti-inflammatory effect of a-MSH.Part I : The study to the long term ocular anti-inflammatory effect of topical alpha-melanocyte hormone to rabbits after extracapsule lensextractionObjectiveTo evaluate the anti-inflammatory effects of alpha-melanocyte stimulating hormone (a-MSH) on the ocular inflammation in pigmented rabbit eyes, by comparing the flare and cell number in aqueous humor (AqH), and ocular signs including intraocular pressure (IOP) with dexamethasone and diclofenac sodium for 4 weeks after extracapsule lens extraction (ECLE). MethodsPigmented rabbit eyes after ECLE were threated by 10"8M a -MSH or 0.1% dexamethasone sodium phosphate or 0.1% diclofenac sodium q.i.d topically for 4 weeks. The value of flare and the number of cells in AqH was determined by laser flare cell meter at 1 day before surgery and 1 day, 3 days, 1 week, 2 weeks , 3 weeks and 4 weeks after surgery. The IOP of the rabbit eyes were measured every week after surgery. ResultsThere was no significant difference in the flare between a-MSH or dexamethasone or diclofenac treated rabbit eyes after surgery in following-up (P>0.05). However, the inhibitory effect to infiltrating cell in AqH of a-MSH was almost twice as good as dexamethasone and diclofenac at 3 days, 1 week and 2 weeks after surgery (PO.005). The IOP of dexamethasone treated rabbit eye was signigicantly higher than a -MSH or diclofenac treated eye at 3 weeks or 4 weeks after surgery (P<0.05), and there was no difference in IOP between a-MSH anddiclofenac treated eye in the whole course of following up (P>0.05). ConclusionThe results suggest that a-MSH can reduce ocular inflammation in eyes after ECLE effectively without side effects including adverse influence to IOP, and its efficacy of inhibiting infiltrated cells into AqH efficacy surpassed that of dexamethasone and diclofenac .Part n : The study to the molecular mechanism of ocular anti-inflammatory effect of alpha-melanocyte hormone to rats afterextracapsule lens extractionObjectiveTo investigate the potential mechanism of anti-inflammatory effects of alpha-melanocyte stimulating hormone (a-MSH) on the ocular inflammation in SD rats eyes, by comparing the mRNA expressions of tumor necrosis factor-alpha (TNF-a) and interleukin 6 (IL-6) and the number of activated NF-kappa B-positive cells in the iris/ciliary body in surgery/a-MSH group with surgery/saline group 6 hours after extracapsule lens extraction (ECLE). MethodsSD rat eyes were treated by intraveinous injection of a-MSH (1000 u g/kg) or saline immediately after ECLE. The isolated eye balls or iris/cilliary body were obtained 6 hours after surgery. mRNA expressions of TNF-a and IL-6 in the iris/ciliary body were measured by using a semiquantitative polymerasechain-reaction method. Immunohistochemical staining with a monoclonal antibody against activated NF-kappa B was performed to evaluate the effect of a-MSH on NF-kappaB activation. ResultsThe ICB at 6 hours after ECLE exhibited increased expression of TNF-a and IL-6 mRNAs, which was significantly decreased after a-MSH treatment(P<0.01). The number of activated NF-kappaB-positive cells in the ICB was reduced significantly by the a-MSH treatment (PO.01). ConclusionThese results demonstrated that a-MSH can suppress the mRNA expressions of TNF-a and IL-6 and inhibit the activation of NF-kappa B in the cell of iris/ciliary body, which suggested that the potential mechanism of the ocular anti-inflammatory effect is to downregulate proinflammatory cytokine expression and inhibite the NF-kappaB-dependent signaling pathway.