Function And Mechanism Studies Of Immune-related Peptide ZY12 Treated In Allergic Conjunctivities

Allergic conjunctivitis(AC)is a common disease in the eye,often causes eye redness,itching and other symptoms,even leads to blindness.It has brought a lot of inconvenience to daily life of people.Staphylococcal enterotoxin B(SEB)plays an important role in allergic conjunctivitis.Drugs for treatment of allergic conjunctivitis include antihistamines,mast cell stabilizer,nonsteroidal anti-inflammatory drugs,vasoconstrictor,glucocorticoids,etc.In spite of the diverse choices of drugs,none of them can completely inhibit the development of this allergic disease.So new types of allergy medications need to be further developed.Our laboratory synthesized an antimicrobial peptide ZY12 which can effectively treat conjunctivitis.Antibacterial peptides are important components of the innate immune.Antimicrobial activity of antimicrobial peptides are broad spectrum.These peptides are generally small and most of them are thermal stability.They play an important role in antibacterial,insect-resistant and immune suppression.ZY12 had a good antibacterial effect on Staphylococcus aureus.At the same time,it inhibited lymphocyte proliferation caused by Staphylococcus aureus enterotoxin B.Hemolytic and cell toxicity has important implications for pharmaceutical research.At a concentration of 800 ?g/ml,ZY12 only induced a hemolysis rate of 1.32%.MTT experiment proved that ZY12 had no cytotoxicity on mice macrophages and human kidney epithelial cells.We also determined the acute toxicity of ZY12.When the dose reached at 20 mg/kg,ZY12 had no effect on mice.With the increasing of the doses,the mice appeared tiredness,blepharochalasis,convulsions and death.Its LD50 is 94.05 mg/kg.Because of its good effect on cells and animal models and the low toxicity,ZY12 can be further studied as a new candidate drug.By constructing the model of mice AC,the study shows that ZY12 can also be effective in relieveing the AC symptoms in vivo in a concentration dependent manner.Because the mast cells play an important role in the progress of AC,so this is also investigate the number of total MC and degranulated MC of conjunctival tissue and the therapeutic effect of ZY12 on AC by tissue slice.Compared with the vehicle group,the number of total MC and degranulated MC of the ZY12 treatment groups significantly decreased.Degranulated MC could promote the secretion of cytokines in Th2 lymphocyte,such as interleukin-4(IL-4),interleukin-5(IL-5)and interleukin-13(IL-13).ZY12 can reduce the level of cytokines in Th2 lymphocyte by enzyme-linked immunosorbent assay(ELISA).By the above experiments proved that the ZY12 can effectively treat alleviate conjunctivitis.In order to further study the therapeutic mechanism,we used the experimental method of western blot.Western Blot assay showed that ZY12 could reduce the expression of Toll receptor 2(TLR2),inhibit the phosphorylation of ERK,and has no effect on phosphorylation of Junk and p38 in the whole conjunctivitis tissuse.In summary,our study indicated that ZY12 can relieve the AC symptoms by inhibiting the phosphorylation of ERK and reducing the number of total MC and degranulated MC.This study provides a scientific basis for ZY12 in the treatment of AC.