The Anti-inflammatory Effect And Mechanism Of α-melanocyte-stimulating Hormone In Aspergillus Fumigatus Keratitis

Purpose:To investigate the anti-inflammatory effect and mechanism of α-melanocyte stimulating hormone(α-MSH)in Aspergillus Fumigatus keratitis.Method:1.The cornea of C57BL/6 mice infected with Aspergillus Fumigatus was established.α-MSH(1x10-4mmol/ml)was given by subconjunctival injection at 1 day post infection(p.i.).The same volume PBS was applied to control group in a same way.After 3 days infection,the photo of corneas was taken by a slit lamp.2.PCR was used to evaluate the m RNA level of IL-1β、TNF-α、IL-6、MIP-2、iNOS、LOX-1 and Dectin-1;Western blot was used to detect the protein level of IL-1β、TNF-α、IL-6 and Dectin-1;the number of neutrophils and macrophage was tested by immunofluorescence staining.3.The RAW264.7 cell was pretreated with α-MSH(10n M、100n M、1000n M)for 24 h,and then tested by CCK8 kits.4.The RAW264.7 cells was pretreated by different concentration of α-MSH(10nM、100n M、1000n M)for 30 minutes,then stimulated with inactivated mycelium of Aspergillus Fumigatus for 8h and 24 h.PCR was used to evaluate the m RNA level of IL-1β、TNF-α、IL-6、MIP-2、iNOS、LOX-1 and Dectin-1;Western blot was used to detect the protein level of IL-1β、TNF-α、IL-6 and Dectin-1.5.Western blot was also used to detect the MPKA protein level including Tatal-JNK(T-JNK)、Phosphorylated-JNK(P-JNK)、Tatal-ERK(T-ERK)and Phosphorylated-ERK(P-ERK).Results:1.The inflammatory response was alleviated and clinical score was decreased in the group of α-MSH combined with Aspergillus Fumigatus group compared to PBS group.2.α-MSH reduced the mRNA level of IL-1β、TNF-α、IL-6、MIP-2、LOX-1、the i NOS and Dectin-1,α-MSH reduced the protein level of IL-1β、TNF-α、IL-6 and Dectin-1.3.The number of neutrophils and macrophage was reduced apparently compared to PBS group.4.α-MSH reduced the m RNA level of IL-1β、TNF-α、IL-6、MIP-2、LOX-1,the i NOS and Dectin-1 down regulated when the concentration of α-MSH were 10 n M and 100 n M.However,the concentration of 1000 n M had no differences in α-MSH combined with Aspergillus Fumigatus group and PBS group.5.The ratio of P-JNK/T-JNK and P-ERK/T-ERK was down regulated in α-MSH combined with Aspergillus Fumigatus group compared to PBS group.Conclusion:α-MSH could alleviate the severity of Aspergillus Fumigatus keratitis in mice,and the migration of neutrophils and macrophage to corneal stroma was restrained.α-MSH could relieved the inflammatory response to Aspergillus Fumigatus infection by the JNK/ERK pathway.