Wound healing in FAT-1 mice: involvement of the cholinergic anti-inflammatory pathway

Author(s)

Thamiris Candreva

• Document type: Master's Dissertation
• Press: Limeira, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Aplicadas
• Defense date: 2017-11-17

Examining board members

Hosana Gomes Rodrigues; Maria Cláudia Gonçalves de Oliveira; Eliana Pereira de Araujo

• Advisor: Hosana Gomes Rodrigues

Abstract

Inflammation is the first stage of the wound healing process and can be divided into initial and late inflammatory response. The late inflammatory response is essential for the control of pro-inflammatory mediators and resolution of inflammation, and is decisive at the beginning of the next phase of tissue formation or proliferation. Polyunsaturated fatty acids of 'omega'-6 and'omega'-3 families, modulate the inflammatory profile, being increasingly studied in the tissue repair process. Thus, to understand the role of these fatty acids in the late inflammatory phase of the wound healing process, we used FAT-1 transgenic mice capable of endogenously producing 'omega'-3 fatty acids from 'omega'-6. To verify the lipid composition of these mice, we evaluated by gas chromatography, plasma and scar tissue 3 days after wound induction. In the plasma, transgenic mice reduced arachidonic acid (AA) concentration and increased the concentration of alpha-linolenic acid (ALA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids, reducing the ù-6/ù-3 ratio in 32,9%, when compared to the wild type (WT) group. In the scar tissue, these mice had a reduction in the percentage of AA in the lipidic tissue classes of phosphatidylcholine (PC) and phosphatidylalonamine (PE) and, increase in the fatty acids docosapentaenoic acid (DPA) and DHA in the lipidic tissue classes of PC, PE and triacylglycerol (TAG). Thus, the transgenic mice obtained a reduction of 'omega'-6/'omega'-3 ratio, equivalent to 56,4% in PC, 55,1% in PE and 39,1% in TAG, in relation to the WT group. In the analysis of wound closure by the Image J program, FAT-1 mice maintained the major wound area from the 1st day after induction, and on the last day of analysis (14th day), the area of the wound was two times greater than that of the WT group (p<0.01). In the analysis of myeloperoxidase activity (MPO) by spectrophotometry, the transgenic mice showed increased activity in the cicatricial tissue of 3 days, when compared to the WT group (p<0.05). Considering this same time of wound for all other analyzes, we observed through PCR real time, that FAT-1 mice increased the gene expression of CD206 and CD280 cell markers (p<0.05), representative of M2 macrophages. However, by the ELISA method, these mice were found to increase the production of mediators: IL-6 (p<0.05), CXCL1 (p<0.05), CXCL2 (p<0.01), TIMP-1 (p<0.01) and VEGF (p<0.05) and reduced IL-10 (p<0.05), indicating a modulation of the late inflammatory response. In order, to investigate whether the cholinergic anti-inflammatory pathway was influencing this response, we analyzed the production of acetylcholine (ACh) and free choline by colorimetric method, acetylcholinesterase activity (AChE) by spectrophotometry and expression of 'alpha'7nAChR, STAT3 and pSTAT3 proteins, by western blotting. FAT-1 mice increased the protein expression of 'alpha'7nAChR (p<0.001) and reduced pSTAT3 (p<0.05), exhibiting impairment of the propagation of the anti-inflammatory signal, in the intracellular environment. To analyze the impact of this late inflammation exacerbated, we analyzed by PCR array, the expression of genes present throughout the wound healing process. In FAT-1 group, genes essential for the formation and maintenance of the extracellular matrix (ECM) were negatively modulated. Thus, we concluded that the non-control of the late inflammatory response by the FAT-1 mice, may have impaired the ECM functions and contributed to delayed tissue repair (AU)

• FAPESP's process: 16/02021-3 - Wound healing in FAT -1 mice: involvement of anti- inflammatory cholinergic pathway
• Grantee: Thamiris Candreva Robles
• Support Opportunities: Scholarships in Brazil - Master