Intestinal Ischemia-reperfusion Injury In Lymph Cell Culture And Polyunsaturated Fatty Acids Intervention Study

Background:Intestinal ischemia-reperfusion(I/R) injury is the common pathway of shock. In the stress situation of severe trauma, surgery, due to the redistribution of blood, it will cause intestinal I/R damage, which leds to the distant tissue damage, organ failure and even death. In recent years, studies show that intestinal lymph is the important connection between visceral I/R and organ damage."Gut-lymph pathway"plays an important role in intestinal I/R injury causing distant tissue damage. However, the special factors in the lymph have not been identified.Objective:In this study, the intestinal lymph during rats intestinal I/R injury in vitro culture monocyte-macrophage, as well as ω-3PUFAs stimulated. To further identify the active factors in the intestinal I/R injury which cause distant tissue damage and inflammation. To explore the intervention of ω-3PUFAs.Methods:48healthy Sprague-Dawley (SD) male rats (SPF grade) were randomly divided into2groups with24rats in each group:normal drainage (N+D) group and ischemia-reperfusion+draining (I/R+D) group. The rats in (I/R+D) group subjected to60min ischemia by clapping the superior mesenteric artery, followed by120min reperfusion, draining lymph180min; The rats in (N+D) were just drained lymph180min. The two groups of lymph made into the stimulated culture medium. Each group was further divided into7sub-groups:untreated group (A1, B1), removal protein group (A2, B2), removal endotoxin group (A3, B3), removal protein+endotoxin group (A4, B4), EPA+untreated lymph group (A5, B5), DHA+untreated lymph group (A6, B6), DHA+EPA+untreated lymph group (A7, B7). Detect the concentration of endotoxin, TC, TG, HDL-C, MCP-1, MIP-2, TLR4and HMGB1in serum and lymph; Detect TNF-a, IL-1β, IL-6, sICAM-1, MCP-1, MIP-2, HMGB1of monocyte-macrophage cultured by intestinal lymph and co-3PUFAs; Measure the expression of TLR4, HMGB,NF-κB, MCP-1, MIP-2.Results:The endotoxin, MCP-1、MIP-2、HMGB1、TLR4in the serum and lymph of the I/R+D group were significantly higher than the N+D group after intestinal ischemia for60mins and reperfusion for120min. The level of TC, TG, HDL-C in serum were significantly higher than those in the N+D group (p<0.05). The levels of TNF-α, IL-1β, IL-6, sICAM-1, TLR4, HMGB1and NF-κB were significantly increased after culture monocyte-macrophage cells stimulated by intestinal I/R lymph; The levels of those factors were significantly decreased after removing the protein in intestinal I/R lymph; Added EPA, DHA and EPA+DHA to the culture could significantly reduce the above factors’levels (p<0.05); Stimulated the cells with EPA and EPA+DHA could significantly decrease the expression of TLR4、NF-κB、MCP-1、 MIP-2(p<0.05); The effect of EPA+DHA could be better(p<0.05).Conclusions:Intestinal I/R injury could increase the level of HMGB1, endotoxin, cytokine in the serum and lymph significantly; The lymph after I/R injury in vitro could elevate inflammatory cytokines, chemotacticfactor and the expression of TLR4and HMGB1, NF-κB which indicated that the lymph after intestinal I/R injury in vitro can cause inflammation; Removing the protein could significantly reduce the above active substances. Removal the protein may be a more effective way to use; ω-3PUFAs can significantly reduce the inflammatory factors and the expression of TLR4and NF-κB in vitro. It indicated that co-3PUFAs in vitro could inhibit the inflammatory reaction, possibly through the inhibition of TLR4and NF-κB.