Role of ulinastatin in protecting the injured intestinal tract of septic rats via inhibiting TLR4/NF-κB inflammatory signaling pathway
Objective To explore the protective role of different doses of ulinastatin(UTI)in injured intestinal tract and the influence on TLR4/NF-κB inflammatory signaling pathway in septic rats.Methods Forty SPF healthy male SD rats were randomly divided into sham-operation group,sepsis group,low-dose UTI group,and high-dose UTI group,with 10 rats in each group.Septic rats models were prepared by cecum ligation and perforation method in sepsis group,low-dose UTI group and high-dose UTI group,and cecum exploratory surgery was performed after openning the abdomen in sham-operation group.Within 24 h after modeling,low-and high-dose UTI groups were given respectively intraperitoneal injections of 50 000 u/kg and 100 000 u/kg of UTI every 6 h,and sepsis and sham-operation groups were given an equivalent volume of normal saline intraperitoneally.The abdominal aortic blood was collected after anesthesia.ELISA technique was used to detect the serum levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),intestinal fatty acid binding protein(I-FABP)and diamine oxidase(DAO).The rats were executed to obtain the small intestinal tissues.The histopathological changes were observed by HE staining,and Chiu's scoring was done to evaluate the intestinal mucosal injury.Immunohistochemical staining was used to detect the percentages of positive areas of claudin-1 and occludin,and the percentages of positive cells of TLR4 and MyD88.The percentage of p-NF-κB p65 positive cells was detected by immunofluorescence.The relative expressions of claudin-1,occludin,TLR4,MyD88 and p-NF-κB p65 proteins were detected by Western blot in the small intestinal tissues of rats in each group.Results(1)The serum levels of TNF-α,IL-6,I-FABP and DAO were higher in sepsis group and low-and high-dose UTI groups than those in sham-operation group(P＜0.05),higher in sepsis group than those in low-and high-dose UTI groups(P＜0.05),and higher in low-dose UTI group than those in high-dose UTI group(P＜0.05).(2)The Chiu's score was higher in sepsis group[5(5,5)],low-dose UTI group[3(3,3)]and high-dose UTI group[2(2,2)]than that in sham-operation group(0)(P＜0.05),higher in sepsis group than that in low-and high-dose UTI groups(P＜0.05),and higher in low-dose UTI group than that in high-dose UTI group(P＜0.05).(3)The percentages of TLR4,MyD88 and p-NF-κB p65 positive cells and proteins relative expressions were higher in sepsis group and low-and high-dose UTI groups than those in sham-operation group(P＜0.05),higher in sepsis group than those in low-and high-dose UTI groups(P＜0.05),and higher in low-dose UTI group than those in high-dose UTI group(P＜0.05).(4)The percentages of positive areas and proteins relative expressions of claudin-1 and occludin were lower in sepsis group and low-and high-dose UTI groups than those in sham-operation group(P＜0.05),lower in sepsis group than those in low-and high-dose UTI groups(P＜0.05),and lower in low-dose UTI group than those in high-dose UTI group(P＜0.05).Conclusion UTI can dose-dependently repair the injured intestinal mucosal barrier in septic rats probably by suppressing the inflammatory response and indirectly up-regulating the expressions of intestinal tight junction proteins claudin-1 and occludin via inhibiting the activation of the TLR4/NF-κB inflammatory signaling pathway.
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