Intervention Effect Of Ulinastatin On Immune Dysfunction Post Scald Injury

Objective:Sepsis is a main complication and primary cause of death in severely burnedpatients. Immune function obstacle closely related with the development of sepsis.Excessive inflammation play important roles in the development of immunedysfunction. Ulinastatin has been used to combat excessively systemicinflamation in clinical treatment of burn and tramatic injury. However, its effectson immune dysfunction post burn are still unveiled. This project was aimed toinvestigate the effects of ulinastatin on immune dysfunction in animal experimentand clinical study as following.Animal experiment: Scald rat model was used to observe the effects ofulinastatin on spleen expression of high mobility group box-1protein (HMGB1)and percentage of regulatory T cells (CD4+CD25+) in peripheral blood, to explorethe function polarization effects and possible mechanisms of ulinastatin oneffector T cells, and to obseve the improvement of the antigen presentationfunction of monocytes.Clinical study: Severely burned patients were dynamically monitored todefine the changes of Treg cells proportion in peripheral blood, to investigate theinfluence of ulinastatin on Treg cells proportion in peripheral blood and itspossible mechanisms, to observe the function polarization status of effector Tcells in burned patients and the effect of ulinastatin on such status of effector Tcells, and to observe the improvement of the antigen presentation function ofmonocytes.Methods:Animal experiment: A total of120Wistar rats were randomly divided intothree groups: sham scald(S) group, scald (B) group and scald+ulinastatin (BU) group (n=40). The latter two groups were subjected to30%TBSA full-thicknessscald on back, immediately followed by intraperitoneal injection of Ringer’ssolution (40ml/kg) in B group or ulinastatin (4×10~4U/kg) and Ringer’s solution(40ml/kg) in BU group respectively, and sterilization of wound with iodine. Therats in S Group were sham scalded at37℃for12s. On1d,3d,5d,7d after scaldor sham scald, the rats in S group and B group received intraperitoneal injectionof4mL Ringer’s solution at each time point, and the rats in BU group receivedintraperitoneal injection of4×10~4U/kg ulinastatin and4mL Ringer’s solution.The rats were sacrificed for collecting arterial blood and spleen at1d,3d,5d,7d,14d after scald or sham scald. HMGB1expression was analyzed in splenic tissueby western blotting. Percentage of Treg cells and expression of Ia antigen inperipheral blood were determined by flow cytometry, serum IL-4and IFN-γconcentration by ELISA.Clinical study: Thirty severely burned patients (burn over30%TBSA) wererandomly divided into two groups: conventional treatment group (13cases), andulinastatin+conventional treatment group (17cases). Conventional treatmentincluded fluid resuscitation, use of antibiotics, wound dressing change, scarexcision, skin transplantation, nutrition support, etc. Patients in ulinasstatintreatment group received both the conventional treatment methods and i.v.injection of80×10~4U ulinasstatin in0.9%normal saline twice a day for7days.Blood samples were taken from peripheral venous before treatment and at1d,3d,5d,7d, and14d after treatment respectively. Venous blood form20healthy caseswere used as control. Percentage of Treg cells in peripheral blood and HLA-DRexpression on CD14+monocytes were determined by flow cytometry, serumlevels of IL-4and IFN-γ by ELISA.Results: Animal experiment:1. Compared with that in S group, HMGB1expressionin B group elevated significantly (P<0.01) concerning each time point, andpeaked on day3post injury. HMGB1expression in BU group was markedlylower than that in B group concerning each time point (P<0.01), and it reached thelowest level on day5post injury.2. Compared with that in S group, the percentage of Treg cells graduallyincreased and peaked on day3post injury and lasted to the last time point. Thepercentage of Treg cells in BU group was obviously lower than that in B group.3. Serum concentration of IFN-γ in B group were decreased comparing withS group from1d through7d post injury, while IL-4level were higher than that inS group. Th2polarization shifting in Th1/Th2ratio occurred in B group. Afterulinastatin treatment, the serum level of IL-4decreased obviously on day1, day2,and day5post injury. However the serum level of IFN-γ increased notably fromday1to day7.4. The expression of Ia antigen on monycytes maintained a higher level on1d-14d in S group, decreased obviously in B group. There was a slow increasefrom day1post burn in B group, but was still lower than that in S group duringthe experimental period. The Ia expression was increased in BU group comparingwith that in B group.Clinical study:1. The percentage of Treg cells in peripheral blood increasedimmediately in the burned patients group, higher than that in the healthy controlgroup during all the time points. Compared with that in conventional treatmentgroup, the percentage of Treg cells decreased in ulinastatin treatment group.2. The serum concentrations of IL-4and IFN-γ were relatively low in healthypersons, but the serum level of IL-4increased and the serum level of IFN-γdecreased obviously in the conventional treatment group. However, in the ulinastatin+conventional treatment group, the serum level of IL-4decreased, theserum level of IFN-γ increased obviously comparing with that in conventionaltreatment group at1d-5d post injury.3. Compared with that in the healthy persons, the expression level of HLA-DR on CD14+monocytes from peripheral blood decreased during all the timepoints. In the ulinastatin+conventional treatment group, HLA-DR expression onthe CD14+monocytes increased especially on day14, compared to that inconventional treatment group.Conclusions:It is concluded that, after severe burn, the expression of HMGB1increaseslately and durably, the percentage of Treg cells in peripheral blood increased and Th2cells direction shifting from T lymphocyte in peripheral blood, the abnormalsecretion of IFN-γ and IL-4, and decreased expression of Ia or HLA-DR onmonocytes leading to abnormal antigen-presenting function. As one kind of anti-inflammatory drug, ulinastatin can markedly reduce HMGB1expression, whichmight inhibit maturation of Treg cells, then improve the function polarizationstatus of effector T cells after severe burn.