Role Of Bruton's Tyrosine Kinase In The Burn Sepsis-induced Intestinal Injury Of Mice

1.Backgrounds Sepsis is one of the most common clinical critical illnesses.It is an imbalance of the body's response to infection,which causes life-threatening organ damage.Due to the imbalance of the host's immune response,the body attempts to eliminate invasive microorganisms,so the release of a large number of inflammatory mediators leads to systemic inflammatory response syndrome(SIRS),which continues to develop into multiple organ dysfunction syndrome(MODS)and even threaten one's life.Due to the presence of extensive necrotic tissue and the invasion of pathogenic microorganisms in patients with severe burns,the pro-inflammatory cytokine of the body expression increases,thereby releasing a large number of inflammatory mediators,triggering a systemic inflammatory response,also causing organ damage and sepsis at the same time.It is the most common cause of death in patients with large body surface area deep burns.2.Objectives To observe the activation of Bruton's tyrosine kinase(Btk)in the intestinal tissue of burn sepsis mice,we use Btk specific inhibitor(LFM-A13)to intervene so as to we could explore the role and molecular mechanism of Btk signaling pathway in intestinal injury of burn sepsis.3.Methods In the experiment,80 male C57 mice were randomly divided into 4 groups.There were8 in the sham operation group(SHAM group),8 in the burned group(BURN group),32 in the burn + LPS group,called the burn sepsis group(SEP group),and burned +LPS+Btk specific inhibitor(LFM-A13)Group of 32 mice.All were prepared and anesthetized.BURN group,SEP group and LFM-A13 group mice were exposed towater bath(100 ?)for 15 seconds at constant temperature,and the scalded area was calculated to be 10%TBSA III°degree.LPS10mg/kg was injected intraperitoneally in order to prepare mouse sepsis model.In the LFM-A13 group,LFM-A13(10 mg / kg BW)was injected intraperitoneally before the mice burned.SHAM group,BURN group were killed immediately after injury,SEP group and LFM-A3 group were sacrificed after 0h,8h,12 h,24h,8 ratsls at each time point.After the mice were sacrificed,the small intestine tissues were quickly removed and frozen in liquid nitrogen,and the expressions of Btk total protein and phospho Btk protein in intestinal tissues were detected by Western blot.Observe and score the pathological changes of intestinal tissue under HE staining light microscope;TUNEL method to detect apoptosis and calculate the apoptotic index(AI);enzyme-linked immunosorbent assay(ELISA)to detect IL-4,IL-6 and TNF-? levels and myeloperoxidase(MPO)activity in serum and intestinal tissue.4.Results The results of Western results showed that the expression of Btk protein in the intestinal tissue of mice in the SHAM group and the 10% BURN group had no significant change;while in burned + LPS-induced burn sepsis(SEP)group,the expression level of Btk protein in mouse intestinal tissue also showed no significant change at different time points(0h,8h,12 h,24h).The same method was used to further detect the expression of phospho-Btk(phosphorylated Btk)protein in the intestinal tissue of each group of mice.The results showed that the expression level of p-Btk protein in the intestinal tissue of the SHAM group and the BURN group was also low,while the expression level of Btk protein also continued to increase over time,reaching a peak at 12 hours and then decreasing.The expression of p-Btk protein in the intestinal tissues of SEP group at 24 hours was also slightly lower than that at 12 hours.The p-Btk protein of LFM-A13 mice showed a continuous upward trend over time,reaching a peak at 12 h,butcompared p-Btk at 12 h and 24 h,The expression of 24 h was lower in intestine slightly.Compared with the SEP group,except for 0h,the p-Btk protein expression levels at the corresponding time points(8h,12 h,24h)were significantly reduced.In addition,HE staining results of pathological changes in the intestine of mice are as followed.No obvious pathological changes were seen in the BURN group and the SHAM group.The glands were densely packed,the epithelial cells were arranged in columns,the goblet cells were abundant,and the LPS-induced In the SEP group,there were obvious pathological changes.Intestinal epithelium was thinned,edema was obvious,part of the glands were atrophied,and goblet cells were reduced or disappeared.The degree of damage gradually increased with time.The LFM-A13 group was stained with HE In addition to the 0h time point,it can be seen that the pathological changes at the corresponding time point were significantly reduced;we also used the TUNEL method to detect the apoptosis of the intestinal epithelial cells and the apoptotic index,and the intestinal sections of the SHAM group were basically not seen apoptotic cells;a small number of apoptotic cells can be seen in the BURN group;many apoptotic cells can be seen in the SEP group,and the number of apoptotic cells continues to increase with time.Compared with the corresponding time point in the SEP group,the brown granules,that is,apoptotic cells were significantly reduced,and the apoptotic index(AI)was significantly decreased.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of inflammatory cytokines IL-4,IL-6,TNF-a and myeloperoxidase(MPO)activity in serum and intestinal tissues in each group.The expression levels of inflammatory factors were significantly higher than those in the other two groups.Compared with the SEP group,the levels of inflammatory indicators in serum at the corresponding time points were significantly reduced after burn in the LFM-A13 group except for 0h.Similarly,experimental results confirm that changes in the levels of inflammatory cytokines in intestinal tissues are basically consistent with changes intheir expression levels in serum.Similarly,the MPO activity of the intestinal tissue in the SEP group was significantly higher than that in the other two groups.Compared with the SEP group,the LPM-A13 group had significant differences in intestinal tissue MPO activity at the corresponding time points except that there was no significant difference at 0h after burn.5.Conclusion Btk signaling pathway plays an important regulatory role which mediates the production of inflammatory cytokines IL-4,IL-6 and TNF-a,participates in intestinal oxidative stress,mediates intestinal cell apoptosis,and develops intestinal injury in burn sepsis.