The Alteration Of Intestinal SIgA Level And The Altering Mechanisms In Ischemia/reperfusion Injury And The Effects Of N-3 PUFAs On The Injury

Intestinal ischemia/reperfusion(I/R)injury is particularly seen during trauma,hemorrhage and shock states,as well as in the course of sepsis and severe acute pancreatitis.As a result,the barrier function of bowel was disrupted followed with bacterial translocation,which is the main cause of systemic inflammatory response syndrome(SIRS)and multiple organs dysfunction syndrome(MODS).The significantly decreasing of intestinal sIgA level has been found in many clinical diseases complicating with intestinal I/R injury.SIgA is generated in gut associated lymphoid tissue(GALT),it can neutralize microbial toxins and pathogens using a high-affinity binding system,and prevent commensal bacteria from breaching the mucosal surface applying a low-affinity binding system.SIgA is the first line of immune protection at mucosal surfaces and promote the establishment of a sustainable immune homeostasis.However,the mechanisms resulted in the decrease of intestinal sIgA level remain unclear.Meanwhile,there is none definite treatment to avoid the decrease of intestinal sIgA level in I/R injury.n-3PUFAs,enriched in fish oils,are the essential fatty acids.Eicosapentaenoic acid(EPA)(C20:5),and docosahexaenoic acid(DHA)(C22:6)are the main components.Despite increasing knowledge of the beneficial effects of fish oils,such as the regulation of Th1/Th2 factors,little is known about the effect of n-3PUFAs on IgA-secreting cells during intestinal I/R injury.Here we study the alternation of both the intestinal sIgA level and the IgA-secreting cells during I/R injury.Simultaneously,we investigate the mechanisms of the alternation.Moreover,we hypothesized that n-3PUFAs have the protest effect against I/R injury.Therefore,the aim of this study is to test our hypothesis in a murine I/R injury model.Part Ⅰ The alternation of intestinal sIgA level and the potensial mechanismsResearch 1.1 To set up a murine intestinal I/R injury modelObjectives:To set up a murine intestinal I/R injury model and to define the suitable ischemic time.Methods:Thirty Balb/c mice were randomly assigned to 5 groups according to different ischemic time(15min、30min、45min、60min、90min)through occluding SMA.The definite ischemic time was defined by 5 days survival.Data were analyzed by Bivariate correlation and the significance level was accepted at P<0.05.Results:The mortality of mice in ischemia time of 45min、60min and 90min groups was over 50%,which was significant comparing with I/R group(16.67%,P<0.05).There was no significance between ischemic 30min group and ischemic 15min group with the mortality 16.67%and 0 respectively(P>0.05).Conclusions:The suitable ischemia time is 30 min.Research 1.2 The alteration of intestinal IgA level during I/R injuryObjectives:In this part we investigated the alternation of the intestinal sIgA level during I/R injury and compared the correlation of sIgA level and histological injury score as well as the incidence of bacterial translocation.Methods:Forty-eight mice were randomly assigned to 6 groups in accordance with different reperfusion duration(2h、6h、12h、24h、72h)including one sham group.The bacterial translocation rate of lung,spleen and mesenteric lymph nodes was displayed.The Distal ileum was analyzed for histological injury.The level of total small intestinal sIgA was measured by enzyme-linked immunosorbent assay(ELISA).All data were expressed as Mean ± SD.Statistical analysis was carried out by Dunnett-t test,Bivariate correlation.The significance level was accepted at P<0.05.Results:The bacterial translocation rate and the histological score of ileum were increasing while the intestinal sIgA level was decreasing along with I/R injury.The intestinal sIgA level was negatively correlated with histological score of ileum and the bacterial translocation(r2= 0.761 and r2= 0.729 respectively,P<0.01).Conclusions:Our results showed that the intestinal sIgA level declined during I/R injury and was negatively correlated with histological score of ileum and the incidence of bacterial translocation.Research 1.3 The alteration of IgM+/IgA+B cells ratio in lymphocytes of GALT during I/R injury.Objectives:To investigated the alternation of IgM+/IgA+B cells ratio in lymphocytes of GALT during I/R injury.Methods:Lymphocytes were isolated from Peyer’s patches and lamina propria of total small intestine and were stained with specific antibodies for B cells by flow cytometric analysis.The number of IgA/IgM containing cells in the Peyer’s patches and lamina propria was determined by immunohistochemistry.All data are presented as Mean ± SD and data were analyzed by Dunnett-t test.The significance level was accepted at P<0.05.Results:With the alteration of severity of intestinal I/R injury,the ratio of IgM+B220+B cells of PPL/LPL was increasing while the proportion of IgA+B220+B cells was less.The most of IgM+B220+B cells and the lest of IgA+B220+B cells of PPL were found in R6h group(56.37±5.34 vs 34.15±4.34/3.08±0.33 vs 6.17±0.38,P<0.01 vs sham group)and it was found in R12h group when pointing to LP B-cell subpopulations(5.97±0.42 vs 2.26±0.53/0.20±0.05 vs 1.44±0.20,P<0.01 vs sham group).Conclusions:The ratio of IgM+ B cells increased and that of IgA+ B cells reduced during intestinal I/R injury.Research 1.4 The alteration of AID mRNA expression in lymphocytes of GALTObjectives:To investigate the alteration of the AID mRNA expression in lymphocytes of the affector and effector sites during I/R injury.Methods:The expression of activation-induced cytidine deaminase(AID)mRNA in lymphocytes isolated from Peyer’s patches and lamina propria was quantitatively analyzed by RT-PCR.Data were analyzed by commercial software package SPSS 13.0.All data are presented as Mean ± SD and data were analyzed by independent t test.Values of P<0.05 were regarded as significant.Results:The expression of AID mRNA inhibited in lymphocytes isolated from both Peyer’s patches and lamina propria during I/R injury(PPL:27.0±3.16 vs 50.75±4.03,LPL:14.50±2.08 vs 34.25±3.30,P<0.01 vs sham group).Conclusions:Our results showed that I/R injury resulted in the decreased AID mRNA expression in both affector and effector sites.Part II The effect of n-3PUFAs on the alternation of intestinal sIgA owing to I/R injury and it’s mechanismsResearch 2.1 The effect of n-3PUFAs on the alternation of intestinal sIgA level and IgA-secreting cells owing to I/R injuryObjectives:n-3PUFAs has been proofed having protective effect on T cells in I/R injury,however,the effect on B cells in I/R injury remains unclear.The aim of this part is to observe the effect of n-3PUFAs on the alternation of intestinal slgA level and PP/LP B-cell subpopulations owing to I/R injury.Methods:Thirty-six male Balb/c mice,6-to 8-weeks old,were randomized into three groups including sham,I/R and FO groups.At the beginning of reperfusion,animals received a different intravenous fluid via tail vein injection(Omegaven in FO group,1 g/kg;the same volume of saline in sham and I/R groups).After 12-hour’s reperfusion,bacterial translocation rate,the intestinal sIgA level and the ratio of IgM+/IgA+B220+B cells of PPL and LPL were determined.Data were analyzed via commercial software package SPSS 13.0.All data are presented as Mean ±SD and data were analyzed by One-Way ANOVA(LSD).The significance level was accepted at P<0.05.Results:The results showed that n-3PUFAs can reduce the occurrence of bacterial translocation(12.5%vs 75%,P<0.01)and ameliorate the reduction of intestinal sIgA level due to I/R injury(28.50±4.32 vs 15.17±2.32,P<0.01).The subpopulations of PP/LP B cells analyzed by FCM showed that the proportion of IgM+B220+Bcells in FO group were significantly decreased compared with I/R group(42.87±4.82 vs 53.33±4.65/4.08±0.45 vs 5.97±0.42;P<0.05)while the ratio of IgA+B220+B cells were markedly increased(5.17±0.42 vs 3.90±0.38/0.61±0.06 vs 0.20±0.05,P<0.05).Conclusions:The results showed that n-3PUFAs can avoid the alteration of both intestinal sIgA level and IgM+/IgA+B220+B cells’ proportion owing to I/R.n-3PUFAs has the protective effect against the I/R injury in the aspect of intestinal sIgA level and IgM+/IgA+B220+B cells’ proportion.Research 2.2 The mechanisms of the protective effect of n-3PUFAs against I/R injuryObjectives:We hypothesized n-3PUFAs can protect I/R injury in the aspect of intestinal sIgA level through altering the expression of AID mRNA in PPL/LPL and A-proliferation and inducing ligand(APRIL)as well as B cell-activating factor(BAFF)proteins in intestinal mucosa.The aim of this part is to test the hypothesis.In addition,we investigate whether n-3PUFAs can avoid total apoptosis ratio of PPL.Methods:Thirty-six male Balb/c mice were randomized into three groups including sham,I/R and FO groups.On the beginning of reperfusion,animals received a different intravenous fluid via tail vein injection(Omegaven in FO group,1 grams/kg;the same volume of saline in sham and I/R groups).After 12-hour’s reperfusion,the expression of AID mRNA in PPL/LPL,the expression of APRIL/BAFF proteins in intestinal mucosa were measured by RT-PCR and western blot respectively.Furthermore,Allophycocyanin(APC)-conjugated Annexin-V and propidium iodide(PI)double-staining cells were analysed by FCM for apoptotic ratio of lymphocyts in peyer’s patches.Data were analyzed via commercial software package SPSS 13.0.All data are presented as Mean ± SD and data were analyzed by Dunnett t test.The significance level was accepted at P<0.05.Results:n-3PUFAs can increase the expression of AID mRNA in PPL/LPL(27.0±3.16 vs 50.75±4.03/14.50±2.08 vs 34.25±3.30,P<0.05)and ameliorate the inhibition of APRIL/BAFF proteins expression due to I/R injury(24.50 ±2.65 vs 49.75±5.56/16.50±2.08 vs 43.57±5.45,P<0.05).There was significance in total apoptosis ratio of PPL between FO group and I/R group(10.44± 1.12 vs 17.69±3.04,P<0.05).Conclusions:The mechanisms of protective effect of n-3PUFAs against intestinal I/R injury might include:Firstly,n-3PUFAs can avoid the apoptosis of PPL.Secondly,n-3PUFAs promotes the class-switching to IgA through increasing AID mRNA.Thirdly,n-3PUFAs promotes the differentiation to IgA-antibody-secreting cells by increasing two factors of APRIL and B AFF.