Endoplasmic Reticulum Stress And Splenic Lymphocyte Abnormal Apoptosis In Sepsis

Objective:This study was performed cecal ligation and puncture(CLP)on mouse to induce sepsis and observed consecutively the effect of splenic tissue.Observed weather there was extensive apoptosis of lymphocytes in the clinically relevant model of sepsis. Detected their change along with the change of the mRNA and protein expression of GRP78 and CHOP genes so as to better understand the roles that endoplasmic reticulum stress plays in the splenic lymphocyte apoptosis of sepsis,and give some intervention method over sepsis in the future.Methods:60 male C57BL/6 mice whose body weight ranged from 17 to 25 grams were utilized and assigned randomly to two groups:sham operated group(Sham)30,cecal ligation and puncture group(CLP)30.All mice ate nothing 12 hours before operation. For all surgical procedures,ether complete anesthesia was used.Mice in sham group, the cecum was located but neither ligated nor puncture.To mouse in CLP group,the cecum was ligated proximal to the cecal pole with a 1 prolene thread without stricture of the ileocecal valve.The ligated cecum was punctured once with an 18-gauge needle,then the cecum was slightly compressed until a small drop of stool appeared. Distribute it uniformly over the surface of the ligation cecum.Closed the abdominal and 0.5ml of sterile saline solution was used for fluid resuscitation.Sepsis models were finished.Mice in each group were divided into two groups randomly,half were used for viewing the general state of health and seven days survival rate,the other were used for experiment detection.Twenty-four hours after surgery,mice per experimental group were sacrificed and photo documentation of the situs was performed.Spleen was removed rapidly,a portion of the spleen was frozen in liquid nitrogen and stored at -80℃for later use in tissue homogenate.A second portion of the tissue sample was placed in 4%neutral buffered paraformaldehyde for hematoxylin and eosin,TUNEL and immunohistochemical staining.Liver,lung and bowels were resected for histopathologic examination.The technique of terminal deoxynucleotidyl transferase-mediated dUTP nick end labling(TUNEL)was used to detected spleen lymphocyte apoptosis.The expression of GRP78 and CHOP protein were detected by immunohistochemical and Western blot methods.The mRNA expression of the genes GRP78 and CHOP were detected by RT-PCR.Statistical analysis was performed using SPSS 11.5 for windows software.Experiment data was indicated with mean±standard.A significance level of 0.01 or 0.05 was determined for all calculation.Results:1 Affirmation of the succeeded CLP model:After operation,the mice in CLP group appearance depressed,drowsiness,clumsy,loss of appetite and so on.But sham group have the same appearance before or after operation.In CLP group 24 hours survival rate was 93%,72 hours crude mortality ratio was33%.No mice were dead in sham group.Different extent injury in the pathological section examination about liver, lung,bowels,spleen has happened in the CLP group.Comparing with CLP group,no obviously inflammation was found in sham group.2 The changes of splenic lymphocyte apoptosis:In sham group there was rarely apoptosis cell,diffused distribution in the splenic white pulp nodulus lymphaticus, but in the CLP group there were a great quantity,and distributed in focus.The serious of apoptosis was remarked with apoptotic index.Comparison between sham and CLP group,the difference has statistical significance.(P＜0.01)3 The changes of splenic GRP78 levels:The mRNA and protein expressions of splenic tissue GRP78 or the positive stain of GRP78 protein were seen in both sham and CLP group,but in the CLP group it were obviously higher compared with sham group(P＜0.05).Immunohistochemical and Western blot re indicated the same result, the protein expression of GRP78 in the CLP group is significance higher compaired with the Sham group(P＜0.05).4 The changes of splenic CHOP levels:The mRNA and protein expressions of splenic tissue CHOP gene or the positive stain of CHOP protein in CLP group was obviously higher compared with sham group(P＜0.05).Immunohistochemical and Western blot' result indicated that the mRNA and protein expression of CHOP gene has the same change trend.(P＜0.05)Conclusion:1 Sepsis induces extensive lymphocyte apoptosis in the spleen.Apoptosis not only induced loss of lymphocyte but also induced anergy or anti-inflammatory cytokines that impairing the immune cells' function.So lymphocyte apoptosis is an important mechanism of immune suppress in sepsis. 2 In the CLP group,the expression of GRP78's mRNA and protein are increasing.It shows that Endoplasmic Reticulum Stress is started in sepsis induced by hypoxia, oxygen free radical releasing,acidosis and to correct cell functional disorder by up-regulation GRP78.3 The high expression of CHOP shows that Endoplasmic reticulum stress is too severe to compensate.At last cell apoptosis happen4 Endoplasmic reticulum stress may contribute to the pathogenesis of sepsis by inducing apoptosis of splenic lymphocytes,suppress immune function and aggravate pathogenetic condition.