Study On The Roles Of Phosphorylated STAT3 In Rat Spleen Damage Induced By Chronic Restraint Stress

Chronic stress influences livestock production and animal health via HPA axis which connects the nervous,endocrine,and immune systems.As the center of innate and adaptive immunity and control antigen tolerance,the spleen was damaged during chronic stress.However,the exact mechanism leading to this situation remains to be elucidated.STAT3 is a transcription factor involved in cell proliferation and immune signaling.Phosphorylated STAT3(p-STAT3)expression is increased in the spleen during chronic stress.It is unclear whether STAT3 is involved in apoptosis caused by chronic stress.We investigated the role of STAT3 in chronic stress-induced splenocyte apoptosis by using p-STAT3 specific inhibitor S3I-201.It clarified the role of STAT3 and unfolded protein response(UPR)by using p-STAT3 specific inhibitor.This study aims to provide a new theoretical basis for the mechanism of spleen injury by chronic stress,and provide theoretical basis and experimental basis for future treatment.56 healthy male Wistar rats were randomly divided into C group(16 rats),CS group(16 rats),C+S3I-201 group(6 rats),CS+S3I-201 group(6 rats),C+DMSO group(6 rats)and CS+DMSO group(6 rats).The rats of C+S3I-201 group,CS+S3I-201 group,C+DMSO group and CS+DMSO group were treated corresponding drugs by intraperitoneal injection every second day.The rats of CS group,CS+S3I-201 group and CS+DMSO group accepted restraint program every day for 21 days.After 21-day physical restraint,six rats taken from each group were sacrificed to collect blood and spleen simples to further experiment.The remaining 10 rats in C group and CS group were tested by the open-field test on the 22 nd day.The behavior and serum corticosterone levels of rats were detected to verify success of establishing of chronic stress model.Spleen injury was detected by observing pathological section,detecting the expression of apoptosis-associated proteins and TUNEL assay.Proteins of three classical apoptotic pathway were detected to investigate regulatory pathway for the change of apoptosis level in spleens.The results of behavior test showed that the rats in CS group had significant anxiety-like behavior compared with C group(p < 0.01).The level of corticosterone of CS group was increased significantly compared with the level of C group(p < 0.01).The results of Western Blot showed that the expression of IL-10 which didn't affect by inhibiting p-STAT3 was significantly increased than C group(p < 0.01)The expression of IL-6 kept stable which didn't affect by chronic stress or inhibiting p-STAT3(p > 0.05).The expression of p-STAT3 in CS group increased significantly compared with C group(p < 0.01).Meanwhile,the expression of p-STAT3 in CS+S3I-201 group decreased significantly compared with CS group(p < 0.01).Spleens in CS group showed reduction of white pulp and partial disappearance of the spleen marginal zone.Spleens in CS+S3I-201 group lost typical histologic architecture.The expression of cleaved caspase-3 and the result of Tunel assay showed strong apoptosis occurred in CS group compared with C group(p < 0.01).And the level of apoptosis in CS+S3I-201 group was significantly higher than that in CS group(p < 0.01).The expression of Bcl-2,Bax and Bcl-x L were not influenced by chronic stress or p-STAT3 inhibition(p > 0.05).The expression of procaspase-8 promoted significantly in CS group compared with C group(p < 0.01),and expression of CS+S3I-201 group had no obviously changes compared with CS group(p > 0.05).The expression of GRP78 in CS+S3I-201 group was higher significantly than C group(p < 0.01).The expression of p-JNK,procaspase-12 and CHOP in CS+S3I-201 group was significantly higher than the other four groups(p < 0.01),and the expression in the other three groups had no obviously changes compared with C group(p > 0.05).The expression of p-e IF2?(p < 0.01),ATF-6?(p < 0.05)and p-IRE1?(p < 0.01)in CS group were significantly higher than C group.The expression of ATF-6?(p < 0.05)and p-IRE1?(p < 0.01)in CS+S3I-201 group decreased compared with CS group.The above experimental results confirm that splenocyte apoptosis was mediated by death receptor pathway which didn't influence by p-STAT3 inhibition.Also,the body activated UPR in chronic stress,and p-STAT3 inhibitor could inhibit the activation of UPR and mediate the beginning of ER stress to enhance splenocyte apoptosis.Our research indicated that UPR and STAT3 are therapeutic target in treating spleen damage in chronic stress.