| Hemorrhagic shock is a hypovolemic shock caused by severe blood loss.Hemorrhagic shock causes the inhibition of immune function and reduces the ability of the body to resist infection,and CD4~+T lymphocytes play an important role in this process.CD4~+T lymphocytes have classical estrogen receptors(ER)-α,ER-βand membrane receptor G protein-coupled receptor 30(GPR30)receptors.There were significant gender differences in trauma,shock and sepsis.At the same time,T cells in male animals treated with 17β-estradiol(E2)weakened cytokine secretion and cell injury after hemorrhagic shock.Hemorrhagic shock causes endoplasmic reticulum stress(ERS),and organ damage in parenchyma cells.However,it is not clear whether ERS is involved in the role of E2 in improving the function of splenic CD4~+T cells after hemorrhagic shock.Therefore,in this study,we hypothesized that E2 is mediated by ER-α,ER-βor GPR30 in the treatment of splenic tissue injury after hemorrhagic shock and the recovery of splenic CD4~+T cell proliferation and cytokine production,while the beneficial effect of E2 is mediated by ERS.One hundred and fifty-six male Wistar rats underwent trauma(5 cm incision in the midline of the abdomen)and blood loss(mean blood pressure 40 mmHg for 90 minutes),and then fluid resuscitation was performed.When the fluid recovers,subcutaneous administration of E2(2mg/kg),ER-αagonist PPT(5μg/kg)and ER-βagonist DPN(5μg/kg),GPR30 agonist G-1(5μg/kg),E2+ER antagonist ICI 182,780(5μg/kg),E2+GPR30 antagonist G15(5μg/kg),ERS agonists TM(2 mg/kg),E2+TM,PPT+TM,DPN+TM,G-1+TM,ERS inhibitor 4-phenylbutyric acid(4-PBA,5μg/kg)or equal volume subcutaneous injection of solvent(100%DMSO,20μl/kg)at the beginning of resuscitation.Three hours later,the rats were killed and CD4~+T cells were isolated from spleen.Under the stimulation of ConA in vitro,48 hours later,the proliferation of CD4~+T cells was detected by CCK-8 assay,the levels of cytokines IL-2,IL4 and TIPE2 in the supernatant of CD4~+T cell culture medium were detected by ELISA assay,and the histopathological changes of spleen were observed by HE staining.The expression of ERS marker protein(GRP78,ATF6)in tissues was detected by Western Blot.The results showed that the proliferation of CD4~+T cells decreased significantly after hemorrhagic shock,and the inhibitory effect of E2 or PPT on the proliferation of CD4~+T cells in spleen of rats was significantly alleviated by hemorrhagic shock.However,DPN and G-1 had no effect on the proliferation of splenic CD4~+T cells,ICI 182780 or G15 could block the effect of E2 on the proliferation of splenic CD4~+T cells,and 4-PBA significantly restored the proliferation of splenic CD4~+T cells after hemorrhagic shock.TM significantly decreased the proliferation of CD4~+T cells in sham operation group,and the combination of TM abolished the beneficial effect of Shock+E2 group and Shock+PPT group on the proliferation of CD4~+T cells.After hemorrhagic shock,the secretion of IL-2,IL-4 and TIPE2 of splenic CD4~+T cells in rats was significantly decreased,and the secretion of IL-2,IL-4 and TIPE2 of splenic CD4~+T cells in hemorrhagic shock rats was significantly restored by the administration of E2 or PPT.However,DPN and G-1 treatment did not recover.ICI 182780 or G15 could block the ability of E2 to produce and secrete IL-2,IL-4 and TIPE2 by splenic CD4~+T cells.After hemorrhagic shock,4-PBA significantly restored the ability of CD4~+T cells to secrete IL-2,IL-4 and TIPE2 in rat spleen.TM significantly decreased the ability of CD4~+T cells to produce cytokines in sham operation group,and the combination of TM abolished the beneficial effect of CD4~+T cells in Shock+E2 group and Shock+PPT group on the secretion of IL-2,IL-4 and TIPE2.In sham operation group,the structure of spleen tissue was basically normal,the marginal zone of white pulp and red pulp was clear.After hemorrhagic shock,the outline of white pulp of spleen tissue disappeared,irregular shape and significantly increased the splenic histology score.After hemorrhagic shock,Administration of E2,PPT or 4-PBA could restore the outline of white pulp and histology score in rat spleen,but DPN and G-1 had no significant effect on white pulp after hemorrhagic shock.ICI 182,780 and G15 could block the beneficial effect of E2 on splenic tissue.E2+TM or PPT+TM could block the effect of E2 or PPT on spleen tissue.In addition,TM alone could damage the spleen tissue and significantly increased the histology score of sham-operated rats.After hemorrhagic shock,Splenic ERS marker protein GRP78 and ERS pathway marker protein ATF6 were significantly up-regulated,E2 and PPT significantly improved the overexpression of GRP78 and ATF6 in spleen of rats with hemorrhagic shock.E2 combined with ICI 182,780 and G15 abolished the therapeutic effect of E2.DPN and G-1 group was no obvious therapeutic effect.After hemorrhagic shock,the overexpression of GRP78 and ATF6 in spleen of rats with hemorrhagic shock was significantly decreased in 4-PBA group.In sham operation group,TM significantly increased the expression of GRP78 and ATF6 in spleen of rats.At the same time,the combination of TM abolished the therapeutic effect of Shock+E2 group and Shock+PPT group.At the same time,the combination of TM abolished the therapeutic effect of Shock+E2 group and Shock+PPT group.Together,the data suggest that E2 produces salutary effects on CD4~+T lymphocytes function through the attenuation of hemorrhagic shock-induced ERS,which is mediated via ER-αand at least in part GPR30. |
