Journal: Scientific Reports

Article Title: Estradiol-induced inhibition of endoplasmic reticulum stress normalizes splenic CD4 + T lymphocytes following hemorrhagic shock

doi: 10.1038/s41598-021-87159-1

Figure Lengend Snippet: Proliferation of splenic CD4 + T lymphocytes isolated from the rats. The animals were sacrificed at 3 h after resuscitation or each time and treated with vehicle (V), 17-estradiol (E2), propyl pyrazole triol (PPT), diarylpropionitrile (DPN), G-1, E2 and ICI 182,780 (E2 + ICI), E2 + G15, 4-Phenylbutyric acid (4-PBA), tunicamycin (TM), E2 + TM, PPT + TM, DPN + TM, G-1 + TM, respectively. The CD4 + T lymphocytes were harvested from spleen in rats with the method of immunomagnetic beads separation techniques, flow cytometry analysis demonstrated that cells contained > 90% CD4 + T lymphocytes ( A ). The splenic CD4 + T lymphocytes (8 × 10 5 cells/ml) were stimulated with ConA (5 μg/ml) for 48 h and incubated with CCK-8 for 4 h, the proliferation was determined with technical replication of three samples from each splenic tissue, and the proliferative capacity was represented using the optical density measured by the SpectraMax M3 plate reader. Values are mean ± SE of 3 animals in each group ( B – D ). B indicated that estrogen receptors (ERs) were involved the role of E2 enhancing the proliferation of splenic CD4 + T lymphocytes isolated from the hemorrhagic shocked rats. C indicated that the role of hemorrhagic shock decreasing the proliferation of splenic CD4 + T lymphocytes was through the excessive endoplasmic reticulum stress (ERS). D indicated that the beneficial effect of E2 on the proliferation of splenic CD4 + T lymphocytes was related to the ERs-dependent inhibition of ERS following hemorrhagic shock. *p < 0.05 vs. the sham + vehicle group, #p < 0.05 vs. the shock + vehicle group, △p < 0.05 vs. the shock + E2 group, □p < 0.05 vs. the shock + PPT group.

Article Snippet: At the onset of the resuscitation, the rats received E2 (2 mg/kg, Merck KGaA, Darmstadt, Germany), PPT (5 μg/kg, Merck KGaA, Darmstadt, Germany), DPN (5 μg/kg, Merck KGaA, Darmstadt, Germany), G-1 (400 μg/kg, ApexBio, Texas, USA), E2 + ICI 182,780 (150 μg/kg, ApexBio, Texas, USA), E2 + G15 (160 μg/kg, ApexBio, Texas, USA), TM (2 mg/kg, ApexBio, Texas, USA), E2 + TM, PPT + TM, DPN + TM, G-1 + TM, ERS inhibitor 4-PBA (20 mg/kg, Merck KGaA, Darmstadt, Germany) or an equal volume of vehicle (20 μL/kg, 100% dimethyl sulfoxide) subcutaneously, respectively.

Techniques: Isolation, Flow Cytometry, Incubation, CCK-8 Assay, Inhibition

Journal: Scientific Reports

Article Title: Estradiol-induced inhibition of endoplasmic reticulum stress normalizes splenic CD4 + T lymphocytes following hemorrhagic shock

doi: 10.1038/s41598-021-87159-1

Figure Lengend Snippet: Cytokine production of splenic CD4 + T lymphocytes isolated from the rats. The CD4 + T lymphocytes were harvested from spleen in rats at 3 h after resuscitation or each time and treated with vehicle (V), 17-estradiol (E2), propyl pyrazole triol (PPT), diarylpropionitrile (DPN), G-1, E2 and ICI 182,780 (E2 + ICI), E2 + G15, 4-Phenylbutyric acid (4-PBA), tunicamycin (TM), E2 + TM, PPT + TM, DPN + TM, G-1 + TM, respectively. Subsequently, the CD4 + T lymphocytes were cultured in 96-well plates and stimulated with ConA (5 μg/ml) for 48 h, Cytokine levels of interleukin (IL)-2, IL-4, and tumor necrosis factor-α-induced protein 8 like 2 (TIPE2) in culture supernatants were determined by ELISA. Data are mean ± SE of 3 animals in each group. *p < 0.05 vs. the sham + vehicle group, #p < 0.05 vs. the shock + vehicle group, Δp < 0.05 vs. the shock + E2 group, □p < 0.05 vs. the shock + PPT group.

Article Snippet: At the onset of the resuscitation, the rats received E2 (2 mg/kg, Merck KGaA, Darmstadt, Germany), PPT (5 μg/kg, Merck KGaA, Darmstadt, Germany), DPN (5 μg/kg, Merck KGaA, Darmstadt, Germany), G-1 (400 μg/kg, ApexBio, Texas, USA), E2 + ICI 182,780 (150 μg/kg, ApexBio, Texas, USA), E2 + G15 (160 μg/kg, ApexBio, Texas, USA), TM (2 mg/kg, ApexBio, Texas, USA), E2 + TM, PPT + TM, DPN + TM, G-1 + TM, ERS inhibitor 4-PBA (20 mg/kg, Merck KGaA, Darmstadt, Germany) or an equal volume of vehicle (20 μL/kg, 100% dimethyl sulfoxide) subcutaneously, respectively.

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Estradiol-induced inhibition of endoplasmic reticulum stress normalizes splenic CD4 + T lymphocytes following hemorrhagic shock

doi: 10.1038/s41598-021-87159-1

Figure Lengend Snippet: Histological examination of spleen in rats following hemorrhagic shock. The animals were sacrificed at 3 h after resuscitation or each time and treated with vehicle, 17-estradiol (E2), propyl pyrazole triol (PPT), diarylpropionitrile (DPN), G-1, E2 and ICI 182,780 (E2 + ICI), E2 + G15, 4-Phenylbutyric acid (4-PBA), tunicamycin (TM), E2 + TM, PPT + TM, DPN + TM, G-1 + TM, respectively. ( A ) Representative sections of hematoxylin and eosin staining for three animals per group viewed under 20 × objective lens. Arrows indicates the structural change of splenic white pulp from the red pulp regions for the evaluation of splenic structural injury. Scale bar: 100 μm. ( B ) Splenic histology score. Data are mean ± SE of 3 animals in each group. *p < 0.05 vs. the sham + vehicle group, #p < 0.05 vs. the shock + vehicle group, △p < 0.05 vs. the shock + E2 group, □p < 0.05 vs. the shock + PPT group.

Article Snippet: At the onset of the resuscitation, the rats received E2 (2 mg/kg, Merck KGaA, Darmstadt, Germany), PPT (5 μg/kg, Merck KGaA, Darmstadt, Germany), DPN (5 μg/kg, Merck KGaA, Darmstadt, Germany), G-1 (400 μg/kg, ApexBio, Texas, USA), E2 + ICI 182,780 (150 μg/kg, ApexBio, Texas, USA), E2 + G15 (160 μg/kg, ApexBio, Texas, USA), TM (2 mg/kg, ApexBio, Texas, USA), E2 + TM, PPT + TM, DPN + TM, G-1 + TM, ERS inhibitor 4-PBA (20 mg/kg, Merck KGaA, Darmstadt, Germany) or an equal volume of vehicle (20 μL/kg, 100% dimethyl sulfoxide) subcutaneously, respectively.

Techniques: Staining

Journal: Scientific Reports

Article Title: Estradiol-induced inhibition of endoplasmic reticulum stress normalizes splenic CD4 + T lymphocytes following hemorrhagic shock

doi: 10.1038/s41598-021-87159-1

Figure Lengend Snippet: Expressions of GRP78 and ATF6 in spleen. The spleens were harvested from the rats at 3 h after resuscitation or each time and treated with vehicle (V), 17-estradiol (E2), propyl pyrazole triol (PPT), diarylpropionitrile (DPN), G-1, E2 and ICI 182,780 (E2 + ICI), E2 + G15, 4-Phenylbutyric acid (4-PBA), tunicamycin (TM), E2 + TM, respectively. Subsequently, the proteins were extracted and the GRP78 and ATF6 expression were analyzed with the method of Western blotting. GRP78 and ATF6 blots obtained from 3 rats were analyzed using densitometry, and densitometric values were normalized to β-actin and are shown as means ± SD. The figures showed the representative cropped images of western blot analysis of GRP78 and ATF6. Original blots are presented in Supplementary Fig. S1 and Fig. S2. *p < 0.05 vs. the sham + vehicle group, #p < 0.05 vs. the shock + vehicle group, △p < 0.05 vs. the shock + E2 group.

Article Snippet: At the onset of the resuscitation, the rats received E2 (2 mg/kg, Merck KGaA, Darmstadt, Germany), PPT (5 μg/kg, Merck KGaA, Darmstadt, Germany), DPN (5 μg/kg, Merck KGaA, Darmstadt, Germany), G-1 (400 μg/kg, ApexBio, Texas, USA), E2 + ICI 182,780 (150 μg/kg, ApexBio, Texas, USA), E2 + G15 (160 μg/kg, ApexBio, Texas, USA), TM (2 mg/kg, ApexBio, Texas, USA), E2 + TM, PPT + TM, DPN + TM, G-1 + TM, ERS inhibitor 4-PBA (20 mg/kg, Merck KGaA, Darmstadt, Germany) or an equal volume of vehicle (20 μL/kg, 100% dimethyl sulfoxide) subcutaneously, respectively.

Techniques: Expressing, Western Blot