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Vascular hypo-reactivity plays a critical role inducing organ injury during hemorrhagic shock. 17β-estradiol (E2) can induce vasodilation to increase blood flow in various vascular beds. This study observed whether E2 can restore vascular hypo-reactivity induced by hemorrhagic shock, and whether E2 effects are associated with RhoA-Rho kinase (ROCK)-myosin light chain kinase phosphatase (MLCP) pathway. The hemorrhagic shock model (40 ± 2 mm Hg for 1 h, resuscitation for 4 h) was established in ovary intact sham operation (OVI), ovariectomized (OVX), and OVX plus E2 supplement female mice. Intestinal microvascular loop was used to assess blood flow in vivo, mRNA expression and vascular reactivity in vitro. Hemorrhagic shock significantly reduced norepinephrine microvascular reactivity. Decreased microvascular reactivity was exacerbated by OVX and reversed by E2 supplement. U-46619 (RhoA agonist) increased microvascular reactivity, and C3 transferase (an ADP ribosyl transferase that selectively induces RhoA ribosylation) or Y-27632 (ROCK inhibitor) inhibited sham mice microvascular reactivity. Similarly, U-46619 increased microvascular reactivity in OVI and OVX mice following hemorrhagic shock, which was abolished by Y-27632 or concomitant incubation of okadaic acid (OA) (MLCP inhibitor) and Y-27632. In OVX plus E2 supplement mice with hemorrhagic shock, Y-27632 inhibited microvascular reactivity, which was abolished by concomitant U-46619 application. Lastly, hemorrhagic shock remarkably decreased intestinal loop blood flow, RhoA and ROCK mRNA expressions in vascular tissues in OVX females, but not in OVI females, which were reversed by E2 supplement. These results indicate that estrogen improves microvascular reactivity during hemorrhagic shock, and RhoA-ROCK signaling pathway may mediate E2 effects.

Hemorrhagic shock is associated with activation of renin-angiotensin system (RAS) and endoplasmic reticulum stress (ERS). Previous studies demonstrated that central RAS activation produced by various challenges sensitizes angiotensin (Ang) II-elicited hypertension and that ERS contributes to the development of neurogenic hypertension. The present study investigated whether controlled hemorrhage could sensitize Ang II-elicited hypertension and whether the brain RAS and ERS mediate this sensitization. Results showed that hemorrhaged (HEM) rats had a significantly enhanced hypertensive response to a slow-pressor infusion of Ang II when compared to sham HEM rats. Treatment with either angiotensin-converting enzyme (ACE) 1 inhibitor, captopril, or ACE2 activator, diminazene, abolished the HEM-induced sensitization of hypertension. Treatment with the ERS agonist, tunicamycin, in sham HEM rats also sensitized Ang II-elicited hypertension. However, blockade of ERS with 4-phenylbutyric acid in HEM rats did not alter HEM-elicited sensitization of hypertension. Either HEM or ERS activation produced a greater reduction in BP after ganglionic blockade, upregulated mRNA and protein expression of ACE1 in the hypothalamic paraventricular nucleus (PVN), and elevated plasma levels of Ang II but reduced mRNA expression of the Ang-(1-7) receptor, Mas-R, and did not alter plasma levels of Ang-(1-7). Treatment with captopril or diminazene, but not phenylbutyric acid, reversed these changes. No treatments had effects on PVN protein expression of the ERS marker glucose-regulated protein 78. The results indicate that controlled hemorrhage sensitizes Ang II-elicited hypertension by augmenting RAS prohypertensive actions and reducing RAS antihypertensive effects in the brain, which is independent of ERS mechanism.

PurposePost hemorrhagic shock mesenteric lymph (PHSML) return contributes to CD4+ T cell dysfunction, which leads to immune dysfunction and uncontrolled inflammatory response. Tumor necrosis factor α induced protein 8 like-2 (TIPE2) is one of the essential proteins to maintain the immune homeostasis. This study investigated the role of TIPE2 in regulation of CD4+ T lymphocyte function in interaction of PHSML and TLR2/TLR4.MethodsThe splenic CD4+ T cells were isolated from various mice (WT, TLR2-/-, TLR4-/-) by immunomagnetic beads, and stimulated with PHSML, normal lymphatic fluid (NML), respectively. Application of TIPE2-carrying interfering fragments of lentivirus were transfected to WT, TLR4-/-, and TLR2-/- CD4+ T cells, respectively. After interference of TIPE2, they were stimulated with PHSML and NML for the examinations of TIPE2, TLR2, and TLR4 mRNA expressions, proliferation, activation molecules on surface, and cytokine secretion function.ResultsPHSML stimulation significantly upregulated TIPE2, TLR2, and TLR4 mRNA expressions, decreased proliferation, CD25 expression, and IFN-γ secretion, and increased the secretion ability of IL-4 in WT CD4+ T cells. TIPE2 silencing enhanced proliferative capacity, upregulated CD25 expression, and increased IFNγ secretion in CD4+ T cells. PHSML stimulated TLR2-/-CD4+ T or TLR4-/-CD4+ T cells of which TIPE2 were silenced. TLR2 or TLR4 knockout attenuated PHSML-induced CD4+ T cells dysfunction; PHSML stimulation of silent TIPE2-expressing TLR2-/-CD4+ T or TLR4-/-CD4+ T revealed that the coexistence of low TIPE2 expression with lack of TLR2 or TLR4 eliminated this beneficial effect.ConclusionTIPE2 improves the PHSML-mediated CD4+T cells dysfunction by regulating TLR2/TLR4 pathway, providing a new intervention target following hemorrhagic shock-induced immune dysfunction.

Background Intestinal barrier dysfunction is the critical link of distant organ injury caused by hemorrhagic shock. The role of autophagy in ischemic intestinal injury has been paid more and more attention. Prophylactic treatment of stellate ganglion block (SGB) reduces intestinal barrier dysfunction induced by haemorrhagic shock. This study investigated the role of SGB post-processing on improved bowel barrier function and autophagy-related mechanisms of action after haemorrhagic shock. Methods The model of hemorrhagic shock in conscious rats was established, and the rats were treated with SGB, autophagy inhibitor 3- methyladenine (3-MA) at the time of fluid resuscitation. We detected the survival rate, intestinal loop blood flow, intestinal barrier permeability, intestinal morphology, wet/dry ratio (W/D), the intestinal barrier and autophagy marker proteins. Simultaneously, we also observed the effect of autophagy activator rapamycin (RAPA) on SGB. Results SGB postconditioning significantly prolonged the overall survival and enhanced survival rate of 72 hours in rats after hemorrhagic shock. SGB post-processing and 3-MA administration improved the intestinal morphology and intestinal permeability, increased the intestinal loop blood flow and expression of ZO1, Occludin and Claudin-1, and decreased the intestinal W/D and LC3 and Beclin-1 expressions, expected for increased P62. Meanwhile, RAPA partially inhibited the effect of SGB on above indices in haemorrhagic shock rats. Conclusion SGB postconditioning alleviates the intestinal barrier dysfunction caused by haemorrhagic shock, which is related to the inhibition of excessive autophagy.