The current study demonstrated that SGB pretreatment or autophagy inhibitor 3-MA administration inhibited the reduction of proliferation and cytokine secretion ability of splenic CD4 + T cells following hemorrhagic shock, and intravenous of PHSML and autophagy agonist RAPA administration eliminated the beneficial effects of SGB. PHSML decreased the proliferation and cytokine production of CD4 + T cells isolated normal rats, increased expression of autophagy marker proteins, and inhibited the phosphorylation of PI3K and AKT proteins, which was inhibited by 3-MA treatment. More importantly, the effect of PHSML-SGB was opposite to that of PHSML, and the indicators of CD4 + T cells were close to normal. The present findings suggest that SGB improves the proliferation and function of CD4+ T cells after hemorrhagic shock by reducing PHSML-mediated autophagy.
Previous report revealed that the capability of proliferating and producing effector cytokines of CD4 + T cells are the key to performing a variety of effector functions and immune responses . Low proliferative response of CD4 + T cells is involved in the depressed immune responses after trauma-induced hemorrhagic shock . After T cell activation and polarization into various T helper subsets, CD4 + T lymphocytes can produce IL-2 and IL-4 . In the meantime, TIPE-2 is required for maintaining immune homeostasis . In vivo, the hypo-secretion of IL-2, IL-4 and TIPE-2 leads to down-regulation of immune function . Therefore, in this study, we measured the proliferation and cytokines production function of ConA-stimulated CD4 + T lymphocytes, and found that hemorrhagic shock decreased the proliferation capacity and cytokines IL-2, IL-4 and TIPE-2 levels in the culture supernatant of CD4+ T cells stimulated by ConA in vitro, which may be one of the factors that cause the immune system dysfunction and MODS.
Neuromodulation is extremely important for internal environment disturbances caused by hemorrhagic shock. The hypothalamic-pituitary- adrenal (HPA) axis and hypothalamic-sympathetic-adrenal axis are the two key axes for the neuromodulation, as well as affecting the immune response . In recent years, neuro-immune interactions have received increasing attention [20, 21], the role of the sympathetic nervous system in the immune response has been gradually elucidated . For example, adrenergic agonists modulate immune responses in vitro, including cytokine production, lymphocyte proliferation and antibody secretions [23–25], and chemical sympathectomy changes the immune response . Under stress, the body first activates the neuro-endocrine regulatory system, which regulates immune and endocrine system functions through the HPA axis and hypothalamus-sympathetic nerve-adrenal axis . Studies have shown that SGB changes the distribution of lymphocyte subpopulations by blocking excessive excitability of sympathetic nerves, increases the proportion of CD4 + T cells , and inhibits excessive inflammatory responses early , thereby regulating the inflammatory response and immune function homeostasis. Therefore, the implementation of SGB in advance is conducive to reducing the excessive excitement of sympathetic nerves caused by hemorrhagic shock. However, whether SGB affect the function of CD4 + T cells following hemorrhagic shock, it is unknown. The current findings demonstrated that SGB significantly ameliorated the proliferation and cytokine production function of splenic CD4 + T cells in hemorrhage shocked rats, and suggested that SGB pretreatment is beneficial for regulating CD4 + T cell function.
Studies have shown that PHSML return after hemorrhagic shock is a factor that reduces spleen CD4+ T cell function and causes immune dysfunction, reducing PHSML return mitigates spleen histological damage and improve immune function [8, 9]. In order to determine whether the benefits of SGB are achieved through the intestinal lymphatic pathway, we injected PHSML intravenously into SGB-treated hemorrhagic shock rats, and found that the infusion of PHSML abolished the beneficial effects of SGB, and reduced the proliferation and the cytokines secretion of CD4+ T cells. Using splenic CD4+ T cells extracted from normal rats, it was found that PHSML treatment reduced the proliferation activity and ability to secrete cytokines of CD4 + T cells. In contrast, PHSML-SGB treated cells have significantly increases in proliferation and cytokines IL-2, IL-4 and TIPE-2. Hence, the beneficial effects of SGB are related to the intestinal lymphatic pathway.
Autophagy is a highly conservative cellular biological process, and is one of the two major degradation systems in eukaryotic cells. It is involved in cell proliferation and physiological apoptosis. Its main role is to protect cells under stress. However, the excessive autophagy leads to cell injury, and involves in the development of sepsis  and immune response . After hemorrhagic shock, splenocytes damage may be associated with excessive cellular autophagy . The present animal experiments showed that treatment with autophagy inhibitor 3-MA restored the proliferation and the ability to secrete cytokines of CD4+ T cells following hemorrhagic shock. Conversely, the autophagy agonist RAPA offset the beneficial effects of SGB. Therefore, inhibition of excessive autophagy is involved in the role of SGB in improving the function of CD4+ T cells.
During formation of autophagy, the double membrane organelles called autophagosomes transfer cytoplasmic material to lysosomes . In mammalian cells, the formation of autophagosomes is through the release of ATG6 / Beclin-1 from Bcl-2 to form a Vps34-PI3K complex containing Beclin-1, which is essential for the production of autophagosomes . Autophagy can be stimulated by starvation and other stresses, various pathological conditions, or drugs such as rapamycin [35, 36]. When autophagy is excessively induced, it will cause autophagic cell death, so-called type II apoptosis . Therefore, in addition to physiological regulatory functions, autophagy is also closely related to inflammation and the occurrence and development of diseases . Normal levels of autophagy can maintain the homeostasis of the body's environment, while excessive autophagy can cause apoptosis . During autophagosome formation, as a classic marker of autophagosome, the increase of LC3-II represents the beginning of autophagy . At the same time, the expression level of Beclin-1 also reflects the intensity of autophagy and become one of the autophagy marker proteins .
In order to further clarify the mechanism of cell autophagy, in this study, splenic CD4 + T cells from normal rats were cultured with ConA for 48 hours in vitro and then incubated with PHSML. We found that PHSML increased the protein expression of LC3 II/I and Beclin-1 in CD4 + T cells. At the same time, the autophagy inhibitor 3-MA significantly reduced the conversion of LC3 I to LC3 II and the expression of Beclin-1 in CD4 + T cells, suggesting that PHSML activated autophagy of CD4 + T cells. Furthermore, PHSML-SGB treatment significantly reduced the expressions of autophagy marker proteins, suggesting that SGB reduced the autophagy of CD4 + T cells through the intestinal lymphatic pathway.
As is well known that PI3K/AKT pathway is the classic pathway of autophagy. More and more studies showed that PI3K/Akt signaling pathway is a primary pathway that inhibits autophagy [42, 43]. This study further confirmed that PHSML inhibits the phosphorylation of PI3K and Akt in CD4 + T cells, which was reversed by the PHSML-SGB and PHSML + 3-MA treatments. These results indicated that the effects of PHSML inducing autophagy and SGB reducing autophagy were related to the PI3K / AKT signaling pathway.
In summary, our experimental data demonstrated that SGB improves the function of CD4+ T cells after hemorrhagic shock by targeting the PI3K / AKT pathway and reducing cell autophagy, which has a positive significance for maintaining the immune homeostasis, and the role is related to the intestinal lymphatic pathway. However, it is unclear whether SGB also has a therapeutic effect on hemorrhagic shock through other molecular mechanisms. This study focusing on CD4+ T cells, provides a certain basis for enriching the pathogenesis of hemorrhagic shock-induced immune dysfunction, and also provides a new therapeutic target for the prevention and treatment of severe hemorrhagic shock.