The Effects And Mechanisms Of Chronic Vagus Nerve Stimulation Alleviates Vascular Endothelial Impairments And The Inflammatory Profile In Ovariectomized Rats

Background: Cardiovascular diseases(CVDs), one of the most serious diseases in the world currently, are a group of diseases caused by heart and vascular diseases, and half of the cases are estimated in Asia. Its morbidity and mortality have exceeded the neoplastic diseases and ranks first. Cardiovascular risks are lower in premenopausal women than in aged-matched men and postmenopausal women, and this diffidence is associated with the protective effects of estrogen from ovarian. In the early stage of menopause, the endothelial function of healthy women began to decline. And the degree of endothelial dysfunction is closely related to the occurrence and development of CVDs and metabolic diseases. Impairments of vascular endothelial cells participate in the occurrence and development of atherosclerosis by expressing immunoglobulin superfamily adhesion molecules(VCAM-1, ICAM-1, etc) and members of selectin family(E-selectin, P-selectin, etc), and secreting the inflammatory mediators(TNF-a, IL-1, IL-6, IL-8, MIP, MCP-1, etc). In addition, researches have demonstrated that menopause and ovariectomy can cause low-grade systemic inflammation, with increased circulating levels of IL-8, MCP-1,RANTES, and MIP-1?. Clinical and experimental investigations have suggested that disrupted autonomic homeostasis occurs in both premenopausal and postmenopausal women when ovarian function is lost characterized by increased sympathetic nervous activity and reduced parasympathetic activity. As a part of the autonomic nervous system, the vagus nerve, may participate in the regulation of the inflammatory profile of the body. And some scholars have named the neuro-immune regulatory pathway as the “cholinergic anti- inflammatory pathway(CAP)”. My previous studies also found that the increase in vagal activity can significantly reduce the inflammatory profile of colonic mucosa. So we speculated that the activation of endothelial cells and the inflammatory profile in postmenopausal women may be related to the abnormal immune regulation caused by dysfunction of the autonomic nervous system. Acetylcholine(ACh) released from the vagal efferents modulates immune responses via alpha 7 nicotinic receptor(?7n Ach R) on human immune cells that regulates the nuclear translocation of NF-?B/p65, and inhibits the transcription of proinflammatory cytokines and chemokines. In addition, MAPKs signal pathway may directly or indirectly affect the transcription process of the gene encoding proinflammatory cytokines, the phosphorylation of I?B and NF-?B/p65, and nuclear translocation of NF-?B/p65. And the I?B/NF-?B signaling pathway is considered to be one of the most important signal transduction pathways downstream of MAPKs signaling pathway. Our previous studies demonstrated that biofeedback can effectively improve the balance of the autonomic nervous system by enhancing vagal activity and reducing sympathetic activity. Therefore, we propose the hypothesis: biofeedback can effectively improve the balance of the autonomic nervous system, enhance the vagal activity, to a certain extent, activate cholinergic anti-inflammatory pathway(CAP), attenuate vascular endothelial impairments in postmenopausal women, and reduce the morbidity and mortality of CVDs in postmenopausal women by inhibiting the synthesis and release of inflammatory cytokines. And its intrinsic mechanisms may be related to n ACh R, MAPKs and NF-?B signaling pathways. In order to verify the above scientific hypothesis, we need to combine animal experiments and in vitro cell experiments. However, biofeedback is normally based on the active participation of the central nervous system and it still cannot be simulated in animal models due to the limitations of current technology. Therefore, based on the previous research, Professor Wang Tinghuai put forward: enhanced vagal activity may partially simulate body reaction in the process of biofeedback, and as a breakthrough point, and began to study the mechanism of biofeedback in a variety of animal disease models. So in the present study, combining with the basis of our previous work, we adopt the model of chronic vagus nerve stimulation to simulate the effects of enhancing vagal activity of biofeedback in the human body, and explore the effects and mechanisms of enhancing vagal activity on vascular endothelial impairments and the inflammatory profile in ovariectomized rats; and to futher explore the possible mechanisms combining with human umbilical vein endothelial cells(HUVECs) in vitro.Objects: To investigate the protective and anti-inflammatory effects of chronic vagus nerve stimulation on vascular endothelial impairments and the inflammatory profile in ovariectomized rats; and to further explore the role of n ACh R, MAPKs, and NF-?B signaling pathway in this process combining with HUVECs in vitro.Methods and Results: Sprague-Dawley rats(7-8 months old) were randomly assigned to the following four groups: sham-OVX, OVX, OVX + sham-VNS, and OVX + VNS. Throughout the experimental period, the OVX + VNS group received VNS for 3 h(20.0 Hz, 1.0 m A, and 10.00 ms pulse width) at the same time every other day. After 12 weeks of VNS, blood samples and thoracic aortas were collected for further analyses. Light microscopy and electron microscopy analyses showed that chronic VNS prevented endothelial swelling, desquamation and even necrosis in the OVX rats. In addition, it obviously improved endothelial function in the OVX rats by restoring the endothelial nitric oxide synthase(e-NOS) and serum endothelin-1(ET-1) level. Increased the expressions of cell adhesion molecules(VCAM-1, ICAM-1 and E-selectin) in the thoracic aortas and the levels of circulating cytokines(TNF-?, IL-6, MCP-1, and CINC/KC) were also observed in the OVX rats. Chronic VNS significantly restored these detrimental changes partly by increasing the ACh concentrations in vascular walls and blocking NF-?B pathway activity. We also found that ACh(10-9- 10-5 M)blocked adhesion molecule expressions(VCAM-1, ICAM-1, and E-selectin) and the inflammatory cytokines(TNF-?, IL-6, MCP-1, IL-8) production by LPS-treated HUVECs in vitro. And this effect can be blocked by nonspecific n ACh R blockers, rather than specific ?7n ACh R blockers. Western blot also were used to detect expressions of NF-?B/p65?p-NF-?B/p65?I?B?p-I?B?ERK?p-ERK?JNK?p-JNK?p38MAPK?p-p38 MAPK of HUVECs in vitro. We found that ACh(10-6 M) can significantly reduce the LPS(100 ng/ml) induced the phosphorylation of NF-?B/p65, I?B, ERK, JNK, p38 MAPK in HUVECs. And this process may be blocked by n ACh R blocker.Conclusions: 1. Chronic vagus nerve stimulation protects OVX rats from endothelial impairments and dysfunction; and the underlying mechanisms may include up-regulation of e-NOS expression and NO production in the thoracic aortas, a reduction in the serum ET-1 level. 2. Chronic vagus nerve stimulation can significantly reduce the expression of vascular endothelial adhesion molecules(VCAM-1, ICAM-1, E-selectin) and the serum levels of inflammatory cytokines(TNF-?, IL-6, CINC/KC, MCP-1) in ovariectomized rats. These results suggest: chronic vagus nerve stimulation can reduce the inflammatory profile in ovariectomized rats; and the mechanism may be related to inhibition of vascular endothelial cells activation by increased content of local ACh in the blood vessels.3. MAPKs(ERK, JNK, p38MAPK) and NF-?B signaling pathway may be involved in the regulation of chronic vagus nerve stimulation on the inflammatory response in ovariectomized rats, and this process may be mediated by n ACh R.