NF-κB Mediated Inflammatory Responses In Arterial Remodeling Induced By Simulated Microgravity In Rats

When exposed to microgravity during space flight, the cardiovascular system in astronauts presents a series of functional adjustment and structural adaptations. However, when they return to the 1 G gravity environment on the ground or encounter with other orthostatic stress during flight or landing, the astronauts commonly exhibit cardiovascular dysfunction responses which mainly manifested by orthostatic intolerance and reduced upright aerobic capacity. For about half a century, scientists have made much progress in the understanding of the mechanism of postflight cardiovascular dysfunction. Although much work still remains to be implemented to understand the true mechanism, previous and current studies have demonstrated that functional adjustment and structural remodeling of the arterial system may play an important role in the occurrence of postflight orthostatic intolerance.In the environment of microgravity, the transmural pressure in arterial system and blood distribution in venous system present a series of changes as a result of the disappearance of hydrostatic pressure. Compared with the up-right posture at 1 G gravity on the ground, the cerebral artery represents a state of “hypertension”, yet arteries in the lower body represent a state of “hypotension”. In recent years, studies based on the animal model of simulated microgravity rats on the ground have demonstrated that the function and structure of the arterial system exhibit regional specific changes, which showed hypertrophic changes and enhanced contractile responsiveness in the arteries of the fore body, yet atrophic changes and reduced contractile responsiveness in that of the hind body of rats. Besides, the endothelium-dependent vasodilatation of the abdominal aorta, common carotid artery and cerebral artery in simulated microgravity rats have been proved to be decreased significantly, suggesting the possible changes of the endothelial function induced by simulated microgravity in rats. During the process of investigating the mechanism of arterial functional adjustment and structural remodeling induced by hydrostatic pressure changes, together with some other labs, we have found that the local rennin-angiotensin system(L-RAS) in the arterial system also exhibit regional specific changes in simulated microgravity rats, which means that the mRNA and protein expression of the main components of RAS, such as angiotensinogen and angiotensin Ⅱ type 1 receptor, are increased in the fore body, yet decreased in the hind body. In addition, it has also been reported that L-RAS might be involved in the process of arterial functional adjustment and structural adaptations in simulated microgravity rats through the mechanism of oxidative stress. Beyond that, the mechanism of vascular smooth muscle ion channels may participate in the above adaptive changes as well and exert crucially regulative effects. However, the impairment of arterial endothelium-dependent vasodilatation cannot be explained well enough through the mechanisms of L-RAS or vascular smooth muscle ion channels. Our previous studies have shown that simulated microgravity can induce oxidative stress in the arterial system. The production of superoxide anions, the main components of ROS, was significantly increased in both large elastic arteries, including abdominal aorta and common carotid artery, and muscular small arteries, such as cerebral artery. NF-κB is known as a kind of important transcriptional factor, and has been proved to be involved in many cardiovascular physiological and pathological processes, such as inflammatory responses and cell proliferation, differentiation and apoptosis. In the regulatory mechanism of the cardiovascular function and structure, NF-κB is the downstream pathway of both L-RAS and ROS, participating in the pathophysiological processes of many cardiovascular diseases. Under the condition of real/simulated microgravity, whether NF-κB is involved in the process of functional impairment in large elastic arteries and muscular small arteries has not been reported yet. In addition, our previous work includes only preliminary studies involving arterial inflammatory responses and lacks comprehensive realization about the condition of arterial inflammatory responses after simulated microgravity. Thus, whether NF-κB is involved in the remodeling of the endothelial function of the large elastic arteries and muscular small arteries in the fore and hind body of rats induced by simulated microgravity through inflammatory responses mechanism still remains to be further discussed.To answer the above questions, the hindlimb unweighting rats were adopted as the animal model to simulate the effects of microgravity on the cardiovascular system, and work was carried out as follows:1. The changes of protein expression and the activity of NF-κB in the abdominal aorta, common carotid artery and cerebral artery of simulated microgravity rats.Western blot analysis was used to observe the nuclear and cytosolic protein distribution of p65, a subunit of NF-κB, and the protein expression of IκBα, and to detect the impact of simulated microgravity on the translocation of NF-κB from cytoplasm to nucleus and the time point of the most obvious impact of the abdominal aorta, common carotid artery and cerebral artery, in 3-, 14-, and 28-day simulated microgravity rats. Besides, pyrrolidine dithiocarbamate(PDTC, 100 mg/kg/day), the inhibitor of NF-κB, was administered by gavage as an intervening treatment to determine the NF-κB DNA combining ability and the inhibitive effects of PDTC against this ability in the arterial system of rats after simulated microgravity.2. The inflammatory responses and the regulatory effects of NF-κB in the abdominal aorta, common carotid artery and cerebral artery of simulated microgravity ratsEn face immunofluorescent staining and the monocyte adhesive capacity of arterial endothelium in vitro were applied to detect the monocyte adhesive capacity of the endothelium in the abdominal aorta and common carotid artery in simulated microgravity rats. Western blot and immunohistochemistry staining analyses were used to determine the expression of inflammatory responses-related factors, such as vascular cell adhesion molecule 1(VCAM-1), E-selectin, and monocyte chemoattractant protein 1(MCP-1) in the abdominal aorta, common carotid and cerebral artery in simulated microgravity rats. The enzyme linked immunosorbent assay(ELISA) was used to detect the content of interleukin 6(IL-6) in the blood plasma of simulated microgravity rats. In addition, with the chronic administration of PDTC, RT-PCR was applied to examine the impact of NF-κB activation on the mRNA level of VCAM-1, E-selectin and MCP-1 in the abdominal aorta, common carotid artery and cerebral artery in simulated microgravity rats.3. The effects of PDTC, an inhibitor of NF-κB signal pathway, on the vasodilatation of the abdominal aorta, common carotid artery and middle cerebral artery of simulated microgravity ratsThrough chronic interference with administration of PDTC, the isometric force of arterial vasoreactivity measurement was used to observe the influences of PDTC on the vasodilatation of the abdominal aorta, common carotid artery and cerebral artery in simulated microgravity rats.The main findings of the present study are as follows:1. Simulated microgravity leads to an increase in NF-κB activity in the abdominal aorta, common carotid artery and cerebral artery of rats.The results of Western blot analysis have shown that, compared with the control group, the expression of p65, a subunit of NF-κB, in nucleus was increased with the time course of hindlimb unweighting in the abdominal aorta, common carotid artery and cerebral artery of simulated microgravity rats, whereas the expression of p65 in cytoplasm and IκBα was decreased, among which the changes in the abdominal aorta was earlier than that in the common carotid artery and cerebral artery. The results of electrophoretic mobility shift assay have shown that, compared with the control group, the NF-κB DNA combining ability was significantly increased in the abdominal aorta, common carotid artery and cerebral artery of 28-day simulated microgravity rats. The chronic interference with administration of PDTC significantly attenuated the increased NF-κB DNA combining ability in the abdominal aorta, common carotid artery and cerebral artery, while partially reversed this ability in the cerebral artery.2. Simulated microgravity increases the monocyte infiltration and THP-1 adhesion in the abdominal aorta and common carotid artery of rats. NF-κB is associated with the occurrence of inflammatory responses in the abdominal aorta, common carotid artery and cerebral artery of simulated microgravity rats.The results of en face immunofluorescent staining have shown that the monocyte infiltration was increased in the abdominal aorta and common carotid artery of simulated microgravity rats. The results of the monocyte adhesive capacity of arterial endothelium in vitro have shown that adhension of THP-1 to vascular endothelial cells were significantly increased in the abdominal aorta and common carotid artery of simulated microgravity rats. The results of Western blot and immunohistochemistry analyses have shown that the protein expression of E-selectin and MCP-1 were both increased in the abdominal aorta, common carotid artery and cerebral artery in simulated microgravity rats. The expression of VCAM-1 was increased in the common carotid artery and cerebral artery yet exhibited no significant change in the abdominal aorta in the simulated microgravity rats. In addition, the positive expression of inflammatory factors was mainly concentrated on the endothelium and smooth muscle layers. The results of ELISA have shown that the level of IL-6 was significantly increased in the blood plasma of simulated microgravity rats than that of control rats. The results of RT-PCR have shown that, compared with the control group, the mRNA level of VCAM-1, E-selectin and MCP-1 was significantly increased in the abdominal aorta, common carotid artery and cerebral artery of simulated microgravity rats. Chronic interference with administration of PDTC significantly attenuated the increased mRNA level of the above three inflammatory factors in the abdominal aorta and common carotid artery, whereas only reversed the mRNA level of MCP-1 in the cerebral artery in simulated microgravity rats.3. The involvement of NF-κB mediated inflammatory responses in the decline of the endothelium-dependent vasodilatation of the abdominal aorta, common carotid artery and cerebral artery of simulated microgravity ratsThe results of isolated vessel rings isometric force measurement in vitro have shown that, compared with the control group, the endothelium-dependent vasodilatation induced by ACh was significantly decreased in the abdominal aorta, common carotid artery and cerebral artery of simulated microgravity rats. Chronic interference with administration of PDTC significantly ameliorated the impaired endothelium-dependent vasodilatation in the abdominal aorta and common carotid artery, yet exerted only recovery trend in the middle cerebral artery.In conclusion, the present study demonstrates that 28-day simulated microgravity can induce NF-κB activation and inflammatory responses in the abdominal aorta, common carotid artery and cerebral artery of rats. Furthermore, the impairmet in vasodilatory function of the abdominal aorta, common carotid artery and cerebral artery is associated with NF-κB mediated inflammatory response. This study has great significance for the clarity of the mechanism of cardiovascular dysfunction induced by real/simulated microgravity and the proposal of new effective countermeasures.