Effects Of The Simulated Weightlessness On Epithelial Barrier And Immune Barrier In Rat Intestine

The spaceflight has gotten a great achievement along with man's exploring to outspace. Our country has also made a greate progress in space program. The ambitious exploration rises challenges to space medical researchers.Gravity has been a constant physical factor during the evolution and development of life on Earth. But the spaceflight activity is under the weightleness or macrogravity. The physiological changes caused by weightlessness will harm the astronauts. More medical researches should be done to meet the need.Ground-based models play a significant role in space exploration because the real spaceflight researchs have too many limits.Although the absence of gravity can not accurately be simulated on ground, several kinds of models could mimic some responses observed after exposure to microgravity. Tail-suspension or hindlimb unloading in rodents is one of the most commonly used models which can mimics many of the physiological alterations in various organ systems caused by actual spaceflight. The hindlimb unloading technique has been approved by the NASA Ames Research Center Animal Care and Use Committee as a rat model for simulating spaceflight.Many serious adverse physiological changes occur during spaceflight. Some of these include fluid redistribution, increased kidney filtration, sensory input changes, cardiovascular deconditioning, bone deterioration, muscle loss, and impaired immune system function. However, there are scanty research reports on the pathophysiological alterations of digestive system under microgravity. To our knowledge ,the effects of the simulated weightlessness on intestinal barrier have not been elucidated. Accordingly, the present study was designed to document the intestinal epithelial barrier and immune changes in rat under simulated weightlessness and to determine whether there is associated stress response with alteration that microgravity imposed upon intestine.Methods:(1) The experiments were performed after approval by the local Ethics Committee and conducted on Male Wistar rats (Laboratory Animals Center, China Agriculture University). The procedure of tail-suspension described by Chen et al was adopted in the present study. At the end of the experiment, animals were anesthetized with pentobarbital (45 mg/kg) and laparotomy via middle line was made. Heparinized blood sample were drawn from the portal vein or vena cava. A small section of terminal ileum samples obtained from control and suspended animals were processed for transmission electron microscopy evaluation.The other terminal ileums were preserved in zinc-buffered formalin.Subsequently,tissue were processed for routine hematoxylin and eosin staining to assess general architecture and injury. (2) Part one:Male Wistar rats weighted 300±20 g were randomly assigned to 5 experimental groups: suspended for 1w, 2w, 3w, 4w and 0w (control). The ultrastructural alterations of intestinal epithelial cells were assessd under transmission electron microscopy; Endotoxin values were measured by use of kinetic test with ET ELISA Kit; D-lactic acid was measured by using D-lactic acid UV-method.(3) Part two:64 male Wistar rats weighted 280-310 g were randomly divided into simulated weightlessness group and control group, each with 4 subgroups :1-day group;2-day group ;4-day group and 7-day group.The expressions of NF-κB p65 were detected by using immunohistochemistry PV-6001. The apoptosis of intestinal epithelial cells was detected by terminal deoxy-nucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Measurement of tumor necrosis factor-α(TNF-α) was performed in blood samples using the immunoassay kit. (4) Part three:The animals and experimental groups were same as in part two.Lymphocytes apoptosis beneath the intestinal epithelium was measured using TUNEL method. The sIgA expression in intestinal mucosa was detected using immunohistochemistry PV-6001 and the number of CD4 in intestinal mucosa was calculated using immunohistochemistry stain.(5) Part four: 48 male Wistar rats weighted 300±20 g were randomly assigned to 6 experimental groups: suspended for 6 h, 12 h, 24 h, 48 h, 96 h and 0 h (control). Hsp70 expression in intestinal mucosa was detected in the first 96h also using immunohistochemistry method.Results:(1) Ultrastructural alterations in the intestine epthilial cells were observed under transmission electron microscopy. They consisted of dilatation of the endoplasmic reticulum, mitochondrial swelling and dilatations within the tight junctions.Endotoxin values in 1w-group were singnificantly higher than control group. Though it slightly declined then, the values in 2w-, 3w-,4w- groups were still in high level. The density of D-lactic in 1w-group was higher then control group, then declined gradually, the level in 4w-group approach the control group's. (2)Under the simulated macrogravity ,the plasma TNF-αlevel rised in 2-day subgroup, reached its peak in 4-day subgroup then declined to level of control groups in 7-day. The tail-suspension significantly increased intestine NF-κB expression in the rats, compared with the control(P<0.01), with peak expression in 2-day subgroups, followed by the slight declining, but the expression in 7-day subgroup was still higher. By TUNEL method, an increase in the percentages of apoptosis index was noticed in the intestine epithelial cells of all experimental rats under simulated microgravity compared with controls. The maximum percentage increase of apoptotic index was observed in 1-day group of suspension.(3) The sIgA expressions were reduced in experimental groups especially in 1-day, 2-day groups compared with control groups. It gradually elevated in 4-day,7-day groups. The percentage of apoptosis index of lymphocyte increased in all experimental groups, the maximum was in 1-day group. The number of CD4 in intestinal mucusa decreased markedly in 1-day group, then gradully increase in 2-day,4-day group.It near normal in 7-day goup.(4) The tail-suspension significantly increased Hsp70 expression levels in intestine, moreover it mainteined at high level in 96h-group.Conclusions: These findings suggest that: (1) Simulated microgravity by tail-suspension increased the intestine permeability which can be inferred from increased endotoxin value and D-lactic level; (2) The ultrastructural alteration and apoptosis of intestinal epithelial cells promoted by simulated microgravity may be the fundamental reason for increased intestinal permeability. (3) The tail-suspension has the effects of inducing activation of NF-κB and elevation of TNF-αin intestine especially in the early stage which suggests that NF-κB plays important roles in the inflammatory reactions of intestine to the weightlessness stress. (4) The simulated weightlessness promotes lymphocyte apoptosis and reduces sIgA secretion and CD4 cells which lead to lower the intestinal immune function.(5) The simulated weightlessness acts as a typical cellular stressor to induce high Hsp70 expression in intestine.