Effects Of The Simulated Weightlessness On Stress Response And Ultrastructural Alterations In Rat Liver

Background and Objective:Gravity has been a constant physical factor during the evolution and development of life on Earth. 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. Antiorthostatic bed rest in humans and tail-suspension or hindlimb unloading in rodents are two of the most commonly used models which can mimics many of the physiological alterations in various organ systems caused by actual spaceflight including fluid shifts, muscle atrophy, bone demineralization, and depressed cellular immunity.With great progress made in space program in recent years, especially after Shenzhou 7 milestone mission of carrying out the country's first spacewalk, China is now pursuing the next plans to build space laboratory or space station and then to land man on the moon. The ambitious exploration plans rise challenges to space medical researches which will ensure the astronauts to take spaceflight for longer periods of time in the near future. However, there are scanty research reports on the pathophysiological alterations of digestive system under microgravity in Chinese literatures, and the effects of the simulated weightlessness on stress response and ultrastructural alteration in rat liver have not been elucidated. Accordingly, the present study was designed to document the experssion features of Hsp70, Hsp70mRNA, NF-κB, apoptosis, p53 and morphological changes in rat liver under simulated weightlessness and to determine whether there is associated stress response and ultrastructural alteration that microgravity imposed upon liver.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. The right lobes of the livers obtained from control and suspended animals were immediately frozen in liquid nitrogen at -80℃until analysis, and the left lobes of the rat livers were preserved in zinc-buffered formalin. The samples from left lobes were processed for transmission electron microscopy evaluation. (2) Part one: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 protein and mRNA expressions in the liver were then detected by using Western blot analysis and RT-PCR respectively. (3) Part two:84 male Wistar rats weighted 280-310 g were randomly divided into simulated weightlessness group and control group, each with 7 subgroups from 1 to 7 according time sequence in day unit. The expressions of NF-κB p65 were detected by using Western blot analysis and immunohistochemistry PV-6001 respectively. (4) Part three : the animals and experimental groups were same as in part two. Hepatocyte apoptosis was quantitated using the terminal deoxy-nucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, and p53 expression was detected using immunohistochemistry assay. Liver samples were examined to assess the ultrastructural alteration under transmission electron microscope.Results:(1) We first studied whether Hsp70mRNA is expressed in rat liver under simulated weightlessness using RT-PCR. PCR products around the predicted size of 324 bp were detected in the liver tissues of both suspended and control animals. Compared with non-suspended animals, however, the tail-suspension significantly increased Hsp70mRNA expression levels in rat liver (P<0.05). The semi-quantitative PCR showed that Hsp70mRNA was up-regulated as early as 6 hours of suspension, with peak value at 12 hours, and persisted above the control level in all suspension subgroups. A fluctuation of Hsp70mRNA expression existed among suspended groups but without significant difference. Western blot analysis indicated that Hsp70 protein (approximately 70 kDa) was expressed in livers of all suspension subgroups as well as control group animals, and significantly up-regulated in the early stage of suspension as compared with controls (P<0.05). The level of Hsp70 expression appeared peak at 6 hours, followed by a declining tendency, and decreased to below the level of the control group at 96 hours suspension with no significant difference. (2) The tail-suspension significantly increased liver NF-κB expression in the rats, compared with the control(P<0.01), with peak expression in 1-day and 2-day subgroups, followed by the gradually declining to near the normal level in 5-day to 7-day experimental subgroups. NF-κB expression in rat liver stained as brown particles were detected in the hepatocytes, also in the infiltrated cells and kupffer cells. Three types of intracellular expression were noticed according to the location of positive NF-κB particles, i.e. cytoplasm, nucleus, and cytoplasm plus nucleus, uniformed or co-existed in rat liver. (3) By TUNEL assay, it was shown that an increase in the percentages of apoptosis index was noticed in the liver samples of all experimental rats under simulated microgravity compared with controls. The p53 expression was up-regulated in the rats at the early phase of simulated microgravity. Immunohistochemical stain indicated that the highest increment of p53 protein content was noticed in the first day of suspension whereas the maximum percentage increase of apoptotic index was observed 2-d group of suspension. Ultrastructural alterations in the liver cells were observed under transmission electron microscopy. They consisted of marked condensation of chromatin around the perinuclear membrane, moderate dilatation of the endoplasmic reticulum, mitochondrial swelling and an increased number of liver cells undergoing apoptosis.Conclusions: These findings suggest that: (1) the simulated weightlessness acts as a cellular insult or stressor that elevates liver Hsp70 expression both at protein and mRNA levels; (2) the tail-suspension has the effects of inducing activation of NF-κB in liver especially in the early stage of the experimental microgravity and suggests that NF-κB plays important roles in the cascade reactions of the liver to the weightlessness stress through the process of transcription; (3) the simulated weightlessness promotes apoptosis along with upregulation of p53 expression and results in ultrastructural alterations in rat liver in its early phase. The microgravity stress produced may lead to liver cell injury, and the function of apoptosis may not be limited only to the elimination of harmful, damaged, and unwanted cells. Further investigations upon this preliminary study are needed to clarify the cellular and molecular mechanisms responsible for the weightlessness stress response and to develop effective countermeasures in the weightless tolerance training and spaceflight.