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. 2015 May 22;10(5):e0127047.
doi: 10.1371/journal.pone.0127047. eCollection 2015.

Morphology and Molecular Mechanisms of Hepatic Injury in Rats under Simulated Weightlessness and the Protective Effects of Resistance Training

Fang Du  1 , Ye Ding  1 , Jun Zou  2 , Zhili Li  3 , Jijing Tian  1 , Ruiping She  1 , Desheng Wang  3 , Huijuan Wang  3 , Dongqiang Lv  1 , Lingling Chang  1
Affiliations

Morphology and Molecular Mechanisms of Hepatic Injury in Rats under Simulated Weightlessness and the Protective Effects of Resistance Training

Fang Du et al. PLoS One. .

Abstract

This study investigated the effects of long-term simulated weightlessness on liver morphology, enzymes, glycogen, and apoptosis related proteins by using two-month rat-tail suspension model (TS), and liver injury improvement by rat-tail suspension with resistance training model (TS&RT). Microscopically the livers of TS rats showed massive granular degeneration, chronic inflammation, and portal fibrosis. Mitochondrial and endoplasmic reticulum swelling and loss of membrane integrity were observed by transmission electron microscopy (TEM). The similar, but milder, morphological changes were observed in the livers of TS&RT rats. Serum biochemistry analysis revealed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher (p<0.05) in TS rats than in controls. The levels of ALT and AST in TS&RT rats were slightly lower than in RT rats, but they were insignificantly higher than in controls. However, both TS and TS&RT rats had significantly lower levels (p<0.05) of serum glucose and hepatic glycogen than in controls. Immunohistochemistry demonstrated that the expressions of Bax, Bcl-2, and active caspase-3 were higher in TS rats than in TS&RT and control rats. Real-time polymerase chain reaction (real-time PCR) showed that TS rats had higher mRNA levels (P < 0.05) of glucose-regulated protein 78 (GRP78) and caspase-12 transcription than in control rats; whereas mRNA expressions of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) were slightly higher in TS rats. TS&RT rats showed no significant differences of above 4 mRNAs compared with the control group. Our results demonstrated that long-term weightlessness caused hepatic injury, and may trigger hepatic apoptosis. Resistance training slightly improved hepatic damage.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Levels of ALT, AST and glucose in serum.
A: Serum ALT and AST levels: ALT and AST levels in the TS group were significantly increased compared with the control group, and ALT and AST levels in the TS&RT group were increased compared with the control group but reduced compared with the TS group; however, these differences were not significant. B: Serum glucose levels: serum glucose levels in the TS and TS&RT groups were significantly decreased compared with the control group (Data are expressed as the mean ± SD. * P <0.05, significantly different from control group).
Fig 2
Fig 2. The analysis of liver glycogen by PAS staining.
A-C: Liver glycogen as demonstrated by PAS staining (purple-red, 100×). A: Liver glycogen in the control group: large areas of hepatic cells around the central vein with positive PAS staining. B: Liver glycogen in the TS group: PAS-positive cells were significantly reduced, mainly at the edge of the portal area, with only scattered, sporadic purple liver cells surrounding the central vein. C: Liver glycogen in the TS&RT group: PAS-positive cells remained mainly in the area around the portal vein, and the number of PAS-positive cells increased around the central vein. D: A semi-quantitative analysis of liver glucose in the PAS-stained slides confirmed the results obtained for the serum glucose levels. Each value represents the percentage of the mean area of glycogen density in each group. n = 150 fields (10 rats) for each value. (Data are expressed as the mean ± SD. ** P <0.01, significantly different from control group.).
Fig 3
Fig 3. Histopathological analysis of rat liver paraffin sections stained with hematoxylin and eosin (H&E), Mallory’s trichrome stain, and Sirius Red stain.
A: Morphological changes in the liver: the normal structure of the rat liver in the control group (A1). In the TS group, we observed mass granular degeneration and the swelling of hepatic cells around the central vein, vague cell boundaries, liver cell cord derangement, edema and the proliferation of bile ducts, and inflammation in liver portal areas (arrow) (A2). In the TS&RT group, pathological changes included the slight vacuolar degeneration of hepatic cells with a completed structure and clear boundaries and fibrosis in the portal areas of the liver in some of the rats (A3). B&C: Sections stained with Mallory’s trichrome (B) and Sirius Red (C) displayed various levels of the proliferation of fibrous liver tissue in the control (B1, C1), TS (B2, C2), and TS&RT groups (B3, C3). Collagen fibers stained blue with Mallory’s trichrome and red with Sirius Red stain (arrow). 400×
Fig 4
Fig 4. Transmission electron micrographs of liver in control, TS and TS&RT group.
A: Liver tissue in control group, which was structurally intact with clearly visible mitochondrial cristae (star) and smooth endoplasmic reticulum cisternae (arrow). B-D: Liver tissue in TS group showed numerous mitochondria in hepatic cells with different degrees of swelling, and mitochondrial cristae displayed cavitation and even disappeared (star). The endoplasmic reticulum was expansive (arrow), and the liver cell matrix was loose. E, F: Liver tissue in TS&RT group showed that the degree of mitochondrial swelling and vacuolation was reversed (star), and the cytoplasmic matrix was more intact compared with TS group (A, C, D: 50000×; B, E: 20000×; F: 40000×).
Fig 5
Fig 5. Immunohistochemical and real-time PCR analysis of Bax and Bcl-2.
A1-C1: Histological sections of livers in control, TS and TS&RT group were stained with Bax antibody, and the positive signals were mainly located around the central veins in the livers. A2-C2: Histological sections of livers in control, TS and TS&RT group were stained with PBS to replace the Bax antibody as blank control; no positive signals were observed. D1-FI: Histological sections of livers in control, TS and TS&RT groups were stained with Bcl-2 antibody, and the location of the Bcl-2 positive signals was similar to the Bax positive signals. D2-F2: Histological sections of livers in control, TS and TS&RT groups were stained with PBS to replace the Bcl-2 antibody as a blank control; no positive signals were observed. G: A semi-quantitative analysis of the ratio of Bax positive staining to the total field. H: A semi-quantitative analysis of the ratio of Bcl-2 positive staining to the total field. I: Relative mRNA levels of Bax and Bcl-2. (Data are expressed as the mean ± SD. * P <0.05, ** P <0.01, significantly different from control group. 400×).
Fig 6
Fig 6. Immunohistochemical and real-time PCR analysis active of caspase-3.
A1-C1: Histological sections of livers in control, TS and TS&RT group were stained with active caspase-3 antibody. A2-C2: Histological sections of livers in control, TS and TS&RT groups were stained with PBS to replace the caspase-3 antibody as blank control. D: A semi-quantitative analysis of the ratio of active caspase-3 positive staining to the total field. E: Relative mRNA levels of caspase-3 in control, TS and TS&RT rat livers (Data are expressed as the mean ± SD. * P <0.05, significantly different from control group. 400×).
Fig 7
Fig 7. Real-time PCR analysis of changes in ER-associated apoptosis proteins.
GRP78 transcription in TS group was significantly increased compared with control group, and GRP78 expression in TS&RT group was significantly reduced compared with TS group and similar to control group. Caspase-12 mRNA was significantly increased in TS group and decreased with no significant differences in TS&RT group. CHOP and JNK transcription was slightly increased in TS group compared with the control group, which was reduced compared with TS group, but no significant differences were noted (Data are expressed as the mean ± SD. * P <0.05, significantly different from control group. # P <0.05, significantly different from TS group.).
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Grants and funding

This work was supported by Advanced Space Medico-Engineering Research Project of China (grant NO. SJ200802).