Omi/HtrA2 Participates in Age-Related Autophagic Deficiency in Rat Liver
Xu Jiahui, Jiao Kun, Liu Xin, Sun Qi, Wang Ke, Xu Haibo, Zhang Shangyue, Wu Ye, Wu Linguo, Liu Dan, Wang Wen*, Liu Huirong*
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, and Beijing Key Laboratory of Metabolic Disorders Related Cardiovascular Diseases, Capital Medical University, Beijing, China.
Liver is a vital organ with many important functions, and the maintenance of normal hepatic function is necessary for health. As an essential mechanism for maintaining cellular homeostasis, autophagy plays an important role in ensuring normal organ function. Studies have indicated that the degeneration of hepatic function is associated with autophagic deficiency in aging liver. However, the underlying mechanisms still remain unclear. The serine protease Omi/HtrA2 belongs to the HtrA family and promotes apoptosis through either the caspase-dependent or caspase-independent pathway. Mice lacking Omi/HtrA2 exhibited progeria symptoms (premature aging), which were similar to the characteristics of autophagic insufficiency. In this study, we demonstrated that both the protein level of Omi/HtrA2 in liver and hepatic function were reduced as rats aged, and there was a positive correlation between them. Furthermore, several autophagy-related proteins (LC3II/I, Beclin-1 and LAMP2) in rat liver were decreased significantly with the increasing of age. Finally, inhibition of Omi/HtrA2 resulted in reduced autophagy and hepatic dysfunction. In conclusion, these results suggest that Omi/HtrA2 participates in age-related autophagic deficiency in rat liver. This study may offer a novel insight into the mechanism involved in liver aging.
Figure 1. Both the level of aging-relative proteins and oxidative stress were increased in natural aging rat models. (A-D) The results of immunostaining and immunoblotting indicated that the expression of p53, p21 and β-gal was increased with growth of age. **P<0.01vs. 3 months, ##P<0.01 vs.9 month, #P<0.05 vs.9-month, n=6-8. (E-F) Compared with 3 and 9-month groups, there were decreased SOD activity and increased MDA level in 22-month group. **P<0.01vs. 3 months, #P<0.05 vs.9-month, n=6-8. SOD, Superoxide dismutase; MDA, Malondialdehyde.
Figure 2. The morphology and function of liver were declined during the natural aging process. (A) The liver morphology of 3, 9, 22-month-old rats was detected by H&E staining, bar=50 μm. (B) The hepatic function was evaluated by serum biochemical detection. (C-F) With the growth of age, both AST and GLB were increased significantly, the level of ALB and the ALB/GLB ratio were decreased. **P<0.01vs. 3 months, #P<0.05 vs.9 month, &P<0.05 vs.3 month. n=8-10. ALB, albumin; GLB, globin; AST, aspartate aminotransferase.
Figure 3. Decreasing of Omi/HtrA2 was accompanied with hepatic dysfunction in the natural aging process. (A) The protein level of Omi/HtrA2 in the liver of 3, 9, 22-month-old rats. Immunostaining, bar=200 μm. (B) Immunoblotting results indicated that the level of Omi/HtrA2 was decreased with growth of age. **P<0.01vs. 3 months, #P<0.05 vs.9 month. n=6-8. (C) The mRNA level of Omi/HtrA2 was detected by quantitative RT-PCR. (D-H) Correlation analysis of Omi/HtrA2 and hepatic function. There was a positive correlation between the protein level of Omi/HtrA2 in liver and the index of hepatic function. ALB, albumin; GLB, globin; AST, aspartate aminotransferase; ALT, alanine aminotransferase.
Figure 4. The level of autophagy in liver was decreased with aging. (A) The immunostaining of LC3II, bar=100 μm. (B) Immunoblotting results indicated that the level of autophagy was decreased with growth of age. **P<0.01vs. 3 months, #P<0.05 vs.9 month. n=6-8.
Figure 5. Inhibition of Omi/HtrA2 resulted in reduced autophagy in liver. (A-B) After treatment with ucf-101, the autophagy-related protein levels in 3 and 9-month-old rat’s liver were detected by immunoblotting. *P<0.05 vs. 3-month control, &P<0.05 vs. 9-month control. n=8-10. (C-D) The eGFP-mRFP-LC3 assays in vitro. The autophagy flux was reduced when cells were treated with ucf-101 (9.5μmol/L, 48h). Representative images showed LC3 staining in different groups of 7701 cells infected with GFP-RFP-LC3 adenovirus for 24 h. Acidified autophagosomes (red arrowheads in merged images) indicated active flux, yellow arrowheads pointed to immature autophagosomes. bar=25 μm. *P<0.05, #P<0.05. n=8. CQ, chloroquine.
Figure 6. Inhibition of Omi/HtrA2 attenuated hepatic function. The effect of Omi/HtrA2 inhibitor ucf-101 on hepatic function from different rats was evaluated by serum biochemical detection. *P<0.05, **P<0.01. n=8-10.
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