Young plasma reverses age-dependent alterations in hepatic function through the restoration of autophagy

Abstract

Recent studies showing the therapeutic effect of young blood on aging-associated deterioration of organs point to young blood as the solution for clinical problems related to old age. Given that defective autophagy has been implicated in aging and aging-associated organ injuries, this study was designed to determine the effect of young blood on aging-induced alterations in hepatic function and underlying mechanisms, with a focus on autophagy. Aged rats (22 months) were treated with pooled plasma (1 ml, intravenously) collected from young (3 months) or aged rats three times per week for 4 weeks, and 3-methyladenine or wortmannin was used to inhibit young blood-induced autophagy. Aging was associated with elevated levels of alanine transaminase and aspartate aminotransferase, lipofuscin accumulation, steatosis, fibrosis, and defective liver regeneration after partial hepatectomy, which were significantly attenuated by young plasma injections. Young plasma could also restore aging-impaired autophagy activity. Inhibition of the young plasma-restored autophagic activity abrogated the beneficial effect of young plasma against hepatic injury with aging. In vitro, young serum could protect old hepatocytes from senescence, and the antisenescence effect of young serum was abrogated by 3-methyladenine, wortmannin, or small interfering RNA to autophagy-related protein 7. Collectively, our data indicate that young plasma could ameliorate age-dependent alterations in hepatic function partially via the restoration of autophagy.

Keywords: aging; autophagy; hepatocyte; liver injury; liver regeneration; rat; senescence; young blood.

Figure 1

Young plasma improves liver function in aged rats. (a) Serum samples were collected for measuring AST and ALT. The data are shown as mean ± SD, n = 8–10 per group. (b) Representative micrographs showing hepatic steatosis in liver sections as stained with Oil Red O (original magnification, 400×). (c) Representative micrographs displaying hepatic fibrosis in liver sections as stained with Masson's trichrome (original magnification, 400×). (D) Quantitative analysis of liver steatosis area and fibrosis area. The data are shown as mean ± SD, n = 4 per group

Figure 2

Young plasma prevents hepatic senescence and endoplasmic reticulum stress in aged rats. (a) Representative micrographs showing SA‐β‐gal activity in hepatic tissues (original magnification, 400×). (b) Representative micrographs exhibiting lipofuscin levels in hepatic tissues (original magnification, 400×). (c) Quantitative analysis of the SA‐β‐gal area and lipofuscin levels. The data are shown as mean ± SD. n = 4 per group. (d) Representative Western blotting of p16, p21, GRP78, and GRP94 in livers. (e) Densitometric analysis of p16, p21, GRP78, and GRP94. The data are shown as mean ± SD, n = 4 per group

Figure 3

Young plasma rescues defective liver regeneration capacity in aged rats. (a) Quantification of serum AST and ALT levels. The data are shown as mean ± SD, n = 4 per group. (b) Representative micrographs exhibiting Ki67‐positive hepatocytes in hepatic tissues obtained from prepartial hepatectomy (PH) and 24 hr post‐PH (original magnification, 400×). (c) The numbers of Ki67‐positive hepatocytes were determined. The data are shown as mean ± SD, n = 4 per group

Figure 4

Young plasma restores aging‐induced suppression in hepatic autophagy. (a) Representative Western blotting of LC3B and p62. (b) Densitometric analysis of LC3B‐II and p62. (c) Representative transmission electron microphotographs showing autophagosomes in hepatic tissues (original magnification, 5000×). Autophagosomes are indicated by arrows. (d) The numbers of autophagosomes were calculated. The data are shown as mean ± SD, n = 4 per group

Figure 5

Young plasma prevents liver aging via the restoration of autophagy. Aged rats were treated with pooled young plasma (1 ml, IV) three times per week for 4 weeks. 3‐methyladenine (3‐MA, 30 mg/kg, IP) or wortmannin (Wor, 0.6 mg/kg, IV) was then given to the rats 3 times per week for 2 weeks before harvest. (a) Representative micrographs showing hepatic steatosis in liver sections as stained by Oil Red O (original magnification, 400×). (b) Representative micrographs displaying hepatic fibrosis in liver sections as stained with Masson's trichrome (original magnification, 400×). (c) Representative micrographs showing SA‐β‐gal activity in hepatic tissues (original magnification, 400×). (d) Representative micrographs exhibiting lipofuscin levels in liver sections (original magnification, 400×). (e) Quantitative analysis of the liver steatosis area, fibrosis area, SA‐β‐gal area, and lipofuscin levels. Data are shown as mean ± SD, n = 3 per group

Figure 6

Inhibition of autophagy abrogates the antisenescence effect of young serum. Old hepatocytes were treated with young serum in the absence or presence of vehicle (Veh), 3‐MA (10 mM), or wortmannin (Wor, 1 μM) for 72 hr. (a) Representative Western blotting of LC3B, p16, p21, GRP78, and GRP94 protein expression. (b) Densitometric analysis of LC3B‐II, p16, p21, GRP78, and GRP94. The experiment was performed in triplicate with similar results. The data are shown as mean ± SD. (c) Representative micrographs of SA‐β‐gal staining in hepatocytes with or without young serum, 3‐MA, or Wor (original magnification, 400×). (d) Representative micrographs of Oil Red O staining in hepatocytes in the absence or presence of young serum, 3‐MA, or Wor (original magnification, 400×). (e) Quantitative analysis of SA‐β‐gal and lipid accumulation. The experiment was performed in triplicate with similar results. The data are shown as mean ± SD