Blood from young animals can positively affect aged animals. Aging is commonly associated with defective autophagic activity, which is a causal mechanism for the age‐dependent liver IRI. Furthermore, age‐impaired autophagy could be restored by young plasma ( 13 ). Therefore, we hypothesized that young plasma could attenuate IRI via restoration of autophagy. In this study, we investigated the role of autophagy in young plasma–afforded protection on IRI in aged livers, as well as the regulatory mechanisms of autophagy, particularly its linkage to the AMPK/ULK1 axis.

Autophagy plays a major role in the maintenance of cellular homeostasis, protein quality control, and adaptation to environmental challenges by degrading of intracellular components by lysosome‐dependent machinery ( 14 , 15 ). It has been demonstrated that autophagy has a protective role in liver diseases ( 16 – 18 ), and induction of autophagic activity is able to reduce liver IRI ( 19 – 22 ). Autophagy also plays a critical role in aging and aging‐related organ injuries, and restoration of autophagy can promote longevity in experimental models and ameliorate aging‐associated anomalies ( 23 – 26 ). It has been reported that autophagic activity normally decreases in the liver with age ( 27 – 29 ). AMPK, a central cellular energy and nutrient status sensor, has been reported to be one of the major autophagy regulators. It is well known that AMPK can regulate that initiation of autophagy by directly phosphorylation of nc‐51–like autophagy activating kinase (ULK)‐1 ( 30 – 32 ). Several studies have demonstrated that aging reduces the activation capacity of AMPK signaling, which impairs the maintenance of efficient cellular homeostasis and augments the aging process ( 33 , 34 ). We previously demonstrated that young plasma could restore the age‐impaired autophagic activity ( 13 ). However, it is unclear whether AMPK/ULK1‐mediated autophagy is a mechanism by which young plasma restores autophagy in aged livers.

Several recent studies show that infusion of blood obtained from young mice into old mice has therapeutic effect on aging‐associated deterioration of organs, including muscle ( 5 , 6 ), brain ( 7 , 8 ), spinal cord ( 9 ), heart ( 10 ), and pancreas ( 11 ). Young plasma also ameliorates pathology and cognition in a mouse model for Alzheimer disease ( 12 ). A similar rejuvenating effect of young plasma has also been demonstrated in aged liver by us. We showed that young plasma reversed age‐dependent alterations in hepatic function ( 13 ). These observations suggest that exposure to a young environment could prevent or reverse the age‐dependent decline in the function of critical organ systems.

Aging is often associated with the development of tumors and chronic liver diseases that require surgery such as liver resection and transplantation. Ischemia reperfusion injury (IRI) occurs when the blood flow to an organ is transiently interrupted. IRI can be a serious complication of major liver resection and liver transplantation. The risk for postoperative complications in response to liver transplantation is increased in elderly patients ( 1 , 2 ), and aged livers exhibit more susceptibility to IRI compared with young ones ( 3 , 4 ). Therefore, developing protective strategies to reduce IRI remains of paramount importance, and is even more important for aged livers with an increased sensitivity to this injurious pathway.

Western blot analysis was performed as previously described ( 35 ). Liver tissues and cultured hepatocytes were homogenized in RIPA lysis buffer (AR0105; BosterBio, Wuhan, China), supplemented with protease and phosphatase inhibitors (05892953001 and 05892970001; Roche, Basel, Switzerland). Homogenates were centrifuged at 12,000 g for 15 min at 4°C, and the supernatants were collected. Protein was quantified with a Bicinchoninic Acid Protein Assay Kit (23225; Thermo Fisher Scientific). After electrophoresis, proteins were transferred to PVDF membranes (IPVH00010; MilliporeSigma). The membranes were blocked and incubated overnight at 4°C with suitably diluted primary antibodies: polyclonal rabbit anti‐LC3B‐II (1:1000, ab48394) and rabbit anti‐ULK1 (1:1000, ab128859) (Abcam, Cambridge, MA, USA); rabbit anti‐Atg7 (1:500, 8558), rabbit anti‐beclin1 (1:500, 3738), rabbit anti‐phospho‐AMPKα (Thr172, 1:500, 2535), and rabbit anti‐AMPKα (1:1000,2532) (Cell Signaling Technology, Danvers, MA, USA); rabbit anti‐p62 (1:1000, P0067) rabbit anti‐phospho‐ULK1 (Ser555, 1:1000, ABC124), and rabbit anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody (1:20,000; G9545; MilliporeSigma) were used for immunoblot analyses. Blots were developed with a chemiluminescent substrate (WBKLS0500; MilliporeSigma) and visualized on the Kodak Image Station (Carestream Health, Rochester, NY, USA). Quantification of protein bands was achieved by densitometric analysis using ImageJ.

Hepatocytes were infected with adenovirus encoding green fluorescent protein‐ microtubule–associated protein 1 light chain 3 (GFP‐LC3; Hanbio, Shanghai, China) at a multiplicity of infection of 20. The culture medium containing the virus was replaced with fresh medium 2 h after infection. After treatment, the hepatocytes were stained with DAPI and visualized with a BX‐51 fluorescence microscope (Olympus, Tokyo, Japan).

An H/R model in vitro was performed as previously described ( 35 , 36 ). In brief, primary hepatocytes were placed into serum‐free Opti‐MEM I equilibrated with 1% O 2 ,5% CO 2 , and 94% N 2 and placed in a hypoxia chamber (Thermo Fisher Scientific) flushed with the same hypoxic gas mixture. After incubation under hypoxia for 20 h, the medium was then replaced with fresh Opti‐MEM I medium with 5% serum, and cells were incubated under normoxic conditions (air / 5% CO 2 ) for 2 h.

Primary rat hepatocytes were isolated and cultured as previously described ( 35 , 36 ). In brief, rats were anesthetized with pentobarbital sodium, and the liver was perfused in situ with warm basic DMEM (11995065; Thermo Fisher Scientific) containing 0.75 mg/ml collagenase D (C5138; MilliporeSigma) for 15 min. Then, the liver was excised, minced, and strained. Hepatocytes were obtained by centrifugation at 30 g for 5 min at 4°C and washed 3 times with basic DMEM. The freshly obtained hepatocytes were seeded on plates coated with rat tail collagen (C3867; MilliporeSigma) in Opti‐MEM I reduced‐serum medium (31985062; Thermo Fisher Scientific) supplemented with 5% serum from young rats or old rats.

Serum aspartate transaminase (AST) and alanine transaminase (ALT) levels were measured with an automated chemical analyzer (Hitachi Co., Tokyo, Japan). Formalin‐fixed, paraffin‐embedded liver sections were cut to 4‐µm thickness with a Leica microtome (Leica Microsystems, Buffalo Grove, IL, USA). The liver sections were stained with hematoxylin and eosin (H&E), and pathologic changes were evaluated in a blinded fashion by 2 independent pathologists. The histologic necrosis area was manually marked and measured with ImageJ (NIH; http://imagej.nih.gov/ij ). The necrotic area was counted in 5 fields per section and averaged for each section.

An established rat model of segmental (70%) hepatic IRI was generated as previously described ( 20 , 35 ). In brief, the rats were completely anesthetized with pentobarbital sodium, and a microvascular clamp was used to interrupt the arterial and portal venous blood supply to the left lateral and median liver lobes. The clamp was removed after 60 min of warm ischemia, thereby initiating hepatic reperfusion. The animals were euthanized 6 h after reperfusion, and a blood sample was collected from the inferior vena cava. Serum was obtained from the blood by centrifugation at 1000 g for 5 min and stored in aliquots at −80°C until use. The left lateral lobe was collected for histologic and protein analyses.

Plasma collection and treatment were performed as described by Liu et al . ( 13 ). Blood samples were collected from the inferior vena cava of rats under pentobarbital sodium anesthesia. Plasma and serum were obtained by centrifugation of the collected blood, with or without heparin, at 1000 g for 5 min. All plasma and serum aliquots were stored at −80°C until use, and the storage time was <3 mo. Aged rats were treated with pooled plasma (1 ml, i.v.) collected from young or aged rats 3 times per week (Monday, Wednesday, and Friday) for 4 wk.

Young male inbred Sprague‐Dawley rats, 3 mo of age and weighing 200–250 g, and 22 mo of age, weighing 600–800 g, were purchased from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). All of the animals were housed under standard animal care conditions and had free access to water and food. All of the animal protocols were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. All of the animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals [National Institutes of Health (NIH), Bethesda, MD, USA].

To investigate whether young serum could protect old hepatocytes from hypoxia/reoxygenation (H/R) insult via the restoration of autophagy in vitro , primary old hepatocytes were cultured for 72 h with young serum or old serum, and then subjected to 20 h hypoxia and 2 h reoxygenation injury. For autophagic flux, hepatocytes were treated with 10 µM chloroquine for 6 h before harvest. Young serum‐restored autophagy was inhibited by 3‐MA (10 mM), wortmannin (1 µM) or autophagy‐related gene (Atg)7 small interfering RNA (siRNA, 50 nM; 259550; Thermo Fisher Scientific, Waltham, MA, USA), respectively.

To determine autophagic flux, rats received chloroquine (60 mg/kg, i.p., C6628; MilliporeSigma, Burlington, MA, USA) 6 h before euthanasia. To evaluate whether young plasma could reduce age‐dependent liver IRI by the restoration of autophagy, 3‐methyladenine (3‐MA, 30 mg/kg, i.p.; 13242; Cayman Chemicals, Ann Arbor, MI, USA) or wortmannin (0.6 mg/kg, i.v., 10010591; Cayman Chemicals) was given to rats 0.5 h before ischemia. Rats were euthanized 6 h after reperfusion, and liver injury was analyzed.

Young serum‐restored autophagy is dependent on AMPK/ULK1 signaling. Old hepatocytes were treated with young serum for 72 h in the absence or presence of vehicle, compound C (10 µM), or AICAR (500 µM) and then subjected to 20 h hypoxia and 2 h reoxygenation injury. A ) Representative Western blot of phospho‐AMPKα, AMPKα, phospho‐ULK1 ULK1, LC3B, and p62 protein expression. B ) Densitometric analysis of phospho‐AMPKα, phospho‐ULK1, LC3B‐II, and p62. C ) Quantification of cell viability and LDH release of primary old hepatocytes after H/R insult. The experiment was performed in triplicate with similar results. The data are means ± sd .

In line with in vivo studies, the levels of phosphorylation of AMPKα and phosphorylation of ULK1 in old hepatocytes cultured with young serum were significantly higher than those in old hepatocytes cultured with old serum, both at baseline and after H/R insult (Supplemental Fig. S3). Inhibition of AMPK activation by compound C led to a decreased phosphorylation of ULK1 and attenuated the young serum‐induced increases in LC3B‐II levels and decreases in p62 levels in old hepatocytes, whereas the AMPK activator AICAR potentiated the effects of young serum (Fig. 6A , B ). Furthermore, cellular injury was increased in compound C–treated old hepatocytes, whereas AICAR reduced cell injury after H/R insult when compared to the vehicle group (Fig. 6C ).

Young plasma induces AMPK and ULK1 activation. Aged rats were treated with pooled young or old plasma (1 ml, i.v.) 3 times per week for 4 wk and then subjected to 60 min of warm ischemia and 6 h of reperfusion. A ) Representative Western blotting of phospho‐AMPKα, AMPKα, phospho‐ULK1, and ULK1 protein expression. B ) Densitometric analysis of phospho‐AMPKα and phospho‐ULK1. Data are means ± sd ( n = 6/group).

To evaluate the possible autophagy signaling pathway through which young plasma exerts its regulatory action on autophagy, AMPK signaling was examined. AMPK was markedly inactivated in old livers, as indicated by lower hepatic phosphorylation of AMPKα levels compared with that of the young controls. However, young‐plasma treatment blunted the aging‐impaired AMPKα activation, both at baseline and after IRI (Fig. 5 ) . To determine whether AMPK could promote autophagy via activation of ULK1, phosphorylation of ULK1 was examined and found to be markedly decreased in old livers when compared to those in young controls. However, young‐plasma administration significantly attenuated age‐dependent decreases in hepatic phosphorylation of ULK1 levels.

Inhibition of autophagy abrogates the protective effect of young serum against H/R insult. A ) Primary old hepatocytes were infected with adenovirus encoding GFP‐LC3 for 24 h and then subjected to 20 h hypoxia and 2 h reoxygenation injury in the absence or presence of vehicle, 3‐MA (10 mM), or wortmannin (1 µM). Representative images of fluorescent LC3 puncta are shown. Original magnification, ×400; scale bars, 30 µm. B ) Quantification of cell viability and LDH release of primary hepatocytes. C ) Old hepatocytes were pretreated with Atg7 siRNA (50 nM) before H/R insult in the presence of young serum. Representative Western blot of Atg7 and p62 protein expression. D ) Densitometric analysis of Atg7 and p62. E ) Quantification of cell viability and LDH release of primary old hepatocytes. The experiment was performed in triplicate with similar results. The data are shown as means ± sd .

We then investigated whether young serum could reduce cell injury via restoration of autophagy. Treatment of old hepatocytes with young serum significantly increased the number of autophagosomes in the cytoplasm of hepatocytes, both at baseline and after H/R insult (Fig. 3B ). Young serum significantly increased LC3B‐II, Atg7, and beclin1 protein expression levels, whereas it decreased p62 protein expression levels. Furthermore, young serum significantly increased autophagic flux, as shown by higher expression levels of LC3B‐II and p62 in old hepatocytes by chloroquine, both at baseline and after H/R insult (Supplemental Fig. S2), indicating that young serum restores autophagic activity in old hepatocytes. To address the role of autophagy in the protective effect of young serum against H/R injury, 3‐MA, or wortmannin was used to inhibit young serum induced‐autophagy. 3‐MA or wortmannin treatment significantly reduced the number of autophagosomes in the cytoplasm of hepatocytes (Fig. 4 A ), and blocked the protective effect offered by young serum, as indicated by higher LDH release and less cell survival after H/R (Fig. 4 B ). Similar results were observed in old hepatocytes with Atg7 knockdown. Atg7 knockdown abrogated the protective effect of young serum against H/R insult (Fig. 4 C – E ).

Young serum restores autophagic activity in primary old hepatocytes. Primary old hepatocytes were cultured for 72 h with young or old serum and then subjected to 20 h hypoxia and 2 h reoxygenation injury. A ) Quantification of cell viability and LDH release of primary hepatocytes. B ) Primary old hepatocytes were infected with adenovirus encoding GFP‐LC3 for 24 h, and then subjected to H/R insult in the absence or presence of young serum. Representative images of fluorescent LC3 puncta are shown. Original magnification, ×400; scale bars, 30 µm. C ) Representative Western blot of LC3B, Atg7, beclin1, and p62 protein expression. D ) Densitometric analysis of LC3B, Atg7, beclin1, and p62. The experiment was performed in triplicate with similar results. The data are shown as means ± sd .

To determine whether the protective effect of young plasma on liver IRI in vivo could be replicated in vitro H/R system, hepatocytes were isolated from old rats and cultured in the presence of young or old rat serum. Consistent with the date from in vivo , young serum exhibited less LDH release, and higher cell survival after H/R compared to the old hepatocytes cultured in old serum (Fig. 3A ) , demonstrating the protective effect of young serum on H/R insult.

To further evaluate the contribution of autophagy to the young‐plasma–afforded protection, rats were pretreated with the pharmacologic autophagy inhibitor 3‐MA or wortmannin before ischemia. 3‐MA or wortmannin treatment abolished the protection conferred by young plasma (Fig. 2C–G ). The serum levels of AST and ALT were significantly increased in 3‐MA—or wortmannin‐treated rats when compared with the vehicle control group. Similarly, a histologic analysis revealed that more severe IRI‐associated histopathologic changes in liver tissues of 3‐MA or wortmannin‐treated‐rats than those in the vehicle control group. These data indicate that the restoration of autophagy may contribute to young‐plasma–provided protection against liver IRI.

Young plasma decreases IRI through the restoration of aging‐impaired autophagy activity. A ) Representative Western blot analysis of LC3B, Atg7, beclin1, and p62 protein expression. B ) Densitometric analysis of LC3B, Atg7, beclin1, and p62. C ) Young‐plasma–pretreated aged rats were administrated with 3‐MA (30 mg/kg, i.p.) or wortmannin (0.6 mg/kg, i.v.) 0.5 h before ischemia and euthanized 6 h after reperfusion. Representative Western blots of p62 protein expression. D ) Densitometric analysis of p62. E ) Quantification of serum AST and ALT levels. F ) Representative images of H&E‐stained liver paraffin‐embedded sections. Dashed lines indicate necrotic areas. Original magnification, ×400; scale bars, 50 µm. G) Quantitative analysis of hepatic necrosis area. Data are means ± sd ( n = 4/group).

To determine whether autophagy plays an important role in the young‐plasma–induced beneficial effect on liver IRI, Western blot analysis was performed to evaluate the change in several autophagy indicators. LC3B‐II, Atg7, and beclin1 protein expression levels were significantly decreased in livers obtained from rats at an old age when compared with those in livers at a young age (Fig. 2A , B ). However, administration of young plasma partially reversed the aging‐induced suppression in LC3B‐II, Atg7, and beclin1 protein expression levels. Aging markedly elevated p62 protein expression in the liver, whereas young plasma attenuated the age‐associated increase in hepatic p62 protein expression. The young‐plasma–pretreated rats also showed significantly higher hepatic LC3B‐II, Atg7, and beclin1 protein levels, but lower p62 protein levels than those in old controls after IRI. Furthermore, young plasma restored the aging‐impaired autophagic flux, as shown by marked increases in LC3B‐II and p62 protein expression in aged livers by chloroquine after IRI (Supplemental Fig. S1).

Young plasma attenuates IRI in aged rats. Aged rats were treated with pooled young plasma or old plasma (1 ml, i.v.) 3 times per week for 4 wk and then subjected to 60 min of warm ischemia and 6 h of reperfusion. A ) Serum samples were collected for measuring AST and ALT. The data are shown as means ± sd ( n = 6 per group). B ) Liver tissues were fixed and processed, and H&E staining was performed. Dashed lines indicate necrotic areas. Original magnification, ×400. Scale bars, 50 µm. C ) Quantitative analysis of hepatic necrosis area. The data are means ± sd ( n = 6/group).

To evaluate the effect of young plasma on IRI in aged livers, we pretreated rats with young plasma, and evaluated liver injuries 6 h after reperfusion. The serum levels of AST and ALT in aged rats were significantly higher than those in young rats, both at baseline and after liver IRI (Fig. 1A ) . However, administration of young plasma inhibited the age‐induced increases in serum levels of AST and ALT. Old plasma did not prevent the age‐dependent increases in liver enzymes. Histologic analysis of the liver revealed severe hepatocellular necrosis in old livers after IRI. Hepatocellular necrosis was significantly reduced in liver tissues obtained from rats pretreated with young plasma. Administration of old plasma did not ameliorate IRI‐associated histopathologic changes (Fig. 1 B , C ).

DISCUSSION

Aging is often associated with a decreased autophagic activity (27–29), which contributes to the high sensitivity of aged livers to IRI. The therapeutic effect of young blood has been demonstrated in several recent studies (5–13). However, whether young blood could protect aged livers from IRI by induction of autophagy remains unknown. Our results showed that young plasma attenuated age‐dependent liver IRI. Young plasma restored the aging‐induced suppression in hepatic autophagic activity, and inhibition of autophagy abrogated the protective effect of young plasma against liver IRI. In vitro, primary old hepatocytes treated with young serum were less sensitive to H/R injury, and inhibition of young‐serum–restored autophagic activity diminished the protective action of young serum, suggesting that young plasma may attenuate age‐dependent liver IRI. Furthermore, we demonstrated that young plasma restores autophagic activity, at least in part, via AMPK/ULK1 signaling.

Exposure to young‐blood circulation through parabiosis or administration of young plasma can positively affect multiple tissues in aged animals. Therefore, it is possible that young blood also has a rejuvenating effect on aged livers and subsequently reduces its injury under stress conditions. In fact, young blood improves stem cells function in liver in aged animals (5). We also recently demonstrated that young plasma administrations could reverse age‐dependent alterations in hepatic function (13). We found that young plasma administrations could attenuate age‐dependent liver IRI. This finding further supports the concept that supplementation with young blood could improve age‐related disorders.

Young blood attenuates age‐related disorders, but the underlying mechanisms are not fully understood. Conboy et al. (5) found that exposure to youthful systemic environment enhanced the regenerative capacity of aged muscle via restoration of normal signaling of the Delta‐Notch pathway. Brack et al. (6) demonstrated that young blood substantially reduced age‐dependent increase in tissue fibrosis via inhibition of the Wnt signaling pathway in aged myogenic progenitors. Moreover, young blood improved age‐related cognitive impairments, in part via activation of the cAMP response element binding protein in aged hippocampus (8).

Aging is associated with decreased autophagic activity, and defective autophagy has been linked to age‐dependent accumulation of damaged intracellular components and subsequent disruption of cellular homeostasis and dysfunction in aging (23–26). Restoration of chaperone‐mediated autophagy in aged liver has been shown to decrease accumulation of damaged proteins and improve liver function (37). Wang et al. (38) recently demonstrated that impaired autophagy in aged livers increased their sensitivity to IRI, and increasing autophagy may ameliorate liver injury. In this study, we showed that young plasma could reduce age‐dependent liver IRI. Our experiments suggest that there are systemic factors in young plasma that could modulate the cell survival signaling pathways. Because autophagy exerts mainly pro‐survival activity, allowing the cell to cope with nutrient starvation and anoxia, and induction of autophagy activity reduces liver IRI (19–22), we observed the effect of autophagy on young‐plasma—afforded protection. In consistent with our previous results, aged livers or old hepatocytes exhibited a marked reduction of autophagic activity, which was reversed by young plasma in vivo or by young serum in vitro, respectively. We further demonstrated that inhibition of autophagic activity abolished the young‐plasma–afforded protection against liver IRI in vivo or young‐serum–provided protection on H/R injury in vitro. These findings strongly support the view that young plasma could protect age‐dependent IRI by restoration of autophagy.

AMPK is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. It has been shown that the activation capacity of AMPK signaling declines with age, which impairs the maintenance of efficient cellular homeostasis and enhances the aging process (33, 34). In contrast, AMPK activation prevents aging process and enhances longevity (39–41). Furthermore, the activation AMPK is necessary to trigger autophagy (30–32). Because mammalian target of rapamycin complex 1 (mTORC)1 activation is the principal mechanism that inhibits autophagy, one of the mechanisms of AMPK‐dependent induction of autophagy is inhibition of mTORC1 activity by phosphorylating tuberous sclerosis complex 2 (42) and raptor (43). ULK1, the most upstream component of the core autophagy machinery forms a complex with Atg13 and FIP200 and regulates autophagy induction. Under energy starvation, AMPK can associate with the ULK1 complex and phosphorylate ULK1 at multiple sites, including Ser317, Ser555, and Ser777 to activate autophagy (30–32). Under nutrient sufficiency, high mTORC1 activity suppresses autophagy by phosphory‐lating ULK1 at Ser757 and affecting interaction between ULK1 and AMPK (44). In addition, AMPK promotes autophagy by directly activating beclin1 phosphorylation at Thr388 in glucose starvation conditions (45). These findings show that autophagy could be activated by AMPK through multiple mechanisms. Our results exhibited that young plasma increased the levels of phosphorylation of AMPK and ULK1. Inhibition of AMPK prevented young‐serum–mediated activation in ULK1 and induction in autophagy in old hepatocytes, whereas activation of AMPK could potentiate the effects of young serum. These results indicate that the AMPK/ULK1 pathway may take part in young‐plasma–restored autophagy.

Decreased autophagic activity with age has been reported extensively in a variety of systems (23–25). Regarding autophagy in aged liver, decreased but also equivalent levels of basal autophagy has been reported (27–29, 38). Wang et al. (38) showed that the baseline levels of autophagy‐related proteins and the basal autophagic flux are indiscernible between age groups. In contrast, age‐associated decreases in autophagic proteolysis have been detected in rat liver and in vitro in rat isolated hepatocytes (28, 29). Uddin et al. (27) demonstrated that autophagosome formation ability in the liver has greatly decreased with mouse age. Moreover, aging is accompanied by a decrease in several autophagy gene transcripts in rat liver (46, 47), as well as with a decrease in autophagic activity in Caenorhabditis elegans (48). In agreement with this view, our results demonstrated that basal autophagy decreased in liver with age. Moreover, AMPK, which plays an important role in autophagy and aging, was inactivated in aged livers, suggesting a decrease in autophagy with age. In addition, we (13) previously demonstrated that the aging liver is usually associated with increased accumulation of lipid droplets, impaired liver regeneration, and increased hepatocellular senescence. The recently elucidated beneficial roles for autophagy in these conditions further suggest the concept of a decrease in autophagy during aging.

In summary, the age‐related autophagy defect in liver may be attributable to the increased sensitivity to liver IRI. Young plasma restored autophagy, at least in part, via the AMPK/ ULK1 axis, and subsequently attenuated age‐dependent liver IRL These findings further support that young blood may afford a potential therapeutic role in the management of aging‐associated deterioration of organs.