Jian-Hui Li ,Jun-Jun Jia ,Ning He ,Xue-Lian Zhou ,Yin-Biao Qiao ,Hai-Yang Xie ,Lin Zhou ,Shu-Sen Zheng a,,,?

a Department of Hepatobiliary and Pancreatic Surgery, Department of Liver Transplantation, Shulan (Hangzhou) Hospital, Zhejiang Shuren University School of Medicine, Hangzhou 310022, China

b NHC Key Laboratory of Combined Multi-organ Transplantation, Hangzhou 310022, China

c Division of Hepatobiliary Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310 0 03, China

d Department of Endocrinology, Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310052, China

Keywords: Exosome P-Akt Ischemia-reperfusion injury Remote ischemic perconditioning

ABSTRACT Background: Remote ischemic perconditioning (RIPerC) has been demonstrated to protect grafts from hepatic ischemia-reperfusion injury (IRI). This study investigated the role of exosomes in RIPerC of liver grafts in rats.Methods: Twenty-five rats (including 10 donors) were randomly divided into five groups ( n = 5 each group): five rats were used as sham-operated controls (Sham),ten rats were for orthotopic liver transplantation (OLT,5 donors and 5 recipients) and ten rats were for OLT + RIPerC (5 donors and 5 recipients). Liver architecture and function were evaluated.Results: Compared to the OLT group,the OLT + RIPerC group exhibited significantly improved liver graft histopathology and liver function ( P < 0.05). Furthermore,the number of exosomes and the level of P-Akt were increased in the OLT + RIPerC group.Conclusions: RIPerC effectively improves graft architecture and function,and this protective effect may be related to the increased number of exosomes. The upregulation of P-Akt may be involved in underlying mechanisms.

Introduction

With advancements in surgical techniques,immunosuppressive agents,and organ preservation technologies,liver transplantation(LT) has become the most effective treatment for end-stage liver disease. However,organ shortage is still the main obstacle to LT.Marginal grafts,such as grafts from donation-after-cardiac-death(DCD) donors,donors with fatty liver and older donors,have been used to expand the sources of donor livers. However,an increase in postoperative complications,including early graft failure and biliary stricture,has been observed,and ischemia-reperfusion injury(IRI) is considered to be critically involved in these unfavorable processes [1] . A variety of therapeutic strategies have been proposed to alleviate graft IRI,among which remote ischemic perconditioning (RIPerC),a modified ischemic preconditioning (IPC) approach,has been demonstrated to effectively protect grafts against hepatic IRI [2] . RIPerC is a method used to enhance the tolerance of distant organs by repeated,nonfatal ischemia and perfusion in remote organs,usually in the limbs. In our previous studies,we found that RIPerC alleviated IRI of liver graft in rats,which might have been related to the activation of the PI3K/Akt/eNOS/NO pathway [ 3,4 ],but the specific protective mechanism has not been elucidated.

Exosomes were originally thought to be cellular metabolic waste. In 2007,they were discovered to contain mRNA and miRNA and participate in the communication between cells [5] . Since then,research on exosomes has rapidly increased,and they are now known to be critically involved in intercellular communication. Exosomes are vesicles of endosomal origin with a diameter of 30-100 nm,and exosomes are characterized by the presence of surface markers,including CD63 and CD81. Exosomes can transfer their biochemical contents (proteins,miRNAs,mRNAs,DNA,and lipids) directly into target cells locally or at a distance through endocytosis or membrane receptors. Recent research has found that exosomes are involved in organ protection during IRI. Stem cellderived exosomes were first isolated in 2013 and shown to protect ischemic/reperfused hearts of rats via decreasing oxidative stress and activating the PI3K/Akt pathway [6] . These beneficial effects were also observed in the kidney and in limb ischemia. Moreover,the exosome concentration was found to be increased dramatically after IPC in humans. Thus,IPC-induced exosomes were suggested protecting the heart against IRI by activating pro-survival kinases [phosphorylation of extracellular signal-regulated kinase(ERK)] and transferring miR-24,playing a role in reducing apoptosis by downregulating Bim expression [7] . Nonetheless,no research has been conducted on the protective effect of exosomes on ischemia/reperfusion in LT. Here,we investigated the role of exosomes in limb RIPerC of liver grafts in rats.

Methods

Animals

Adult male Sprague Dawley rats (weighing 250-300 g,aged 8-9 weeks old) were used in our experiments. The animals were housed under constant humidity (50%-60%) and temperature (25-30 °C) and fed a standard diet with free access to water. This study was approved by the Ethics Committee for the Care and Use of Experimental Animals of the First Affiliated Hospital,Zhejiang University School of Medicine (Hangzhou,China). Twenty-five rats were randomly divided into 5 groups (n= 5): five rats were used as sham-operated controls (Sham),ten rats were for orthotopic liver transplantation (OLT,5 donors and 5 recipients) and ten rats were for OLT + RIPerC (5 donors and 5 recipients). The development of the OLT and RIPerC models has been previously reported by our team [3] . Briefly,both donor and recipient were anesthetized by intraperitoneal injection of 4% chloral hydrate anesthesia (Shanghai No. 1 Biochemical & Pharmaceutical,China) after 12 h-fasting.Graft from donor was perfused through the portal vein with cold saline containing 25 U/mL heparin and then placed into cold saline(0-4 °C) for about 40 min before graft being transplanted into the recipient. As the previous method,anastomosis of portal vein,suprahepatic inferior vena cava,hepatic artery infrahepatic inferior vena cava and bile duct was performed sequentially. Saline was injected through the penile vein of the recipient after the operation.The RIPerC model was developed by a standard tourniquet which was performed in recipient. RIPerC protocols were performed by the designed cycles of reperfusion and reocclusion of the hind limb applied immediately at the onset of recipient anhepatic phase with 1 kg weight both sides. In the Sham group,the abdomen was just opened for 75 min (the mean total ischemic time during OLT in our center) and then closed under anesthesia. The rats were sacrificed at 3 h after the portal vein of the recipients was opened under anesthesia,then liver tissues were obtained and fixed in 10% neutral formalin for histological studies. Fresh liver tissue was stored at -80 °C for further examination. Blood samples were obtained from the portal vein and centrifuged at 3000 × g for 10 min at 4 °C to collect the serum for liver function tests and exosome isolation.

Histopathological examination

Rat livers were dehydrated and embedded in paraffin and sectioned to a thickness of 3μm. Then,the sections were gradually deparaffinized and hydrated,followed by hematoxylin and eosin (H&E) staining. Morphological assessment was performed by liver pathologist in a blind fashion. The histopathological evaluation based on parenchymal cell necrosis and inflammatory infiltration was performed with a grading scale of 0-4 and 0-3 respectively according to the Suzuki classification [8] and our previous study [9] .

Liver function tests

Liver function was evaluated based on the levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT),which were measured by standard laboratory methods using a Hitachi 7600 automatic analyzer (Hitachi High-Technologies Corporation,Tokyo,Japan).

Exosome isolation and identification

The total exosomes were isolated from the serum of the different groups using the Total Exosome Isolation Kit (Life Technologies,Carlsbad,CA,USA) following the manufacturer’s instructions.In brief,each sample was subjected to centrifugation at 3000 × g(at room temperature) for 30 min. Next,the supernatant was collected,and 0.2 volume of the Total Exosome Isolation reagent(based on serum volume) was added. The mixtures were then vortexed and incubated at 4 °C for up to 30 min,followed by centrifugation at 10000 × g for 10 min at room temperature to precipitate the exosome pellets. The exosome fraction was resuspended using 1 × phosphate buffered saline (PBS) and stored at -80 °C for further analysis. The exosome concentrations and sizes were determined by transmission electron microscopy (TEM). CD63 and CD81 were used to mark the exosomes and detected by Western blotting.

TEM

Exosomes from the serum were examined by standard technical procedures used in TEM [10] . Briefly,the exosomes were carefully fixed with 3% glutaraldehyde (4 °C,pH 7.4),placed onto a copper grid,and stained with 2% uranyl acetate. The grid was viewed at 45000 magnification using an electron microscope (Philips,Eindhoven,the Netherlands).

Western blotting for exosome markers

The proteins were isolated from exosomes or tissues and lysed with RIPA lysis buffer (Santa Cruz Biotechnology,Santa Cruz,CA,USA) containing a protease inhibitor. The protein concentration of the concentrated exosomes was measured using a bicinchoninic acid protein kit (Thermo Scientific). Denatured proteins were next resolved with 10%/12% SDS-PAGE gels (Thermo Scientific,Madison,WI,USA) and electrotransferred onto nitrocellulose membranes. The membranes were blocked with skim milk for 1 h and then incubated with the following primary antibodies overnight at 4 °C: 1:1000 anti-Akt,1:2000 anti-phospho-Akt (Cell Signaling Technology,Beverly,MA,USA); 1:300 anti-CD63 (Abcam Inc.,Cambridge,MA,USA); and 1:300 anti-CD81 (Santa Cruz Biotechnology). After washing,the blots were incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies for 1.5 h at room temperature. Furthermore,the membranes were visualized by enhanced chemiluminescence (ECL)using an ECL kit (Pierce Biotechnology,Rockford,MD,USA). Betaactin was utilized as the loading control. ImageJ software (Version 1.48,NIH) was used to analyze the intensity of the immunoreactive bands.

Statistical analysis

All the data were presented as mean ± standard deviation (SD).Statistical analysis was performed using SPSS (Version 22.0,SPSS Inc.,Chicago,IL,USA) and GraphPad Prism (Version 7.0,GraphPad Software,San Diego,USA). The differences between the experimental groups were determined by ANOVA,followed by Bonferroni’s test for multiple comparisons. Statistical significance was set atP<0.05.

Results

RIPerC treatment improved histopathology

H&E-stained liver sections are depicted in Fig. 1 . Inflammatory infiltration and necrosis were hardly observed in the Sham group,whereas massive hepatic necrosis and inflammatory cell infiltration were detected in the OLT group. Importantly,the OLT + RIPerC group exhibited reduced necrosis and inflammation compared to the OLT group,and thus,OLT + RIPerC improved the posttransplantation histopathology.

RIPerC treatment alleviated liver injury

As shown in Fig. 2,the serum ALT and AST levels were significantly increased in the OLT group and OLT + RIPerC group (P<0.05) but not in the Sham group. The serum ALT and AST levels of the OLT + RIPerC group were significantly lower than those of the OLT group (P<0.05). Altogether,these results revealed that RIPerC treatment alleviated liver injury.

Fig. 2. RIPerC treatment improved post-transplantation liver function (serum ALT and AST). The serum ALT and AST levels were reduced in the OLT + RIPerC group compared with those in the OLT group. ?: P < 0.05 compared with the Sham group; #: P < 0.05 compared with the OLT group. OLT: orthotopic liver transplantation; RIPerC: remote ischemic perconditioning; ALT: alanine aminotransferase; AST: aspartate aminotransferase.

RIPerC increased the number of exosomes

Exosomes had rounded morphologies under TEM with approximate sizes of 100 nm ( Fig. 3 ). The number of exosomes in the OLT group was lower than that in the Sham group (P<0.05). However,RIPerC increased the number of exosomes compared to OLT (P<0.05). Then,exosome markers,including CD63 and CD81,were detected by Western blotting. The data obtained were consistent with the TEM results ( Fig. 4 ).

Fig. 3. RIPerC increased the number of exosomes in the liver. A,B,C: serum exosomes of the Sham,OLT and OLT + RIPerC groups examined by TEM; D: statistical analysis of exosomes. ?: P < 0.05 compared with the Sham group; #: P < 0.05 compared with the OLT group. OLT: orthotopic liver transplantation; RIPerC: remote ischemic perconditioning; TEM: transmission electron microscopy.

Fig. 4. RIPerC upregulated P-Akt expression in exosomes. A : Western blotting analysis showed the expression of the exosome markers of CD63 and CD81 in all the groups.B and C : Statistical analysis of exosome markers CD63 and CD81. D: The expression levels of P-Akt in the exosomes were higher in the OLT + RIPerC group than in the OLT and the Sham groups. E: Statistical analysis of expression levels of P-Akt. ?: P < 0.05 compared with the Sham group; #: P < 0.05 compared with the OLT group. OLT:orthotopic liver transplantation; RIPerC: remote ischemic perconditioning.

RIPerC treatment upregulated Akt levels in exosomes

We adjusted the exosome loading amount and measured the protein expression levels of P-Akt by Western blotting. The results revealed that the expression levels of P-Akt in the exosomes in the OLT and OLT + RIPerC groups were significantly higher than those in the Sham group ( Fig. 4 D and E). Furthermore,we found that the P-Akt protein levels were notably higher in the OLT + RIPerC group than in the OLT group (P<0.05).

Discussion

We found that OLT induced liver damage and decreased exosome secretion compared to the Sham operation. RIPerC increased the number of exosomes compared with OLT alone and alleviated liver damage. However,RIPerC treatment did not achieve the levels observed in the Sham group. The P-Akt levels in the exosomes were consistent with the number of exosomes. All these results indicated that exosome may be involved in the protective effect of RIPerC after LT.

The concept of remote ischemic conditioning (RIC) was originally developed by Przyklenk et al. [11],who discovered that brief ischemia in one organ conferred protection to important distant organs. RIC overcomes the main limitation of local ischemic conditioning (pre- and postischemic conditioning) by increasing the total ischemic time and can thus be applied before ischemia [remote ischemic preconditioning (R-IPC)],after ischemia,before perfusion[remote ischemic perconditioning (RIPerC)],or after the onset of reperfusion [remote ischemic postconditioning (RIPostC)]. Considerable effort has been made in translating RIPerC from bench to bedside. Mounting evidence has shown that RIPerC exerts strong protective effects against IRI,including cardiac [ 12,13 ],liver [2],kidney [ 14,15 ],brain [ 16,17 ] and lung [18] injury. RIPerC is thought to activate both neuronal and humoral signaling pathways. In our previous work,we established a novel model of RIPerC in rat LT and optimized the procedure (5 min × 3) [3] . Then,we found that inhibition of oxidative stress [3] and activation of the PI3K/Akt/eNOS/NO pathway [4] and Mfn2-MICUs axis [19] were involved in the protective effect of RIPerC. However,the underlying mechanisms have not been completely elucidated. In this experiment,we further investigated the effects of exosomes on the mechanism of RIPerC and found that exosome-derived P-Akt might be involved in the protective effect of RIPerC.

Exosomes are smaller than 150 nm in diameter and are enriched in components including RNA,miRNAs,proteins,and lipids.Exosomes have important functions and are known as key mediators of intercellular communication. Many circulating exosomes from transplanted grafts alleviate IRI. Yang et al.evaluated the role of exosome secretion in hepatic IRI and discovered that interferon regulatory factor 1 (IRF-1) regulated Rab27a transcription and exosome secretion,reducing hepatic IRI [20] . Sun et al. in rat experiments found that combined treatment with melatonin and mesenchymal stem cell-derived exosomes offered superior protection against liver IRI [21] . Gangadaran et al. provided evidence that exosomes from mesenchymal stem cells activated vascular endothelial growth factor (VEGF) receptors and protected against hind limb IRI [22] . Wen et al. found that RIPerC-induced exosomes reduced myocardial IRI and that miR-24 in exosomes played a central role in mediating the protective effects of RIPerC [7] . Our results showed that RIPerC in the hind limb can provide protection against LT-induced injury,and exosomes play an important role in regulating this protective effect,which is consistent with previous findings [ 7,12 ]. Furthermore,we found that the P-Akt protein levels derived from exosomes were increased in the RIPerC group compared to the OLT group,indicating that P-Akt may be involved in this protective effect. PIPerC on graft is associated with Akt activations of the exosomes. However,further experiments are needed to verify the mechanism.

Our findings shed light on the mechanism of RIPerC in LT.However,there are some limitations in our study. First,we confirmed that exosome-derived Akt was critically involved in RIPerC,but more experiments are required to explore which types of cell secrete these exosomes. Second,we did not carry out gain/loss-of-function experiments in coculture systems using a hypoxia-reoxygenation model,and no direct evidence showed that exosome-derived P-Akt mediates the protective effect of RIPerC.

In conclusion,we confirmed that RIPerC effectively improves graft architecture and function and discovered that exosomes are involved in this protective effect. Our results suggest that the upregulation of P-Akt may be involved in the protective effect of RIPerC.

Acknowledgments

None.

CRediT authorship contribution statement

Jian-HuiLi:Funding acquisition,Methodology,Writing – original draft,Writing – review & editing.Jun-JunJia:Data curation,Formal analysis,Funding acquisition,Methodology,Writing – original draft,Writing – review & editing.NingHe:Data curation,Formal analysis,Writing – original draft.Xue-LianZhou:Writing –original draft.Yin-BiaoQiao:Writing – original draft.Hai-Yang Xie:Conceptualization,Writing – review & editing.LinZhou:Conceptualization,Writing – review & editing.Shu-SenZheng:Conceptualization,Funding acquisition,Supervision,Writing – review& editing.

Funding

This study was supported by the Public Projects of Zhejiang Province (LGF21H030006),the Major Science and Technology Projects of Hainan province (ZDKJ2019009),Research Project of Jinan Microecological Biomedicine Shandong Laboratory(JNL-2022002A,JNL-2022023C),Research Unit Project of Chinese Academy of Medical Sciences (2019-I2M-5-030),Innovative Research Groups of National Natural Science Foundation of China( 81721091 ).

Ethical approval

This study was approved by the Ethics Committee for the Care and Use of Experimental Animals of the First Affiliated Hospital,Zhejiang University School of Medicine (Hangzhou,China).

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.