hucMSC Exosome-Derived GPX1 Is Required for the Recovery of Hepatic Oxidant Injury

Exosomes are small biological membrane vesicles secreted by various cells, including mesenchymal stem cells (MSCs). We previously reported that MSC-derived exosomes (MSC-Ex) can elicit hepatoprotective effects against toxicant-induced injury. However, the success of MSC-Ex-based therapy for treatment of liver diseases and the underlying mechanisms have not been well characterized. We used human umbilical cord MSC-derived exosome (hucMSC-Ex) administrated by tail vein or oral gavage at different doses and, in engrafted liver mouse models, noted antioxidant and anti-apoptotic effects and rescue from liver failure. A single systemic administration of hucMSC-Ex (16 mg/kg) effectively rescued the recipient mice from carbon tetrachloride (CCl4)-induced liver failure. Moreover, hucMSC-Ex-derived glutathione peroxidase1 (GPX1), which detoxifies CCl4 and H2O2, reduced oxidative stress and apoptosis. Knockdown of GPX1 in hucMSCs abrogated antioxidant and anti-apoptotic abilities of hucMSC-Ex and diminished the hepatoprotective effects of hucMSC-Ex in vitro and in vivo. Thus, hucMSC-Ex promote the recovery of hepatic oxidant injury through the delivery of GPX1. Exosomes are small biological membrane vesicles secreted by various cells, including mesenchymal stem cells (MSCs). We previously reported that MSC-derived exosomes (MSC-Ex) can elicit hepatoprotective effects against toxicant-induced injury. However, the success of MSC-Ex-based therapy for treatment of liver diseases and the underlying mechanisms have not been well characterized. We used human umbilical cord MSC-derived exosome (hucMSC-Ex) administrated by tail vein or oral gavage at different doses and, in engrafted liver mouse models, noted antioxidant and anti-apoptotic effects and rescue from liver failure. A single systemic administration of hucMSC-Ex (16 mg/kg) effectively rescued the recipient mice from carbon tetrachloride (CCl4)-induced liver failure. Moreover, hucMSC-Ex-derived glutathione peroxidase1 (GPX1), which detoxifies CCl4 and H2O2, reduced oxidative stress and apoptosis. Knockdown of GPX1 in hucMSCs abrogated antioxidant and anti-apoptotic abilities of hucMSC-Ex and diminished the hepatoprotective effects of hucMSC-Ex in vitro and in vivo. Thus, hucMSC-Ex promote the recovery of hepatic oxidant injury through the delivery of GPX1. Liver injury often occurs in response to various chronic injuries, such as viral hepatitis, alcohol, drugs, metabolic diseases, and autoimmune hepatic cell insult. Drug- and toxicant-induced liver injury is a major reason for liver transplantation, and at present, no ideal treatments are available for liver injury.1Fisher K. Vuppalanchi R. Saxena R. Drug-induced liver injury.Arch. Pathol. Lab. Med. 2015; 139: 876-887Crossref PubMed Scopus (93) Google Scholar Thus, better therapies are needed to prevent additional hepatic damage. Increasing evidence shows that mesenchymal stem cell (MSC) transplantation may be a promising new therapeutic approach for treating liver injury. Currently, approximately 36 MSC-based clinical trials are being conducted for different types of liver injuries, such as autoimmune hepatitis, alcoholic liver cirrhosis, and liver fibrosis. Human umbilical cord is a transplantable source of MSCs and human umbilical cord-derived MSCs (hucMSCs) are of interest in regenerative medicine for liver injury because of low cost, minimal invasiveness, convenient isolation, low immunogenicity and immunomodulatory activity, and differentiation multipotency.2Phinney D.G. Prockop D.J. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views.Stem Cells. 2007; 25: 2896-2902Crossref PubMed Scopus (1551) Google Scholar, 3Kolf C.M. Cho E. Tuan R.S. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation.Arthritis Res. Ther. 2007; 9: 204Crossref PubMed Scopus (739) Google Scholar, 4Stoltz J.F. de Isla N. Li Y.P. Bensoussan D. Zhang L. Huselstein C. Chen Y. Decot V. Magdalou J. Li N. et al.Stem cells and regenerative medicine: myth or reality of the 21th century.Stem Cells Int. 2015; 2015: 734731Crossref PubMed Scopus (113) Google Scholar Results from phase I and II clinical studies of hucMSC transplantation for patients with decompensated liver cirrhosis showed that hucMSCs may improve liver function and clinical symptoms (https://ClinicalTrials.gov identifier NCT0134225). Although advances in hucMSC transplantation offer great potential for treating liver injury, long-term safety is a concern.5Xu X. Qian H. Zhu W. Zhang X. Yan Y. Wang M. Xu W. Isolation of cancer stem cells from transformed human mesenchymal stem cell line F6.J. Mol. Med. (Berl.). 2010; 88: 1181-1190Crossref PubMed Scopus (8) Google Scholar Recent work suggests that transplantation of native MSCs can alleviate liver injury through paracrine effects.6Kuo T.K. Hung S.P. Chuang C.H. Chen C.T. Shih Y.R. Fang S.C. et al.Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells.Gastroenterology. 2008; 134: 2111-2121Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar MSC-derived exosomes (MSC-Ex), membrane-enclosed vesicles found in MSC-conditioned medium (MSC-CM), have been reported to contribute to angiogenesis, neurite outgrowth, and skeletal muscle regeneration.7Xin H. Li Y. Buller B. Katakowski M. Zhang Y. Wang X. Shang X. Zhang Z.G. Chopp M. Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth.Stem Cells. 2012; 30: 1556-1564Crossref PubMed Scopus (655) Google Scholar, 8Hu G.W. Li Q. Niu X. Hu B. Liu J. Zhou S.M. Guo S.C. Lang H.L. Zhang C.Q. Wang Y. Deng Z.F. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.Stem Cell Res. Ther. 2015; 6: 10Crossref PubMed Scopus (255) Google Scholar, 9Nakamura Y. Miyaki S. Ishitobi H. Matsuyama S. Nakasa T. Kamei N. Akimoto T. Higashi Y. Ochi M. Mesenchymal-stem-cell-derived exosomes accelerate skeletal muscle regeneration.FEBS Lett. 2015; 589: 1257-1265Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar We previously demonstrated that hucMSCs and hucMSC-Ex could alleviate liver fibrosis, promote liver and renal injury repair, and improve wound healing.10Li T. Yan Y. Wang B. Qian H. Zhang X. Shen L. Wang M. Zhou Y. Zhu W. Li W. Xu W. Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis.Stem Cells Dev. 2013; 22: 845-854Crossref PubMed Scopus (605) Google Scholar, 11Zhou Y. Xu H. Xu W. Wang B. Wu H. Tao Y. Zhang B. Wang M. Mao F. Yan Y. et al.Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro.Stem Cell Res. Ther. 2013; 4: 34Crossref PubMed Scopus (452) Google Scholar, 12Zhang B. Wang M. Gong A. Zhang X. Wu X. Zhu Y. Shi H. Wu L. Zhu W. Qian H. Xu W. HucMSC-Exosome Mediated-Wnt4 Signaling Is Required for Cutaneous Wound Healing.Stem Cells. 2015; 33: 2158-2168Crossref PubMed Scopus (471) Google Scholar hucMSC-Ex may have similar protective and reparative properties as their cellular counterparts in tissue repair.13Lai R.C. Chen T.S. Lim S.K. Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease.Regen. Med. 2011; 6: 481-492Crossref PubMed Scopus (426) Google Scholar Compared with MSC transplantation, hucMSC-Ex therapies are preferred because of fewer immune responses; increased safety; and ease of storage, shipment, and administration.13Lai R.C. Chen T.S. Lim S.K. Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease.Regen. Med. 2011; 6: 481-492Crossref PubMed Scopus (426) Google Scholar Therefore, hucMSC-Ex may be ideal for treating liver injuries and diseases, but mechanisms underlying their therapeutic effects are unclear. Exosomes contain functional mRNA, microRNA, and proteins that can alter the cellular environment to enhance tissue repair.12Zhang B. Wang M. Gong A. Zhang X. Wu X. Zhu Y. Shi H. Wu L. Zhu W. Qian H. Xu W. HucMSC-Exosome Mediated-Wnt4 Signaling Is Required for Cutaneous Wound Healing.Stem Cells. 2015; 33: 2158-2168Crossref PubMed Scopus (471) Google Scholar, 14Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8870) Google Scholar Thus, it is necessary to determine key factors in hucMSC-Ex that mediate hepatoprotective effects. Oxidative stress is often involved in liver diseases and may contribute to the development of viral hepatitis, alcoholic liver disease, non-alcoholic steatohepatitis (NASH), Wilson’s disease, and hepatocellular carcinoma (HCC).15Ivanov A.V. Smirnova O.A. Petrushanko I.Y. Ivanova O.N. Karpenko I.L. Alekseeva E. Sominskaya I. Makarov A.A. Bartosch B. Kochetkov S.N. Isaguliants M.G. HCV core protein uses multiple mechanisms to induce oxidative stress in human hepatoma Huh7 cells.Viruses. 2015; 7: 2745-2770Crossref PubMed Scopus (63) Google Scholar, 16Li H. Zhu W. Zhang L. Lei H. Wu X. Guo L. Chen X. Wang Y. Tang H. The metabolic responses to hepatitis B virus infection shed new light on pathogenesis and targets for treatment.Sci. Rep. 2015; 5: 8421Crossref PubMed Scopus (99) Google Scholar, 17Yu Y. Guerrero C.R. Liu S. Amato N.J. Sharma Y. Gupta S. et al.Comprehensive assessment of oxidatively induced modifications of DNA in a rat model of human Wilson’s disease.Mol. Cell. Proteomics. 2015; 15: 810-817Crossref PubMed Scopus (32) Google Scholar, 18Gentric G. Maillet V. Paradis V. Couton D. L’Hermitte A. Panasyuk G. Fromenty B. Celton-Morizur S. Desdouets C. Oxidative stress promotes pathologic polyploidization in nonalcoholic fatty liver disease.J. Clin. Invest. 2015; 125: 981-992Crossref PubMed Scopus (141) Google Scholar, 19Takaki A. Yamamoto K. Control of oxidative stress in hepatocellular carcinoma: Helpful or harmful?.World J. Hepatol. 2015; 7: 968-979Crossref PubMed Scopus (69) Google Scholar Antioxidant therapy has been considered for treatment of liver diseases, and previous work suggests that transplanted MSCs can restore liver function.6Kuo T.K. Hung S.P. Chuang C.H. Chen C.T. Shih Y.R. Fang S.C. et al.Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells.Gastroenterology. 2008; 134: 2111-2121Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 20Singal A.K. Jampana S.C. Weinman S.A. Antioxidants as therapeutic agents for liver disease.Liver Int. 2011; 31: 1432-1448Crossref PubMed Scopus (168) Google Scholar However, how MSC-Ex modulate oxidative stress in liver injury repair is unclear. Glutathione peroxidase 1 (GPX1), a critical human antioxidant,21Brigelius-Flohé R. Maiorino M. Glutathione peroxidases.Biochim. Biophys. Acta. 2013; 1830: 3289-3303Crossref PubMed Scopus (1157) Google Scholar detoxifies hydrogen peroxide and upregulates GPX1 activity to promote cell survival, but whether hucMSC-Ex-mediated delivery of GPX1 can restore liver function is unknown. In this study, we assessed the use of hucMSC-Ex for treatment of liver disease and assessed efficacy and mechanism of action. Antioxidant and anti-apoptotic effects of hucMSCs-Ex on CCl4- and H2O2-induced hepatic injury in vitro and in vivo were also investigated, and hucMSC-Ex-derived GPX1 promoted detoxification of CCl4 and H2O2 and inhibited oxidative stress and apoptosis in vitro and in vivo. Our results provide a better understanding for using hucMSC-Ex for treating liver injury. To evaluate the lethality of CCl4 in mice, CCl4/kg (0.15–0.35 mL, intraperitoneal) was tested for hepatotoxicity and lethality. CCl4 at 0.15 and 0.2 mL/kg body weight did not induce sufficient lethality, but 0.3 and 0.35 mL/kg body weight caused rapid death. Thus, 0.25 mL/kg was used for subsequent experiments (Figure S1). hucMSC-Ex was prepared and purified as described previously.22Zhu W. Huang L. Li Y. Zhang X. Gu J. Yan Y. Xu X. Wang M. Qian H. Xu W. Exosomes derived from human bone marrow mesenchymal stem cells promote tumor growth in vivo.Cancer Lett. 2012; 315: 28-37Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) analyses confirmed spheroid morphology of hucMSC-Ex (diameter 30–100 nm; Figures S2A and S2B). Western blot results confirmed expression of exosomal markers CD9, CD61, and CD63 in hucMSC-Ex (Figure S2C). In vivo fluorescent imaging and human CD63 staining results showed that CM-Dir-labeled hucMSC-Ex administered by tail vein or oral gavage targeted injured and normal livers at 24 hr post-injection (Figures 1A, S3A, and S3B). Hematoxylin and eosin (H&E) staining confirmed large areas of fatty degeneration and portal hepatocyte necrosis after PBS and 8 mg/kg hucMSC-Ex treatment, while 16 and 32 mg/kg hucMSC-Ex significantly inhibited hepatocyte denaturation and hepatic lobule destruction (Figures 1B and S4). To assess the therapeutic potential of hucMSC-Ex, 8, 16, and 32 mg/kg (tail vein or oral gavage) was given 24 hr after the administration of CCl4. For the tail vein administration of hucMSC-Ex, all animals infused with 8 mg/kg died of liver failure, and half survived after treatment with 16 mg/kg. All mice were rescued by 32 mg/kg hucMSC-Ex (Figure 1C). A dose-dependent effect was also observed after treatment with hucMSC-Ex from oral gavage administration of hucMSC-Ex. Specifically, 8 mg/kg hucMSC-Ex failed to rescue recipient animals from liver failure, but 60% and 90% of mice recovered from 16 and 32 mg/kg of hucMSC-Ex treatment, respectively (Figure 1D). Also, serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) decreased in animals given 8 mg/kg hucMSC-Ex at 72 hr post-injection, confirming that hucMSC-Ex reduced acute extensive liver injury by CCl4 (Figures 1E and 1F; n = 5; *p < 0.05, **p < 0.01, ***p < 0.001). CCl4 induces hepatotoxic effects via the CYP 450-dependent monooxygenase system to release reactive free radicals, and hucMSC-Ex had antioxidant effects on CCl4-induced liver failure. To illustrate the antioxidant effects of hucMSC-Ex, human lung fibroblast (HFL-1)-derived exosomes (HFL-Ex) were used as controls. First, reactive oxygen species (ROS) were measured in hepatocytes isolated from hucMSC-Ex-treated livers using a DCF-DA probe. Imaging flow cytometry indicated that the percentage of DCF-positive cells and fluorescent intensity decreased after hucMSC-Ex treatment (Figures 2Ai and 2Aii ; n = 4; **p < 0.01). The oxidative stress product 8-OHdG and TUNEL-positive cells were decreased in hucMSC-Ex cells compared with PBS and HFL-Ex groups (Figures 2B and 2C; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). MDA was also reduced in the hucMSC-Ex group (Figure 2D; n = 3; *p < 0.05). Luminex analysis confirmed that pro-inflammatory cytokines (G-CSF, interleukin [IL]-1α, IL-6, monocyte chemoattractant protein-1 [MCP-1], and tumor necrosis factor-α [TNF-α]) were reduced in the hucMSC-Ex group (Figure 2E; n = 3; *p < 0.05, **p < 0.01). Thus, hucMSC-Ex can inhibit oxidative stress and apoptosis in CCl4-injured mouse livers and promote hepatic recovery. To measure distribution of exosomes in L02 cells, exosomes and L02 cells were labeled with fluorescent carbocyanine dyes CM-Dil (red) and CM-Dio (green) respectively. After 24 hr co-incubation, the uptake efficiency was measured by imaging flow cytometry, and results showed that ∼75% of cells were double-stained (Figure 3Ai), the CM-Dil labeled HFL-Ex and hucMSC-Ex could be imaged in L02 cells (Figure 3Aii). CCl4 and H2O2 treatment-induced ROS, oxidative stress, and hepatotoxicity20Singal A.K. Jampana S.C. Weinman S.A. Antioxidants as therapeutic agents for liver disease.Liver Int. 2011; 31: 1432-1448Crossref PubMed Scopus (168) Google Scholar, 23Hafez M.M. Al-Shabanah O.A. Al-Harbi N.O. Al-Harbi M.M. Al-Rejaie S.S. Alsurayea S.M. Sayed-Ahmed M.M. Association between paraoxonases gene expression and oxidative stress in hepatotoxicity induced by CCl4.Oxid. Med. Cell. Longev. 2014; 2014: 893212Crossref PubMed Scopus (46) Google Scholar was assessed by measuring antioxidant activity with DCF-DA 24 hr after hucMSC-Ex co-incubation. ROS were decreased after treatment with MSC-conditioned medium (MSC-CM) and hucMSC-Ex compared with HFL-conditional medium (HFL-CM), HFL-Ex, and PBS (Figure 3B; n = 5; ***p < 0.001). MDA level was also reduced in MSC-CM and hucMSC-Ex groups (Figure 3C; n = 3; ***p < 0.001). We next assessed the effect of hucMSC-Ex on hepatocytes survival. Cell viability were reduced in CCl4- and H2O2-injured L02 cells, and were increased in a dose-dependent fashion in hucMSC-Ex-treated cells. In contrast, HFL-Ex had little effect on cell viability (Figure 3D). Thus, hucMSC-Ex may have antioxidant activity that can improve cell viability. Oxidative stress can induce hepatic apoptosis.24Wang K. Molecular mechanisms of hepatic apoptosis.Cell Death Dis. 2014; 5: e996Crossref PubMed Scopus (228) Google Scholar We found that at 24 hr after treatment, apoptotic cells decreased (2-fold) in the hucMSC-Ex group compared with the PBS and HFL-Ex groups (Figure 4A; n = 3; *p < 0.05, **p < 0.01). Hoechst 33342 staining revealed fewer apoptotic cells (half-moon nuclei) in the hucMSC-Ex group compared with the PBS and HFL-Ex groups (Figure 4B; n = 3; *p < 0.05, **p < 0.01). Mitochondria can undergo apoptosis because of oxidative stress.24Wang K. Molecular mechanisms of hepatic apoptosis.Cell Death Dis. 2014; 5: e996Crossref PubMed Scopus (228) Google Scholar Thus, we measured changes in mitochondrial membrane potential in L02 cells treated with or without hucMSC-Ex using JC-1 staining. More cells were fluorescent green in the PBS and HFL-Ex groups, but more cells were fluorescent red in the hucMSC-Ex group (Figure 4Ci). The ratio of green to red fluorescence was significantly decreased in L02 cells treated with hucMSC-Ex, indicating a reversal of oxidative stress-induced perturbation of mitochondrial membrane potential (Figure 4Cii; n = 3; **p < 0.01). ERK1/2 MAPK and Bcl2 mediate protection against apoptosis,25Guégan J.P. Frémin C. Baffet G. The MAPK MEK1/2-ERK1/2 pathway and its implication in hepatocyte cell cycle control.Int. J. Hepatol. 2012; 2012: 328372Crossref PubMed Google Scholar, 26Lei K. Nimnual A. Zong W.X. Kennedy N.J. Flavell R.A. Thompson C.B. Bar-Sagi D. Davis R.J. The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase.Mol. Cell. Biol. 2002; 22: 4929-4942Crossref PubMed Scopus (441) Google Scholar and we noted that hucMSC-Ex induced ERK1/2 phosphorylation and Bcl2 expression at 12 and 24 hr after treatment with hucMSC-Ex in H2O2- or CCl4-injured L02 cells (Figure 5A). Oxidative stress-induced apoptosis is usually associated with caspase activation.27Riedl S.J. Shi Y. Molecular mechanisms of caspase regulation during apoptosis.Nat. Rev. Mol. Cell Biol. 2004; 5: 897-907Crossref PubMed Scopus (1567) Google Scholar Thus, we measured the effect of hucMSC-Ex on the IKKB/NFkB/casp-9/-3 signaling pathway and noted that IKKB and NFkB phosphorylation was inhibited at 24 hr after hucMSC-Ex treatment (Figure 5A). Casp-9 and Casp-3 expression decreased after hucMSC-Ex treatment. hucMSC-Ex dose-dependently inhibited the IKKB/NFkB/casp-9/-3 signaling pathway and pNFkB nuclear translocation and induced Bcl2 expression and ERK1/2 phosphorylation in CCl4-injured L02 cells (Figures 5B and S5). Compared with recovery by z-vad-FMK, a Road caspase inhibitor, hucMSC-Ex was more effective (Figure 5C). Furthermore, casp-3 activity was dose-dependently inhibited (Figure 5D; n = 3; *p < 0.05, **p < 0.01). Cleaved casp-3 expression in CCl4-injured L02 cells (Figure 5Ei) and livers were inhibited (Figure 5Eii). Therefore, hucMSC-Ex induces ERK1/2 phosphorylation and Bcl2 expression and reverses oxidative stress-induced apoptosis. To understand which hucMSC-Ex component offered antioxidant effects, we measured GPX1 and superoxide dismutase (SOD) in L02 cells after exposure to CCl4 and hucMSC-Ex treatment. GPX1 increased with hucMSC-Ex treatment, and glutathione-S-transferase (GST) activity was also reduced in the hucMSC-Ex group compared with the PBS group (Figure 6A; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). hucMSC-Ex did not change SOD activity in L02 cells (Figure 6A) and had no effect on GPX1 mRNA expression in L02 cells (Figure 6B). GPX1 protein was detected in hucMSCs and hucMSC-Ex, and it was greater in hucMSC-Ex compared with HFL-Ex cells (Figure 6C). To investigate the role of GPX1 in hucMSC-Ex-mediated antioxidant activity, we knocked down GPX1 in hucMSCs with small interfering RNA (siRNA) and noted that GPX1 expression was reduced in GPX1 siRNA transfected hucMSCs and hucMSC-Ex (GPX1-siRNA-Ex) (Figure 6D). Gpx activity promotion by hucMSC-Ex treatment was reversed by GPX1 knockdown (Figure 6E; n = 3; **p < 0.01). Less MDA and ROS in hucMSC-Ex-treated L02 cells were also reversed by GPX1 knockdown (Figures 6F and 6G; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). hucMSC-Ex-mediated reduction of L02 cell apoptosis, NFkB P65 phosphorylation, and increase of Bcl2 expression were abolished by GPX1 knockdown in vitro (Figures 7A and 7B ; n = 3; *p < 0.05, **p < 0.01). hucMSC-Ex-mediated inhibition of inflammatory infiltration, 8-OHdG expression, and hepatocyte apoptosis was also interfered by GPX1 knockdown in vivo (Figures 7C and 7D; n = 3; *p < 0.05, **p < 0.01). Furthermore, GPX1 knockdown reduced the Gpx activity promotion of ctr-siRNA-Ex in ex vivo hepatocytes (n = 3; *p < 0.05) (Figure S7A). GPX1 knockdown also attenuated the rescue of hucMSC-Ex on CCl4-induced liver failure (n = 10; *p < 0.05) (Figure S7B). Thus, GPX1 knockdown delayed hucMSC-Ex-induced recovery from acute liver injury, so GPX1 is an important factor for hucMSC-Ex-mediated antioxidant activity and liver protection. MSC transplantation has been investigated in clinical trials as potential treatment for liver diseases,28Meier R.P. Müller Y.D. Morel P. Gonelle-Gispert C. Bühler L.H. Transplantation of mesenchymal stem cells for the treatment of liver diseases, is there enough evidence?.Stem Cell Res. (Amst.). 2013; 11: 1348-1364Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 29Houlihan D.D. Hopkins L.J. Suresh S.X. Armstrong M.J. Newsome P.N. Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes.Hepatology. 2011; 54: 1891-1892Crossref PubMed Scopus (15) Google Scholar, 30Zhang Z. Lin H. Shi M. Xu R. Fu J. Lv J. Chen L. Lv S. Li Y. Yu S. et al.Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients.J. Gastroenterol. Hepatol. 2012; 27: 112-120Crossref PubMed Scopus (269) Google Scholar because systemic injection has been shown to be effective in experimental research.31van Poll D. Parekkadan B. Cho C.H. Berthiaume F. Nahmias Y. Tilles A.W. Yarmush M.L. Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo.Hepatology. 2008; 47: 1634-1643Crossref PubMed Scopus (442) Google Scholar, 32Meier R.P. Mahou R. Morel P. Meyer J. Montanari E. Muller Y.D. Christofilopoulos P. Wandrey C. Gonelle-Gispert C. Bühler L.H. Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice.J. Hepatol. 2015; 62: 634-641Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 33Park M. Kim Y.H. Woo S.Y. Lee H.J. Yu Y. Kim H.S. Park Y.S. Jo I. Park J.W. Jung S.C. et al.Tonsil-derived mesenchymal stem cells ameliorate CCl4-induced liver fibrosis in mice via autophagy activation.Sci. Rep. 2015; 5: 8616Crossref PubMed Scopus (88) Google Scholar This therapeutic effect may include transdifferentiation of MSCs into hepatocyte-like cells, secretion of anti-inflammatory factors, and hepatocyte regeneration promotion at the site of injury.34Sato Y. Araki H. Kato J. Nakamura K. Kawano Y. Kobune M. Sato T. Miyanishi K. Takayama T. Takahashi M. et al.Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion.Blood. 2005; 106: 756-763Crossref PubMed Scopus (557) Google Scholar, 35Mei S.H. Haitsma J.J. Dos Santos C.C. Deng Y. Lai P.F. Slutsky A.S. Liles W.C. Stewart D.J. Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis.Am. J. Respir. Crit. Care Med. 2010; 182: 1047-1057Crossref PubMed Scopus (520) Google Scholar, 36Feng J. Mantesso A. De Bari C. Nishiyama A. Sharpe P.T. Dual origin of mesenchymal stem cells contributing to organ growth and repair.Proc. Natl. Acad. Sci. U S A. 2011; 108: 6503-6508Crossref PubMed Scopus (309) Google Scholar Interestingly, Bruno’s group confirmed that microvesicles derived from MSCs could protect against acute renal tubular injury via cell-to-cell communication.37Gatti S. Bruno S. Deregibus M.C. Sordi A. Cantaluppi V. Tetta C. Camussi G. Microvesicles derived from human adult mesenchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury.Nephrol. Dial. Transplant. 2011; 26: 1474-1483Crossref PubMed Scopus (627) Google Scholar, 38Bruno S. Grange C. Deregibus M.C. Calogero R.A. Saviozzi S. Collino F. Morando L. Busca A. Falda M. Bussolati B. et al.Mesenchymal stem cell-derived microvesicles protect against acute tubular injury.J. Am. Soc. Nephrol. 2009; 20: 1053-1067Crossref PubMed Scopus (998) Google Scholar Exosomes/microvesicles contain mRNAs, microRNAs, and proteins, and our previous work indicated that hucMSCs and hucMSC-Ex can alleviate CCl4-inducued liver injury as that observed in hucMSCs,10Li T. Yan Y. Wang B. Qian H. Zhang X. Shen L. Wang M. Zhou Y. Zhu W. Li W. Xu W. Exosomes derived from human umbilical cord mesenchymal stem cells alleviate liver fibrosis.Stem Cells Dev. 2013; 22: 845-854Crossref PubMed Scopus (605) Google Scholar, 39Yan Y. Xu W. Qian H. Si Y. Zhu W. Cao H. et al.Mesenchymal stem cells from human umbilical cords ameliorate mouse hepatic injury in vivo.Liver Int. 2009; 29: 356-365Crossref PubMed Scopus (125) Google Scholar suggesting that MSC-derived exosomes may be critical to reversing liver injury. Thus, we sought to understand the route, dose, and mechanism for the hepatoprotective role of hucMSC-Ex. Using a murine model of CCl4-induced acute liver failure, we investigated the route and dose that govern the success and efficacy of using hucMSC-Ex for treatment of liver disease. Our data showed that a single systemic administration of hucMSC-Ex as little as 16 mg/kg body weight effectively rescued the recipient mice from CCl4-induced liver failure. hucMSC-Ex administrated by the intragastric route have a similar effect as those administered by the tail vein route. hucMSC-Ex administration by the intragastric route may be preferred because it is noninvasive compared with the intrasplenic or intravenous approach commonly used clinically. We measured the antioxidant activity and the hepatoprotection of hucMSC-Ex. We found that hucMSC-Ex reduced ROS and MDA and increased cell viability in L02 cells exposed to CCl4 or H2O2. Similar data were observed in hucMSC-Ex-treated CCl4-injured mouse livers. hucMSC-Ex can reverse oxidative stress-induced NFkB apoptosis. Superoxide dismutase (SOD) and glutathione peroxidase 1 (GPX1) are the major endogenous antioxidant enzymes. To understand which exosome component mediated antioxidant effects, SOD and GPX1 were measured in L02 cells after exposure to CCl4 and hucMSC-Ex treatment. Results showed that GPX1 activity was increased dose-dependently, and SOD activity was not changed in hucMSC-Ex-treated L02 cells. GPX1 is important to hucMSC-Ex-mediated antioxidant and hepatoprotection. Thus, hucMSC-Ex-derived GPX1 was chosen for further analysis. CCl4 and H2O2 are model hepatotoxicants.32Meier R.P. Mahou R. Morel P. Meyer J. Montanari E. Muller Y.D. Christofilopoulos P. Wandrey C. Gonelle-Gispert C. Bühler L.H. Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice.J. Hepatol. 2015; 62: 634-641Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 40Conde de la Rosa L. Schoemaker M.H. Vrenken T.E. Buist-Homan M. Havinga R. Jansen P.L. Moshage H. Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases.J. Hepatol. 2006; 44: 918-929Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar They were used to develop a mouse liver injury model and study the antioxidant effects of hucMSC-Ex. We measured the levels of ROS, MDA, 8-OHdG, cell viability, and cell apoptosis. Histopathology and serum chemistry confirmed that hucMSC-Ex could repair the injured livers. Tetraspanin molecules CD9, CD63, and CD81 are often used to measure exosomes.41Clayton A. Turkes A. Dewitt S. Steadman R. Mason M.D. Hallett M.B. Adhesion and signaling by B cell-derived exosomes: the role of integrins.FASEB J. 2004; 18: 977-979Crossref PubMed Scopus (246) Google Scholar TEM was used to confirm the size and morphology of exosomes derived from hucMSCs, and western blotting was used to quantify CD9 and CD63 expression in hucMSC-Ex. Oxidative stress is caused by an imbalance between ROS production and antioxidant defenses that neutralize reactive intermediates, triggering damage. Oxidative stress is associated with liver injury and may arise after CCl4, H2O2, and acetaminophen treatment.40Conde de