Umbilical Cord MSCs Reverse D-Galactose-Induced Hepatic Mitochondrial Dysfunction via Activation of Nrf2/HO-1 Pathway

Abstract

Mitochondria are the central hubs for cellular bioenergetics and are crucial to cell survival. It is well accepted that compromised mitochondrial function is linked with hepatocytes injury and contribute to progression of liver diseases. Despite the therapeutic potential of mesenchymal stem cells (MSCs) transplantation on hepatic disorders have been extensively investigated, the effects of MSCs on mitochondrial function in liver injury models remain unknown. Here we investigated the effects of treatment with umbilical cord (UC) MSC in a rat model of D-galactose (D-Gal) induced liver injury, characterized by organ damage, oxidative stress and mitochondrial dysfunction. Our results showed that UC-MSCs treatment significantly alleviated histological lesion and attenuated the elevation of liver biochemical markers, demonstrating its protective effects on D-Gal induced hepatic disorders. Mitochondria isolated from the liver of D-Gal models exhibited decreased antioxidant capacity as well as compromised bioenergetics functions, as shown by a loss of mitochondrial membrane potential, elevation of reactive oxygen species (ROS) production, reduction of mitochondrial respiration complexes and ATP decrement. Treatment of rats with UC-MSCs remarkably blunted these changes and rescued mitochondrial efficiency. Mechanistically, we found that the protective potential of UC-MSCs administration was mediated by nuclear factor-E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway, but not FOXO3a pathway. In conclusion, the attenuating effects of UC-MSCs on hepatic damage partially rely on normalizing mitochondrial function and preventing a state of energetic deficit via activation of Nrf2/HO-1 pathway.

Chronic hepatic disorders are a major public health concern worldwide. Despite tremendous recent advances in medical technology, the prevention and treatment options for hepatic diseases remain limited. Multiple studies have shown that oxidative stress, which can be defined as an imbalance between reactive oxygen species (ROS) and antioxidant agents, is implicated in the pathogenesis of chronic hepatic diseases, regardless of the cause. Mitochondria are thought to play a critical role in the development and pathogenesis of chronic liver disorders, due not only to their role as the main source of endogenous ROS, but also due to the role as the target of ROS attack.¹⁾ Augmented free radical generation leads to mitochondrial impairment and stimulates additional ROS production, cytokine release and cell apoptosis,²⁾ which in turn leads to elevated oxidative stress that promotes further deterioration of liver function.

Mesenchymal stem cells (MSCs), deriving from the stroma, can be obtained from multiple human tissues, such as bone marrow (BM), adipose tissue, skeletal muscle tissue, and the umbilical cord (UC).³⁾ As a multi-potent cell type, MSCs have self-renewing potential and multi-directional differentiation abilities.⁴⁾ Therapeutic effects of MSCs have been confirmed in numerous diseases, particularly in the area of liver function improvement. Animal and clinical studies have shown that MSC transfusion can contribute to regression of liver fibrosis and can alleviate fulminant hepatitis damage that has the capacity to differentiate into hepatocytes.^5,6) In addition, compelling evidence suggest that MSCs is a potential antioxidative candidate and exerts its hepatoprotective effects by redox-signaling pathways.^7,8) However, the underlying mechanism is not fully elucidated. Previous studies have shown that D-galactose (D-Gal) overload can mimic hepatic failure in clinical settings,^9,10) and the mechanisms are associated with accumulation of free radicals, antioxidant defensive ability impairment and mitochondrial dysfunction.¹¹⁾ In the present study, we demonstrate for the first time that umbilical cord MSCs (UC-MSCs) protected against liver injury induced by D-Gal via the preservation of mitochondrial function through inhibiting the overproduction of intracellular ROS and activation of nuclear factor-E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats with a body weight of 200–220 g each were purchased from the Shandong University Experimental Animal Center. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Shandong University. Rats were maintained in the animal facility at the Qilu Hospital of Shandong University and had free access to water and rodent food. They were maintained under stable conditions (23±1°C and 60% humidity) and were housed in cages with a 12 h light/dark cycle.

Generation and Administration of UC-MSCs

Experimental protocols were submitted to the Qilu Hospital’s Human Research Ethics Committee, which approved all research. The UCs (n=10) were the products of clinically normal pregnancies. In order to remove excess blood, each UC was excised and was then washed in a 0.1 mol/L phosphate buffer (pH 7.4). The cords were dissected and the blood vessels removed. After this, remaining tissues were cut into small pieces (1–2 mm³) and placed onto plates treated with low-glucose Dulbecco-modified Eagle medium (L-DMEM) (Gibco-BRL, Grand Island, NY, U.S.A.). The medium in these plates was supplemented with a 10% fetal bovine serum (FBS, Gibco-BRL), 2 ng/mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, U.S.A.), 2 ng/mL epidermal growth factor (EGF; R&D Systems), 2 ng/mL fibroblast growth factor (FGF; R&D Systems), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco-BRL), and then maintained at 37°C in a humidified atmosphere of 5% CO₂. The media were renewed at intervals of 3–4 d. Subsequent to the beginning of the incubation period, adherent cells were proliferated from individual explanted tissues at 7–12 d. At the time of cell proliferation, the small tissue bits were removed from the culture and the adherent fibroblast-like cells were cultured to confluence, a process that required an additional 2–3 weeks. 0.25% trypsin (Gibco-BRL) was then utilized to trypsinize the resulting cells, and these cells were passaged at 1×10⁴ cells/cm², in the above-described medium. The cells were deemed ready for experimental use after five-plus passages.

Cell Surface Antigen Phenotyping

Fifth- to sixth-passage UCs were collected and were treated with 0.25% trypsin, after which phycoerythrin-conjugated monoclonal antibodies in 100 µL phosphate buffers were used to stain the cells for 15 min at room temperature, following the manufacturer’s instructions. The antibodies were used to target human antigens CD29, CD31, CD34, CD44, CD45, CD73, CD90 and CD105, and CD271 (SeroTec, Raleigh, NC, U.S.A.). Flow cytometry (Cytometer 1.0, Cytomics™ FC500, Beckman Coulter) was used for purposes of cell analysis. A count was made of positive cells, and these were compared to the signal of corresponding immunoglobulin isotypes.

Experimental Design

After acclimatization to laboratory conditions, rats were randomly assigned into one of three groups: saline control group, D-Gal group, and UC-MSCs+D-Gal group (n=12 per group). Except for the control group, rats received daily subcutaneous (s.c.) injections of D-Gal (Sigma-Aldrich, St. Louis, MD, U.S.A.) at a dosage of 300 mg/kg for 8 weeks, after which rats from D-Gal or UC-MSCs+D-Gal group were injected with the infusion of UC-MSCs [1×10⁶ cells in 1000 µL of normal saline (NS)] or saline (0.9%), via the tail vein at each time point (2, 4, 6 and 8 weeks during injection of D-Gal), 4 times each. Those in the control group underwent injections of a like volume of saline. At the end of the experimental period of 2 months, all animals were anesthetized and dissected, hepatic samples of the right lobe were collected immediately for hematoxylin–eosin (H&E) and β-galactosidase staining examinations, and then the left lobe of the liver was snap frozen and stored at −80°C for other assays. Serum was obtained from the blood via centrifugation at 3000×g for 20 min at 4°C. Ethylenediaminetetraacetic acid (EDTA)-containing blood (50 µL of 100 mmol/L EDTA solution for each 1 mL of blood) was centrifuged at 2500×g for 5 min to separate plasma.

Histological Evaluations

Liver tissues were fixed in 4% paraformaldehyde solution, paraffin embedded and cut into 2 µm thickness sections. H&E staining was performed following standard procedure. The histopathological results were recorded under an IX71 Olympus microscope (Olympus, Japan).

Senescence-associated β-galactosidase (SA-β-Gal) activity was detected with a commercial kit following the manufacturer’s protocol. Briefly, liver tissues were perfused with OTC solution and immediately frozen in liquid nitrogen. Ten micrometers frozen sections were cut by a Leica CM1950 freezing microtome (Leica, Germany), then incubated overnight in β-galactosidase staining solution (Beyotime Institute of Biotechnology, Haimen, China) at 37°C. The following day, the samples were washed with phosphate-buffered saline and immediately imaged with an IX71 Olympus microscope (Olympus, Japan).

Serum Aminotransferase Level Examination

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBIL) and direct bilirubin (DBIL) activities were assessed by commercial kits (Nanjing Jiancheng Bioengineering Institute, China).

Preparation of Hepatic Mitochondria

Hepatic mitochondria were isolated by differential centrifugation as previously described.¹²⁾ After isolation, mitochondria were validated by measurement of levels of the mitochondrial enzyme marker succinate dehydrogenase (Sigma-Aldrich). For each experiment, all steps were strictly conducted on ice and mitochondria were prepared fresh and used within 4h after isolation to maintain the activities of mitochondrial function. Protein concentration was determined by use of a Bradford Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute).

Measurement of Hepatic Malondialdehyde (MDA) Levels

Lipid peroxidation was determined by MDA concentration, using a malondialdehyde assay kit (Nanjing Jiancheng Bioengineering Institute).

Measurement of Hepatic Antioxidant System

Superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) levels in the liver were determined by commercial kits (Nanjing Jiancheng Bioengineering Institute).

Determination of Mitochondrial ROS Levels

The 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) method was used to evaluate mitochondrial ROS levels by means of a commercial kit (Beyotime Biotech., China), and the DCF fluorescence was then read on a fluorescence spectrophotometer (Hitachi F-7000, Japan) at the excitation and emission wavelengths of 488 and 525 nm, respectively. ROS formation was quantified from a DCF standard curve, and the data were expressed as fluorescence/min/mg protein.

Measurement of Mitochondrial Membrane Potential (MMP)

Rhodamine 123 (Beyotime Biotech.) was employed for the determination of mitochondrial membrane potential. Previously-described protocols and methods were followed and measurements were performed on flow cytometer equipped with a 503 nm argon ion laser, and the signals were obtained using a 527 nm bandpass filter.

Measurement of Mitochondrial Enzymatic Activity

The activity of succinate dehydrogenase was determined by commercial kit (Sigma-Aldrich). Cytochrome-c oxidase activity was determined by polarographical method as previously described.¹³⁾ The process was carried out at 25°C in 1.4 mL of standard respiratory medium supplemented with 2 µM rotenone, 10 µM oxidized cytochrome-c, 0.3 mg Triton X-100 and freeze-thawed mitochondria (0.25 mg). After this phase, the reaction was initiated by adding 5 mM ascorbate plus 0.25 mM tetra methylphenylene–diamine (TMPD). ATP-ase activity was determined spectrophotometrically at 660 nm, in association with ATP hydrolysis.¹⁴⁾ In summary, the reaction was carried out at 37°C, in 2 mL reaction medium (125 mM sucrose, 65 mM KCl, 2.5 mM MgCl₂ and 0.5 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4). Subsequent to the addition of 0.25 mg of freeze-thawed mitochondria, the reaction was initiated by means of adding 2 mM Mg²⁺-ATP, both in the presence or the absence of oligomycin (1 µg/mg protein). After 10 min, the reaction was ended by the addition of 1 mL of 40% trichloroacetic acid, and the samples were centrifuged for 5 min at 3000 rpm. At this point, a solution containing 2 mL of ammonium molybdate mixed into 2 mL H₂O was added to 1 mL of supernatant. ATP-ase activity was calculated as total absorbance minus absorbance in the presence of oligomycin.

ATP Assay

Endogenous mitochondrial ATP was extracted using a commercially available kit (Sigma-Aldrich). The extraction was effected by an alkaline extraction procedure, and results were measured according to instructions provided by the kit’s manufacturer.

Western Blot Analysis

Total protein extracted from the liver was resolved by means of electrophoresis and was electroblotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, U.S.A.). The membranes were incubated with rat Nrf2, HO-1, FOXO3a, p-FOXO3a and β-actin (Cell Signaling Technology, U.S.A.) antibodies overnight at 4°C respectively, following incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific, U.S.A.) at room temperature for 1 h. The membranes were washed and the protein signals were detected by enhanced chemiluminescence methods (Millipore, Billerica, MA, U.S.A.).

Data Analysis

Data are presented as the mean±standard deviation (S.D.). All statistical analyses were conducted using the Statistical Program for Social Sciences 19.0 software program (SPSS Inc., Chicago, IL, U.S.A.). Statistical analysis for multiple group comparisons was performed by one-way ANOVA followed by Tukey’s post hoc tests. A value of p<0.05 was considered to be statistically significant.

RESULTS

Characterization of UC-MSCs

UC-derived MSCs were characterized by flow cytometry after undergoing several passages. As seen in Fig. 1A, a monolayer of typical fibroblastic and plastic-adherent cells was formed by adherent cells from UC. Spindle-shaped cells can be seen in Fig. 1B, as shown by H&E staining. The results of flow cytometry demonstrated that the UC-derived cells shared most of their immunophenotypes with MSCs. These shared immunophenotypes included the expression of positive stromal markers (CD29, CD44, CD73, CD90 and CD105); the expression of negative hematopoietic markers (CD34 and CD45); endothelial cell marker CD31 and differentiated activated effector cell marker CD271 (Fig. 1C). These results indicated that the cells were undifferentiated and that they possessed the characteristics of stem cells.

Fig. 1. Characterization of UC-Derived MSCs

A. Bright-field image. B. H&E staining image. C. Immunophenotype of MSCs at passage 5 by flow cytometry. UC-derived cells shared most of their immunophenotypes with MSCs, including positive expression of stromal markers (CD29, CD44, CD73, CD90, and CD105) and expression of negative hematopoietic markers (CD34 and CD45), endothelial cell marker CD31 and differentiated activated effector cell marker. Scale bars represent 200 µm.

UC-MSCs Transplantation Attenuates D-Gal-Induced Hepatic Impairment

As shown in Fig. 2B, hepatic histology in the group given D-Gal demonstrated evidence of liver injury with visible histological changes including structural damage and necrosis of hepatocytes. Whereas, UC-MSCs treatment alleviated the liver impairment in D-Gal-treated rats (Fig. 2C). Hepatic parameters were also assessed for the evaluation of liver function and the results were summarized in Table 1. D-Gal treatment caused an elevation of hepatic marker enzymes, while UC-MSCs treatment significantly suppressed the effects of D-Gal on serum AST, TBIL, DBIL and ALP activities.

Fig. 2. H&E Staining and SA-β-Gal Assessment in Rat Livers

D-Gal models received daily subcutaneous (s.c.) injections of 300 mg/kg D-Gal for 8 weeks, while the control group was treated with normal saline. D-Gal+UC-MSCs group was injected with the infusion of UC-MSCs [1×10⁶ cells in 1000 µL of normal saline (NS)] at each time point (2, 4, 6 and 8 weeks during injection of D-Gal), four times each. H&E staining in (A) control group, (B) D-Gal group, (C) D-Gal+UC-MSCs group. Black arrows indicate apoptotic hepatocytes. (D) Quantification of SA-β-Gal positive cells relative to the total number of cells. Scale bars: 100 µm. Error bars indicate the S.D. for n=5 per group; statistical analysis: ** p<0.05 vs. control group; ^## p<0.05 vs. D-Gal group.

Table 1. Effects of UC-MSCs on Liver Functions in D-Gal Models

Hepatic parameters	Groups
	Control	D-Gal	D-Gal+MSCs
ALT (U/L)	43.2±4.5	41.8±7.7	47.4±10.6
AST (U/L)	82.1±7.8	128.9±18.9**	98.8±20.1#
TBIL (mmol/L)	3.0±0.4	4.2±0.7*	3.6±0.7#
DBIL (mmol/L)	1.0±0.1	1.8±0.4*	1.3±0.4#
ALP (U/L)	72.9±5.7	97.9±10.8*	80.9±9.1#

Each value is represented as the mean±S.D., n=8 for each group. * p<0.05 and ** p<0.01 versus control group, ^# p<0.05.

Chronic D-Gal administration promoted cellular oxidative stress, which ultimately induced typical age-related changes in the liver. Given that β-galactosidase is a reliable and commonly used biomarker for cellular senescence, we stained hepatic samples for β-galactosidase detection. D-Gal administration accumulated the SA-β-Gal activity in the hepatic cells. However, this accumulation were attenuated by UC-MSCs treatment. The percentage of SA-β-Gal-positive hepatocytes per field were calculated and the result was shown in Fig. 2D. Increased SA-β-Gal expression in hepatocytes strongly confirmed D-Gal-induced cellular senescence, and this was suppressed by UC-MSCs. Taken together, these results indicate that UC-MSCs treatment effectively antagonized D-Gal-induced hepatotoxicity and facilitated the recovery of hepatic function.

UC-MSCs Restored Hepatic Mitochondrial Antioxidative Capacities

Antioxidant enzymes (SOD, GPx and GSH) levels and MDA content were examined in order to assess the protective effect of UC-MSCs treatment on hepatic mitochondrial redox state. As shown in Table 2, chronic administration of D-Gal caused a significant decline in SOD, GPx activities and GSH level. Mitochondrial MDA contents, which are the byproduct of lipid peroxidation, were significantly increased in D-Gal models. In contrast, treatment with UC-MSCs could restore mitochondrial ROS scavenging abilities, which is demonstrated by the recovery of antioxidant systems and the reduction in MDA contents.

Table 2. Effects of UC-MSCs on Mitochondrial SOD, GPx Activities, GSH Level and MDA Content in D-Gal Models

Redox parameters	Groups
	Control	D-Gal	D-Gal+MSCs
SOD (U/mg protein)	120.4±15.7	70.7±19.0**	97.5±17.4#
GPx (U/mg protein)	47.6±5.3	20.7±6.8**	38.3±7.3##
GSH (mg/g protein)	0.54±0.09	0.28±0.10**	0.41±0.10#
MDA (nmol/mg protein)	2.47±0.31	5.52±0.91*	3.01±0.87#

Each value is represented as the mean±S.D., n=8, for each group, * p<0.05 and ** p<0.01 versus control group, ^# p<0.05 and ^## p<0.01 versus D-Gal group.

UC-MSCs Treatment Alleviates Mitochondrial Dysfunction in D-Gal Models

Mitochondrial dysfunction, revealed as enhanced ROS production, decreased mitochondrial membrane potential and impaired ATP synthase, contributes to the deterioration of hepatic damage. As shown in Figs. 3A and B, the D-Gal models have obvious mitochondrial defects, exhibiting a 51.7% reduction of MMP and a 2.08-fold increase in ROS production when compared with the control group. Treatment with UC-MSCs, however, demonstrated a significant recovery of mitochondrial damage as indicated by increasing MMP and decreasing ROS production by 83.2% and 1.48-fold, respectively.

Fig. 3. Effect of UC-MSCs on D-Gal-Induced Alteration in Hepatic Mitochondrial Functional Markers

A. ROS production was measured with DCFH-DA fluorescence. B. Mitochondrial membrane potential was detected with Rhodamine 123 using flow cytometry. C. Succinate dehydrogenase activity. D. Cytochrome-c oxidase activity. E. ATP-ase activity. F. ATP quantification. All data are represented as the mean±S.D., n=8 per group. * p<0.05 and ** p<0.01 versus control group, ^# p<0.05 and ^## p<0.01 versus D-Gal group.

Mitochondrial ATP is the energy currency that maintains biological functions in all living systems. Respiratory chain components, coupled with ATP synthase, are essential in the process of mitochondrial ATP generation, and the alteration of respiratory chain complexes is the indicative markers of mitochondrial dysfunction. Here, cytochrome-c oxidase (COX) and succinate dehydrogenase (SDH) activities were evaluated, and this revealed an impairment of a component in the mitochondrial respiratory chain as a result of D-Gal administration (Figs. 3C, D). The activity of another element of the mitochondrial oxidative phosphorylation system, the ATP synthase (ATP-ase) was also evaluated. The result showed that D-Gal caused a significant reduction in ATP synthase activity. UC-MSCs treatment rescued D-Gal-induced mitochondrial dysfunction, as seen with the restoration of the mitochondrial ATP-ase (Fig. 3E) and respiratory chain complexes such as SDH and COX. Corresponding to decreased mitochondrial function, shrinkage of mitochondrial ATP content was also observed in D-Gal models, and the effect of such shrinkage was rescued by UC-MSCs treatment (Fig. 3F).

Nrf2 Pathway, not FOXO3a Pathway, Is Involved in Protective Effects of UC-MSCs on D-Gal-Induced Mitochondrial Dysfunction

In addition to serving as a master regulator that induces activation of the antioxidant response element (ARE) pathway, Nrf2 can also affect the mitochondrial membrane potential, fatty acid oxidation, availability of substrates for respiration and ATP synthesis. It is an important player in the maintenance of mitochondrial homeostasis and structural integrity. As shown in Fig. 4B, D-Gal treatment significantly inhibited the protein accumulations of Nrf2, with respect to which UC-MSCs treatment showed effective protective mechanisms. This result indicates the involvement of the Nrf2 pathway in the mitochondrial protective process of UC-MSCs. Parallel to the analysis of Nrf2 expression, a similar pattern of regulation was also observed at the Nrf2 downstream component HO-1 (Fig. 4C). For this enzyme, a significant down-regulation was observed in D-Gal models as compared to the control group. In contrast, the UC-MSCs treatment protected against the loss of HO-1 expression. These results suggest that the Nrf2 pathway may be a target of the UC-MSCs treatment in the protective process. By enhancing the Nrf2 expression, UC-MSCs supplementation increases downstream antioxidant HO-1 expression, thereby contributing to the recovery of hepatic function and attenuation of mitochondrial impairment.

Fig. 4. Nrf2 Pathway Not FOXO3a Pathway Is Involved in Protective Effects of UC-MSCs on D-Gal-Induced Mitochondrial Dysfunction

A. Expression of Nrf2, HO-1, FOXO3a and p-FOXO3a protein was determined by Western blot analysis. β-Actin served as the standard. B and C. Fold change in relative density analysis of Nrf2 and HO-1 protein bands. β-Actin was probed as an internal control in relative density analysis of the protein bands. D. The relative density is expressed as the p-FOXO3a/FOXO3a ratio. The bands were analyzed with Image Pro Plus and β-actin was probed as an internal control. Values are averages from three independent experiments. All data are represented as mean±S.D., n=4 per group. * p<0.05 and ** p<0.01 versus control group, ^## p<0.01 versus D-Gal group.

FOXO3a also regulates detoxification of ROS through up-regulation of mitochondrial antioxidative systems. Normally, FOXO3a binds to the DNA and transcriptionally induces targeted gene expressions. Phosphorylation of FOXO3a can cause the inactivation of its transcriptional function. Here, we further detected the FOXO3a expression. As shown in Fig. 4D, D-Gal treatment led to no significant alterations in FOXO3a protein levels, but an up-regulation of p-FOXO3a was observed. However, UC-MSCs supplementation had no effect on the D-Gal-induced disturbance of the p-FOXO3a/FOXO3a ratio, suggesting that FOXO3a activity was not contributing to the protective effects of UC-MSCs on mitochondrial function.

DISCUSSION

The therapeutic effects of MSCs transplantation on hepatic disorders have been extensively investigated in animal and pre-clinical studies.^15,16) Umbilical cord-derived mesenchymal stem cells (UC-MSCs), as a source of MSCs, provide a promising approach for hepatic disorder therapy, due to advantages that include high self-renewal capacity and pluripotency, having similar gene profiles to those of embryonic stem cells, and less ethical restrictions.¹⁷⁾ Accumulating evidence proved that mitochondrial dysfunction, resulting in the accumulation of ROS and the reduction of respiratory complex activity, plays an important role in the pathogenesis of chronic hepatic disorders.¹⁸⁾ However, little is known about the potential effects of MSC transplantation on mitochondrial function in chronic hepatic disease.

D-Gal is known as a hepatotoxin that is metabolized mainly in hepatocytes. Research has demonstrated that D-Gal promotes oxidative damage in the liver of rodents, followed by impaired hepatic function and an accelerated aging process.¹⁹⁾ In the present study, increasing serum enzyme values, including levels of AST, TBIL, DBIL and ALP, were found in D-Gal models. In addition, alteration of SA-β-Gal activity, which is a biomarker in senescent cells, was also observed. UC-MSCs treatment remarkably attenuated D-Gal-induced hepatopathy, revealed as the reduction of serum AST, TBIL, DBIL, ALP levels and SA-β-Gal activities, thus suggesting the hepatoprotective characteristic of UC-MSCs against D-Gal-induced chronic liver injury.

It has been accepted that promotion of ROS generation is an important mechanism for D-Gal induced hepatotoxicity. Mitochondria, as the primary intracellular site of oxygen consumption and the major site of ROS generation, are also susceptible to oxidative attack, due to the high content of polyunsaturated fatty acids (PUFAs) in the membrane. There is increasing evidence that chronic D-Gal administration disturbs cellular mitochondrial redox homeostasis and ultimately induces mitochondrial deficiency.^20,21) In the current study, we also found that chronic exposure to D-Gal resulted in hepatic mitochondrial oxidative damage, revealed as depletion of antioxidative capacity, loss of membrane potential and the elevation of ROS generation, which is consistent with findings in previous studies.²²⁾ Interestingly, UC-MSCs treatment strongly elevated the activities of SOD, GPx and GSH, decreased MDA content, attenuated ROS overgeneration and enhanced mitochondrial membrane potential in D-Gal models, indicating that UC-MSCs treatment reduced D-Gal-induced mitochondrial oxidative stress. Since mitochondria are critical to normal cell and organ function due to their central role in energy production, the mitochondrial bioenergetic functions were also discussed here. In addition to balancing the redox state, UC-MSCs also ameliorated D-Gal-induced mitochondrial bioenergetic dysfunction, causing an improvement in the activities of COX, SDH and ATPase, and preventing a decline of ATP production following D-Gal exposure. It is well known that MSCs precipitate potent antioxidative activity and exhibit therapeutic effects on the restoration of ROS-damaged tissue,^23,24) and studies have proven the antioxidative mechanism of MSCs on damaged tissue is related to paracrine action.²⁵⁾ Zhou et al.²³⁾ observed that exosomes derived from UC-MSCs can repair cisplatin-induced acute kidney injury in rats by ameliorating oxidative stress. In previous work from our institution, hepatogenic differentiation of MSCs has been revealed.²⁶⁾ Differentiated hepatocyte-like cells (HLCs) from MSCs were able to engraft into the injured liver and restore hepatic functions in vivo.^27,28) Here the restoration of mitochondrial activity and serum enzyme levels as the effects of UC-MSCs are clear indicators of improvement of hepatic function, which proves that UC-MSCs exerts its hepatic protective effects through maintenance of mitochondria. From the present results, we infer that effects of UC-MSCs treatment on liver injury can not only directly replace impaired hepatocytes but can also maintain mitochondrial function.

Recent reports have shown that Nrf2/HO-1 system can stimulate mitochondrial bioenergetics which may rely on modulating the availability of substrates²⁹⁾ and protection on mitochondrial respiration complexes.³⁰⁾ In the present study, the synchronous decreases or increases of mitochondrial antioxidative enzymes by D-Gal treatment or UC-MSCs supplementation underlined the involvement of Nrf2/HO-1. Indeed, the Western-blot results demonstrated that the Nrf2/HO-1 activity was inhibited in D-Gal models, on which UC-MSCs treatment showed effective protection on Nrf2/HO-1, suggesting that Nrf2/HO-1 pathway was one target of UC-MSCs in the mitochondrial protective process. Although several publications have revealed that MSCs evoke a cellular adaptive response against oxidative damage via activation of the Nrf2 signaling pathway,^7,31) the mechanism controlling this activation is still poorly understood. Normally, the cellular distribution and stability of Nrf2 is tightly regulated by its inhibitor Kelch-like ECH-associated protein 1 (Keap1). Keap1 not only acts to retain Nrf2 in the cytoplasm, but also promotes its degradation by the ubiquitin–proteasome system.³²⁾ Electrophiles and ROS are known as as the canonical stimuli for Nrf2 activation, which they accomplish by means of disrupting the Keap1–Nrf2 complex. Here, we demonstrated an inconsistent pattern in Nrf2 expression and dynamics change of ROS production. This apparent paradox can be explained by the existence of alternative mechanisms for Nrf2 induction including transcriptional regulation and post-translational modifications of either the Keap1 or the Nrf2.³³⁾ A number of studies have revealed that upstream kinases, for example, phosphatidylinositol 3-kinase (PI3K),³⁴⁾ can phosphorylate Nrf2 at Ser40, which is a residue located in the Neh2 domain that binds Keap1, and ultimately induce the disturbance of Keap1–Nrf2 interaction. Evidence has demonstrated that MSCs-derived exosomes could rapidly restore bioenergetics and reduce oxidative stress in myocardial ischemic/reperfusion (I/R) injury models via activation of the PI3K/Akt pathway,³⁵⁾ suggesting that enhancement of MSCs on Nrf2 stabilization may be partially dependent on kinase pathways. In addition, as a critical component of the tumor microenvironment, MSCs was reported to cause a dose-dependent inhibition of cancer cells due to upregulated p21 expression.³⁶⁾ Several studies have revealed that p21 could bind with Nrf2 at the DLG and ETGE domains, and cause the dissociation of the Keap1–Nrf2 complex.³⁷⁾ From the present results, we infer that a multifaceted joint comprehensive activation mechanism is involved. The mechanisms of Nrf2 activation were previously considered to be the result of ROS stimulation. However, these are now considered to be the result of a multifaceted joint comprehensive mechanism. Former studies proved that FOXO3a, which is another key transcription factor in mitochondrial gene expression and mitochondrial activity regulation, can bind to mitochondrial DNA (mtDNA) and stimulate the transcription of mitochondrial-encoded core or catalytic subunits of the OXPHOS machinery to increase respiration, thus sustaining energy metabolism.³⁸⁾ However, our study observed failure of the UC-MSCs treatment to normalize expression of the FOXO3a, suggesting that FoxO3a is not involved in a recovery mechanism of UC-MSCs.

In conclusion, the present study demonstrates that UC-MSCs treatment has the potential to alleviate D-Gal induced liver injury and to reverse mitochondrial dysfunction in rat models. Our data also highlighted the fact that attenuation effects of UC-MSCs on hepatic damage was partially dependent on the mitochondrial bioenergetic functions via activation of Nrf2/HO-1 pathway, but not FOXO3a pathway, which suggest that UC-MSCs based cellular therapies could be a viable therapeutic approach to liver damage.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Pal PB, Sinha K, Sil PC. Mangiferin, a natural xanthone, protects murine liver in Pb(II) induced hepatic damage and cell death via MAP kinase, NF-κB and mitochondria dependent pathways. PLOS ONE, 8, e56894 (2013).
• 2) Cichoż-Lach H, Michalak A. Oxidative stress as a crucial factor in liver diseases. World Journal of Gastroenterology: WJG, 20, 8082–8091 (2014).
• 3) Corselli M, Chen CW, Crisan M, Lazzari L, Péault B. Perivascular ancestors of adult multipotent stem cells. Arterioscler. Thromb. Vasc. Biol., 30, 1104–1109 (2010).
• 4) Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells, 25, 2739–2749 (2007).
• 5) Rabani V, Shahsavani M, Gharavi M, Piryaei A, Azhdari Z, Baharvand H. Mesenchymal stem cell infusion therapy in a carbon tetrachloride-induced liver fibrosis model affects matrix metalloproteinase expression. Cell Biol. Int., 34, 601–605 (2010).
• 6) Parekkadan B, Van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, Yarmush ML. Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS ONE, 2, e941 (2007).
• 7) Cho KA, Woo SY, Seoh JY, Han HS, Ryu KH. Mesenchymal stem cells restore CCl 4-induced liver injury by an antioxidative process. Cell Biol. Int., 36, 1267–1274 (2012).
• 8) Francois S, Mouiseddine M, Allenet-Lepage B, Voswinkel J, Douay L, Benderitter M, Chapel A. Human mesenchymal stem cells provide protection against radiation-induced liver injury by antioxidative process, vasculature protection, hepatocyte differentiation and trophic effects. Biomed. Res. Int., 2013, 151679 (2013).
• 9) Pushpavalli G, Veeramani C, Pugalendi KV. Influence of chrysin on hepatic marker enzymes and lipid profile against D-galactosamine-induced hepatotoxicity rats. Food & Chemical Toxicology an International Journal Published for the British Industrial Biological Research Association, 48, 1654–1659 (2010).
• 10) Ruan Q, Liu F, Gao Z, Kong D, Hu X, Shi D, Bao Z, Yu Z. The anti-inflamm-aging and hepatoprotective effects of huperzine A in D-galactose-treated rats. Mech. Ageing Dev., 134, 89–97 (2013).
• 11) Chen HL, Wang CH, Kuo YW, Tsai CH. Antioxidative and hepatoprotective effects of fructo-oligosaccharide in D-galactose-treated Balb/cJ mice. Br. J. Nutr., 105, 805–809 (2011).
• 12) Emaus RK, Grunwald R, Lemasters JJ. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim. Biophys. Acta, 850, 436–448 (1986).
• 13) Brautigan DL, Ferguson-Miller S, Tarr GE, Margoliash E. Definition of cytochrome c binding domains by chemical modification. II. Identification and properties of singly substituted carboxydinitrophenyl cytochromes c at lysines 8, 13, 22, 27, 39, 60, 72, 87, and 99. Journal of Biological Chemistry, 253, 140–148 (1978).
• 14) Gaballo A, Zanotti F, Raho G, Papa S. Disulfide cross-linking of subunits F(1)-gamma and F(0)I-PVP(b) results in asymmetric effects on proton translocation in the mitochondrial ATP synthase. FEBS Lett., 463, 7–11 (1999).
• 15) Dinarvand P, Hashemi SM, Soleimani M. Effect of transplantation of mesenchymal stem cells induced into early hepatic cells in streptozotocin-induced diabetic mice. Biol. Pharm. Bull., 33, 1212–1217 (2010).
• 16) Prasajak P, Leeanansaksirl W. Mesenchymal stem cells: current clinical applications and therapeutic potential in liver diseases. Journal of Bone Marrow Research, 02, 137–146 (2014).
• 17) Otsuru S, Gordon PL, Shimono K, Jethva R, Marino R, Phillips CL, Hofmann TJ, Veronesi E, Dominici M, Iwamoto M, Horwitz EM. Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms. Blood, 120, 1933–1941 (2012).
• 18) Auger C, Alhasawi A, Contavadoo M, Appanna VD. Dysfunctional mitochondrial bioenergetics and the pathogenesis of hepatic disorders. Frontiers in Cell & Developmental Biology, 3, 825–830 (2015).
• 19) Cakatay U, Aydın S, Atukeren P, Yanar K, Sitar ME, Dalo E, Uslu E. Increased protein oxidation and loss of protein-bound sialic acid in hepatic tissues of D-galactose induced aged rats. Current Aging Science, 6, 135–141 (2013).
• 20) Long J, Wang X, Gao H, Liu Z, Liu C, Miao M, Cui X, Packer L, Liu J. D-Galactose toxicity in mice is associated with mitochondrial dysfunction: protecting effects of mitochondrial nutrient R-alpha-lipoic acid. Biogerontology, 8, 373–381 (2007).
• 21) Kumar A, Prakash A, Dogra S. Naringin alleviates cognitive impairment, mitochondrial dysfunction and oxidative stress induced by D-galactose in mice. Food & Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association, 48, 626–632 (2010).
• 22) Zhang J, Xu L, Zhang L, Ying Z, Su W, Wang T. Curcumin attenuates D-galactosamine/lipopolysaccharide-induced liver injury and mitochondrial dysfunction in mice. J. Nutr., 144, 1211–1218 (2014).
• 23) Zhou Y, Xu H, Xu W, Wang B, Wu H, Tao Y, Zhang B, Wang M, Mao F, Yan Y, Gao S, Gu H, Zhu W, Qian H. Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis. Stem Cell Research & Therapy, 4, 34 (2013).
• 24) Tichon A, Eitan E, Kurkalli BG, Braiman A, Gazit A, Slavin S, Beith-Yannai E, Priel E. Oxidative stress protection by novel telomerase activators in mesenchymal stem cells derived from healthy and diseased individuals. Curr. Mol. Med., 13, 1010–1022 (2013).
• 25) Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H, Noiseux N, Zhang L, Pratt RE, Ingwall JS, Dzau VJ. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat. Med., 11, 367–368 (2005).
• 26) Ju X, Li D, Gao N, Shi Q, Hou H. Hepatogenic differentiation of mesenchymal stem cells using microfluidic chips. Biotechnol. J., 3, 383–391 (2008).
• 27) Sato Y, Araki H, Kato J, Nakamura K, Kawano Y, Kobune M, Sato T, Miyanishi K, Takayama T, Takahashi M, Takimoto R, Iyama S, Matsunaga T, Ohtani S, Matsuura A, Hamada H, Niitsu Y. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood, 106, 756–763 (2005).
• 28) Stock P, Brückner S, Ebensing S, Hempel M, Dollinger MM, Christ B. The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver. Nat. Protoc., 5, 617–627 (2010).
• 29) Ludtmann MH, Angelova PR, Zhang Y, Abramov AY, Dinkovakostova AT. Nrf2 affects the efficiency of mitochondrial fatty acid oxidation. Biochem. J., 457, 415–424 (2014).
• 30) Miller DM, Singh IN, Wang JA, Hall ED. Administration of the Nrf2–ARE activators slforaphane and carnosic acid attenuate 4-hydroxy-2-nonenal induced mitochondrial dysfunction ex vivo. Free Radic. Biol. Med., 57, 1–9 (2013).
• 31) Ni S, Wang D, Qiu X, Pang L, Song Z, Guo K. Bone marrow mesenchymal stem cells protect against bleomycin-induced pulmonary fibrosis in rat by activating Nrf2 signaling. Int. J. Clin. Exp. Pathol., 8, 7752–7761 (2015).
• 32) Huang Y, Li W, Su ZY, Kong ANT. The complexity of the Nrf2 pathway: Beyond the antioxidant response. J. Nutr. Biochem., 26, 1401–1413 (2015).
• 33) Hayes JD, Dinkova-Kostova AT. The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem. Sci., 39, 199–218 (2014).
• 34) Wang P, Peng X, Wei ZF, Wei FY, Wang W, Ma WD, Yao LP, Fu YJ, Zu YG. Geraniin exerts cytoprotective effect against cellular oxidative stress by upregulation of Nrf2-mediated antioxidant enzyme expression via PI3K/AKT and ERK1/2 pathway. Biochim. Biophys. Acta, 1850, 1751–1761 (2015).
• 35) Arslan F, Lai RC, Smeets MB, Akeroyd L, Choo A, Aguor EN, Timmers L, van Rijen HV, Doevendans PA, Pasterkamp G, Lim SK, de Kleijn DP. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res., 10, 301–312 (2013).
• 36) Lu Y, Yuan Y, Wang X, Wei L, Chen Y, Cong C, Li S, Long D, Tan W, Mao Y, Zhang J, Li YP, Cheng JQ. The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo. Cancer Biol. Ther., 7, 245–251 (2008).
• 37) Chen W, Sun Z, Wang XJ, Jiang T, Huang Z, Fang D, Zhang DD. Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Mol. Cell, 34, 663–673 (2009).
• 38) Peserico A, Chiacchiera F, Grossi V, Matrone A, Latorre D, Simonatto M, Fusella A, Ryall JG, Finley LW, Haigis MC, Villani G, Puri PL, Sartorelli V, Simone C. A novel AMPK-dependent FoxO3A–SIRT3 intramitochondrial complex sensing glucose levels. Cell. Mol. Life Sci., 70, 2015–2029 (2013).