The Protective Effects Of Umbilical Cord MSCs On D-Galactose Induced Hepatic Mitochondrial Dysfunction

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.Augmented free radical generation leads to mitochondrial impairment and stimulates additional ROS production,cytokine release and cell apoptosis,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).As a multi-potent cell type,MSCs have self-renewing potential and multi-directional differentiation abilities.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.In addition,compelling evidence suggest that MSCs is a potential antioxidative candidate and exerts its hepatoprotective effects by redox-signaling pathways.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,and the mechanisms are associated with accumulation of free radicals,antioxidant defensive ability impairment and mitochondrial dysfunction.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 Nrf2/HO-1 pathway.PurposeTo establish chronic liver injury models and to evaluate the therapeutic effect of UCMSCs in this model;to investigate the protective effects of UCMSCs on mitochondrial function in D-Gal model.Methods1.Each UC was cut into small pieces and placed onto plates treated with low-glucose Dulbecco-modified Eagle medium(L-DMEM).0.25%trypsin was then utilized to trypsinize the resulting cells,and these cells were passaged at 1×104 cells/cm2,in the above-described medium.The immune phenotype of UCMSCs was identificated by:flow cytometry,and then differentiation potential of UCMSC was identified in vitro.2.After acclimatization to laboratory conditions,rats were randomly assigned into one of three groups:saline control group,D-Galactose(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,USA)at adosage 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 ?L 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.3.ROS production was measured with DCFH-DA fluorescence.Mitochondrial membrane potential was detected with Rhodamine 123 using flow cytometry.Protein expression of Nrf2?HO-1?FOXO3a?p-FOX03a were analyzed by western blot.Results1.UC-derived MSCs were characterized by flow cytometry after undergoing several passages.A monolayer of typical fibroblastic and plastic-adherent cells was formed by adherent cells from UC.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 CD 105);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.2.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.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.3.Chronic D-Gal administration promoted cellular oxidative stress,which ultimately induced typical age-related changes in the liver.Given that ?-galactosidase is a reliable and commonly used biomarker for cellular senescence,we stained hepatic samples for p-galactosidase detection.D-Gal administration accumulated the SA-?-Gal activity in the hepatic cells.However,this accumulation were attenuated by UC-MSCs treatment.Increased SA-?-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.4.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.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 contrasty 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.5.Mitochondrial dysfunction,revealed as enhanced ROS production,decreased mitochondrial membrane potential and impaired APTP synthase,contributes to the deterioration of hepatic damage.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.6.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.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).7.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.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.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.8.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 FOX03a expression.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.