| Non-alcoholic steatohepatitis(NASH)is a more severe stage in the progression of non-alcoholic fatty liver disease(NAFLD),characterized by hepatocellular ballooning,inflammation and fibrosis.The incidence of NASH is rapidly increasing and can lead to end-stage liver complications such as cirrhosis and hepatocellular carcinoma,as well as increasing the risk of extra-hepatic diseases such as cardiovascular disease,chronic kidney disease and type 2 diabetes.Therefore,further exploration and studies on the pathogenesis and therapeutic drugs would be helpful for the early prevention and treatment of NASH with better disease prognosis.The pathogenesis of NAFLD is not well understood,and it is generally considered that oxidative stress plays a crucial role in the pathogenesis of NAFLD,and it is believed that oxidative stress is the key factor in the progression of simple fatty liver to NASH.Excessive lipid deposition in hepatocytes will accelerate beta-oxidation in mitochondria,which leads to excessive production of reactive oxygen species(ROS)in cells,enhances oxidative stress in the liver,promotes the generation of inflammatory factors such as tumor necrosis factor(TNF)-αand interleukin(IL)-6,and finally accelerates hepatocyte injury.Nuclear factor erythroid 2-related factor 2(Nrf2)is thought to be an essential transcription factor for defending against oxidative stress.When cells are exposed to oxidative stress,Nrf2 translocates from the cytoplasm to the nucleus and activates the expression of a series of antioxidant protein genes,such as NAD(P)H dehy-drogenase,quinone 1(NQO1),super-oxide dismutase(SOD),glutathione(GSH).Meanwhile,the activation of Nrf2 can reduce fatty acid oxidation and lipogenesis and inhibit the production of inflammatory factors.Therefore,Nrf2 activation inhibits oxidative stress in hepatocytes and may contribute to alleviate the onset and progression of NASH.Exosomes are membranous vesicles with diameters of approximately40-160nm that are secreted from the cells via the paracrine pathway.Exosomes carry the same bioactive components and can communicate information between cells.Mesenchymal stem cells exosomes(MSCs-EXO)exhibit the advantages of high safety,low immunogenicity,and non-tumorigenicity,and perform the functions of inflammation suppression,immune modulation,and tissue restoration.MSCs-EXO derived from different tissues have played therapeutic roles in NAFLD/NASH,hepatic ischemia-reperfusion injury,liver failure,acute liver injury,liver fibrosis by inhibiting inflammation,anti-oxidative stress,immune regulation and anti-apoptosis.It has been shown that different sources of MSCs-EXO regulated Nrf2-associated signaling pathways to inhibit oxidative stress in skin damage,osteoporosis,and cognitive impairment.However,it is unclear whether exosomes derived from human umbilical cord mesenchymal stem cells(h UC-MSCs)could be useful in treating NASH by inhibiting oxidative stress.In addition,the potential mechanism of MSCs-EXO in regulating oxidative stress in NASH is also uncertain.In this experiment,two NASH mice models were established using high fat and high cholesterol diet(HFHC)and methionine-choline deficiency diet(MCD).To develop two steatosis cell models,Hep G2 cells were cultured with palmitic acid(PA)and methionine-choline deficient medium was used to culture AML12 cells.The models were given h UC-MSCs exosomes and we observed the effects of h UC-MSCs exosomes on hepatocellular injury,inflammatory response,lipid metabolism,oxidative stress,etc.Meanwhile,the Nrf2-specific blocker ML385 was applied to study the role of Nrf2 pathway in NASH treated by h UC-MSCs exosomes.This experiment includes the following four parts:Part one:Human umbilical cord-derived mesenchymal stem cell exosomes reduced hepatic steatosis in NASHObjective:We extracted h UC-MSCs exosomes and explored the effects of h UC-MSCs exosomes on on steatosis of NASH in vivo and in vitro.Methods:1)Exosomes were extracted from the culture supernatants of human umbilical cord-derived mesenchymal stem cells by ultrafiltration combined with chemical precipitation.The morphology,size and surface markers of exosomes were observed by transmission electron microscopy,particle size analysis and Western blot.2)Animal part:HFHC diet and MCD diet were used to establish two NASH animal models,which were randomly divided into:Control group,HFHC group,HFHC-EXO group;MCS group,MCD group,MCD-EXO group.After the treatment of h UC-MSCs exosomes by tail vein injection,the color and morphology of liver were observed,and lipid droplet deposition in liver cells were determined by oil red"O"staining.The triglyceride(TG)in liver tissue was measured by GPO-PAP enzymatic method.The expressions of SREBP-1c,PPAR-α,Fabp5,CPT-1α,ACOXs and FAS were examined by Western blot and Real-time q PCR.3)Cell part:two adipocyte models were prepared with Hep G2 cells intervened by PA and AML12 cells cultured with MCD medium,then divided into:Control group,PA group,EXO100μg/ml,EXO200μg/ml;Control group,MCD group,EXO100μg/ml,EXO200μg/ml.Nile red staining was used to detect lipid deposition in cells under confocal microscopy,and the TG contents in cells were detected by GPO-PAP enzyme assay.Results:1)The exosomes of h UC-MSCs were disk-shaped vesicles under transmission electron microscope.The average diameter of exosomes was about 72.22nm.Western blot showed that the exosomes of h UC-MSCs expressed characteristic surface markers CD9,CD63 and TSG101,but did not express Calnexin.2)NASH mouse models induced by HFHC diet and MCD diet were successfully established.3)In NASH models,the livers of mice in the HFHC and MCD groups were grayish yellow and greasy.After treatment with h UC-MSCs exosomes,the livers of mice were redder and greasier,and the greasiness was significantly reduced.Compared with Control and MCS groups,the livers of mice in HFHC and MCD groups showed significant lipid deposition,which were significantly reduced by h UC-MSCs exosome treatment,the oil red"O"positive area were also lowered(P<0.05).4)Compared with Control group and MCS group,the hepatic TG content in HFHC group and MCD group were significantly higher(P<0.05).After treatment,the hepatic TG content in HFHC-EXO group and MCD-EXO group were significantly lower than those in HFHC group and MCD group(P<0.05).5)Exosomes of h UC-MSCs decreased the levels of SREBP-1c protein and FAS m RNA in liver tissues of NASH mice and increased the expression of PPAR-αprotein and FABp5,CPT1αand ACOX m RNA in liver tissues(P<0.05).6)h UC-MSCs exosomes could attenuate lipid deposition in steatotic cells with Nile red staining in vitro for further confirmation.Also,h UC-MSCs exosomes reduced the TG content of steatotic cells in the EXO100μg/ml and EXO200μg/ml groups compared with PA and MCD groups(P<0.05).Part two:Human umbilical cord-derived mesenchymal stem cell exosomes alleviated liver inflammatory injury in NASHObjective:To investigate the effect of human umbilical cord-derived MSCs-EXO on liver inflammatory injury in NASH mice.Methods:1)Two NASH mouse models were established with HFHC diet and MCD diet,then randomly divided into:Control group,HFHC group,HFHC-EXO group;MCS group,MCD group,MCD-EXO group.h UC-MSCs exosomes was administered by tail vein injection.2)The pathological changes of liver tissue were determined by H&E,and the NAS score was calculated.3)Serum ALT was measured by biochemical method.4)The levels of TNF-αand IL-6 in liver and serum were detected by Real-time q PCR and ELISA.5)Flow cytometry and Real-time q PCR were used to detect the changes of macrophage F4/80,CD11c and CD206 in liver tissue.Results:1)Compared with Control and MCS groups,the livers of mice in HFHC and MCD groups showed significant steatosis,hepatocyte swelling and inflammatory cell infiltration,which were significantly reduced by h UC-MSCs exosome treatment,the NAS score were also lowered(P<0.05).2)Compared with Control group and MCS group,the serum ALT level in HFHC group and MCD group were significantly higher(P<0.05).After treatment,the serum ALT content in HFHC-EXO group and MCD-EXO group were significantly lower than those in HFHC group and MCD group(P<0.05).3)Compared with Control and MCS groups,the serum levels and hepatic m RNA expression of TNF-αand IL-6 were significantly higher in mice of HFHC and MCD groups(P<0.05),while h UC-MSCs exosomes decreased the levels of TNF-αand IL-6 in serum and reduced the liver m RNA expression of TNF-αand IL-6 in NASH mice(P<0.05).4)Compared with HFHC group and MCD group,exosomes of h UC-MSCs significantly reduced the expression of F4/80~+macrophages and M1 macrophage marker CD11c~+,and increased the proportion of M2 macrophage marker CD206~+in liver tissue of NASH mice in HFHC-EXO and MCD-EXO groups(P<0.05).Real-time q PCR showed that h UC-MSCs exosomes also significantly decreased the expression of CD11c m RNA and increased the expression of CD206 m RNA in the liver of NASH mice(P<0.05).Part three:Human umbilical cord-derived mesenchymal stem cell exosomes inhibited oxidative stress in NASHObjective:To investigate the effects of h UC-MSCs exosomes on oxidative stress in NASH in vivo and in vitro.Methods:1)Animal models and drug interventions are the same as Part I.The expression of CYP2E1 protein and m RNA in the liver tissues of the mice was measured by Western blot and Real-time q PCR.Liver GSH was analyzed by microplate method,the content of malondialdehyde(MDA)of liver was measured by thiobarbituric acid method,and the activity of hepatic SOD was measured by WST-1 method.2)Cellular models and drug interventions were performed as in part I.Cellular ROS were observed with DHE fluorescent probe staining under fluorescence microscopy.Results:1)Compared with Control and MCS groups,liver SOD activity and GSH levels of mice in HFHC and MCD groups decreased(P<0.05).but h UC-MSCs exosomes significantly increased the liver SOD activity and GSH levels of NASH mice after the intervention(P<0.05).2)Compared with HFHC and MCD groups,hepatic MDA levels,the expression of liver CYP2E1 protein and m RNA were significantly lower in HFHC-EXO and MCD-EXO groups of NASH mice(P<0.05).3)In vitro,ROS were significantly increased in Hep G2 cells intervened by PA and AML12 cells cultured in MCD medium compared with Control group(P<0.05),but ROS levels were reduced in EXO100μg/ml and EXO200μg/ml groups compared with PA and MCD groups(P<0.05).Part four:Human umbilical cord-derived mesenchymal stem cell exosomes activated the Nrf2/NQO-1 pathway to inhibit oxidative stress for ameliorating NASH liver injuryObjective:To understand the effects of h UC-MSCs exosomes on the Nrf2/NQO-1 pathway in vivo and in vitro,Nrf2-specific blocker ML385 was applied to verify whether h UC-MSCs exosomes exert anti-oxidative stress effects through the Nrf2/NQO-1 pathway.Methods:1)Differential genes in liver tissues of NASH patients and healthy controls were analyzed by bioinformatics,and gene pathway enrichment analysis was performed.2)The animal models and drug interventions were the same as in Part I.The protein expression of p-Nrf2,Nrf2,NQO-1,p-AMPK and AMPK was detected by Western blot in liver tissues of each group.3)The cell models and drug interventions were the same as in the first part,and the protein expression of p-Nrf2,Nrf2,NQO-1,p-AMPK,and AMPK in each group of cells was detected by Western blot.4)PA was applied to Hep G2 cells and AML12 cells were cultured with MCD medium to induce two adipocyte models.h UC-MSCs exosome was administered after pretreatment with Nrf2-specific blocker ML385 for 3 h.The groups were divided as follows:Control group,PA group,PA+EXO200μg/ml group,PA+EXO200μg/ml+ML385 ML385 group;Control group,MCD group,MCD+EXO200μg/ml group,MCD+EXO200μg/ml+ML385 group,cellular ROS were observed using DHE fluorescent probe staining under fluorescence microscope,and the expression of p-Nrf2,Nrf2,NQO-1 in the cells of each group was detected by Western blot.Results:1)Bioinformatics analysis showed that Nrf2-related pathways were down-regulated in liver tissues of NASH patients.2)Compared with Control and MCS groups,the protein ratios of liver p-Nrf2/Nrf2,p-AMPK/AMPK and the protein expression of NQO-1 were decreased by Western blot in HFHC and MCD groups(P<0.05).3)In cellular experiments,Western blot results showed that the protein expression of cellular p-Nrf2,p-AMPK,and NQO-1was reduced in PA and MCD groups compared with Control groups.After intervened with h UC-MSCs exosome,the protein expression of p-Nrf2,p-AMPK,and NQO-1 was increased in a dose-dependent manner in the cells.Compared with MCD+EXO200μg/ml group,h UC-MSCs exosomes were unable to up-regulate the protein expression of p-Nrf2 and NQO-1 in steatotic cells and failed to reduce the level of ROS in steatotic cells which were pretreated with ML385(P<0.05).Conclusions:1.Human umbilical cord-derived MSCs-EXO could regulate genes related to hepatic lipid metabolism and reduce lipid deposition in hepatocytes of NASH mice;promote macrophage M2 polarization,inhibit the release of inflammatory factors and alleviate inflammatory damage in the hepatocytes.2.Human umbilical cord-derived MSCs-EXO could inhibit hepatic oxidative stress and improve hepatic antioxidant capacity in NASH mice.3.We demonstrated that human umbilical cord-derived MSCs-EXO could promote the phosphorylation of Nrf2,upregulate the protein expression of NQO-1,which is a target gene of Nrf2,then inhibit the oxidative stress and alleviate the liver injury in NASH mice. |
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