The Effect Of Mesenchymal Stem Cell-conditioned Medium On Nonalcoholic Fatty Liver Disease Through SIRT1

BackgroundDiabetes is a recognized chronic metabolic disorder syndrome,which is classified as a disease characterized by chronic hyperglycemia.Diabetes is one of the biggest epidemics in the world in both developed and developing countries.At present,there are about 463 million sick people in the world.According to the latest estimation of the International Diabetes Federation(IDF),this number will rise to 642 million by 2040.The increasing prevalence rate makes diabetes one of the most severe public health challenges in the 21st century.90%of diabetic patients are classified as type 2 diabetes mellitus(T2DM),which is the main cause of the global epidemic of diabetes.The liver plays an important role in the regulation of glucose metabolism,and its main function is to keep blood sugar constant.Non-alcoholic fatty liver disease(NAFLD)is defined as a disease with excessive liver fat caused by non-alcoholic causes.NAFLD consists of benign non-alcoholic fatty liver disease(NAFL)and severe non-alcoholic steatohepatitis(NASH).Obesity,insulin resistance,and diabetes are all closely related to excessive lipid accumulation in the liver parenchyma.In the past few decades,the prevalence of NAFLD has risen sharply.Studies have shown that NAFLD has a strong correlation with diabetes risk,and up to 70%of diabetic patients have NAFLD.NASH is related to overweight and metabolic syndrome.The latest findings in 22 countries show that there is obesity(>80%),dyslipidemia(72%),and type 2 diabetes mellitus(44%)in NASH patients,suggesting that NASH is closely related to metabolic syndrome.Most of the increase in the prevalence of fatty liver is due to its association with the pathophysiology of T2DM and obesity.Recent studies have extensively described the relationship between mitochondrial function and NAFLD.Studies have shown that mitochondrial dysfunction(especially oxidative respiratory chain defects)plays an important role in the occurrence and progression of NAFLD diseases.Oxidative stress is the key to the pathogenesis of NAFLD,and its main feature is the production of reactive oxygen species(ROS).In NAFLD,mitochondrial dysfunction is the main source of ROS.Oxidative respiratory chain damage and lipid peroxidation caused by mitochondrial dysfunction will further increase the production of ROS,which in turn aggravates lipid peroxidation and releases highly reactive aldehydes(such as malondialdehyde)which have various harmful effects on hepatocytes and other cells,further forming a vicious circle and possibly destroying mitochondrial proteins and mitochondrial DNA.Therefore,mitochondrial dysfunction may aggravate the severity of NAFLD.Therefore,improving mitochondrial function has become a potential treatment to reduce NAFLD.In the past decade,mesenchymal stem cells(MSCs)have become the most potential resources for cell transplantation because of their immunomodulation,paracrine,and dedifferentiation.Extensive studies have shown that umbilical cord mesenchymal stem cells can reduce the degree of liver fibrosis in rats and improve NAFLD in obese type 2 diabetic mice.It highlights their ability to reduce liver cell damage.However,the known MSCs have their limitations,including the inability to effectively reside in target organs,the lack of effective utilization,the low survival rate in vivo,and the potential tumorigenicity of infused MSCs,which may hinder the development of stem cell therapy.However,the cell-free mesenchymal stem cell-conditioned medium(MSC-CM)is rich in nutrients,such as growth factors and cytokines,and is easy to obtain,which makes the application of MSC-CM another treatment option.The related research based on MSC-CM shows that MSC-CM can improve the endothelial dysfunction of diabetes and reduce renal fibrosis.However,the potential protective effect of MSC-CM on diabetes mellitus complicated with NAFLD,and its exact mechanism are still unclear.Sirtuin 1(SIRT1),the first member of the class Ⅲ histone deacetylase family,is an NAD+-dependent enzyme,which is widely involved in gene regulation,genome stability maintenance,apoptosis,autophagy,aging,proliferation,aging,and tumorigenesis.SIRT1 can mediate the deacetylation of downstream target proteins such as peroxisome proliferator-activated receptor γ coactivator 1α(PGC1α),thus participating in fatty acid oxidation,mitochondrial biosynthesis,and function.Up-regulation of SIRT1 can improve NAFLD,obesity,and insulin resistance in rodents.On the contrary,down-regulation or complete silence of SIRT1 will aggravate fatty liver and liver inflammation.The expression level of SIRT1 was decreased in plasma and liver of obese and NAFLD patients.PurposeThe purpose of this study is to explore the effect of MSC-CM on type 2 diabetes mellitus complicated with nonalcoholic fatty liver disease and to further clarify the relationship among MSC-CM,NAFLD,and SIRT1.Methods1.Isolation and identification of Hu-MSCs:extracting Hu-MSCs from the fresh umbilical cord after cesarean section,fusing the second or third generation to 70-80%MSCS for flow cytometry analysis,and detecting the expression of surface markers CD34,CD73,CD 105 and HLA-DR;After 1-2 weeks of adipogenesis induction,the formation of lipid droplets in Hu-MSCs was observed under oil red O staining microscope After 2-3 weeks of osteogenesis induction,the formation of calcium nodules in Hu-MSCs was observed by alizarin red staining.2.MSC-CM was prepared from the supernatant of Hu-MSCs.Hu-MSCs were inoculated into the culture flask.When MSCs reached 70-80%fusion,the culture medium was changed to serum-free medium and the culture was continued for 24 hours.Then,the supernatant was collected and ultrafiltered with a centrifugal filter to produce MSC-CM,which was filtered with a 0.22μm filter and stored in a refrigerator at-80℃ for use.3.The model of NAFLD in T2DM mice was induced by high-fat diet and STZ injection.Mice in the T2DM group were randomly divided into two groups,which were infused with PBS or MSC-CM via tail vein respectively,and mice in the normal diet group were infused with PBS as control,and the changes of body weight and blood glucose of mice were detected every week.4.After the intervention of PBS and MSC-CM,abdominal glucose tolerance test(IPGTT)and abdominal insulin tolerance test(IPITT)were performed,and the changes of body fat content in mice were detected.5.Western blot detection of MSC-CM intervention on the expression levels of SIRT1 and PGC1α in the liver of type 2 diabetic mice,maintaining mitochondrial biogenesis and function-related factors(NRF1 and TFAM),inflammatory factors(TNF-α,IL-1β and IL-6))And the expression of apoptosis-related indicators(BAX,Bcl-2,cleaved caspase3,caspase3,cleaved PARP,PARP).HE staining to observe changes in liver tissue structure,PAS staining to detect glycogen accumulation,oil red O staining to detect lipid deposition,TUNEL staining to detect liver cell apoptosis,kit to detect liver cell total antioxidant capacity,ELISA method to detect serum Medium liver function level(ALT and AST)and blood lipid level(TG and TC).6.Effects of MSC-CM on PA-induced steatohepatitis in L-O2 cells:L-O2 cells were stimulated with 0.4 mmol/L PA for 48 h,and MSC-CM was added at the same time as PA stimulation.The expressions of SIRT1 and PGCla in cells were detected by Western blot,and the expressions of mitochondrial biosynthesis and functional related factors(NRF1 and TFAM),inflammatory factors(TNF-α,IL-1β,and IL-6),and apoptosis-related indicators(BAX,Bcl-2,cleaved caspase3,caspase3,cleaved PARP,PARP)were maintained.ROS content in cells was detected by DCFH-DA fluorescent probe,apoptosis of L-O2 cells was detected by TUNEL staining,and total antioxidant capacity of L-O2 cells was detected by the kit.7.Discussion on the mechanism of action of MSC-CM:To verify whether SIRT1 plays a key role in the protective effect of MSC-CM on L-O2 after PA stimulation.SIRT1 of L-O2 cells was knocked down with small interfering RNA(siRNA)and then stimulated with 0.4 mmol/L PA or PA combined with MSC-CM for 48 h.The expression of SIRT1 and PGC1α in cells was detected by Western blot,and the expressions of mitochondrial biosynthesis and functional related factors(NRF1 and TFAM),inflammatory factors(TNF-α,IL-1β,and IL-6),and apoptosis-related indicators(BAX,Bcl-2,cleaved caspase3,caspase3,cleaved PARP,PARP)were maintained.The ROS content in cells was detected by DCFH-DA fluorescent probe,and the apoptosis of L-O2 cells was detected by TUNEL staining.The total antioxidant capacity of L-O2 cells was determined by the kit.Results1.The shape of the isolated HU-MSCs was arranged in a long fusiform spiral shape under a microscope.The expression of CD 105 and CD73 on the cell surface was positive,while the expression of CD34 and HLA-DR was negative,which was consistent with the surface marker characteristics of umbilical cord mesenchymal stem cells.After induction of directed differentiation,the cells differentiated into osteocytes containing calcium nodules and adipocytes containing a large number of lipid droplets.2.A mouse model of T2DM complicated with NAFLD was established by combining high-fat diet with STZ injection.The results showed that compared with the T2DM group,mice with MSC-CM intervention had relatively less body weight and lower body fat content.IPGTT and IPITT experiments suggested that MSC-CM intervention significantly improved impaired glucose tolerance and insulin sensitivity in T2DM mice.3.Western blot results showed that the protein levels of SIRT1,PGC1α,NRF1 and TFAM were increased,the protein levels of inflammatory factors(TNF-α,EL-1βand IL-6)were decreased,and the apoptotic indexes of liver cells(BAX/Bcl-2,c-cas3/cas3,c-PARP/PARP)were decreased after MSC-CM treatment compared with those in the T2DM group.TUNEL staining also showed that MSC-CM improved the apoptosis of liver cells in T2DM mice,and MSC-CM also increased the total antioxidant capacity of the liver.HE staining showed that MSC-CM treatment improved the disordered liver structure in T2DM mice and reduced the area of hepatic steatosis.Glycogen staining showed that MSC-CM increased glycogen accumulation.Oil red O staining showed that MSC-CM could reduce lipid deposition.ELISA results showed that serum indexes of impaired liver function(ALT and AST)and blood lipid levels(TC and TG)were significantly increased in T2DM mice,while liver function and blood lipid levels were improved after MSC-CM intervention.4.The use of palmitic acid(PA)in vitro stimulation L-O2 cell-induced adipose sex hepatitis model,the experimental results show that the PA can reduce L-O2 cell SIRT1,PGC1α,NRF1,TFAM protein levels,increase inflammation index(TNF-α,IL-1β and IL-6)and apoptosis index(BAX/Bcl-2,c-cas3/cas3,c-PARP/PARP),TUNEL staining showed a significant increase in apoptosis L-O2 cell,cell ROS produced increases and total antioxidant capacity reduces.After treatment with MSC-CM,the protein levels of SIRT1,PGC1α,NRF1 and TFAM in L-O2 cells were increased,inflammatory indexes(TNF-α,IL-1β and IL-6)and apoptotic indexes(BAX/Bcl-2,c-cas3/cas3,c-PARP/PARP)were decreased,TUNEL staining showed that the apoptotic L-O2 cells were significantly decreased,ROS production was decreased and total antioxidant capacity was enhanced.5.After siRT1 siRNA transfection,SIRT1 expression in L-O2 cells was silenced,and the positive therapeutic effect of MSC-CM was partially or completely offset.After SIRT1 knockdown,MSC-CM could not increase the protein levels of PGC1α,NRF1 and TFAM.Inflammation indexes(TNF-α,IL-1β and IL-6)and apoptosis indexes(BAX/Bcl-2,c-cas3/cas3,c-PARP/PARP)were increased,TUNEL staining showed that L-O2 cell apoptosis could not be reduced,ROS production and total antioxidant capacity of cells could not be improved.ConclusionMSC-CM can improve glucose tolerance and insulin sensitivity,improve impaired liver function and reduce lipid levels in T2DM mice.In both in vivo and in vitro,MSC-CM can improve the impaired mitochondrial function,reduce inflammatory cytokines and apoptosis,and increase the total antioxidant capacity of liver cells in non-alcoholic steatohepatitis.The positive therapeutic effect of MSC-CM depended on the expression of SIRT1,and the therapeutic effect of MSC-CM was partially or completely offset after silencing SIRT1.