| Diabetes mellitus(DM)is a metabolic disorder characterized by high blood glucose levels resulting directly from insulin resistance,insufficient insulin secretion,or excessive glucagon secretion.According to the 10th edition of the Diabetes Atlas published by the International Diabetes Federation(IDF),approximately 783 million adults aged 20-79 worldwide will have DM by 2045.Currently,anti diabetic medications have been widely accepted and utilized,but their application in the field of diabetes treatment is limited by side effects such as diarrhea,nausea,vomiting,and anemia,highlighting the urgent need for the development of novel therapeutic approaches.The discovery of stem cells has brought hope for chronic diseases,including DM.Mesenchymal stem cells(MSCs),as multipotent stromal cells,possess advantages such as multidirectional differentiation potential,high self-renewal capacity,immunomodulation,and low immunogenicity,making them widely applicable in various cell therapies.Among them,human umbilical cord-mesenchymal stem cells(HUC-MSCs)have unique advantages in DM treatment due to their abundant source,minimal ethical controversy,low risk of infection,strong proliferation and differentiation abilities,and extremely low immunogenicity.Although regenerative medicine centered on MSC therapy may become a new strategy for DM treatment beyond traditional therapies,the complex mechanisms of action of MSCs and the pathogenesis of DM necessitate further research to determine the efficacy of HUC-MSC transplantation.To enhance the survival rate of transplanted cells,various methods have been explored,including genetic engineering,tissue engineering,and pretreatment of cells with biological factors such as growth factors,drugs,and hypoxia.Among these methods,hypoxic preconditioning is considered one of the most effective ways to activate stem cells.In order to improve the therapeutic efficacy of stem cells in tissue repair,research has shown that MSCs cultured under standard conditions need to adapt from 21%oxygen in the culture dish to less than 1%oxygen in ischemic tissues,which can induce the expression of hypoxia inducible factor-1α(HIF-1α).HIF-1α is the main transcriptional regulator that tissues and cells use to respond to hypoxia,serving as the primary responsive protein to hypoxic stimuli in the body.By regulating oxygen uptake and transport,HIF-1α mediates cellular responses to oxygen levels in the body,maintaining oxygen homeostasis and enhancing the adaptive response of stem cells to hypoxia,thereby exerting a protective effect.Mitochondria,as the primary consumers of oxygen in cells,are severely affected by reduced oxygen supply.Under hypoxic conditions,cells undergo crucial adaptive responses.Mitochondria regulate the survival and growth of stem cells by influencing the generation of adenosine triphosphate(ATP)and reactive oxygen species(ROS),the tricarboxylic acid(TCA)cycle,the NAD+/NADH ratio,the glyoxylate cycle,and mitochondrial dynamics.Approximately 99%of the human mitochondrial proteome is encoded by nuclear genes,with the remaining 1%encoded by mitochondrial genes,indicating that cross-talk between mitochondria and the nucleus is one of the major responses of stem cells to stress,including hypoxia.The mitochondrial unfolded protein response(UPRmt)is a mitochondrial protein quality control mechanism that helps transmit signals from mitochondria to the nucleus to maintain mitochondrial protein homeostasis and cell survival,thus playing a critical role in maintaining cellular homeostasis.UPRmt can be activated by endogenous stress(protein homeostasis imbalance,mitochondrial DNA(mtDNA)damage,inhibition of mitochondrial protein synthesis,and inhibition of oxidative phosphorylation(OXPHOS))and exogenous stress(hypoxia,pathogen infection,and exogenous substances).In stressful conditions,the accumulation of unfolded or misfolded proteins within mammalian mitochondria can upregulate the expression of activating transcription factor 4(ATF4),C/EBP-homologous protein(CHOP),and activating transcription factor 5(ATF5).ATF4 enters the nucleus and,together with CHOP,binds to the ATF5 promoter,thereby activating ATF5 transcription.ATF5,by upregulating the expression of heat shock protein 60(HSP60),heat shock protein 10(HSP10),and lon peptidase 1(LONP1),initiates a transcriptional program to maintain mitochondrial protein homeostasis,improve mitochondrial function,enhance mitochondrial adaptability to stress,and increase cell survival rates.Therefore,from the perspective of the UPRmt,the potential mechanisms underlying the activation of stem cell function under hypoxia can be further elucidated.HSP60,as a molecular chaperone protein,can form symmetric functional heterodimers with its co-chaperone protein HSP10,enhancing protein folding in an ATP-dependent manner to maintain mitochondrial protein homeostasis.Studies have found that the function of HSP60 is dependent on its subcellular localization within the cytoplasm or mitochondria and the cell type.In mitochondria,HSP60 assists in the folding and transport of other proteins and exhibits anti-apoptotic functions,while in the cytoplasm,it has dual functions of both anti-apoptosis and pro-apoptosis.Mitochondrial OXPHOS is central to cellular metabolism and is crucial for energy production in eukaryotes.It relies on the mitochondrial respiratory chain composed of five mitochondrial enzyme complexes and two mobile electron carriers.Complex Iserves as the starting point of the electron transport chain(ETC)and is the largest(970 kDa)multi-subunit complex,playing a pivotal role throughout the OXPHOS process.Additionally,research suggests that Complex I may be associated with various integrated stress responses mediated by mitochondria.Therefore,by further exploring the relationship between HSP60’s coordination of mitochondrial Complex I and mitochondrial protein homeostasis and function,new research directions may be provided for enhancing the cellular activity of HUC-MSCs.Therefore,in-depth investigation from a mitochondrial perspective into the effects of pre-culturing HUC-MSCs under hypoxic conditions(1%O2)and changes in their mitochondrial function can elucidate how hypoxic preconditioning may serve as a novel strategy for improving Type 2 diabetes mellitus(T2DM)cell therapy with HUC-MSCs.Methods:1.Hypoxia-induced activation of hypoxia-inducible factor 1-alpha(HIF-1α)promotes the mitochondrial unfolded protein response(UPRmt)and enhances the cellular activity of HUC-MSCs(1)To assess the impact of hypoxia on the cellular activity of HDUC-MSCs,HUC-MSCs were treated with 1%O2 for 0h,3h,6h,and 12h.Cellular activity was measured using the MTT assay.Additionally,changes in the protein levels of HIF-1αand mRNA levels were evaluated through Western blotting and qPCR,respectively.(2)To investigate the effect of hypoxia on the mitochondrial function of HUC-MSCs,HUC-MSCs were exposed to 1%O2 for 0h,3h,6h,and 12h.The ATP level,ROS level,and changes in mitochondrial membrane potential were assessed using ATP and ROS assay kits,as well as JC-1 staining,respectively.(3)To assess the impact of hypoxia on the UPRmt in HUC-MSCs,HUC-MSCs were exposed to 1%O2 for 0h and 12h.The mitochondrial proteome was analyzed using mitochondrial proteomics to detect changes in KEGG functional classification and expression of proteins related to mitochondrial protein homeostasis.Additionally,the protein levels and mRNA expression of ATF4,CHOP,ATF5,LONP1,and HSP60 were evaluated using Western blotting and qPCR,respectively.(4)To investigate the impact of hypoxia on the mitochondrial respiratory chain complexes in HUC-MSCs,HUC-MSCs were exposed to 1%O2 for 0h and 12h.The expression changes of most subunits of mitochondrial respiratory chain complexes Ⅰ-Ⅴwere assessed using mitochondrial proteomics.To validate the improvement of mitochondrial function in HUC-MSCs during hypoxic preconditioning mediated by complex I,we conducted an experiment using the complex I inhibitor,metformin,in conjunction with hypoxic preconditioning on HUC-MSCs.2.Hypoxia activates HSP60 to coordinate complex I,promoting the viability of HUC-MSCs(1)To assess the impact of metformin under hypoxia on the viability of HUC-MSCs,we treated the cells with different concentrations of metformin for 24 hours and measured cell viability using the MTT assay.Additionally,we treated HUC-MSCs with 100μM of metformin for 24 hours,simultaneously exposing them to 1%O2 for 0,3,6,and 12 hours.Cell viability was determined using the MTT assay.(2)To assess the impact of metformin under hypoxia on the mitochondrial function of HUC-MSCs,we treated the cells with 100μM of metformin for 24 hours,concurrently exposing them to 1%O2 for 12 hours.We evaluated the ATP levels,ROS levels,and changes in mitochondrial membrane potential using ATP and ROS assay kits,as well as JC-1 staining.(3)Detecting the effect of metformin under hypoxia on the unfolded protein response of HUC-MSCs mitochondria.HUC-MSCs were treated with 100μM of metformin for 24 hours,concurrently exposed to 1%O2 for 12 hours.Mitochondrial proteomics was employed to investigate changes in KEGG functional classification and mitochondrial protein homeostasis-related protein expression.Western blot analysis was conducted to measure the expression levels of ATF4,CHOP,ATF5,LONP1,and HSP60 proteins.Additionally,Western blot was utilized to detect the expression of HSP60 protein in mitochondria.(4)Detecting the impact of metformin under hypoxia on the mitochondrial respiratory chain complexes of HUC-MSCs.HUC-MSCs were treated with 100μM of metformin for 24 hours,concurrently exposed to 1%O2 for 12 hours.Mitochondrial proteomics was utilized to investigate changes in the expression of most subunits of respiratory chain complexes Ⅰ-Ⅴ in HUC-MSCs mitochondria.Western blot was employed to validate the expression of respiratory chain complexes.(5)Assessing the effect of HSP60 knockdown on HUC-MSCs mitochondrial function.Transfection efficiency of HSP60-siRNA was evaluated through qPCR and Western blot.ATP and ROS assay kits,along with JC-1 staining,were used to measure ATP levels,ROS levels,and changes in mitochondrial membrane potential in HUC-MSCs after HSP60 knockdown.(6)MTT assay was performed to detect changes in cell viability of HUC-MSCs after HSP60 knockdown.HSP60-siRNA was transfected into HUC-MSCs using transfection reagents,and cell viability was measured using MTT assay.3.Preconditioning with hypoxia enhances the therapeutic effects of HUC-MSCs on T2DM mice(1)Establishment of the T2DM mouse model:Male C57BL/6N mice aged 6-8 weeks,weighing 18-20g,were randomly divided into a control diet group(CD)and a high-fat diet group(HFD).Mice in the HFD group were fed a high-fat diet for 12 weeks,followed by intraperitoneal injection of streptozotocin(STZ)at a dose of 50mg/kg for 5 consecutive days.Starting from the 3rd day after injection,random blood glucose levels were measured every other day,and on the 7th day,oral glucose tolerance test(OGTT)and intraperitoneal insulin tolerance test(IPITT)were performed.Mice with random blood glucose levels exceeding 16.7mmol/L for three times and impaired glucose and insulin tolerance in OGTT and IPITT were considered to have successfully developed T2DM.(2)Experimental grouping and administration:After successful modeling of T2DM mice,they were randomly divided into 3 groups(5 mice per group),ensuring no significant differences in body weight and blood glucose between groups.The groups included the T2DM group(DM),the HUC-MSCs treatment group(DM+Nor),and the 1%hypoxia preconditioned HUC-MSCs treatment group(DM+Hyp).To investigate whether HUC-MSCs had any serious effects on healthy mice,we compared them with mice in the control diet group,which were randomly divided into 3 groups(5 mice per group)with no significant differences in body weight and blood glucose between groups.These groups included the healthy control group(Con),the HUC-MSCs treatment group(Con+Nor),and the 1%hypoxia preconditioned HUC-MSCs treatment group(Con+Hyp).HUC-MSCs were transplanted into mice in the DM+Nor group and Con+Nor group via tail vein injection,while 1%hypoxia preconditioned HUC-MSCs were transplanted into mice in the DM+Hyp group and Con+Hyp group.PBS was injected into mice in the Con group and DM group.HUC-MSCs were suspended in PBS at a dose of 0.2 ml/mouse,with 1×106 HUC-MSCs/mouse,administered every 10 days for a total of 3 times.(3)After receiving 3 doses of HUC-MSCs treatment,mice were euthanized,and blood and tissue samples were collected and processed.Fresh pancreas,liver,and de-encapsulated kidney tissues were harvested from mice,fixed in formalin overnight,embedded in paraffin,and sectioned for HE staining and PAS staining.Immunofluorescence staining was performed on pancreatic tissues using specific antibodies,and pathological morphology was observed under a microscope.Results:1.Low oxygen induces HIF-la activation and promotes UPRmt to enhance the activity of HUC-MSCs(1)MTT results show:With the extension of the treatment time under 1%O2 concentration,the activity of HUC-MSCs cells shows an increasing trend.At 12 hours of treatment with 1%O2 concentration,the activity of HUC-MSCs cells is significantly higher compared to the control group.At 12 hours of treatment with 1%O2 concentration,the levels of HIF-1α protein and mRNA in HUC-MSCs are significantly higher compared to the control group.(2)Mitochondrial function detection results show:After 12 hours of treatment with 1%O2 concentration,there is a significant increase in mitochondrial membrane potential,an increase in intracellular ROS levels,and a decrease in ATP content in HUC-MSCs.(3)Proteomic analysis results show:After 12 hours of treatment with 1%O2 concentration,KEGG functional classification indicates an upregulation of protein expression related to folding,sorting,and degradation functions;expression of proteins related to HUC-MSCs mitochondrial unfolded protein response and the mitochondrial inner membrane TIM system is upregulated;expression of most subunit proteins of mitochondrial respiratory chain complexes Ⅰ-Ⅴ is upregulated.Further validation by Western blot and qPCR shows an increase in transcription and translation of ATF4 mRNA,CHOP mRNA,ATF5 mRNA,LONP1 mRNA,and HSP60 mRNA.This indicates that low oxygen treatment can promote UPRmt in HUC-MSCs,maintain mitochondrial protein homeostasis,promote upregulation of mitochondrial respiratory chain subunits expression,and establish a novel energy metabolism pattern.2.Low oxygen activates HSP60 to coordinate complex I,promoting the cellular activity of HUC-MSCs(1)MTT results showed that after 24 hours of treatment with 100 μM metformin combined with 12 hours of 1%O2,the cell viability of HUC-MSCs was significantly increased.(2)Mitochondrial function detection results indicated that after 24 hours of treatment with 100μM metformin combined with 12 hours of 1%O2,the mitochondrial membrane potential and intracellular ATP content of HUC-MSCs increased,while the ROS level decreased.(3)Proteomic analysis results demonstrated that after 24 hours of treatment with 100 μM metformin combined with 12 hours of 1%O2,KEGG functional classification revealed upregulation of proteins involved in folding,sorting,and degradation.Proteins related to mitochondrial unfolded protein response and the TIM system in the mitochondrial inner membrane were upregulated,as well as most subunit proteins of mitochondrial respiratory chain complexes Ⅰ-Ⅴ.Western blot validation further confirmed a significant increase in HSP60 protein expression compared to the control group.Additionally,Western blot validation of respiratory chain complexes showed increased expression of subunit proteins in HUC-MSCs.Protein-protein interaction analysis of respiratory chain complexes and the HIF-1α-HSP60 pathway indicated that they do not act independently.HIF-1α promotes the expression of mitochondrial respiratory chain complex subunit proteins by upregulating HSP60,thereby improving mitochondrial function and promoting the cellular activity of HUC-MSCs.(4)Knockdown of HSP60 resulted in decreased mitochondrial membrane potential,decreased intracellular ATP content,and increased ROS levels in HUC-MSCs.MTT results showed that knockdown of HSP60 significantly reduced the cellular activity of HUC-MSCs.3.Therapeutic Effects of Hypoxia-Preconditioned HUC-MSCs on T2DM Mice(1)After successful modeling of T2DM in mice,it was found that the infusion of HUC-MSCs or hypoxia-preconditioned HUC-MSCs at 1%oxygen significantly improved the blood glucose homeostasis and insulin resistance in T2DM mice.Particularly,the therapeutic effect was more pronounced with the infusion of hypoxia-preconditioned HUC-MSCs.HE staining of pancreatic tissue showed improved islet morphology,relatively intact islet structure,and partial restoration of damaged islets following treatment with HUC-MSCs or 1%hypoxia-preconditioned HUC-MSCs,with a more significant improvement observed in the DM+Hyp group.Immunofluorescence results demonstrated that compared to the DM group,the DM+Nor and DM+Hyp groups exhibited increased pancreatic islet volume,enlarged area of insulin-positive red fluorescence,and improved islet cell structure,with the DM+Hyp group showing a more pronounced improvement in islet cell structure.(2)Histopathological analysis of mouse liver tissue showed that treatment with HUC-MSCs or 1%hypoxia-preconditioned HUC-MSCs resulted in varying degrees of restoration of disordered hepatic cord arrangement,reduction in the number and size of intracellular vacuoles,and improvement in hepatic steatosis,with a more significant effect observed in the DM+Hyp group.Western blot analysis of liver tissue revealed increased expression of PI3K and phosphorylated AKT(p-AKT)proteins in the DM+Nor and DM+Hyp groups after treatment with HUC-MSCs or 1%hypoxia-preconditioned HUC-MSCs,with a particularly marked enhancement of AKT phosphorylation in the DM+Hyp group.This suggests that compared to the infusion of HUC-MSCs,the infusion of hypoxia-preconditioned HUC-MSCs at 1%oxygen is more beneficial in activating the PI3K/AKT signaling pathway and improving insulin resistance.(3)HE staining of mouse kidney tissue showed that compared to the DM group,the DM+Nor and DM+Hyp groups exhibited some restoration in glomerular volume and reduced inflammatory cell infiltration.PAS staining of kidney tissue revealed a partial recovery in glomerular volume,significant reduction in mesangial matrix proliferation,and noticeable improvement in cystic cavity count in the DM+Nor and DM+Hyp groups,with a more pronounced pathological improvement observed in the DM+Hyp group.Western blot analysis of kidney tissue showed that infusion of HUC-MSCs or 1%hypoxia-preconditioned HUC-MSCs led to decreased expression of tumor necrosis factor-α(TNF-α)protein in mouse kidneys compared to the DM group,with a more significant reduction observed in the TNF-α protein expression level in the DM+Hyp group.Conclusions:(1)The cellular activity of HUC-MSCs is related to HIF-1α.Hypoxia up-regulates the expression of several mitochondria-related target genes such as HSP60 through activation of UPRmt,maintains mitochondrial protein homeostasis,maintains the function of OXPHOS,and establishes a novel energy metabolism pattern under hypoxic conditions to promote the cellular activity of HUC-MSCs.(2)Knockdown of HSP60 inhibits mitochondrial function in HUC-MSCs,reducing intracellular ATP levels and mitochondrial membrane potential,increasing ROS levels,and suppressing cellular activity.(3)HSP60 may coordinate with complex I to increase protective effects,suggesting that HSP60 is an important target for activating HUC-MSCs and enhancing their protective effects on multi-organ damage in T2DM mice. |
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