The Mechanism Of Nrf2 Regulating Cellular Senescence And Immunosuppressive Ability Of UC-MSCs And Its Role In BPD

Bronchopulmonary dysplasia(BPD)is the most common chronic lung disease in newborns,especially in premature infants.BPD with high morbidity and mortality seriously threatens the lives of infants,especially premature infants.Although perinatal and neonatal medicine has made remarkable advances in improving the survival rate of premature infants,limited progress has been made in the treatment of BPD.In the field of traditional medicine,innovation and exploration of clinical intervention and treatment methods still cannot fundamentally promote the prognosis of severe BPD.Therefore,new and effective intervention and therapy are urgently needed to settle this problems.Recently,the transplantation of mesenchymal stem cell(MSCs)has provided a new treatment strategy for BPD.Umbilical cord mesenchymal stem cells(UC-MSCs)are a kind of pluripotent stem cells with high self-renewal ability and multi-directional differentiation.Their low immunogenicity and immunoregulatory properties make UC-MSCs become a sort of excellent cells sources inMSCs transplantation of immune-related diseases.Although early clinical studies have showed the safety of MSCs,their clinical effectiveness has been controversial.Therefore,it is the key of developing effective therapeutic strategies to explore the factors that affect the immunomodulatory properties of MSCs.Large-scale amplification in vitro is an inevitable process for clinical applications of MSCs,and replicative senescence may be the important element affecting effectiveness of MSCs in clinical applications.Studies have showed that replicative senescent MSCs display lower immunoregulatory ability,thus,slowing down cellular senescence may be of benefit to their immunosuppressive ability.Nuclear factor erythroid 2-related factor 2(Nrf2)is an evolutionarily conservative transcription factor that interacts with antioxidant responsive element(ARE)to regulate the coding of antioxidant protein.Nrf2 / ARE pathway is essential for body's self-defense mechanism,and it plays decisive roles in different respects including reducing apoptosis,delaying aging,reducing organ damage and so on.Studies have found that disruption of Nrf2-mediated cellular antioxidant pathways is one of important driving forces resulting in cell senescence,and activation of Nrf2 can delay the senescence of MSCs.This study will investigate whether Nrf2 participates in regulating the therapeutic effect of UC-MSCs in treatment of BPD induced by hyperoxia in neonatal rats.And initially explore the role of Nrf2 in the process ofreplicative senescence and immunosuppressive ability of UC-MSCs,in order to further understand the involved mechanism of Nrf2 in immunosuppressive ability of UC-MSCs.Part I knock-down of Nrf2 in UC-MSCs attenuates the therapeutic effect on neonatal rats with bronchopulmonary dysplasiaObjective: To explore the role of Nrf2 on treatment of UC-MSCs in BPD induced by hyperoxia in rat pups.Methods: Cell experiment: UC-MSCs were cultured in vitro,flow cytometry was used to detect the expression of cell-specific surface markers.Oil red O staining was used to detect adipogenic differentiation,alizarin red staining and ALP staining were used to detect osteogenic differentiation.UC-MSCs treated with siRNA were divided into two groups:negative control group(NC)and Nrf2 knock-down group(siNrf2).Real-time qPCR and WB were used to verify the transfection efficiency of siRNA.Animal experiment: 80% hyperoxia exposure was used to establish BPD model(12h-21 d after birth).Endotracheal intubation was performed on 7th day,and then the NC group or siNrf2 group UC-MSCs(1×106/40?L)or equal volume sterilized PBS were slowly transplanted into rats lungs by intratracheal administration.SD neonatal rats were divided into normoxic solvent control group(NOR+PBS),normoxic negative control treatment group(NOR+NC),normoxic knock-down treatment group(NOR+siNrf2),and BPD model group(BPD+PBS),BPD negative control treatment group(BPD+NC)and BPD knock-down treatment group(BPD+siNrf2).Observethe physical development,activity and deoxygenation tolerance performance of rats in each group during the feeding process.Evaluate the survival rate and weight gain(7d-21d).Collect the samples of BALF and lung tissues at 21 d age and count the total cell number of bronchoalveolar lavage fluid(BALF).Observe the pathological changes of lung tissues by H&E staining and measure the mean linear intercept(MLI).Results: Cell experiment: successfully cultured UC-MSCs in vitro and identified them,UC-MSCs have osteogenic and adipogenic differentiation ability.Real-time qPCR and WB verified that the expression of Nrf2 decreased in UC-MSCs treated by siRNA(**P<0.01,***P<0.001).Animal experiment: compared with all normoxic groups,rats under hyperoxic conditions manifested stunted physical growth,decreased activity,exercise intolerance,and delayed weight gain.At the endpoint of observation,compared with all normoxic groups,extremely rare cases were suffered from death on hyperoxia groups,but those survival rates among all groups had no statistical significance.Calculate the changes in body weight growth from 7th to 21 th day.The weight gain in hyperoxia groups was significantly slower than that in normoxia groups(***P<0.001);Compared with(BPD+PBS)model group,the weight gain of(BPD+NC)treatment group was significant increased(*P<0.05).The weight gain between(BPD+NC)and(BPD+siNrf2)treatment groups had no significant difference.The BALF total cell count in(BPD+PBS)model group wassignificantly increased than all normoxic groups(***P<0.001),and the total cell count in(BPD+NC)treatment group was decreased than(BPD+PBS)model group(*P<0.05),while the number of cell in(BPD+siNrf2)group was significantly increased than(BPD+NC)treatment group(**P<0.01).The H & E staining of lung tissues showed that the alveolar structure of all normoxic groups was complete.The pathology of lung tissues suggested that the alveolar structure was simplified in(BPD+PBS)model group.The alveolar developmental arrest in(BPD+NC)and(BPD+siNrf2)treatment groups had been ameliorated in different degrees compared with(BPD+PBS)model group.The morphometric analysis further showed that the mean linear intercept(MLI)was notably altered in these groups(NOR+PBS: NOR+NC: NOR+siNrf2: BPD+PBS:BPD+NC: BPD+siNrf2 = 49.86±1.605?m: 46.46±1.605?m: 49.12±2.157?m: 100.5±3.033?m: 56.98±1.793?m: 84.12±2.076?m,***P<0.001).The value of MLI in(BPD+NC)group was significantly decreased than(BPD+siNrf2)treatment group(***P<0.001).Conclusion: knock-down of Nrf2 attenuates the therapeutic effect of UC-MSCs on the lung injury of BPD model induced by hyperoxia.Part ? The role of Nrf2 in replicative senescence and immunosuppressive ability of UC-MSCsObjective: To investigate the correlation between UC-MSCs replicative senescence and Nrf2,and whether cellular senescence affects the immunosuppressive ability of UC-MSCs.To further explore the role of Nrf2 in senescence phenotype and immunosuppressive ability of UC-MSCs.Methods: P5 and P18 UC-MSCs were cultured,senescence phenotype were detected by ?-gal staining and HP-1? immunofluorescence.CCK-8,Ki67 immunofluorescence and cell cycle detection were used to detect changes of cell proliferation ability,western blot was used to detect the protein expression of senescence-associated protein p16 and Nrf2 signaling pathway-related molecules Nrf2 and p S40-Nrf2.RT-q PCR and western blot were used to detect the m RNA and protein expression of IDO-1 in P5 and P18 UC-MSCs under the stimulation of IFN-?.Flow cytometry was used to detect the difference of ability of early and late passage UC-MSCs to inhibit PBMC proliferation.UC-MSCs were transfected with si RNA and divided into NC group and si Nrf2 group.?-gal staining was used to detect the difference of ?-gal positive cells in UC-MSCs after 3 days.Ki67 immunofluorescence and Ed U proliferation detection were used to detect the effect of downregulation of Nrf2 on cell proliferation.The growth curves of NC group and si Nrf2 group UC-MSCs transduced with si RNA cultured for 7days were detected by CCK-8.UC-MSCs were treated with IFN-?(10ng /m L)for 12 hours,and then were transduced with si RNA.They were divided into four groups: negative control group I(NC)and knock-down group I(si Nrf2),IFN-? negative control group II(NC+IFN-?)and IFN-?knock-down group II(si Nrf2+IFN-?),two pairs corresponding.After cultured 3 days,RT-q PCR and western blot were used to detect the expression of Nrf2 and IDO-1.Subsequently,PBMC were co-cultured with UC-MSCs and the experiment was divided into PBMC control group ?(PHA(-)),PBMC control group ?(PHA(+)),and co-culture negative control group ?(NC+PHA(+))and co-culture Nrf2 knock-down group(si Nrf2+PHA(+)).Co-culture images were collected using light microscope,and PBMC proliferation was detected by flow cytometry.Results: Compared with P5 UC-MSCs,the number of ?-gal staining positive cells and protein expression of heterochromatin 1?(HP-1?)were notably increased in P18 UC-MSCs.CCK-8 analysis indicated that the cell proliferation ability of P18 UC-MSCs was decreased(*P<0.05).The proportion of Ki67 positive cells was decreased in P18 UC-MSCs by immunofluorescence staining(***P<0.001).Cell cycle detection showed that G0/G1 phase cells were significantly increased,and the proportion of S and G2/M phase cells was decreased.Western blot showed that the protein expression of p16 was increased and the protein expression of Nrf2 and p S40-Nrf2 was decreased in P18 UC-MSCs.(***P<0.001).Under the treatment of IFN-? with different concentrations,the m RNA expression of IDO-1 was considerably decreased at P18 than that at P5UC-MSCs(*P<0.05),and the protein expression of IDO-1 was also reduced under the treatment of 10ng/m L IFN-?(***P<0.001).The proliferation ability of PBMC co-cultured with P5 UC-MSCs was significantly impaired than that with P18(*P<0.05).After UC-MSCs were transduced with si RNA,?-gal staining results showed that the number of ?-gal positive UC-MSCs was significantly increased in si Nrf2 group(***P<0.001).Ki67 immunofluorescence detection showed the proportion of Ki67 positive cells was decreased in si Nrf2 group UC-MSCs(**P<0.01).CCK-8 detection showed that the proliferation of si Nrf2 UC-MSCs was significantly decreased from 4d to 7d(**P<0.01).Ed U proliferation testing also showed that the proliferation ability of UC-MSCs decreased in si Nrf2 group(***P<0.001).RT-q PCR showed that Nrf2 m RNA expression of si Nrf2 group and(si Nrf2+IFN-?)group was decreased compared with according control group.The m RNA expression of IDO-1 was significantly increased in UC-MSCs treated by IFN-?;compared with(NC+IFN-?)group,the m RNA expression of IDO-1was significantly reduced in(si Nrf2+IFN-?)group(**P<0.01).Western blot showed that the protein expression of Nrf2 and IDO-1 was consistent with m RNA expression levels.Subsequently,PBMC were co-cultured with UC-MSCs.Co-culture images and flow cytometry showed that PHA activated PBMC successfully,UC-MSCs remarkably inhibited PHA-induced proliferation of PBMC,and their ability of inhibiting PBMC proliferation was reduced in UC-MSCs transduced with Nrf2 target si RNA.Conclusion: During the ex vivo expansion of UC-MSCs,the immunosuppressive ability is impaired accompanied with increased senescence phenotype and decreased expression of Nrf2.Downregulation of Nrf2 accelerates senescence and reduces cell proliferation of UC-MSCs.And the impaired immunosuppressive properties of UC-MSCs may be associated with Nrf2-mediated IDO-1 expression.Part ? Study on the role of Nrf2 agonist ALA in enhancing the immunosuppressive properties of UC-MSCsObjective: To further investigate the regulatory role of Nrf2 in senescence,proliferation ability and immunosuppressive ability of UC-MSCs.Methods: UC-MSCs were treated with ?-lipoic acid(ALA),an agonist of Nrf2,at 50?mol/L,100?mol/L and 150?mol/L concentrations,respectively.The protein expression of Nrf2 was detected by western blot.CCK-8 was used to detect the difference of cell proliferation ability,and then selected the optimal drug concentration of ALA.Cells were divided into control group and ALA group.Cellular senescence phenotype were detected by ?-gal staining.UC-MSCs were treated with Nrf2 activator oltipraz(OLP),and then the proper concentration of OLP was determined by western blot.Cells were divided into control group and OLP group,the changes of proliferation ability were detected by CCK-8 and Ki67 immunofluorescence.RT-q PCR was used to detect the difference of m RNA expression of IDO-1 and flow cytometry was used to detect the inhibition of UC-MSCs treated with ALA on PBMC proliferation.Results: When the concentration of ALA was increased,the protein expression of Nrf2 in UC-MSCs was increased accordingly(***P<0.001).Adding IFN-?(10ng/m L)co-treated,the protein expression of Nrf2 was the highest expression at 100 ? mol/L concentration of ALA in UC-MSCs(***P<0.001).CCK-8 showed that the proliferation ability of UC-MSCs after 3 days of ALA treatment was especially enhanced at 100?mol/L concentration(**P<0.01).Thus,100?mol/L was used as the optimal concentration of ALA.Compared with control group,the proportion of ?-gal positive UC-MSCs was significantly decreased in ALA group(***P<0.001).30?mol/L was decided as the proper concentration of OLP by western blot.Compared with control group,CCK-8 showed that cell proliferation ability was promoted and Ki67 immunofluorescence showed the proportion of Ki67 positive cells was significantly increased in OLP group(*P<0.05,***P<0.001).After UC-MSCs were treated with ALA,the m RNA expression of IDO-1 was obviously increased.And the inhibitory effect of UC-MSCs on the PHA-induced proliferation of PBMC was promoted in ALA group than that in control group(**P<0.01,***P<0.001).Conclusion: Small molecule compounds promote Nrf2 expression of UC-MSCs,which can delay cellular senescence and enhance proliferative ability.Nrf2 may regulate the immunosuppressive properties of UC-MSCs in vitro via Nrf2-mediated IDO-1 expression.