The Anti-senescent Effect And Expressionai Regulation Of HO-1 In Human Fibroblasts

Heme oxygenase(HO)is a rate-limiting enzyme that catalyzes the breakdown of free heme in cells.Two HO isozymes have been found in mammals,HO-1 and HO-2.The expression of HO-1 is induced by oxidative stress and is expressed at high levels in the spleen and liver,while the expression of HO-2 is mainly in the brain and testis.Heme is a prosthetic group of hemoglobin and is essentially a porphyrin ring containing a ferrous ion.Some hemoglobin releases prosthetic heme when oxidative stress occurs in cells.The ferrous ion in free heme can generate free radicals through the fenton reaction,induce the expression of HO-1,and protect cells.The protective effect of HO-1 on cells is mainly through two ways,one is to protect cells by the product of its decomposition of heme.HO-1 is capable of catalyzing the decomposition of free hemoglobin to produce biliverdin,carbon monoxide(CO)and free iron.Bilirubin forms bilirubin under the action of biliverdin reductase,which has strong anti-oxidation effect and can help cells resist necrosis.It has also been found that bilirubin inhibits NF-?B-mediated transcription of adhesion molecules.This suggests that HO-1 may have an anti-inflammatory effect,during which the free iron-induced ferritin produced by HO-1 decomposition of heme may also play an important role.In our study,NF-?B activation in senescent cells,increased secretion of inflammatory factors,and inhibition of HO-1 expression may further activate NF-?B and promote cell senescence.In addition,inhibition of HO-1 expression results in the blocking of important sources of Fe in cells and impaired cell proliferation.NRF2,which transactivates HO-1,is the most important antioxidant regulator and detoxification factor in mammals.It regulates the cis-acting elements of target gene sequences such as PRDx1,GSR,HO-1,and FTH1.ARE,which initiates transcription of a target gene,plays an extremely important role in cellular oxidative stress.We found that in senescent cells,the ARE binding activity of NRF2 was inhibited,the expression of downstream target genes regulated by NRF2 was significantly reduced,the antioxidant capacity of cells was decreased,and the levels of ROS and Mito-Sox in mitochondria were significantly increased.NF-?B is a classical pro-inflammatory factor that promotes the expression of SASP factors during cellular senescence.When stimulated by pro-inflammatory cytokines such as tumor necrosis factor,it again exhibits cellular anti-apoptotic effects.This may also be closely related to its role in aging.Other studies have found that ROS can activate IKK,release NF-?B into the nucleus and promote the transcription of IL-8 and P53,leading to cell senescence.Studies have found that in cells of premature aging patients,p65 is able to inhibit the activity of Nff2 by competitive binding to the transcriptional coactivator CBP(CREB-binding protein)-p300 complex.We also found that in senescent cells,NF-?B enters the nucleus,P-p65 protein levels increase,and inflammation increases.Thus inhibiting the ARE binding activity of NRF2,destroying the protective mechanism of cell antioxidants,leading to elevated ROS levels,thereby further activating NF-?B,providing a continuous oxidative stress environment for NF-?B activation,further promoting cell senescence.In order to explore the mechanism by which HO-1 protects cells against aging,and the regulation of NRF2 and NF-?B,we carried out the following research work.1.Expression of HO-1 in senescent cellsWe induced senescence of NHF cells with hydrogen peroxide,HU and radiation,respectively.The expression of HO-1 was down-regulated by Western blot in several aging models.At the same time,we also detected that the expression of HO-1 in the NHF and DPSCs cells of passage aging was also significantly down-regulated.2.HO-1 knockdown leads to cellular senescenceIn order to explore the causal relationship between down-regulation of HO-1 expression and cellular senescence,after interfering with HO-1,through EdUIncorporation experiments have found that cell proliferation ability is significantly reduced.It was found by SA-P-gal aging staining that the proportion of senescence staining increased significantly.After interfering with HO-1,cell ROS levels were measured by flow cytometry and ROS levels were elevated.Treatment of NHF cells with Hemin for 6 h,detection of ROS found that ROS levels increased significantly.Normal NHF cells and NHF cells interfering with HO-1 were treated with Hemin for 6 days,and SA-?-gal senescence staining revealed that the senescence staining ratio was up-regulated.It indicates that the lack of HO-1 in cells leads to an increase in ROS,and the accumulation of heme produces cytotoxicity and promotes cell senescence.Treatment of NHF cells interfering with HO-1 with DFO revealed a significant increase in the aging rate of cells.Treatment of NHF cells interfering with HO-1 with FAS revealed that the aging ratio of cells was significantly down-regulated.It is indicated that iron deficiency is an important mechanism of HO-1 deficiency leading to cell senescence.3.Heme induces ROS-dependent HO-1 expressionTreatment of NHF cells with Hemin for 6,12,24,and 48 hours revealed that the protein level of HO-1 was significantly up-regulated.Treatment of NHF cells with NAC in combination with Hemin showed that NAC significantly attenuated HO-1 upregulation induced by Hemin.This indicates that elevated heme by ROS can induce HO-1 up-regulation which can protect cells.4.Inhibition of NRF2 activity in senescent cells is responsible for the down-regulation of HO-1 expressionThe stress-induced senescent NHF cells were treated with hydrogen peroxide and Hemin,respectively,and the protein levels of HO-1 were determined.The results showed that the induction of HO-1 by hydrogen peroxide or Hemin was significantly weakened in senescent cells.Next,we further tested the expression of other NRF2 target genes in senescent cells.The cellular senescence was induced by subjecting NHF cells to hydrogen peroxide and HU for 6 days,respectively.The transcription levels of NRF2 and NRF2 target genes were determined by RT-PCR.The results showed that The mRNA levels of NQO1,GSTM4,GCLC,GCLM,GSR,TxNDR1,TALDO,and TKT decreased significantly,while the expression level of NRF2 itself did not change significantly.The NRF2-ARE luciferase reporter assay showed that the NRF2-ARE activity in senescent cells was significantly decreased,indicating that NRF2-ARE binding activity is impaired.NRF2 target genes aren downregulated in senescent cells.5.NF-?B activation contributesd to oxidative stress by inhibiting NRF2 activity in senescent cells.p-p65 was up-regulated in replicative senescent cells as determined by Western blot,RNA interference of p65 in senescent cells led to an upregulation of of HO-1.The intracellular ROS levels were reduced by p65 RNAi.At the same time,treatment of senescent cells with NF-?B inhibitor Bay can also reduce ROS levels in senescent cells.This indicates that NF-?B plays a pro-oxidative role in senescent cells by inhibiting NRF2 activity.6.Maintenance of NF-kB activity in senescent cells requires sustained oxidative stressThe ROS levels in hydrogen peroxide-induced NHF senescent cells and passaged senescent NHF cells were determined by flow cytometry.It was found that the ROS level in senescent cells were significantly higher than those in young cells.The expression of inflammatory factors was determined after the senescent cells were treated with NAC.The expression of inflammatory factors in senescent cells was significantly decreased after NAC treatment,indicating that NF-?B activity maintenance in senescent cells requires sustained oxidative stress.