| Background:Acute kidney injury(AKI)is a common clinical complication.Due to its high morbidity,high initial mortality rate and a large amount of medical costs,it becomes a great burden on the health of patients.The pathophysiological mechanisms of AKI are mainly inflammation,neurochemical stress,mitochondrial dysfunction and cell senescence.At present cell senescence is known to play an important role in kidney diseases,and some studies even believe that AKI is a state of acute cell senescence,indicating that cell senescence is closely related to AKI.Cellular senescence is not only a state of decreased cell function with increasing age,but also stress senescence of cells can be found in many diseases.Stress senescence of cells is very similar to cell senescence,mainly manifested by cell cycle arrest and increased expression of senescence related proteins.Senescence associated secretory phenotype(SASP)has the greatest impact on surrounding tissue cells from a variety of cytokines,inflammatory cytokines and chemokines secreted by senescent cells.In the kidney,the cytokines secreted by age-related secretion phenotypes mainly include pro-inflammatory cytokines and pro-fibrosis cytokines,such as Interleukin 6(IL-6),Human monocyte chemotactic protein-1(Human monocyte chemotactic protein-1).Transforming growth factor beta 1(Transforming growth factor beta 1,TGF-beta 1),α-smooth muscle actin(α-SMA),vascular endothelial growth factor(VEGF),etc.The production and release of these cytokines will further lead to damage to cell function,resulting in cell and tissue damage,and further aggravate cell aging,forming a vicious cycle.Kidney tubular epithelial cells play an important role in the transformation of AKI to Chronic kidney disease(CKD).In this process,renal tubular epithelial cells will be damaged,incomplete repair after injury and continuous inflammatory reaction,and eventually lead to the development of CKD.And even End stage renal disease(ESRD).It can be seen that renal tubular cell damage and cell senescence are present in AKI,but whether there is a correlation between the two and which factors are involved is still unclear.GSK3β is an important substance in organ development and tissue repair.In normal adults,GSK3β is not only involved in glucose metabolism,but also in a variety of physiological and pathological pathways.Recent literature has shown that over-activation of GSK3β is associated with age-related diseases,such as Alzheimer’s disease,cognitive dysfunction,osteoporosis,etc.,and found that it may play an important role in cellular senescence.However,it is still unknown whether GSK3β is directly related to renal tubular senescence.Above all,renal tubular cell injury in vitro factors,will appear at the same time accompany aging cell excitability,cell morphological and functional changes,appear even secretion of age-related phenotypic cell factors secreted by further changes such as cell normal state,thus further increase cell damage,form a vicious circle,kidney health cause significant harm to human body.GSK3β,as a kinase associated with a variety of renal diseases and also involved in a variety of cell senescence related diseases,may play an important role in renal tubular cell injury and stress aging.This study aimed to investigate the occurrence of stress senescence and cell damage in AKI and the role of GSK3β in this process.To clarify the role of GSK3β in renal cell injury and stress aging provides a new possibility for delaying renal tubular cell aging and preventing and treating renal tubular cell injury.Method:In this study,human renal tubular epithelial cells(HK2)were selected as the study object.1.After digestion,the cultured HK2 cells with good growth status were inoculated in a petri dish or culture bottle at an appropriate concentration.After 24 hours,the cell attachment was set to t=0.The concentration of Hydrogen peroxide was used to induce the senescence of renal tubule cells,and the concentration of Hydrogen peroxide(H2O2)was 400μm,to stimulate HK2 cells for 12 and 24 hours.2.Damage of HK2 cells induced by H2O2:Cell immunofluorescence(IF)was used to detect the expression of cadherin E-cadherin in epithelial cells.Western blot was used to detect cadherin E-cadherin expression in epithelial cells.WB)to detect the expression of epithelial cell damage protein a-SMA and Neutrophil gelatinase-associated lipocalin(NGAL).3.The expression level of p-GSK3β(Ser9)was detected by Western blotting.4.H2O2-induced senescence of HK2 cells:the morphological changes of senescence cells were observed under light microscope,mainly nuclear changes;β-galactosidase(SA-β-gal)kit was used to detect cell senescence.The expression of DNA damage marker protein yH2AX was detected by IF.Cell cycle arrest was detected by flow cytometry.5.72h after transfection with GSK3β knockdown lentivirus,HK2 cells were stimulated by H2O2(400 pM,24 h).6.Cell injury related indicators were detected:Cell immunofluorescence was used to detect the expression of epithelial marker proteins,and protein immunoblotting was used to detect the expression of renal tubular epithelial injury molecules.7.Detection of cell senescence related indicators:the kit was used to detectβ-galactosidase expression;Furthermore,the expression of age-related secreted phenotypes IL-6,MCP-1,TGF-β1 were detected by cell immunofluorescence assay and Western blot.Meanwhile,the expression levels of age-related proteins p16,p21,p53 and p-Rb were detected by Western blot.8.72h after transfection of GSK3β overexpressing lentivirus,HK2 cells were stimulated by H2O2(400μM,24 h).9.Detection of cell injury-related indicators:Cell immunofluorescence was used to detect the expression of epithelial marker proteins,and Western blot was used to detect the expression of renal tubular epithelial injury molecules.10.Detection of cell senescence related indicators:the kit was used to detectβ-galactosidase expression;Furthermore,the expression of age-related secreted phenotypes IL-6,MCP-1,TGF-β1 were detected by cell immunofluorescence assay and Western blot Meanwhile,the expression levels of age-related proteins p16,p21,p53 and p-Rb were detected by Western blot.Result:1.After 12 and 24h of H2O2 stimulation,HK2 cells in both groups showed cell damage,and the damage was stronger at 24h than at 12h:the expression of epithelial marker protein E-cadherin decreased,and the senescence related protein a-SMA and NGAL increased(24h less than 12h).2.The activity of GSK3β increased,that is,p-GSK3 β(Ser9)decreased.3.H2O2-induced cell senescence,and the senescence degree at 24h was greater than that at 12h:binuclear morphological changes of cells were observed under optical microscope;Increased activity of β-galactosidase,a marker of aging,was enhanced.γH2AX expression increased;The secretion of SASP and senescence related proteins in senescent cells were increased by WB detection.Flow cytometry was used to detect significant cell cycle arrest,mainly in G2 phase.4.After GSK3β specific knockdown,H2O2-induced cell injury was reduced:epithelial marker E-cadherin was increased,and the expression of injury-related proteins α-SMA and NGAL was decreased.5.After GSK3β specific knockdown,H2O2-induced cell senescence was reduced:senescence related markers β-galactosidase and yH2AX were decreased,senescence related secretion phenotype SASP was decreased,and secretion of pro-inflammatory cytokines IL-6 and MPC-1 were decreased.And the expression of age-related proteins(p16,p21,p53)decreased,and the expression of p-Rb increased.6.After GSK3β overexpression,H2O2-induced cell injury was increased:epithelial marker E-cadherin was decreased,and injury-related proteins a-SMA and NGAL were increased.7.After the overexpression of GSK3p,H2O2-induced cell senescence increased:senescence marker β-galactosidase increased,yH2AX increased,senescence related secretion phenotype Sasp increased,secretion of pro-inflammatory cytokines IL-6 and MCP-1 increased;At the same time the expression of age-related proteins(p16,p21,p53)was increased,and the expression of p-Rb was decreased.Conclusion:GSK3β is involved in H2O2-induced cellular senescence and injury of HK2 cells. |
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