The Effects Of NOX4-NLRP3 Signaling Pathway In Aging-related Renal Injury And The Regulation Of Ginsenoside Rg1 In Mice

The kidney is one of the most vulnerable organs to aging,a series of structural lesions will occur in the kidney during the aging process.Abnormal fiber formation will damage the balance of the structure and the function of kidney,leading to end-stage renal disease,and ultimately leading to renal failure,which is harmful to health.However,the exact mechanism of aging-related renal damage remains unclear.Oxidative stress and inflammation play a key role in the etiology of senescence-related disease.This study investigated the effect of NADPH oxidase 4(NOX4)and nucleotide-binding oligomolytic domain containing protein(NOD)like receptor protein 3(NLRP3)on senescence-related renal injury.Ginsenoside Rg1 has a wide range of anti-aging effects.The purpose of this study is to investigate the role of NOX4-NLRP3 signaling pathway in renal injury of mice during aging and the regulation of ginsenoside Rg1.Part One The effects of NOX4-NLRP3 signaling pathway on renal fibrosis in natural aging miceObjective To research the changes of NOX4 and NLRP3 inflammasome activation in the process of renal aging in naturally aging mice,and to explore the possible time points and targets of anti-renal aging fibrosis.Methods 6 months old male Kunming mice were randomly divided into groups and normally bred to 16 months,20 months and 24 months old,with 6 months as the young control group.Serum blood urea nitrogen and serum creatinine were used to detect changes in renal function in naturally aging mice;senescence-related β-galactosidase staining was used to detect the aging of kidney tissue;HE and PAS staining was used to detect renal histopathological changes;Masson,IHC staining(collagen IV)to detect collagen fiber changes in kidney tissue;DHE staining to detect the expression of ROS in kidney tissues;Western Blot to detect the expressions of NOX4,p22 phox,p47phox in kidney tissues of naturally aging mice;Western Blot to detect the expressions of NLRP3,ASC,caspase-1,and IL-1β in the kidney tissue of naturally aging mice;IHC staining was used to detect the localized expression of NLRP3 in kidney tissues of naturally aging mice.Results The levels of SCr and BUN were lower in 6 months mice,higher in 16 months mice,significantly higher in 20 and 24 months mice.The expression of β-Gal was lower in 6 months,increased in 16 months,significantly increased in 20 and 24 months.The results of HE and PAS staining showed that there were no abnormal pathological changes in the kidney of 6 months,with regular glomerular morphology and clear structure,normal mesangial cells and stroma,no mesangial hyperplasia in the glomerulus,and complete and clear renal tubules;mesangial hyperplasia and increased glomerular volume in the glomeruli,no obvious pathological damage in the renal tubules at 16 months;while the glomeruli and renal tubules showed obvious pathological damage at 20 and 24 months.The results of Masson,IHC(collagen IV)staining showed that there was no significant collagen formation in 6 months,there was significant collagen deposition in the glomerulus,but only a little collagen was produced in the renal tubulointerstitium at 16 months;while the collagen deposition was significantly increased in the glomerulus and renal tubules at 20 and 24 months,especially 24 month.The results of DHE staining showed that the expression of ROS was lower in 6 months,increased significantly in 16,20 and 24 months.Western Blot results showed that the expression of NOX4,p22 phox and p47 phox was less in 6 months;the expression of p22 phox and p47 phox increased significantly in 16 months,while the expression of NOX4 increased,but there was no significant difference;the expression of NOX4,p22 phox and p47 phox were significantly increased in 20 and 24 months mice.Western Blot results showed that the expression of NLRP3,ASC,caspase-1 and IL-1β were lower in 6 months;the expression of NLRP3,caspase-1 and IL-1β increased significantly in 16 months mice,while the expression of ASC increased,but there was no significant difference;the expression of NLRP3,ASC,caspase-1 and IL-1β increased significantly in 20 and 24 months.The results of IHC(NLRP3)staining showed that the expression of NLRP3 was lower in 6 months,increased in the glomerulus of 16 months,significantly increased in the glomerular and tubule interstitium of 20 and 24 months.Part Two Protective effect of ginsenoside Rg1 on senescent-related renal fibrosis in SAMP8 miceObjective The purpose of this study was to investigate the protective effect of Rg1 on aging-related renal fibrosis in SAMP8 mice and the effect on NOX4-NLRP3 signaling pathway.Methods Male 6 months old SAMP8 mice and SAMR1 mice.Mice were randomly divided into 6 groups:(1)SAMR1 control group;(2)SAMP8 model group;(3)SAMP8 + Tempol(50mg/kg)group;(4)SAMP8 + Apocynin group(50 mg/kg);(5)SAMP8 + Rg1 (5mg/kg)group;(6)SAMP8 + Rg1(10mg/kg)group.Tempol,Apocynin,Rg1 as treatment group,the mice were administered intragastrically(0.1 ml/10g/day)with Tempol,Apocynin and Rg1 for 9 weeks,respectively.Serum urea nitrogen and serum creatinine were used to detect the changes of renal function in mice.β-galactosidase staining was used to detect the aging of kidney tissue in mice.HE and PAS staining was used to detect the renal histomorphological changes in mice.Masson and IHC staining(collagen IV)was used to detect the changes of collagen fibers in the kidney tissues of mice.The expression of ROS in kidney tissues was detected by DHE staining.The expressions of NOX4,p22 phox and p47 phox in renal tissues were detected by Western Blot.Western Blot was used to detect the expression of NLRP3,ASC,caspase-1 and IL-1β in renal tissues of mice.IHC staining was used to detect the localized expression of NLRP3 in renal tissues of mice.Results The levels of BUN and SCr in SAMP8 mice were significantly higher than in control mice,Tempol,Apocynin and Rg1(5,10 mg/kg)treatment were significantly decreased the levels of BUN and SCr.The expression of β-Gal in SAMP8 mice was significantly increased compared with control mice,while were reduced sharply after the Tempol,Apocynin and Rg1(5,10 mg/kg)treatment.The results of HE and PAS staining showed that the kidney of SAMP8 mice showed significant pathological damage,Tempol,Apocynin and Rg1(5,10 mg/kg)group were significantly improved kidney damage in SAMP8 mice.Masson,IHC(collagen IV)staining results showed that the significant collagen deposition in the glomerular and tubulointerstitium of SAMP8 mice,Tempol,Apocynin and Rg1(5,10 mg/kg)treatment were significantly reduced the collagen deposition.DHE staining results showed that the expression of ROS increased significantly in SAMP8 mice,Tempol,Apocynin and Rg1(5,10 mg/kg)treatment significantly reduced the ROS expression.Western Blot results showed that the expression levels of NOX4,p22 phox and p47 phox were significantly increased in SAMP8 mice,Rg1(10 mg/kg)treatment significantly reduced the expression of NOX4,p22 phox and p47 phox,Rg1(5 mg/kg)and Apocynin can reduce the expression of NOX4 and p22 phox,but had no significant effect on p47 phox expression,Tempol can reduce the expression of NOX4,while had no significant effect on the expression of p22 phox and p47 phox.Western Blot results showed that the expression of NLRP3,ASC,caspase-1 and IL-1β were significantly increased in SAMP8 mice,Rg1(5,10 mg/kg)treatment significantly reduced the expression of NLRP3,ASC,caspase-1 and IL-1β,Tempol and Apocynin treatment can obviously reduce the expression of NLRP3,caspase-1 and IL-1β,but had no obvious effect on the expression of ASC.IHC(NLRP3)staining results showed that the expression of NLRP3 in the glomeruli and tubules of the SAMP8 mice was significantly increased,Tempol and Apocynin treatment can decrease the expression of glomerular and renal tubular NLRP3 SAMP8 mice,especially the glomerulus,Rg1(5,10 mg/kg)can significantly decreased the expression of NLRP3 in glomerular and renal tubular of SAMP8 mice.Conclusion The results showed that there were significant senescence related renal injury and fibrosis in mice.NOX4 and NLRP3 inflammasomes were significantly activated during renal senescence,especially in mice aged 20 and 24 months.NOX4-mediated excessive production of ROS plays an important role in the activation of NLRP3 inflammasome.The NOX4-NLRP3 signaling pathway is closely related to renal injury and fibrosis in naturally aged mice,which may be an important target for the prevention of senescence related renal injury,and the age of 16-20 months may be an important period.Ginsenoside Rg1 improved senescence-related renal injury and fibrosis in SAMP8 mice.Rg1 has obvious antioxidant effect and reduces the expression of NOX4.In addition,Rg1 significantly inhibited the activation of NLRP3 inflammasome in SAMP8 mice.The results showed that Rg1 could delay renal senescence and reduce senescence-related renal injury and fibrosis by inhibiting NOX4-mediated ROS oxidative stress and NLRP3 inflammasome activation.