| Objectives: At present,diabetic nephropathy(DN)has gradually become one of the main causes of end-stage renal disease.Data show that the prevalence of diabetes in China has reached 11.6%(nearly 200 million people),and about 20%-40% of diabetic patients are complicated with diabetic nephropathy.It brings huge economic burden to the whole society.Therefore,it has always been the focus of exploring the pathogenesis of DN and find effective drugs and methods to prevent and cure DN.The pathogenesis of DN is very complex.It was found that high glucose stress induced the production of Reactive oxygen species(ROS)in mitochondria,which played a key role in the occurrence and development of DN.Renal tubular injury is a key link in the progression of DN.At the early stage of DN,renal tubular epithelial cells show a unique process of hyperplasia,hypertrophy and then senescence under high glucose stimulation,which is called stress-induced senescence.This process is manifested by upregulation of p21,p16,cytoplasmic SA-β-gal expression,telomere shortening,cell cycle stagnation,and SAHF accumulation in the cell nucleus.It causes structural and functional damage to the kidneys.It suggests that stress-induced senescence may be a major participant in promoting the development of DN.Therefore,it may be an important research direction to delay the progression of DN by interfering with stress-induced senescence of renal tubular epithelial cells.Sirt3 is one of the homologous protein families expressed by silencing information regulator 2(Sir2),which is mainly located in the mitochondria of cells.It has the activity of deacetylase and plays an important role in the regulation of cellular oxidative stress,cell senescence and cell apoptosis.Current studies suggest that Sirt3 can reduce the production of ROS by binding and regulating the acetylation level and activity of Complex I and Complex III.It may also increase the scavenging of mitochondrial ROS by regulating the antioxidants SOD2,GPx and Prx3.During the development of DN,our previous study found that Sirt3 can reduce the oxidative stress and apoptosis of HK-2 cells stimulated by high glucose,and Sirt3 may be a new target for the intervention of DN.In recent years,Sirt3 has been found to play an important role in oxidative stress-induced cell senescence through its antioxidant mechanism.Antioxidant hydrogen inhibits retinal aging by upregulation of Sirt3 to reduce oxidative stress and down-regulate the expression of P21,P53 and P16.In summary,Sirt3 plays an important role in anti-cell senescence by affecting oxidative stress.The Keap1/Nrf2 pathway is an important signaling pathway that regulates the REDOX process in vivo.Previous studies have shown that Nrf2 is decoupled with Keap1 after cells are stimulated by reactive oxygen species,and then Nrf2 is transported into the nucleus.The disorder of Nrf2 pathway mediates cell senescence.In MSCs,reactivation of Nrf2 can effectively reverse the phenotype of premature enescence and restore the activity of MSCs.Pyrroquinoline quinone(PQQ),the third water-soluble coenzyme of oxidoreductase,is a new B vitamin independent of flavin adenine dinucleotide and niacinamide adenine dinucleotide phosphate.Previous studies have shown that PQQ has a variety of biological functions,including neurological and cardiovascular protection,promoting cell growth and reproduction,and acting as an antioxidant to protect cells from oxidative stress,and to reduce inflammation and cellular aging.Another study found that PQQ increased the expression and activity of Sirt1 and Sirt3 genes in Hep G2 cells.It is suggested that the protective effect of PQQ may be related to Sirt3.In this study,we first observed the expression of Sirt3 and stress-induced senescence in diabetic kidney and renal tubular epithelial cells stimulated by high glucose.Further,plasmid transfection was used to overexpress Sirt3.We observed the changes of oxidative stress and stress-induced senescence in renal tubular epithelial cells stimulated by high glucose,and further explored the role and mechanism of Sirt3 in diabetic nephropathy.In addition,we also explored the role of Sirt3 in anti-oxidative stress and cell senescence of pyrroloquinoline quinone,which provides a scientific basis for finding effective drugs for the treatment of DN.Methods:1.Expression and significance of Sirt3 in diabetic patients renal tissue and renal tubular epithelial cells stimulated by high glucose.Ten patients diagnosed as developed diabetic nephropathy by renal biopsy and clinical data in this study.The renal tissues(n=10)obtained from distant portions of kidneys surgically excised because of the presence of a localized neoplasm were used as control.Renal tissue was conventionally paraffin-embedded,and 4 μm sections were obtained.They were used to detect the expression of Sirt3 and P16 in each group by immunohistochemistry,and the co-expression of Sirt3 and P16 in DN group was detected by immunofluorescence co-staining.Human renal tubular epithelial cells were divided into normal control group(NG,5.5 m M D-glucose),mannitol control group(24.5m M mannitol concentration,5.5m M D-glucose)and the high glucose group(HG,30 m M D-glucose).After the specified time stimulated(6 h,12 h,24 h,48 h,72 h),SA-β-Gal was used to detect cell senescence,total protein and RNA were extracted.The expression of Sirt3,P16 and P21 proteins were detected by Western blot or real-time PCR.2.Role of Sirt3 on stress-induced senescence of renal tubular epithelial cells in high glucose environment through Keap1/Nrf2 pathwayTransfection plasmids were transfected with Lipofectamin 3000 and P3000,HK-2 cells were transferred to a six-well plate and transfected with different proportions.Cells were collected after 48 h,and the transfection effect of p CMV3-Sirt3 was detected by Western blot.In order to observe the effects of Sirt3 over-expression on oxidative stress,stress-induced senescence and related pathways in HK-2 cells under high glucose stimulation,the cells were divided into the normal group(NG,5.5m M D-glucose),high glucose group(HG,30 m M D-glucose),high-glucose plus p CMV3-vector Control group(HG+Control,30 m M D-glucose + p CMV3-vector),high-glucose plus p CMV3-Sirt3 group(HG+Sirt3,30 m M D-glucose + p CMV3-Sirt3).After 48 h stimulation,ROS and membrane potential changes in mitochondria were detected by immunofluorescence,and SOD2 and CAT protein expressions were detected by Western blot.In order to further investigate whether the Keap1/Nrf2 pathway is involved in the protective effect of Sirt3 on renal tubular epithelial cells under high glucose stimulation,Nrf2-specific inhibitor(ML385)was added.After blocking the Keap1/Nrf2 pathway,the effection of Sirt3 over-expression on oxidative stress,stress-induced senescence and downstream related protein expression of HK-2 cells in high-glucose environment were observed.The cells were divided into normal control group(NG,5.5m M D-glucose),high glucose group(HG,30 m M D-glucose),and high glucose +p CMV3-Sirt3 group(HG+Sirt3,30 m M D-glucose + p CMV3-Sirt3),high glucose + p CMV3-Sirt3+ML385 group(HG+Sirt3+ML385,30 m M D-glucose + p CMV3-Sirt3 +ML385 5μM).All groups were stimulated for 48 h.Western blot was used to detect the expression of Nrf2 and the downstream proteins HO-1 and NQO-1,as well as the expression of SOD2,CAT,P16 and P21.3.Effect and mechanism of pyrroquinoline quinone on stress-induced senescence of HK-2 cells under high glucose stimulationIn order to determine the effect of pyrroloquinoline quinone on the activity of HK-2 cells in high-glucose environment,appropriate concentration was selected for subsequent experiments.HK-2 cells were divided into normal control group(NC,5.5mm D-glucose)and high glucose group(HG,30 m M D-glucose),high glucose plus different pyrroquinoline quinone concentration(1nmol/L,10nmol/L,100nmol/L,500nmol/L,1000nmol/L,10000nmol/L,10000nmol/L,100000nmol/L).After adherence to the wall,the original medium was replaced with a high glucose medium containing the corresponding pyrroquinoline quinone concentration.After 48 h,experiments were conducted.In order to study the effection of pyrroquinoline quinone on oxidative stress,stress-induced senescence and related pathways of HK-2 cells under high glucose,the cells were divided into normal control group(NG,5.5m M D-glucose),normal glucose plus pyrroquinoline quinone group(NG+PQQ,5.5m M D-glucose +PQQ 100 n M),high glucose group(HG,30 m M D-glucose),and high glucose plus pyrroquinoline quinone group(HG+PQQ,30 m M D-glucose +PQQ 100 n M).After 48 h,immunofluorescence was performed in each group,proteins and RNA were collected to detect the expression of proteins SOD2,CAT,P16,P21 by Western blot.In order to study the effection of pyrroquinoline quinone after Sirt3 silence,the cells were divided into normal control group(NG,5.5m M D-glucose),high glucose group(HG,30 m M D-glucose),high glucose plus pyrroquinoline quinone group(HG+PQQ,30 m M D-glucose +PQQ 100 n M),high glucose plus pyrroquinoline quinone plus Sirt3-si RNA group(HG+PQQ+si RNA,30 m M D-glucose +PQQ 100 n M+ Sirt3-si RNA).All groups were stimulated for 48 h.The expression of SOD2,CAT,P16,P21 and other proteins was detected by Western blot.Results:1.Expression of Sirt3 in renal tissue of diabetic nephropathy patientsImmunohistochemical results showed that Sirt3 was significantly expressed in the kidney tissues of the control group,and the expression of Sirt3 was significantly decreased in the DN group compared with the control group.Sirt3 was mainly distributed in renal tubular epithelial cells,and the expression of glomerular and renal interstitium was not obvious.The expression of P16 protein in renal tissues of the control group was low.Compared with the control group,it was significantly increased in DN group.P16 was expressed in glomerulus,renal tubules and renal interstitium.The results of Immunofluorescence co-staining showed that P16 was less expressed in renal tubular epithelial cells with more Sirt3 expression,while P16 was significantly increased in those with less Sirt3 expression.2.Expression and significance of Sirt3 in renal tubular epithelial cells under high glucose1)Immunofluorescence results showed that Sirt3 was mainly located in the cytoplasm.Compared with NG group,the expression of Sirt3 was significantly reduced after 48 h under high glucose.Results of Western blot and real-time PCR showed that the expression of Sirt3 changed under high glucose with the stimulation time.Compared with the NG group,it was no significant difference in the in the M group.For 6h stimulated under high glucose,the expression of Sirt3 increased,reached the maximum expression at 12 h,and then gradually decreased,significantly decreased at 48h(P<0.05).The difference was statistically significant.2)Compared with the control group,the proteins expression of P16 and P21 in HK-2 cells began to increase at 12 h after high-glucose stimulation,and basically reached a peak at 48 h.They all showed a time-dependent trend(P < 0.05).results of SA-β-gal staining showed that,compared with the control group,the positive expression of SA-β-gal increased significantly after 48 h under high glucose environment.3.Sirt3 alleviates stress-induced senescence in renal tubular epithelial cells under high glucose stimulation through the Keap1/Nrf2 pathway1)HK-2 cells were transfected with Lipofectamin 3000 3.75 ul,P3000 5ul and p CMV3-Sirt3 2ug for optimal transfection efficiency.2)Western blot results showed that the proteins expression of SOD2 and CAT induced by high glucose changed with the stimulus time,and both showed a trend of increasing and then decreasing(P<0.05).Sirt3 over-expression could increase the expression of proteins SOD2 and CAT.Compared with NG group,the expression of intracellular and mitochondrial ROS was significantly increased in HG group,and Sirt3 over-expression could reduce the accumulation of total ROS and intracellular ROS.Results of JC-1 showed that red fluorescence was significantly stronger than green fluorescence in HK-2 cells in NG group,while JC-1 green fluorescence was significantly enhanced and red fluorescence was significantly reduced in HG group after 48 h under high glucose stimulation.After Sirt3 over-expression,red fluorescence in cells was enhanced in high-glucose environment,suggesting that high-glucose stimulation can significantly reduce mitochondrial membrane potential in HK-2 cells,while Sirt3 over-expression can restore the decrease of mitochondrial membrane potential induced by high-glucose stimulation.3)Western blot showed that,compared with NG group,the proteins expression of P16 and P21 were significantly increased in HG group(P < 0.05),while Sirt3 over-expression reduced the expression of P16 and P21 in HK-2 cells under high glucose environment.The results of SA-β-gal showed that the positive expression cells induced by high glucose decreased significantly after over-expression of Sirt3.4)Western blot showed that the expressions of Keap1,Nrf2 and their downstream proteins NQO-1 and HO-1 changed with the stimulus time.Nrf2,NQO-1 and HO-1 all increased first and then decreased with the time of high glucose stimulation,and their expression was significantly reduced after 48 h under high glucose stimulation.5)Compared with the NG group,the expressions of protein Nrf2,NQO-1 and HO-1 in HG group were significantly reduced,while the expression of protein Keap1 was increased.Compared with HG group,Sirt3 over-expression significantly increased the proteins expression of Nrf2,NQO-1,HO-1,and nuclear Nrf2 in HK-2 cells under high glucose environment,while the protein expression of Keap1 decreased(P < 0.05).6)After ML385 was added,the enhancement of Sirt3 over-expressed on the Keap1/Nrf2 signaling pathway and its downstream NQO-1 and HO-1 were significantly inhibited.Western blot results showed that,compared with HG+Sirt3 group,the expressions of antioxidant proteins SOD2 and CAT were significantly decreased after the addition of ML385,while the proteins expressions of P16 and P21 were significantly increased.4.Effect and mechanism of pyrroquinoline quinone on stress-induced senescence of HK-2 cells under high glucose stimulation1)Under NG culture conditions,pyrroquinoline quinone at 10,100,500,1000 and 10000 n M could significantly improve the activity of HK-2 cells,whereas at 100000 n M,pyrroquinoline quinone inhibited the activity of HK-2 cells.The activity of HK-2 cells was significantly inhibited cultured for 48 h in high glucose.Pyrroquinoline quinone at concentrations of 10,100,500,1000,and 10000 n M protected HK-2 cells from high glucose-induced reduction in cell activity(P<0.05).Pyrroquinoline quinone at concentrations of 100 n M was used for further study.2)Compared with the NG group,the proteins expressions of SOD2 and CAT in HG group were significantly decreased,the activity of SOD was significantly decreased,and the levels of cells and mitochondria ROS were increased.Compared with HG group,the expression of antioxidant protein was significantly up-regulated in HG+PQQ group(P<0.05),and the accumulation of cells and mitochondria ROS was reduced.3)Compared with NG group,the proteins expression of P16 and P21 increased significantly after 48 h in hk-2 cells under high glucose stimulation(P<0.05).The number of positive cells increased.Compared with HG group,the expressions of P16 and P21 were both decreased in HG+PQQ group(P<0.05),and the positive cells were reduced.4)Immunofluorescence showed that,compared with HG group,the expression of Sirt3 was significantly increased in HG+PQQ group.Western blot and real-time PCR results showed that compared with NG group,protein and m RNA levels of Sirt3 were both decreased in HG group(P<0.05).In HG+PQQ group,the inhibitory effect of high glucose on Sirt3 was partially reversed(P<0.05).5)The protein expression of Sirt3 was significantly inhibited after transfection of sirt3-si RNA in HK-2 cells.Compared with HG+PQQ group,the proteins expressions of SOD2 and CAT in HK-2 cells were decreased under the co-culture condition of high glucose and pyrroloquinoline quinone after Sirt3 silencing(P<0.05),the expressions of P16 and P21 were significantly increased(P<0.05),and cells and mitochondria ROS were increased.6)Compared with NG group,the proteins expression of Nrf2,GPx-3,GST,HO-1 and NQO-1 in HK-2 cells were significantly down-regulated in HG group(P<0.05).Immunofluorescence showed a decreased Nrf2 signal.Compared with HG group,the expression of Nrf2,its downstream protein and nuclear Nrf2 in HK-2 cells were significantly increased after co-culture of high glucose and pyrroquinoline quinone.Results of Realtime PCR also suggested that pyrroquinoline quinone could up-regulate the level of Nrf2 m RNA.Conclusions:1.The expression of Sirt3 in renal tissues of diabetic patients and HK-2 cells under high glucose environment is significantly reduced,and the expression of stress-induced senescence proteins is increased.Sirt3 may be involved in the process of stress-induced senescence in diabetic nephropathy.2.Sirt3 over-expression can significantly reduce stress-induced senescence in HK-2 cells stimulated by high glucose.3.Sirt3 plays the role of antioxidant stress and anti-senescence through the Keap1/Nrf2 signaling pathway in HK-2 cells under high glucose environment.4.Pyrroquinoline quinone can reduce oxidative stress and stress-induced senescence in HK-2 cells under high glucose conditions.5.Pyrroquinoline quinone plays an important role in anti-oxidative stress and anti-senescence by up-regulating the expression of Sirt3 and the activity of the Keap1/Nrf2 pathway. |
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