The Mechanism Of STAT3/mTOR Regulation Of Autophagy In Angiotensin Ⅱ Induced Human Glomerular Mesangial Cell Senescence

Objective:Kidney is one of the fastest aging organs.The aging of kidney is particularly important,has become the main diseases affecting the health of the national.Therefore,exploring the mechanism of kidney aging,delaying the aging process of the kidney,has caused the attention of researchers.Cell senescence is the key link in the age related diseases.Multiple signal transduction pathways regulate renal cell senescence.In previous studies,the JAK2/STAT3 pathway was involved in the senescence induced by high glucose and angiotensin Ⅱ.But the unique role and mechanism of STAT3 activation in human glomerular mesangial cells induced by angiotensin Ⅱ is not clear.This paper discusses the effect of STAT3 on the senescence of human glomerular mesangial cells induced by angiotensin Ⅱ.As autophagy is an important mechanism for regulating senescence,STAT3 can activat or inhibite autophagy in transcription dependent or independent form depending on cell types or environments.This paper focuses on the mechanism of autophagy in regulating cell senescence.Angiotensin Ⅱ receptor antagonist agent losartan and antioxidant probucol is used to explore the role in delaying glomerular mesangial cell senescence,to provide a method of prevention and treatment of kidney diseases and aging.Materials and methods:1.Effects of autophagy on the regulation of cell senescence of human glomerular mesangial cell induced by angiotensin ⅡFirst we establish an cell senescence model using angiotensin Ⅱ in human glomerular mesangial cell.We conducted morphological assay,β-galactosidase staining and western blot detecting aging related protein p53,p21 protein expression.At the same time,the autophagy activity was detected by electron microscope and westernblot for LC3 Ⅱ and p62 protein.Autophagy inhibitors 3MA and autophagy inducer rapamycin will be used for evaluating the effects on senescence.2.The role of STAT3/mTOR in the regulation of autophagy on angiotensin Ⅱinduced senescence in human glomerular mesangial cellWestern blot is used for detecting the STAT3/mTOR.In senescent cells specific STAT3 inhibitor S3I-201 and STAT3 siRNA were given for observing the number of autophagosomes,β-galactosidase staining and expression of STAT3,p-STAT3,LC3 Ⅱ,p62,p53,p21,mTOR,,p-mTOR.3.The effects of losartan and probucol on autophagy and cell senescence induced by angiotensin Ⅱ of HGMC through STAT3/mTORLosartan and probuco pre-treatment of human glomerular mesangial cell,and then treated with angiotensin Ⅱ.We observe the number of autophagosomes,β-galactosidase staining,intracellular reactive oxygen species(ROS)and expression of STAT3,p-STAT3,LC3 Ⅱ,p62,p53,p21,mTOR,p-mTOR.Result:1.Effects of autophagy on the regulation of cell senescence of human glomerular mesangial cell induced by angiotensin ⅡThe positive rate of β-galactosidase staining in angiotensin Ⅱ induced human glomerular mesangial cells was 83.74±5.37%,and normal control group was 23.15±5.39%.Aging related protein p53,p21 was significantly higher than the normal control group,During the senescence of human glomerular mesangial cells induced by angiotensin,LC3Ⅱincreased significantly,and the ratio of LC3Ⅱ/LC3 Ⅰ was significantly higher than control group.The expression of p62/SQSTM1 was gradually decreased.The number of autophagosomes was significantly more than that in normal control group(43.50±9.42vs23.00±12.25,P<0.01)in 72 hours.Compared with angiotensin Ⅱ group,3mA reduced autophagosome formation(17.50±3.49 vs43.50±9.42,P<0.01),reducedβ-galactosidase staining positive rate(34.95±9.18 vs68.52±7.45,P<0.01),reduced LC3Ⅱ and the ratio of LC3Ⅱ/LC3 Ⅰ,reduced p53 and p21.The p62 protein expression increased significantly.Although Rapamycin increased the expression of LC3Ⅱ and autophagosome formation,not influence for p62,p53 and p21 protein expression and beta galactosidase staining.2.The role of STAT3/mTOR in the regulation of autophagy on angiotensin Ⅱinduced senescence in human glomerular mesangial cellAngiotensin Ⅱ on human mesangial cell increased the expression of STAT3 and p-STAT3,with mTOR pathway activity gradually reduced.The number of autophagosomes in angiotensin Ⅱ+S3Ⅰ-201group,angiotensin Ⅱ+STAT3-siRNA group and angiotensin Ⅱ group were 22.0±10.71,16.50±9.39 and 43.50±9.42,respectively,(P<0.01).The β-galactosidase staining positive rate in angiotensin Ⅱ+S3Ⅰ-201group,angiotensin Ⅱ+STAT3-siRNA group and angiotensin Ⅱ group were 55.26±12.53%,52.90±7.81%and 68.1±7.45%(P<0.05).Compared with the Ang Ⅱ group,using the S3Ⅰ-201 and transfection of STAT3 siRNA could significantly down regulated LC3Ⅱexpression,reduce ratio of LC3Ⅱ/LC3 Ⅰ,p53 and p21 expression,and increase p62 protein expression,but the mTOR and p-mTOR expression were increased.3.The effects of losartan and probucol on autophagy and cell senescence induced by angiotensin Ⅱ of HGMC through STAT3/mTORThe number of autophagosomes in the losartan group and the probucol group were 14.00±13.57 and 20.00±11.60,respectively,which were significantly lower than that of the angiotensin Ⅱ group 43.50±9.42(p<0.01).The positive rates of β-galactosidase staining were 54.87±7.83%,48.11±6.13%,which were significantly lower than that of the angiotensin Ⅱ group 68.10±7.45%.NAC,losartan,and probucol pretreatment significantly inhibite the production of reactive oxygen species(P<0.01).Simple application of losartan and probucol on normal cells had no significant effect on the expression of LC3Ⅱ,p62,p53 and p21,but in angiotensin Ⅱ stimulated for 72 hours,losartan,probucol can be significantly reduced protein LC3 Ⅱ expression and reduce the ratio of LC3Ⅱ/LC3 Ⅰ,inhibit angiotensin Ⅱ leads to p53 and p21 protein expression,inhibite of STAT3 activation,restore the activity of mTOR.