The Effect Of FBW7 On Regulation Autophagy Signaling In Messagial Cells Induced By High Glucose

bjective: 20% ~ 40% patients with diabetes may develop diabetic nephropathy,and finally develop renal failure.Intensive glucose control can delay the onset of diabetic nephropathy,but the mechanism of hyperglycemia induced renal disease is not yet clear.Recent studies have shown that autophagy is involved in the development and progression of diabetic nephropathy,which can degrade senescent organelles,redundant proteins and harmful cytoplasmic components.Recent studies have shown that abnormal regulation of autophagy leads to the development of diabetic nephropathy.The level of autophagy in diabetic nephropathy was lower than that in normal mice.The most common negative regulation signal pathway is P13K-Akt-mTOR.mTOR can be used as the substrate protein of FBW7,which can be modified by ubiquitination and degraded by proteasome.FBW7 is SCF(Skp1-Cullin1-F-box protein)recognition subunit of the E3 ubiquitin ligase.What exactly is the expression of mTOR signaling molecules in diabetes mellitus,and whether FBW7 is involved in the regulation of mTOR signaling pathway in the pathogenesis of diabetic nephropathy? For this reason,we cultured mesangial cells in vitro to study the effect of high glucose on FBW7 and its effects on the regulation of autophagy.Methods :(1)The expression of FBW7,LC3-II/I,mTOR,p-mTOR,Col-I,IL-1β,caspase-1 and autolysosome in renal mesangial cell induced by different concentrations of glucose:The cells was cultured in vitro for 72 h and was grouped as follows(1)NC group;(2)OP group;(3)HG1group;(4)HG2 group;(5)HG3 group.The expression of FBW7,mTOR,p-mTOR and LC3-II/I were detected by Western blot;The mRNA of FBW7 was detected by RT-PCR,using the levels of autophagy lysosome weredetected by mRFP-GFP-LC3.ELISA was used to detect the expression of Col-I,IL-1 β,caspase-1.(2)The expression of FBW7,LC3-II/I,mTOR,p-mTOR,Col-I,IL-1 β,caspase-1and autolysosome in renal mesangial cell induced by glucose at different times:The 5.6 mmol / L glucose was used as control group,and 2h,6h,12 h,24h,48 h,72h were respectively induced by 30 mmol / L glucose,Using the above techniques to detect the levels of related proteins and autophagy.(3)The expression of FBW7,LC3-II/I,mTOR,p-mTOR,Col-I,IL-1 β,caspase-1 and autolysosome in renal mesangial cell induced by rapamycin: The cells was cultured in vitro for 72 h and was grouped as follows(1)NC Group;(2)NC-RAPA3 Group;(3)HG3 Group;(4)HG3-RAPA3 Group.Using the above techniques to detect the levels of related proteins and autophagy.(4)The expression of FBW7,LC3-II/I,mTOR,p-mTOR,Col-I,IL-1 β,caspase-1 and autolysosome in renal mesangial cell induced by gene silencing FBW7: The cells was cultured in vitro and was grouped as follows(1)NC Group;(2)NC-si FBW7 Group;(3)HG3 Group;(4)HG3-si FBW7 Group.Using the above techniques to detect the levels of related proteins and autophagy.(5)The expression of FBW7,LC3-II/I,mTOR,p-mTOR,Col-I,IL-1 β,caspase-1 and autolysosome in renal mesangial cell induced by Overexpression FBW7: The cells was cultured in vitro and was grouped as follows(1)NC-LV-CON238 Group;(2)NC-LV-FBW7 Group(3)HG3-LV-CON238 Group;(4)HG3-LV-FBW7 Group.Using the above techniques to detect the levels of related proteins and autophagy.Results: ⑴With different concentrations of glucose sustained stimulation of renal mesangial cells 72 hours,it was found that the higher the concentration of glucose,the lower the expression of FBW7,LC3-II/I and autolysosome,the higher the expression of mTOR,p-mTOR,Col-I,IL-1 β,caspase-1(P<0.05).⑵2h,6h,12 h,24h,48 h,72h were respectively induced by 30 mmol / L glucosein renal mesangial cells.Compared with the NC group,the expression of FBW7,LC3-II/I and autolysosome were significantly increased in the 12 h group,and mTOR,p-mTOR were significantly decreased in the 12 h group(P<0.05);Compared with the NC group,the expression of FBW7,LC3-II/I and autolysosome were significantly decreased in the 72 h group,and mTOR,p-mTOR,Col-I,IL-1 β,caspase-1 were significantly increased(P<0.05);The best condition of autophagy was 30 mmol / L glucose for about12 hours,and the weakest condition of autophagy was about 30 mmol / L glucose for 72 hours.⑶ The expression of LC3-II/I and autolysosome were significantly increased,mTOR,p-mTOR,Col-I,IL-1 β,caspase-1 were significantly decreased in renal mesangial cell induced by 200 nmol/L rapamycin(P<0.05).⑷ The expression of FBW7,LC3-II/I and autolysosome were significantly decreased,mTOR,p-mTOR,Col-I,IL-1β,caspase-1 were significantly increased in renal mesangial cell induced by gene silencing FBW7(P<0.05).⑸ The expression of FBW7,LC3-II/I and autolysosome were significantly increased,mTOR,p-mTOR,Col-I,IL-1 β,caspase-1 were significantly decreased in renal mesangial cell induced by Overexpression FBW7(P<0.05).Conclusion:1.High glucose can upregulate the expression of FBW7 in a short period,inhibit mTOR protein,which induced mesangial cell stress enhanced autophagic activity,may have a protective effect on the kidney in the early stage of diabetes.2.Long time high glucose can down regulate the expression of FBW7 and activate mTOR,resulting in the decrease of the level of autophagy in renal mesangial cells and the occurrence of renal inflammation and fibrosis.3.FBW7 on regulation autophagy signaling may be involved in the pathogenesis of diabetic nephropathy by induced by high glucose in Messagial Cells.