| BackgroundDiabetic nephropathy (DN) is one of the most common complications of diabetes, which is the chief reason leading to end-stage renal failure (ESRF). The basic pathological changes of early DN include excessive proliferation of mesangial cells (MCs), accumulation of extracellular matrix and incrassation of basement membrane. Excessive proliferation of MCs further promote the secretion of extracellular matrix. DN will eventually evolve to diabetic glomerulosclerosis. So that, excessive proliferation of MCs plays an pivotal role in the development of DN. Mang studies show that the forkhead box transcription factor O1(FoxO1) is the key regulatory factor in a lot of important signaling pathways, which was regulated in nuclear-cytoplasmic shuttling way by phosphatidylinositol-3-hydroxy kinase/protein kinase B(PI3K/Akt), thereby through regulating downstream target genes, play an significant role in glucolipid metabolism, oxidative stress, distribution of cell cycle, cell proliferation, cell apoptosis and tumorigenesis. Our previously studies have confirmed that the activity of FoxO1also decreased in renal cortex of DN rats and RMCs cultured under high glucose conditions, which indicate that excessive proliferation of mesangial cells in DN rat maybe have something to do with the activity levels of FoxO1. Through constructing-the lentiviral vectors, containing the coede sequence of constitutively active FoxO1, which upregulate the expression of constitutively active FoxO1in rat mesangial cells, we explore the roles and molecule mechanism of FoxO1on proliferation in rat mesangial cells cultured under high glucose conditions.ObjectiveTo study the roles and molecule mechanism of FoxO1on proliferation in rat mesangial cells cultured under high glucose conditions.MethodsThe constitutively active FoxO1lentiviral vectors (LV-CA-FoxO1) were constructed with four-plasmids packaging approach. The titer of lentiviral vectors and the optimal MOI were detected. RMCs were divided into four groups:the RMCs cultured in normal glucose (5.6mmol/L) medium served as normal control group (group NG), and the RMCs cultured in high glucose (25mmol/L) medium were divided into three groups:simple high glucose group (group HGO), high glucose+empty lentiviral vectors (group HG1), high glucose+LV-CA-FoxO1(group HG2). After RMCs in each group were cultured for72h, the mRNA expression of FoxO, cyclin kin.ase inhibitor p27(p27Kip1), growth arrest and DNA-damage inducible protein45alpha (Gadd45a) were detected by real-time quantitative PCR (qRT-RCR). The protein expression of FoxO1and phosphorylation FoxO1(p-FoxO1) were detected by western blotting. The proliferation and cell cycles of RMCs were analysed by MTT assay and flow cytometry (FCM) respectively.Results1. LV-CA-FoxO1were successfully constructed by sequencing analysis, the titer of lentiviral vectors was108TU/mL and the optimal MOI was100.2. Compared with group NG, the mRNA expression of p27Kip1was decreased and the protein expression of p-FoxO1was increased in group HGO (t=10.92,10.13, all P<0.05).3. Compared with group HG1, the mRNA expression of FoxO1, p27Kip1, Gadd45a were enhanced (t=439.1,130.9,266.6, all P<0.05) and FoxO1/p-FoxO1protein were increased (t=45.91,14.71, all P<0.05) in group HG2.4. Compared with group NG, the proliferation of RMCs in group HGO was advanced (t=30.28, P<0.05). Compared with group HG1, the proliferation of RMCs in group HG2was inhibited (t=54.31, P<0.05)5. Compared with group HG1, the cell cycles of RMCs in group HG2were arrested in Gl phase (t=14.51, P<0.05)Conclusions1. The transcriptional activity of FoxO1was decreased in RMCs cultivated with high concentration glucose, the proliferation of RMCs was promoted.2. Overexpression of active FoxO1inhibit the rapid proliferation and arrest cell eycle in Gl phase in RMCs cultivated with the high concentration glucose.3. Overexpression of active FoxO1inhibit the rapid proliferation of RMCs maybe through Akt/FoxO1/p27Kip1signaling pathway. |
PDF Full Text Request