| ObjectiveTo investigate the effects of insulin at different concentrations on glomerular mesangial cells growth conditions and to research the regulation of PI3K/AKT/ gsk-3 β signaling pathway and changes in intracellular calcium ions and TRPC6, investigate the protective effect of Astragalosides Ⅳ (As-Ⅳ) on glomerular mesangial cells under high insulin condition and its mechanisms, to provide a reasonable theoretical basis for diabetic nephropathy (DN) prevention and treatment.Methods1. The HMCs were cultured with DMEM medium containing 10% fetal calf serum, and incubated at 37℃ with 5% CO2 in the constant temperature incubator. The HMCs at logarithmic growth phase were digested with 0.25% trypsin and taken for the subsequent experiment.2. The cells were randomly divided and treated as follows:(1) normal group (NG), insulin (INS, respectively treated with 2,4,8,16,32,64,128 nmol/L insulin) group; (2) normal group (NG), high insulin group (HINS,128 nmol/L), As-Ⅳ (12.5,25,50, 100 μmol/L) and Tempol (100 μmol/L), LY294002 (10 μmol/L); (3) normal group (NG), high insulin group (HINS,128 nmol/L), As-IV (25,50,100 μmol/L) and Tempol (100 μmol/L), LY294002 (10 μmol/L)3. The cells were randomly divided and treated as 2 (1), MTT assay was applied to detect the value of OD at different concentrations and time to make sure the optimal concentration and time of insulin were chosen according to the results. Then the cells were randomly divided and treated as 2(2), MTT assay was also applied to detect the value of OD make sure the Optimal drug concentration is selected for the subsequent experiment at the same time.4. The cells were randomly divided and treated as 2(3), by enzyme-linked immunosorbent assay detecting 1-type plasminogen activator inhibitor (PAI-1) and regulated upon activation plasminogen activator (PA) in each group of cell supernatants, and using the 2,7-dichlorodihydro fluorescin diacetate (DCFH-DA) to detecte the content of reactive oxygen species (ROS) in each group. Additionally, using the Western blot to detect the expression of phosphor-protein kinase b (p-PKB, also known as p-Akt) and total akt, phosphorylation of glycogen synthase kinase 3β (p-gsk-3β) and total gsk-3β and the up and down-regulated of TRPC6 protein expression. Flu-3/AM as a fluorescent probe for detecting a packet of intracellular calcium ion concentration in 2 (3) group.Results1. Checking for the microscopic result, compared with the NG, high insulin group HMC increased in number, cell volume and morphological dilation and accompany with an obvious fusion on the edge of HMC which imply an injury of HMC under high insulin condition. Compared with high insulin group, Tempol group, LY294002 group and different concentrations of As-IV (12.5,25,50,100 μmol/L) group can inhibit cell proliferation, and changes in morphology which suggest that As-IV has protective effects on high insulin-induced HMC injury.2. The results of MTT assay show to be investigated that the proliferative effect of high insulin condition on the HMC can be increased by improving action time and concentration. Compared with NG, high insulin group could increase HMC proliferation after a certain period of 12 hours (P<O.01). Compared with high insulin group, Tempol group, LY294002 group and different concentrations of As-IV (12.5,25,50,100 μmol/L) group can inhibit the excessive proliferation of HMC under high insulin condition with varying degrees (p<0.05 or p<0.01). MTT results showed a dose-dependent, the higher concentration of As-Ⅳ, the greater inhibition it does, As-Ⅳ (12.5 μmol/L) group was not statistically significant (p> 0.05).3. The results of ELISA assay show to be investigated that compared with NG, the contents of PAI-1 in HMCs induced by high insulin increased significantly, but the contents of PA and the ratio of PA/PAI-1 was low (p<0.01). Compared with HINS group, Tempol, LY294002, and As-Ⅳ (25,50,100 μmol/L) could regulate the abnormal secretions of PA and PAI-1 induced by high insulin (p<0.05,p<0.01), ELISA results showed a dose-dependent manner, suggesting that As-Ⅳ can effectively control the synthesis and degradation of extracellular matrix components mesangial matrix.4. According DCFH-DA can check the results, compared with the NG, the fluorescence intensity is more significant in high insulin group, suggesting that insulin was significantly increased ROS in cells (p<0.01), compared with the high insulin group, Tempol group, LY294002 group and different concentrations of As-Ⅳ (25,50,100 μmol/L) group weakened fluorescence intensity in varying degrees, suggesting that ROS intracellular content of each group have different degrees of reduction (p<0.05 or p<0.01), as result, As-Ⅳ can effectively reduce the oxidative stress under the hyperinsulinemia HMC environment.5. According to Western blot showed that results can be found, compared with the NG, high insulin group significantly increased protein expression of p-akt and p-gsk-3β, and decreased TRPC6 protein expression (p<0.01); compared with high insulin group, p-akt and p-gsk-3β protein expression were significantly decrease in Tempol group, LY294002 group and different concentrations of As-Ⅳ (25,50,100 μmol/L) group and TRPC6 protein expression was increased (p<0.05 or p<0.01), which reached the best effect is the highest concentration of As-Ⅳ group.6. According to the calcium ion concentration test results showed that, compared with the NG, the fluorescence intensity is weaker in high insulin group, suggesting that a significant reduction of intracellular free calcium concentration in the high insulin (p <0.01), compared with the high insulin group, fluorescence intensity of Tempol group, LY294002 group and different concentrations of As-IV (25,50,100 μ mo1/L) group enhanced in varying degrees, suggesting that intracellular free calcium concentration is in varying degrees of improvement in each group(p<0.05 or p<0.01), obviously, As-IV improve the calcium inflow reduction which induced by high insulin.ConclusionInsulin can promote the proliferation of HMC cultured in vito, and the effect was in a time-and concentration-dependent manners. As-IV has protective effects on HMCs induced by high insulin, and the effect was in a concentration-dependent manner. The protective mechanism of As-IV may on the basis of inhibiting the cell proliferation, improving the statement of cellular oxidative stress, decreasing the high protein expression of p-Akt, p-gsk-3 β, increasing the protein expression of TRPC6 and intracellular free calcium concentration, regulating the abnormal secretion about PA、 PAI-1、 RANTES, then, reducing extracellular matrix accumulation. |
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