Emodin Ameliorates High-glucose Induced Mesangial Cell Hypocontractility

Objective:Early stage diabetic nephropathy is characterized by elevated glomerular filtration. The pathogenesis of diabetic nephropathy involves many respects,but renal mesangial cell proliferation may play an important role in it.p38MAPK is the intersection of cell signaling pathway,which conduct extracellular signal to the intracellular, and mediate multiple cytokines and growth factors signal transduction, activate the corresponding target genes, resulting in biological effects.Recent studies have identified that high-glucose induced p38 MAPK over-activation in mesangial cells. PPARy is a known regulator of the p38 signal pathway, and PPARy activation blocks p38 activation. Some studies have demonstrated that emodin played a significant role in immunity,anti-inflammatory,anti-histiocyte proliferation by suppressing p38 signal pathway.Recently, studies from two groups have demonstrated that emodin has peroxisome proliferator-activated receptor y (PPARy) activation effects. Whether emodin ameliorate high-glucose induced p38MAPK over-activation via activation of PPARy is unknown. We investigated the role of PPARy protective effect in emodin in high-glucose treated mesangial cells.Methods:1. The establishment of high glucose:The DMEM cell-culture mediums with low and high glucose were applied;glucose concentration 5.6mmol/L for the low glucose control group, glucose concentration 30mmol/L for the high glucose experimental group. 2. The groups:mesangial cells were divided into five groups:the low glucose group (glucose 5.6mmol/L),the high glucose group (glucose 30mmol/L),the low-dose emodin group (50mg/L emodin+30mmol/L glucose),the high-dose emodin group (100mg/L emodin+30mmol/L glucose) and GW9662 group (lOμmol/L GW9662+ 30mmol/L glucose+100mg/L emodin).3. Mesangial cell contractility assay:Mesangial cell contractility was evaluated by measuring alternations in the cellular planar surface area. Angiotensionll was used as a contractile agonist. Cells were visualized using an inverted fluorescence microscope and images were captured before and after angiotension IⅡstimulation 30 min.4. Detection of the expression and activity of T-p38,p-p38 protein and PPARγprotein: T-p38,p-p38 protein activity and PPAR Y expresssion was detected by using Western blot.5. Detection of the expression of PPARγmRNA:The level of PPARγmRNA was observed by RT-PCR.Results:1.Effect of high glucose and emodin on contractility of GMCs:Mesangial cells demonstrated a 39%decrease in the planar surface area after angiotensionⅡstimulation. Compared with the NG group, cells cultured using 30 mmol/L glucose (high gucose group, HG) only exhibited a 12% decrease in the planar surface area (P <0.05). Mesangial cells demonstrated by a 22% decrease in the cell planar surface area in the low dose emodin group (LE) (50mg/l of emodin, P<0.05) and a 30% decrease in the high dose emodin group (HE) (100mg/l, P<0.05).2. Effect of emodin on the protein expression of p38MAPK and p-p38MAPK in high glucose:p38 activities were evaluated by measuring the protein levels of p-p38 cells and total p38 using Western blotting.Compared with the NG group, high glucose treatment resulted in a 280% increase in the p-p38 levels (P<0.01) while it did not affect the total p38 levels.Compared with the HG group, administration of 50mg/l and 100mg/l of emodin reduced p-p38 levels by 40%(P< 0.05) and 73%(P< 0.01),respectively.3. Effect of emodin on PPARγin different mesangial cells:Expression of PPARγwas evaluated by measuring mRNA and protein levels using real-time PCR and Western blotting. Compared with the HG group, administration of 50mg/l and 100mg/l of emodin resulted in a 151%(P<0.05) and 177%(P<0.01) increase in the PPARγmRNA levels, respectively. Consistent with these results, the protein content of PPARγwas also elevated by emodin treatment (196% elevation in the LE group and 421% elevation in the HE group, P<0.05).Conclusion:1. Our results showed high-glucose resulted in an increasement in p38MAPK activition associated with significant impairment of mesangial contractility;2. Emodin treatment dose-dependently inhibited high-glucose induced p38MAPK over-activation and mesangial hypocontractility was ameriolated by emodin;3. Both the PPARγmRNA and protein levels were elevated after emodin treatment;4. Inhibition of PPARγusing GW9662 effectively blocked the ameliorating effects of emodin on high-glucose induced p38 over-activation and mesangial hypocontractility.