| Objectives: Diabetic kidney disease(DKD)is the most common microvascular complications and is a leading cause of chronic kidney disease(CKD).The crucial pathology underlying progressive CKD in diabetes including tubular injury,glomeruloslerosis and renal tubulointerstitial inflammation.Thus,there remains an urgent request to prevent or slow the progression of diabetic kidney disease and to protect kidney function of diabetic kidney disease.Many studies demonstrated that peroxisome proliferator-activated receptor alpha(PPAR-?)could increase the expression of liver X receptor alpha(LXR-?)in macrophages,which subsequently mediated lipid metabolism.In addition to their important role in regulating lipid metabolism,PPAR-? and LXR-? exhibit anti-inflammatory effects in many inflammatory and metabolic diseases.Recent studies found that the PPAR-? activator fenofibrate ameliorates inflammation by inhibiting NF-?B activity and suppressing the production of the proinflammatory cytokines ICAM-1,TGF-?1,and MCP-1.The activation of LXR-? also inhibits the production of the proinflammatory cytokines MCP-1,ICAM-1 and TGF-?1.Thus,PPAR-?and LXR-? are physiological regulators that lie at the intersection of lipid metabolism and inflammation.Many studies have shown that anthocyanidin is a kind of widely found in plants in the natural pigment,have strong hypoglycemic,hypolipidemic,anti-apoptosis,anti-cancer effects.Anthocyanins exhibit powerful antioxidative properties in diabetic animal models and also exert protective effects against hyperglycemia-induced kidney injury.Previous studies have shown that anthocyanin acts on the cholesterol transport pathway in renal tubularepithelial cells under diabetic conditions,thus relieving renal lipid deposition in diabetic nephropathy.In addition,many studies suggested that anthocyanins also have anti-inflammatory effects.Purple corn anthocyanins(PCA)prevent HG-induced glomerulosclerosis and renal fibrosis via NF-?B-dependent mechanisms.However,there were no studies have revealed the mechanisms involved in the reciprocal regulation of inflammation and cholesterol efflux by anthocyanins in DKD.In this study,HK-2 cells were used to perform experiments.Cells were collected after 24 or 48 hours of intervention.To observe the effect of anthocyanin on the secretion of inflammatory factors in HK-2 cells stimulated by high glucose and its mechanism.Methods:The HK-2 cells were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum in a 5% CO2,37°C atmosphere.LXR-?-shRNA plasmids were transfected into human renal tubular epithelial HK-2 cells by lipofectamine 2000 transfection reagent.Cells were cultured in selection medium containing 0.3mg/ml G418 before single clones were isolated.After 75% to 85% confluence,the cells were cultured in serum-free medium for 24 h.After this time period,cells were randomly divided into group: normal glucose group(5.6 mmol/L glucose,NG),high glucose group(30mmol/L glucose,HG),high glucose+C3G group(30mmol/L glucose+50?mol/L cyanidin-3-O-?-glucoside chloride,HG+C3G),high glucose+Cy group(30mmol/L glucose +50?mol/L cyanidin chloride,HG +Cy),high glucose + GW6471 group(30mmol/L glucose +25?mol/L GW6471,HG + GW6471),high glucose + LXR-?-shRNA group(30mmol/L glucose +LXR-?-shRNA,HG+LXR-?-shRNA).The groups were cultured for 24 or 48 hours respectively,and then HK-2 cells were harvested.The protein expression of LXR-?,PPAR-?,NF-?B,MCP-1,ICAM-1 and TGF-?1 were detected by Western blot.The mRNA levels of LXR-?,PPAR-?,MCP-1,ICAM-1were evaluated by Real-time PCR.The MCP-1,ICAM-1 and TGF-?1activity were examined by ELISA.Immunocytochemistry was used to detect the expression of NF-?B in HK-2 cells.Results:1 Effects of anthocyanins on HK-2 cell viability.Compared with the control group,the growth of cells in high glucose(HG)was not inhibited significantly.Compared with high glucose,the cell viability of HK-2 cells was inhibited significantly in HG + C3 G or Cy group.That suggested that cell viability of HK-2 cells in HG was significantly effect by anthocyanins.2 Effects of anthocyanins on expression of MCP-1,ICAM-1 and TGF-?1 in HK-2 cells stimulated by high glucose.The results showed that the expression of MCP-1,ICAM-1 and TGF-?1were significantly increased in the high glucose group(HG)compared with the normal control group.Compared with the high glucose group(HG),the expression of MCP-1,ICAM-1 and TGF-?1 in the cells and cell culture supernatant of HG+ C3 G or Cy group decreased significantly,which indicated that anthocyanin could inhibit the proliferation of HK-2 cells in the secretion of inflammatory factors.3 Effects of anthocyanins on expression of NF-?B in HK-2 cells stimulated by high glucose.The results showed that compared with the normal control group,the transfer of NF-?B from cytoplasm to nuclear was significantly higher in the high glucose group.Compared with the high glucose group(HG),the transfer of NF-?B from cytoplasm to nuclear was decreased in HG+C3G or Cy group,indicating that anthocyanin can inhibit the transfer of NF-?B from cytoplasm to nuclear in high glucose stimulated HK-2 cells.4 Effects of anthocyanins on the expression of PPAR-? and LXR-? in HK-2cells stimulated by high glucose.The results showed that the expression of PPAR-? and LXR-? were significantly increased in the high glucose group(HG)compared with the normal control group.Compared with the high glucose group(HG),the expression of PPAR-? and LXR-? were significantly decreased in HG + C3 Gor Cy group.That suggested that anthocyanins could inhibit the expression of PPAR-? and LXR-? in HK-2 cells stimulated by high glucose.5 Effects of inhibited of PPAR-? and LXR-? on expression of MCP-1,ICAM-1 and TGF-?1 in HK-2 cells stimulated by high glucose.The expression of MCP-1,ICAM-1 and TGF-?1 in high glucose group(HG)were significantly higher than those in normal glucose group(HG).Compared with the high glucose group(HG),the expression of MCP-1,ICAM-1 and TGF-?1 were significantly decreased in HG + LXR-?-shRNA,but not in HG + GW6471.That indicating anthocyanin inhibited HK-2 cells in high glucose-stimulated MCP-1,ICAM-1 and TGF-?1 secretion may be achieved by inhibiting LXR-?.6 Effects of inhibited of LXR-? on the expression of NF-?B in HK-2 cells stimulated by LXR-?.The results showed that compared with the normal control group,the transfer of NF-?B from cytoplasm to nuclear was significantly higher in the high glucose group(HG).Compared with the normal high glucose group,the transfer of NF-?B from cytoplasm to nuclear could be inhibited by knockdown of LXR-? in HK-2 cells stimulated by high glucose.Conclusion:1 Anthocyanin have an effect on HK-2 cell activity in high glucose condition.2 Anthocyanin inhibited the secretion of proinflammatory cytokines MCP-1,ICAM-1 and TGF-?1 in HK-2 cells induced by high glucose.3 Anthocyanin inhibited the transfer of NF-?B from cytoplasm to nuclear in HK-2 cells induced by high glucose.4 Anthocyanin inhibited high glucose-stimulated proinflammatory cytokines MCP-1,ICAM-1 and TGF-?1 secretion by inhibiting LXR-?.5 Anthocyanin inhibited the NF-?B from cytoplasm to nuclear in HK-2cells induced by high glucose may be mediated by LXR-? expression. |
PDF Full Text Request