Preparation Of Marein Monoclonal Antibody And The Study Of The Antidiabetic Nephropathy Mechanism Of Marein

Objective:Diabetic nephropathy is the main cause of diabetic nephropathy（DN）and end-stage nephropathy in the world.Marein is one of the effective antidiabetic nephropathy components in Coreopsis tinctoria Nutt,but its mechanism is not clear.The aim of this study is to prepare a monoclonal antibody against marein and establish an indirect competitive enzyme-linked immunosorbent assay（ic-ELISA）for marein detection.Moreover,the protective mechanism of marein on DN was discussed in vitro and vivo.Methods:1.Preparation of marein monoclonal antibody:marein conjugates was prepared by sodium periodate method and mannich method.Cell fusion between mouse splenic cells and SP2/0 cells by PEG method,monoclonal hybridoma cells that can specifically recognized marein were selected.After subcloning,the antibody was prepared by ascites method and purified by saturated ammonium sulfate method.An indirect competitive ELISA method was established for the quantitative detection of marein in Coreopsis tinctoria Nutt extract and compared with HPLC method.2.The absorption mechanism of marein in vitro:MDCK cell monolayer was used to investigate the absorption and efflux transport characteristics of marein,the content of marein was determined by Ultra performance liquid chromatography coupled tandem mass spectrometry（UPLC-MS/MS）.Phloridzin（SGLTs non-selective inhibitor）and phloretin（GLUT2 inhibitors）were used to investigate the effect of sodium glucose transporters（SGLTs）and glucose transporter 2（GLUT2）on the transmembrane transport of marein.3.In vivo experiments:the effect of marein on SGLT2 expression in high glucose-induced HK-2 cells by Western-blot and RT-q PCR.AMPK/ACC signaling pathway,inflammation and fibrosis were also discussed in high glucose-induced HK-2 cells.4.In vivo experiments:db/db diabetic mice were divided into four groups:db/m group,db/db group,db/db empagloflozin（10mg/kg）group and db/db marein（50mg/kg）group,all of them were administrated for 12 weeks continuously.Biochemical,liver and kidney function parameters were measured.Renal histopathological changes were observed by PAS staining,Masson staining and Transmission Electron Microscopy.Western-blot and immunohistochemical were used to detect renal SGLT2 expression,and based on AMPK/ACC/PGC-1 pathway to discuss the renal protective mechanism of marein.Results:1.Preparation of marein monoclonal antibody:marein conjugates were prepared by Na IO₄and Mannich method,respectivly.The Thin layer chromatography results showed that both methods were successful,but only marein conjugates prepared by Mannich method produced marein specific antibody in mice.After fusing splenic cells of mice and myeloma cells,4B11 of marein monoclonal cell line was obtained by subcloning.And an indirect competitive ELISA was established to determine marein in the range of 156.25 to 5000 ng/m L（R²=0.991）,and the ic-ELISA had a good correlation with HPLC.2.The permeability coefficient of marein absorption and efflux transport on MDCK monolayer cells was in 1-4×10^-6cm·s^-1.When marein concentration was 60μg/m L,the transepithelial transport from the apical to basolateral（A-B）reached saturated.SGLTs inhibitor phloridzin could significantly decreased the absorbtion transport amount of marein,but GLUT2 inhibitor phloretin had no effect.3.In vivo experiments:marein could significantly inhibit 2-NBDG uptake by decreasing SGLT2 expression in HK-2 cells.In high glucose-induced HK-2 cells,marein decreased SGLT2 expression to normal level.Dapagliflozin had no effect on SGLT2 expression in normal or high glucose-induced HK-2 cells,but dapagliflozin could reverse the high glucose-induced increase on SGLT2 m RNA expression.Dapagliflozin,phloridzin and marein activated phosphorylated AMPK and phosphorylated ACC,and dapagliflozin could increase the m RNA expression of AMPK and ACC more significantly.Phloridzin and marein inhibited high glucose-induced renal extracellular matrices（COL1 and FN）and proinflammatory factors（MCP-1 and IL-6）expression.Moreover,marein could significantly inhibit the increase of SGLT2 expression on SGLT2-overexpressed HK-2 cell.4.In vivo experiments:marein and empagliflozin had no effect on body weight and kidney weight in db/db mice,but both of them significantly reduced fasting blood glucose and improved insulin resistance.Moverover,marein and empagliflozin could reduced serum triglycerides,cholesterol,and low-density lipoprotein in diabetic mice,improved the disorder of lipid metabolism by increasing serum adiponectin.Both of them decreased serum proinflammatory factor IL-6 and MCP-1 in diabetes mice,but had no effect on the total antioxidant capacity.Marein and empagliflozin had no effect on liver function in diabetic mice but could significantly improve renal dysfunction.PAS and Masson staining results showed that both marein and empagliflozin improved glomerular basement membrane thickening,glomerulosclerosis and tubular fibrosis.Empagliflozin and marein inhibited ectopic lipid deposition on renal tubules.SGLT2 expression was significantly increased in renal tubules in db/db mice.Marein and empagliflozin significantly inhibit SGLT2 expression in diabetic mice.Both of them improved renal lipid deposition and reducing renal lipotoxicity by activating p-AMPK/p-ACC/PGC-1 and inhibiting the expression of SREBP-1.Moreover,both marein and empagliflozin can alleviate kidney inflammation and fibrosis caused by diabetes.Conclusion:1.The established indirect competitive ELISA based on marein monoclonal antibody can be used for quantitative detection of marein in plant extracts,and had good relationship with HPLC.2.Marein belongs to a medium absorption compound,SGLTs may mediate transmembrane transport of marein on MDCK monolayer cells.3.Marein inhibits the expression of SGLT2 and glucose uptake in normal HK-2 cells.Marein could inhibit SGLT2 expression in high glucose-induced HK-2 cells and SGLT2-overexpressed HK-2 cells.Moreover,Marein relieves lipotoxicity by activating p-AMPK/p-ACC in high glucose-induced HK-2 cell.4.Marein inhibits the increased expression of SGLT2 on renal tubular in db/db mice.Marein relieves renal lipid ectopic accumulation and lipid toxicity by activating p-AMPK/p-ACC/PGC-1 pathway and inhibiting SREBP-1 expression.Furthermore,marein ameliorates renal injury by inhibiting fibrosis and inflammation.