SCP-88 Prevents High Glucose-induced Extracellular Matrix Proteins Expression In Rat Mesangial Cells

Background:Diabetic nephropathy is a major cause of end stage renal disease (ESRD) and brings heavy burden to the society. The pathology features of DN are characterized by mesangial expansion, glomerular basement membrane thickness and glomerular hypertrophy. The mechanisms by which DN initiate and develop remain indistinct. Mesangial cell is thought to be an important player in DN, because mesangium expansion is consistently observed in DN. The excessive ECM deposition has generally been considered as the central of the DN. Many in vitro investigations found exposing mesangial cells to high glucose and advanced glycation end products induced overproduction of ECM proteins.High glucose in serum is a feature of diabetes. Effect of high glucose on diabetic nephropathy has been concerned about. Study found that glycemic control can delay the development of diabetic nephropathy. The heightened ECM deposition has generally been considered as the central of DN. When the mesangial cells are stimulated by high glucose, the expression of extracellular matrix proteins increase and the enzyme degrades the extracellular matrix decreases. It makes extracellular matrix synthesis and degradation unbalance, leading to excessive accumulation of ECM and glomerular hypertrophy. Signal activated by high glucose plays an important role in diabetic nephropathy formation, especially the mechanism of ECM accumulation. Glucose enters into the mesangial cell mainly through the glucose transporter (GLUT-1) on the surface of cells. Stimulus of high glucose makes the expression of GLUT-1 increased, excessive glucose enter into the cell. The main metabolic pathway of glucose is glycolysis. But if too much glucose enter into cell, there are other pathways, including Polyol pathway, DAG pathway and the Hexosamin pathway. Mesangial cell produces too much sorbitol which is the production of polyol pathway, leading to the NADH/NAD+ratio increase and DAG de nove synthesis. Excessive DAG activates PKC which is an important kinase in the progression of mesangial cell injuried by high glucose. PKC belong to serine-threonine kinase family. Classic PKC include PKCa,β1,β2 andγ. PKC regulates vascular permeability, vasoconstriction, cell proliferation and extracellular matrix synthesis. PKC can activate the transcription factor c-fos and c-jun which could form AP-1 that can increase ECM proteins. It has been proved that PKC inhibitors can inhibit overexpression of extracellular matrix proteins (fibronectin and collagen IV) in mesangial cell exposed to high glucose. Thus, PKC plays a critical role in the progression of high glucose stimulating mesangial cell.Oxidative stress has been found in mesangial cell expsoed to high glucose. Oxidative stress is a condition in which cell is exposed to excessive levels of oxygen (O2) and derivatives of O2, The major one is superoxide (O2-). ROS induced by many factors, including high glucose, AGE, AOPP and so on. Although multiple pathways may result in ROS generation, studies indicate that a multicomponent phagocyte-like NAD(P)H oxidase is a major source of ROS in many nonphagocytic cell, including renal cell such as tubular epithelial cell and glomerular mesangial cell. ROS produced by mesangial cell exposed to high glucose is through PKC dependent NADPH oxidase activation. It is either an injury factor or a mesanger. It found that ROS is an important factor in the progression of ECM accumulation in mesangial cell exposed to high glucose. Using antioxidants, for example vitamin C, can inhibit ROS and ECM proteins expression.Recent studies have found that high glucose stimulates rat mesangial cell through activation of mesangial cell membrane endothelial growth factor receptor (EGFR), which activates PLC-yl and PI3K. PKC as the down-stream signal can increase the expression of ECM.SCP-88 is an extractive of Acorus tatarinowii. Our lab found that it is an antioxidant, could inhibit oxygen free radicals in supernatant of mesangial cell exposed to high glucose. There is a close relationship between ROS and ECM formation. So we study whether SCP-88 could inhibit ECM (fibronectin and collagen IV) expression in rat mesangial cell exposed to high glucose.Method1. Cell cultureRat mesangial cells were cultured in DMEM supplemented with 10%fetal bovin serum (GIBCO, USA),2mM glutamine, 100u/ml penicillin and streptomycin at 37℃, 5%CO2. After confluence reached 80%, mesangial cells were growth-arrested in 0.5% FCS for overnight and use for experiment.2. Determination of fibronectin and collagen IV mRNA by Real-Time PCRAfter confluence mesangial cells rest for 12 hours in serum-free medium, then divided into normal glucose groups and the high glucose groups. Cells of normal glucose groups were treated with 0, 10μM SCP-88 incubation for 1 hour. Cells of high glucose groups were treated with 0,0.1,1,10,50μM SCP-88 and 200u/ml SOD incubation for 1 hour. Then Cells of normal glucose groups were treated with 5.6mM glucose for 12 hours. Cells of high glucose groups were treated with 30mM glucose for 12 hours. Total RNA of RMCs was extracted with TRIZOL reagent. Determine fibronectin and collagen IV mRNA by Real-Time PCR.3. Determination of fibronectin and collagen IV protein by Western BlotAfter confluence mesangial cells rest for 12 hours in serum-free medium, then divided into normal glucose groups and high glucose groups. Cells of normal glucose groups were treated with 0, 10μM SCP-88 incubation for 1 hour. Cells of high glucose groups were treated with 0,0.1,1,10,50μM SCP-88 and 200u/ml SOD incubation for 1 hour. Then cells of normal glucose groups were treated with 5.6 mM glucose for 12 hours. Cells of high glucose groups were treated with 30mM glucose for 12 hours. Collect the protein islation and determinate fibronectin and collagen IV protein by Western Blot.4. Determination of p47pox phosphorylationAfter confluence mesangial cells rest for 12 hours in serum-free medium, then divided into normal glucose groups and high glucose groups. Cells of normal glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Cells of high glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Then cells of normal glucose groups were treated with 5.6mM glucose for 30 minutes. Cells of high glucose groups were treated with 30mM glucose for 30 minutes. Collect the protein and determine p47pox phosphorylation by immunoprecipitation and immunoblotting.5. Determination of p47phox binding to p22phoxAfter confluence mesangial cells rest for 12 hours in serum-free medium, then divided into normal glucose groups and high glucose groups. Cells of normal glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Cells of high glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Then cells of normal glucose groups were treated with 5.6mM glucose for 30 minutes. Cells of high glucose groups were treated with 30mM glucose for 30 minutes. Collect the protein and detect p47phox binding to p22phox by immunoprecipitation and immunoblotting.6. Determination of p47phox and p22phox protein expressionAfter confluence mesangial cells rest for 12 hours in serum-free medium, then divided into normal glucose groups and high glucose groups. Cells of normal glucose groups were treated with 0, lOμM SCP-88 for 1 hour. Cells of high glucose groups were treated with 0, lOμM SCP-88 for 1 hour. Then cells of normal glucose groups were treated with 5.6mM glucose for 12 hours. Cells of high glucose groups were treated with 30mM glucose for 12 hours. Collect protein and determine p47phox and p22phox by Western Blot.7. Determination of PKC phosphorylationAfter confluence mesangial cells rest for 12 hours in serum-free medium, then divided into normal glucose groups and high glucose groups. Cells of normal glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Cells of high glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Then cells of normal glucose groups were treated with 5.6mM glucose for 15 minutes. Cells of high glucose groups were treated with 30mM glucose for 15 minutes. Collect the protein and determine PKC phosphorylation by Western Blot.8. Determination of PLC-yl and PI3K-p85 phosphorylationAfter confluence mesangial cells rest for 12 hours in serum-free medium, then divided into normal glucose groups and high glucose groups. Cells of normal glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Cells of high glucose groups were treated with 0, 10μM SCP-88 for 1 hour. Then cells of normal glucose groups were treated with 5.6mM glucose for 15 minutes. Cells of high glucose groups were treated with 30mM glucose for 15 minutes. Collect the protein and determine PLC-γ1 and PI3K-p85 phosphorylation by Western Blot.Results1. Effect of SCP-88 on the viability of rat mesangial cellsThe result of MTT shows that there is no effect on the vaibility of cells stimulated by SCP-88 for 12 hours. Cells treated with 2,10,50μM SCP-88 for 12 hours have no difference from cells treated with only medium in the viability of cells.2. Effect of SCP-88 on fibronectin2.1 Effect of SCP-88 on fibronectin mRNAThe result of Real-Time PCR shows that fibronectin mRNA increased significantly when cells exposed to high glucose for 12 hours (P<0.05). SCP-88 can inhibit fibronectin mRNA in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on fibronectin mRNA in mesangial cells exposed to normal glucose (P>0.05).2.2 Effect of SCP-88 on fibronectin protein expressionThe result of Western Blot shows that fibronectin protein expression increased significantly when cells exposed to high glucose for 12 hours. SCP-88 can inhibit fibronectin protein expression in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on fibronectin protein expression in mesangial cells exposed to normal glucose (P>0.05).3. Effect of SCP-88 on collagenⅣ3.1 Effect of SCP-88 on collagenⅣmRNAThe result of Real-Time PCR shows that collagenⅣmRNA increased significantly when cells exposed to high glucose for 12 hours (P<0.05). SCP-88 can inhibit collagenⅣmRNA in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on collagen IV mRNA in mesangial cells exposed to normal glucose (P>0.05).3.2 Effect of SCP-88 on collagen IV protein expressionThe result of Western Blot shows that collagen IV protein expression increased significantly when cells exposed to high glucose for 12 hours (P<0.05). SCP-88 can inhibit collagen IV protein expression in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on collagen IV protein in mesangial cells exposed to normal glucose (P>0.05).4. Effect of SCP-88 on NADPH oxidase4.1 Effect of SCP-88 on p47phox phosphorylationCo-immunoprecipitation and Western Blot show that p47phox phosphorylation increased significantly when cells exposed to high glucose for 30 minutes (P<0.05). SCP-88 can inhibit phosph-p47p ox protein expression in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on phosph-P47phox protein expression in mesangial cells exposed to normal glucose (P>0.05).4.2 Effect of SCP-88 on p47phox binding to p22phoxCo-immunoprecipitation and Western Blot show that p47phox binding to p22 phox increased significantly when cells exposed to high glucose for 30 minutes (P<0.05). SCP-88 can inhibit p47phox binding to p22 phox in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on p47phox binding to p22phox in mesangial cells exposed to normal glucose (P>0.05).4.3 Effect of SCP-88 on p47phox and p22phox protein expressionThe result of Western Blot shows that p47phox and p22phox protein expression increased significantly when cells exposed to high glucose for 12 hours (P<0.05). SCP-88 can inhibit p47phox and p22phox protein expression in mesangial cells exposed to high glucose (P<0.05). Yet it have no effect on p47phox and p22 phox protein expression in mesangial cells exposed to normal glucose (P>0.05).5. Effect of SCP-88 on PKC phosphorylationThe result of Western Blot shows that PKCa and PKCβ1 phosphorylation increased significantly when cells exposed to high glucose for 15 minutes (P<0.05). SCP-88 can inhibit phosph-PKCa and phosph-PKCβ1 protein expression in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on phosph-PKCa and phosph-PKCp1 protein expression in mesangial cells exposed to normal glucose (P>0.05).6. Effect of SCP-88 on PLC-yl and PI3K-p85 phosphorylationThe result of Western Blot shows that PLC-yl and PI3K-p85 phosphorylation increased significantly when cells exposed to high glucose for 15 minutes (P<0.05). SCP-88 can inhibit PLC-γ1 and PI3K-p85 phosphorylation in mesangial cells exposed to high glucose (P<0.05). Yet it has no effect on PLC-yl and PI3K-p85 phosphorylation in mesangial cells exposed to normal glucose (P>0.05).ConclusionThis experiment confirmed that SCP-88 can inhibit the high glucose induced ECM overexpression in rat mesangial cells. It protects mesangial cells from high glucose by influencing PLCy1, PI3K-p85, PKC (PKCa and PKCβ1) and NADPH oxidase activation.