Role Of TRB3in The Inhibitory Effect Of Fenofibrate Against High Glucose-Induced Proliferation Of Glomerular Mesangial Cells And Its Mechanism

Background:Diabetes mellitus (DM) is a global disease,and is harmful to human health, with its morbidity is rising year by year.China becomes the second largest diabetes country,only less than India. As the development of the this disease, chronic complications such as eyes, kidneys, nerves, heart, and blood vessels and so on will occur.Diabetic nephropathy (DN)is one of the most common diabetic microvascular diseases. Its early symptom is microscale urinary protein, as the illness worsens, it will gradually develop into diffuse and nodular glomerular sclerosis, eventually lead to end-stage renal failure. Every year about40%of diabetes patients could develop into DN patients in the western developed countries.Diabetic nephropathy is developed by glomerular mesangial cell proliferation and accumulation of matrix secreted protein and collagen protein in mesangial areaThe PI3K/AKT signal pathway widely exists in various kinds of cells. The combination of tyrosine growth factors with its ligand results in a series of reaction that can activate the activity of PI3K, Then PI3K combines with AKT, which can activate the phosphorylation of AKT, phosphorylated AKT can regulate the cell cycle by modulating the expression of cyclin. Amount of a previous study confirmed that high glucose can promote the proliferation of glomerular mesangial cells through PI3K/AKT pathway. Therefore,we think that we can inhibit the proliferation of glomerular mesangial cells by inhibiting PI3K/AKT pathway so as to alleviate diabetic nephropathy. However, in recent years, many studies have found that the inhibitors of P13K lack of specificity and are poisonous, and did not have much clinical practicability. Therefore, the way to inhibit the phosphorylation of AKT is a new direction for the inhibition of PI3K/AKT pathway.Tribbles homolog3(TRB3) is a kind of pseudokinase protein that can regulate cell mitosis. In recent years, many studies have found that TRB3can play a role in regulating cell cycle and promoting cell apoptosis by inhibiting the phosphorylation of AKT. This pathway has been approved the inpancreas, liver, lymphocyte and colon cancer study. In physiological conditions, there is a little expression of TRB3in kidney. Therefore, we hypothesize that we can inhibit the PI3K/AKT pathways by promoting the expression of TRB3which in turn to inhibit the proliferation of glomerular mesangial cell.In recent years,a large number of studies have showed that peroxidase proli-feration activated receptor(PPAR-a) can bind to the sequence between-750with-350of TRB3gene.PPAR-a agonist can play a role in inhibiting the PI3K/AKT pathway by promoting the expression of TRB3which in turn to promote cell apoptosis. Fenofibrate is a synthetic agonists of PPAR-α,Some studies have confirmed that fenofibrate may reduce cell number and cause cell cycle block by the way we mentioned above in lymphocytes and pancreatic cancer.Therefore,we speculate that fenofibrate inhibit the phosphorylation of AKT by augmenting TRB3expression, which in turn reduces the number of glomerular mesangial cells.The proliferation of glomerular mesangial cells is tightly under the regulation of cell cycle protein.Its cell cycle process is strictly accordance with the order of phase G1-S-G2-M.Whether cells can enter into the M phase to complete mitosis or not depends on the successful transition of G1/S phase and G2/M phase. Cell cycle is regulated by cyclin which located in downstream of phosphorylated AKT.We don’t know whether fenofibrate could reduce the number of glomerular mesangial cells by augmenting TRB3expression, which in turn induces cell cycle arrest by regulating the expression of cyclin.So we make use of glomerular mesangial cells as the research object to explore the expression of TRB3in glomerular mesangial cells and to make sure that fenofibrate can inhibit the proliferation of glomerular mesangial cells by augmenting TRB3expression, which in turn lead to cell cycle arrest by inhibiting phosphorylation of AKT.In order to f providing theoretical and clinical basis for fenofibrate in the treatment of DN.ObjectiveIn this project we aim to on the base of proliferation model of mesangial cell induced by higlucose in vitro, to observe the effects of fenofibrate on the expression of TRB3,investigate the effects of fenofibrate on phosphorylation of AKT, also to discuss the effects and mechanisms of fenofibrate on proliferation of glomerular mesangial cell induced by higlucose.Methods1%The preparation of fenofibrateTook360.84mg fenofibrate dissolved in10ml DMSO to make up100mmol/L mother liquor. In accordance with DMEM:fenofibrate=9:1, the mother liquid fenofibrate was diluted into100μmol/L fenofibrate. Then,100μmol/L fenofibrate was diluted into50,10μmol/L fenofibrate according to the proportion of1:1and1:9respectively.2、The cultivation and grouping of glomerular mesangial cells(MC)The glomerular mesangial cells in logarithmic growth phase were vaccinated in6,96,24orifice according to the appropriate proportion and the cells adhered the orifice standing after6hours.The culture medium was replaced with DMEM containing1%fetal bovine serum to synchronizate the cells in phase of GO.The mesangial cells were divided into normal control group (N,5.5mmol/L glucose), the high glucose group (H,25mmom/L glucose), high glucose+10umol/L fenofibrate group, high glucose+50μmol/L fenofibrate group, high glucose+100μmol/L finofibrate group.3、Cell counting kit-8(CCK-8) was used to determine the cells activityThe glomerular mesangial cells was vaccinated of in the96-well plates with density of1×104/ml, and then gave the intervention according to the groups.After incubation of24hour, PBS was used to wash every hole and then added100ul DMEM and10∴lCCK8to each hole. The blank control group contained DMEM and CCK8without cell.we used enzyme standard instrument to measure the absorbance to determine the cell vitality at450nm wavelength.4、Hoechst33258Staining and acridine orange staining were used to determine to determine morphological changes of apoptosis of MCs.The glomerular mesangial cells was vaccinated of in the6-well plates with density of1×105/ml,and then gave the intervention according to the groups.After incubation of24hours, PBS was used to wash every hole twice and4%paraformaldehyde was used to fixed mesangial cell,Hoechst33258or acridine orange staining were added to observed the effect of fenofibrate on glomerular mesangial cell apoptosis with fluorescence microscope.5、Flow cytometry was used to determine the change of cell cycle after cultured with fenofibrateThe glomerular mesangial cells was vaccinated of in the6-well plates with density of1×106/ml,and then gave the intervention according to the groups.After incubation of24hours,Pancreatic enzyme was used to digest cells of different of groups, and collected the cells suspension, centrifuged the cells suspension,and then discarded the supernatant.PBS was used to wash cells, and adjusted cell concentration to1x106/ml, the cells were washed twice with PBS and then fixed with70%ethanol for the night. After centrifuged and washed cell twice with PBS, RNA enzyme were added into centrifuge tube for watering for thirty minutes at37℃, then stained for thirty minutes at4℃with propidium iodide.The testing samples were put into to the flow machine and the Cell Quest software were used to analysis the Cell cycle changes.6、Immunocytochemistry was used to detect the expression of TRB3in different groups.The glomerular mesangial cells was vaccinated in the24-well plates with density of1×104/ml, and then gave the intervention according to the groups.After incubation of24hours, cells were fixed with paraformaldehyde for thirty minutes and washed three times with PBS.The cells were closed endogenous peroxidase with3%H2O2-methanol for ten minutes, washed three times with PBS.The cells were incubated with0.3%of Triton,and then washed cells three times with PBS. The cells were closed in nonimmune rabbit sera wet box for thirty minutes,sucked away excess serum, added TRB3antibody and then incubated in4℃for the night. Added100μ second antibody into cells, the cells were incubated in37℃for thirty minutes,washed three times with PBS and stained with the ABC method.The cells were showed with DAB chromogenic,then were redyed with haematin,dehydrated with gradient alcohol, dropped with mounting agent.Immune fluorescence microscope was used to observe the expression of TRB3.7、The effect of fenofibrate on the expression of TRB3and p-AKT of glomerular mesangial cells Cells were cultured for24hours after intervention,glomerular mesangial cells were harvested and lysed in150μl NP-40lysis buffer, the protein concentration was determined by the BCA method, resolved by SDS-PAGE with100volt for one hour, blotted on PVDF with semi-dry transmembrane device. Membranes were blocked in5%nonfat milk for one hour. After membranes were washed, the TRB3polyclonal antibody (1:500diluted) or P-AKT (1:500diluted) monoclonal antibody were added and incubated in4℃for the night. After washed, the second antibody (1:2000diluted) were added and incubated for one hour. ECL was added, X-ray were exposed in the darkroom, and were developed, fixed and then scaned.The results can be analysed with Image analysis system. The relative expression quantity of the protein was semi-quantitatively analysed with grey value of the target protein and GAPDH as internal protein. The above experiments were repeated3times.8、Statistical MethodsSPSS13.0statistical soft was used to statistical analysis.Statistical analysis results were expressed as mean±SD. Statistical significance of multiple was determined using One-Way ANOVA. Homogeneity of variance analysis was did, comparison between the two groups using LSD test. When the variance of arrhythmia, used of robust estimation Welch, then Dunnett’s T3was used. The test criterion is at a value of P<0.05.Results1. The OD value were (0.80±0.06) in high glucose group, and significantly higher than(0.58±0.05) in control group (P=0.000).The OD value were (0.67±0.02)、(0.56±0.02)、(0.44±0.03), respectively in10、50、100μmol/L fenofibrate group,significantly lower than high glucose group (P=0.000).There were statistically significant difference among groups (F=71.584, P=0.000)2. The apoptotic cells in fenofibrate groups showed topic morphological changes were observed using acridine orange staining and hoechst33258staining. Apoptotic cells were observed as intact round nuclei and fragmented (or condensed) nuclei.3. The proportion of glomerular mesangial cells in G0/G1phase was (71.81+0.92)%in high glucose group,and significantly lower than (85.72±0.35)%in control group (P=0.000).The proportion of glomerular mesangial cells in G0/G1phase was (78.78±4.12)%,(82.04±0.43)%,(84.71±0.21)%respectively in10、50、100μmol/Lfenofibrate group,significantly higher than high glucose group (P=0.000).There proportion of glomerular mesangial cells in GO/G1phase were statistically significant difference among groups (F=115.775,P=0.000)4.There were expression of TRB3in control group-high glucose group and fenofibrate group,The expression of TRB3located in the cytoplasmof glomerular mesangial cells.In both of control group and high glucose group,cytoplasm of glomerular mesangial cells were stained yellow-brown,and the expression of TRB3is weakly positive.In fenofibrate group,cytoplasm of glomerular mesangial cells were stained brown,the expression of TRB3is strongly positive.As the increase of concentration of fenofibrate,the TRB3expression enhanced.5. TRB3protein relative grayscale value was (0.34±0.03) in high glucose group, and grayscale value was (0.37±0.01) in control group,there was no significant difference between both group (P=0.568).The grayscale value were (0.52±0.04),(0.58±0.01),(0.62±0.03) respectively in10、50、100μ×mol/Lfenofibrate group, significantly higher than high glucose group (P=0.000).p-AKT protein relative grayscale value was (1.84±0.02) in high glucose group,and significantly higher than (1.39±0.04) in control group (P=0.000).Grayscale value was (1.83±0.03) in10μmol/L fenofibrate group,there was no significant difference contrast with high glucose group (P=0.079).The grayscale value were (1.58±0.03)、(1.45±0.04) respectively in50、100μmol/Lfenofibrate group,significantly lower than in high glucose group (P=0.000)ConclusionFenofibrate can inhibit the proliferation of mesangial cell, blocked the cell cycle. The mechanism may be that fenofibrate can promote the expression of TRB3which can regulate the expression of cylin through inhibiting phosphorylation of AKT.