The Change Of PEDF And VEGF Expression In Rat Mesangial Cells Cultured With High Glucose And Cordyceps-containing Serum

Objective:Diabetic nephropathy(DN) is one of the major microvascular complications of diabetes mellitus.The early pathological process of DN are characterized by the thickening of glomerular basement membrane and expanded extracellular matrix (ECM), leading to glomerular hyperfiltration and microalbuminuria. It has been confirmed that high glucose can stimulate mesangial cells to synthesize many mesangial matrix, resulting in glomerulosclerosis.Several growth factors have been suggested to be involved in the pathogenesis of DN—specially, transforming growth factor(TGF)-β1,vascular endothelial growth factor(VEGF) and fibroblast growth factor(FGF). Under high-glucose conditions, the expression of VEGF in renal tissue increase, on the one hand, it causes increased vascular permeability, resulting in proteinuria; On the other hand, it increases monocyte-macrophage cell migration activity, the TGF-β1 production and extracellular matrix accumulation, promoting glomerulosclerosis. Researches show that pigment epithlium-derived factor (PEDF) may function as an endogenous inhibitor of TGF-β1 expression and play a protection role in DN.In vitro studies showed that high concentrations of glucose significantly decreased PEDF secretion in primary human glomerular mesangial cells, suggesting that hyperglycemia is a direct cause of the PEDF decrease in the kidney. Therefore, decreased expression of PEDF in diabetic kidneys may contribute to extracellular matrix overproduction and the development of DN.In this study, we use serum pharmacology method, intervening rat mesangial cells in vitro with high glucose and Cordyceps-containing serum. Through the PEDF and VEGF contents was measured, we further study the effects and mechanisms of Cordyceps sinensis (CS) on DN.Methods:1 Preparation of experimental serum:35 male SD rats were randomly divided into 3 groups: a normal control group(n=18), a group treated with low-dose CS[(5 g/ ( kg·d) , n =8)], and a group treated with high-dose CS[10g/ ( kg·d) , n =9]。Cordyceps extract per milliliter contains 0.98g Cordyceps powder. The above groups, gavage administration was used at a fixed time everyday. The group of NC were given normal saline. Administrating 8 days, we collected rats'blood through post-orbital venous plexus after the last administration 2 h later, centrifugated within 6 h, separated serum, inactivated at 56℃30 min, preserved at - 80℃refrigerator . 2 Cell Culture: HBZY-1 cells were inoculated with 10% FBS, 100U/ml penicillin, 100ug/ml streptomycin MEM medium, at 37℃, 5% CO2 conditions. Passaged every 2-3 days, use it when cells entered the logarithmic phase. Experimental sub-groups: ①Normal glucose control group (glucose concentration 5.6mmol/L + normal rat serum, NC)②High glucose group (glucose concentration 30mmol/L + normal rat serum, HG)③Low-drug group(glucose concentration 30mmol/L + low-dose Cordyceps-containing serum, CS-LD)④High-drug group(glucose concentration 30mmol/L + high-dose Cordyceps-containing serum, CS-HD), a total of four groups.Inoculate HBZY-1 cells to25cm2 cultivat bottles by 2×105 per bottle, stagnate them with serum-free MEM culture medium 24 hours after 24 hours adherent, so that synchronize cells to the telogen. Take out the medium, intervente 24,72 hours with four different MEM culture medium, and separately collect cells. Cell apoptosis rate was detected by flow cytometry; PEDF and VEGF mRNA expression in the mesangial cells was analysed with reverse transcription-polymerase chain reaction (RT-PCR);the change of PEDF and VEGF in the mesangial cells supernatants were examined by enzyme-linked immunosorbent assay (ELISA).Result:1 Compared with the NC group, HG glucose inhibited apoptosis by reduceing the percentage of cells in G0/G1 phase and increasing the percentage of cells in S phase (P <0.05). In CS-LD group and CS-HD group, G1 phase cells gradually increased, S phase cells decreased, the apoptosis rate of mesangial cell in the two groups was significantly higher than that in HG group (P <0.05), and that in CS-HD group was higher than that in CS-LD group (P <0.05). 2 RT-PCR results showed that: compared with NC group, the PEDF expression in HG group was significantly decreased (P <0.05) and the VEGF expression was significantly increased (P <0.05); compared with HG group, the PEDF expression in CS-LD group and CS-HD group significantly increased (P <0.05) and VEGF expression significantly decreased (P <0.05). 3 ELISA results showed that①Cells treated with four culture mediums for 24 hours, there was no significant difference of PEDF in the four mesangial cells supernatants (P> 0.05); Cells treated for 72 hours, the content of PEDF in HG group was significantly lower than that in NC group (P <0.05) and the expression of PEDF in CS-LD group and CS-HD group supernatants was higher than that in HG group (P <0.05). Between CS-LD group and CS-HD group, there was no significant difference (P >0.05).②Cultured with high glouse for 24 and 72 hours, the VEGF levels from HBZY-1 were higher than that in NC group (P<0.05). Cultured with Cordyceps-containing serum in two diffierent concentrations for 24 and 72 hours, the VEGF levels from HBZY-1 were decreased than that in HG group (P <0.05). Between CS-LD group and CS-HD group, there was no significant difference (P >0.05).Conclusion : 1 High glucose inhibited HBZY-1 cell apoptosis. Cordyceps sinensis could inhibit the proliferation of HBZY-1 induced by high glucose and promote the mesangial cell apoptosis in a concentration-dependent manner. 2 Under high-glucose conditions, PEDFmRNA expression decreased and VEGFmRNA expression increased in mesangial cells. It probably promote mesangial cell proliferation in diabetic nephropathy, and would be one of the mechanisms of glomerular sclerosis. Cultured with cordyceps, PEDF expression were increased and VEGF expression decreased, suggesting that Cordyceps may affect PEDF and VEGF expression on mRNA level and may play a important role on the inhibition to high glucose-induced mesangial cell proliferation.3 High glouse might increase the production of VEGF in HBZY-1 and decrease the production of PEDF, which could be reversed by cordyceps. It indicate that Cordyceps sinensis may play a protective role through regulating the prodution of cytokines in the mesangial cells.