ABSTRACT
OBJECTIVE: To study the effect of fasudil on inhibiting the Rho/ROCK signaling pathway under high glucose in human mesangial cells (HMCs) inflammation and fibrosis. METHODS: Synchronized HMCs were divided into following groups: (1) Normal glucose control group (NG, 5.5 mmol/L glucose); (2) High glucose group (HG, 30 mmol/L glucose); (3) Mannitol group (Man, 5.5 mmol/L glucose + 24.5 mmol/L mannitol); (4) High glucose + fasudil group (HG + F, the concentrations of fasudil were 25, 50 and 100 µmol/L, respectively). Collect the supernatant and cells at 0, 12, 24, 36, 48 and 72 h respectively, and determine the concentration changes of the RhoA, ROCK-I, connective tissue growth factor (CTGF)mRNA with real-time PCR method in the cells, then used the ELISA method to check the protein content of the fibronectin (FN), CTGF, TNFα in the supernatant. RESULTS: (1) RhoA, ROCK-I and CTGF mRNA of the HMCs cultured under the high glucose expressed significantly higher than those in the normal group, and there was certain time-dependence. Besides, there was no statistic significance by comparing Man and NG. (2)Under the high glucose situation, after the fasudil pretreatment with different concentrations and 24 h or 48 h culture with high glucose, RhoA, ROCK-I, CTGF mRNA expression was significantly decreased in HG + F, compared with HG, and there was certain concentration-dependence. (3) High glucose increased the FN, CTGF, TNFα protein secretion of HMCs in a time-dependent manner, but normal glucose and mannitol had no such effect. (4) After the fasudil pretreatment with different concentrations and culture with high glucose for 12, 24, 36, 48, 72 h, the FN, CTGF, TNFα protein secretion was significantly reduced compared with HG. CONCLUSION: Fasudil can reduce the secretion of downstream inflammatory factors and cytokines by inhibiting high glucose-activated HMCs Rho/ROCK signaling pathway, and reduce the inflammation and fibrosis of HMCs. This provides a new basis for the therapeutic target in the treatment of diabetic nephropathy.
Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Mesangial Cells/drug effects , Mesangial Cells/metabolism , Signal Transduction/drug effects , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Cells, Cultured , Connective Tissue Growth Factor/metabolism , Culture Media/chemistry , Fibronectins/metabolism , Glucose/metabolism , Humans , Inflammation , Tumor Necrosis Factor-alpha/metabolism , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
OBJECTIVE: To investigate urinary excretion of platelet-derived growth factor-BB (PDGF-BB) during the different stages of diabetic nephropathy (DN) in type 2 diabetes mellitus (T2DM) as well as its clinical significance. METHODS: Sixty-five cases with T2DM were divided into three groups: normoalbuminuric group [N-UAlb; urine albumin excretion (UAE) <30 mg/24 h, n=25], microalbuminuric group [M-UAlb; UAE 30-300 mg/24 h, n=20], and macroalbuminuric group [L-UAlb; UAE >300 mg/ 24 h, n=20]. The urinary excretion rates of PDGF-BB were determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) in all the cases and 27 subjects of control. RESULTS: The excretion rates of PDGF-BB in T2DM groups were markedly higher than that in control (P<0.001). Moreover, the excretion rates of PDGF-BB increased with the increase of UAE and there were significant differences among the three groups (P<0.05) except the groups of M-UAlb and L-UAlb. Urinary PDGF-BB was also positively correlated with UAE, triglyceride (TG), cholesterol (CHO), low-density lipoprotein (LDL) and negatively correlated with creatinine clearance (Ccr), high-density lipoprotein (HDL), while had no significance correlated with glycohemoglobin A1c (HbA1c). CONCLUSION: PDGF-BB might play a very important role in the initiation and progression of DN. Measurements of urine PDGF-BB in T2DM could be used for early diagnosis of diabetic renal dysfunction.
