| Early renal mesangial cellular proliferation followed by subsequent fibrosis is a prominent hallmark of proliferative glomerulonephritis, and it may ultimately lead to end-stage renal disease. It is accepted that Phosphorylationtol 3 kinase/protein kinase B/mammalian target of rapamycin is an important pathway in cell life activity, it switchs cell life and death. Cytokine, high glucose, mechanical strain and so on can induce activation and proliferation of mesangial cells through the signaling pathway. Curcumin, a major compound extracted from the rhizomes of Curcuma species, has presents widely biological effects, such as anti inflammatory, anti oxidative, anti cancer and anti fibrosis. However, the research of curcumin on the kidney diseases and fibrosis is not well characterized. It is reported that curcumin can prevents adriamycin nephrotoxicity in rats. It is also reported that curcumin blocks TGF- β's profibrotic actions on renal fibroblasts. In this study, we explored the effect of curcumin on proliferation and fibrogenesis in rat mesangial cells, and possible correlation with intracellular PI3K/Akt/mTOR signaling pathway.The viability and proliferation of mesangial cell was determined with MTT method (tetrazolium salt colorimetry assay); the assay is based on the principle that live cells convert the yellow tetrazolium salt into a blue formazan. After quiescent mesangial cell cultured in vitro were stimulated with 10% fetal bovine serum, curcumin reduced cell proliferation in a concentration-dependent manner. Significant inhibition of cell proliferation was observed at curcumin dosages of 6.25 μM (19.5%) and 100 μM (89.7%), the median inhibition concentration (IC50) of curcumin was approximate 24.92 μM. The specific PI3K inhibitior LY294002 also inhibit cell proliferation in a dose-dependent manner, 50 μM LY294002 can inhibit about 60% cell growth. Rapamycin, the mTOR specific inhibitior, also inhibit cell proliferation in adose-dependent manner; the inhibition effect of 1nM rapamycin achieve 26.7%, but the inhibition effect of 100nM only get 46.3% . The IC50 of rapamycin was 381.14nM. The inhibition effect of curcumin combine rapamycin on mesangial cell is stronger than the effect of curcumin or rapamycin alone, the combination index of curcumin and rapamycin is below 1, this indicating they can exert synergic action. Pretreatment with either LY294002 or rapamycin, they all can weaken the inhibition effect of curcumin. This show the inhibition effect of curcumin on mesangial cells is get across PI3K/Akt/mTOR signal pathway. Flow cytometric analysis of propidium iodide stained mesangial cell demonstrated that cell cycle progression was arrested in G0/G1 phase with curcumin or rapamycin. Curcumin combine rapamycin can strongly induced cell growth arrest in G1 phase than curcumin or rapamycin alone.We performed time course of 10% fetal bovine serum-induced Akt phosphorylation from 0 to 120 min, the phosphorylation peaked at 30 min. Total Akt protein has no change. Either LY294002 or rapamycin reduced the Akt phosphorylation, especially treatment with LY294002. These results suggested that PI3K/Akt/mTOR signal pathway is required for mesangial activation. Curcumin inhibited the expression of Akt phosphorylation in a dose dependent manner, while pretreatment with LY294002 can reduced it. This indicates that PI3K/Akt pathway is involved in growth inhibition of curcumin. Curcumin also reduced the CTGF and Fn mRNA levels. Pretreatment with LY294002 can weaken this effect, but curcumin combine with rapamycin can enhance this effect. When the expression of CTGF and Fn pretein in curcumin induced in mesangial cells was checked, similar findings were observed.These findings suggest that curcumin inhibits mesangial cell proliferation and limits the expression of CTGF and Fn, the mechanism may be involved in the inhibiton of PI3K/Akt/mTOR signal pathway. Thus curcumin might be a potential antifibrotic drug in kidney diseases.
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