| Diabetic nephropathy (DN) is one of the most serious complications of diabetes mellitus (DM). However, the mechanism of this pathological progression has not been fully elucidated, hence there is no satisfactory therapeutic method in treating DM. The early pathological characteristic of DN is glomerular hypertrophy and glomerular extracellular matrix (ECM) accumulation. The glomerulosclerosis and fibrosis is the late pathological feature.A common and key pathological feature is ECM accumulation. To investigate mechanism of ECM accumulation in the pathogenesis of DN is beneficial for clinical treatment of DN.Recent studies indicated that cyclooxygenase-2 (COX-2) is increased in DN kidney, the COX-2 inhibitor can protect kidney damage in DN, but it is not clear the role of COX-2 in the progression of DN. CTGF, as a downstream of transforming growth factor-β(TGF-β), can mediate the effect of TGF-βthat related to the ECM accumulation, NF-κB plays a crucial role in the apoptosis and inflammation in DN conditions, however, the relationship between COX-2 and NF-κB, COX-2 and CTGF in the ECM accumulation under DN condition is not investigated.COX-2 inhibitor can ameliorate the progression of renal disease. However, this inhibitor has significant side effect including cardiovascular disease and acute renal failure, especially in the old patients, it is very important to use non-COX-2 inhibitor, to decrease COX-2 expression in DN to ameliorate progression of DN.Fluvastatin can reduce proteinuria, ameliorate glomeruloscierosis independent of lipid level, but the mechanism underlying is not so clear. It is very important to investigate whether fluvastatin can reduce COX-2, NF-κB, CTGF expression in DN condition that related to the progression of DN.Our purpose is to investigate: (1) expressions of nuclear COX-2 factor (NF)-κB, connective tissue growth factor (CTGF), and fibronectin (FN) in DN; (2) interactions of NF-κB, COX-2, and CTGF in the progress of DN; (3) the relationship between COX-2 and CTGF in DN, to clarify the mechanism of COX-2 in the inducing of ECM accumulation and effects of fluvastatin on the influences of COX-2, NF-κB, CTGF, FN,TypeⅣcollagen ColⅣ(ColⅣ) and its mechanism underlying in DN. (4) to observe expression of COX-2 and total protein in mesangial cell which was stimulated by high concentration of glucose and prostaglandin (PG) E2, further to clarify the relationship between COX-2 and its productions of PGE2, and the interaction of COX-2 and mesangial cell hypertrophy in the pathogenesis of DN.We performed in vivo and in vitro experiment, Part one (in vivo experiment): healthy male wistar rats were randomized into four groups of similar body weight. (1) control group; (2) DN group induced by intraperitoneal injection of 55mg/kg (0.5%) streptozotocin (STZ); (3) DN plus celecoxib treated group; (4) DN plus fluvastatin treated group, then the protein expressions of COX-2, NF-κB, CTGF, ECM, FN and ColⅣwas measured by immunohistochemical and western blotting methods. Finally, the effects of fluvastatin on the kidney structure and function, kidney hypertrophy, accumulation of ECM and the influence of fluvastatin on the expression of COX-2, NF-κB, CTGF, ECM, FN, and ColⅣ were also investigated. 2. Part two (in vitro experiment): the expression of COX-2 and total protein of mesangial cell which was stimulated with normal concentration of glucose, high concentration of glucose, PGE2 or high concentration of glucose combine with PGE2 were analyzed by western blotting and PGE2 secretion in mesangial cell by enzyme-linked immunosorbent assay (ELISA).Our results show that: In DN group, kidney hypertrophy index and proteinuria were significantly increased, glomerular volume, and glomerular filtration rate were markedly increased compared to control group. Compared to DN group, the kidney hypertrophy index and proteinuria significantly decreased in celecoxib and fluvastatin treated group. In DN group, the protein expressions of COX-2, NF-κB, CTGF, FN, ColⅣwere markedly increased. The protein expression of COX-2 was decreased and the expression of CTGF, FN, and ColⅣwere also decreased by treatment of celecoxib. The kidney structural and functional damage induced by STZ was significantly improved by treatment of fluvastatin, the protein expressions of COX-2, NF-κB, CTGF, FN, and ColⅣin DN renal cortex were decreased. The protein expression of COX-2 and expression of total protein of mesangial cell were increased by treatment of high concentration of glucose or PGE2. Treatment of fluvastatin can reduce kidney structural and functional damages, prolongs excretion of proteinuria in DM and decreases protein expressions of COX-2, NF-κB, CTGF, FN, and ColⅣ.These findings suggest that protein expressions of COX-2, NF-κB, CTGF, FN, and ColⅣincreased in renal cortex of DN, increased COX-2 expression possibly mediated by increased NF-κB expression, and concomited by progression of kidney functional changes and glomerular hypertrophy, and accumulation of ECM; PGE2 can increases total protein of mesangial cell, increased expression of COX-2 can induces mesangial cell hypertrophy and resulting DN through over secretion of PGE2. celecoxib can suppress expressions of COX-2 and CTGF, reduces kidney hypertrophy caused index and ECM. Possible mechanism of glomerular hypertrophy and accumulation of ECM caused by COX-2 partially mediated by CTGF.the COX-2, NF-κB, and CTGF participated to pathogenesis of DN; the over expression of COX-2 involved in the accumulation of ECM in DN; COX-2 may plays a important role in DN caused kidney structure and function damage; COX-2 and PGE2 has synergistic effect in the DN caused kidney structural and functional damage; glomerular hypertrophy and ECM accumulation caused by COX-2 may in part mediated by CTGF. Fluvastatin has a protecting effect in DN, possibly through inhibition of protein expression of COX-2, NF-κB, CTGF and decrease of ECM accumulation by fluvastatin.In summary, (1) through investigation of expressions of COX-2 and CTGF and its interactions in DN renal cortex rats and interactions of protein expression of COX-2, kidney hypertrophy, and accumulation of ECM, we concluded that ND kidney hypertrophy, accumulation of ECM closely related to expression of COX-2, there is existence of mediation pathway between COX-2 and CTGF possibly, the one of mechanisms of the accumulation of ECM caused by COX-2 possibly mediated through over expression of CTGF. (2) through investigation of influences of high concentration of glucose, PGE2 on the expression of COX-2 in mesangial cell in vitro, we concluded that that high concentration of glucose or PGE2 can be independent stimulation factor for increasing the protein expression of COX-2, indicate that COX-2 and its production, PGE2 has synergistic effect in the kidney damage of DN. (3) through investigation of influences of COX-2 and its production, PGE2 on the expression total protein in mesangial cell, we concluded that total protein in mesangial cell can be increased by PGE2 indicate that over expression of COX-2 involved mesangial cell hypertrophy formation. (4) through investigation of influences of fluvastatin on the protein expression of COX-2 in rat renal cortex, we concluded that protein expressions of COX-2, NF-κB, and CTGF can be inhibited by fluvastatin and accumulation of ECM ca be decreased by fluvastatin, and kidney structure and function can be improved by fluvastatin, indicate that fluvastatin has kidney protective effect with independent of lipid level through inhibition of COX-2 pathway.
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