| Early pathological changes of diabetic nephropathy is manifested mainly glomerular hypertrophy, and the emergence of extracellular matrix (ECM) increasing. With the lesions progressing, glomerular sclerosis and renal interstitial fibrosis gradually arise, culminating in renal failure. Diabetic nephropathy is the result of many factors and high blood sugar is a prerequisite for kidney damage occurring. Sorbitol metabolism, deposition of advanced glycation end products, protein kinase C activation, hexose amine pathways metabolic abnormalities and RAS activation caused by high blood sugar are the main pathogenesis of diabetic nephropathy, other causes, including genetic factors, renal hemodynamic abnormalities, high blood pressure in diabetic nephropathy may play an important role in the development process. In recent years,12-lipoxygenase and cell cycle regulatory proteins in the pathogenesis of diabetic nephropathy had been increased attention and devoted to become a research hotspot.Lipoxygenase (lipoxygenase,LO) is a polyunsaturated fatty acid oxygenase. LO in mammals according to the similarity of its isomerase genotype were divided into white blood cell types, epithelial cell type and platelet type. Cell membrane phospholipids by the activation of phospholipase A2-catalyzed release of arachidonic acid (Arachidonic acid:AA). LO isomerase with a specific location by adding oxygen molecules can be divided into 5-,8-,12-,15-LO isomerase, to the corresponding Hydroxyeicosatetraenoic acid (HETE) with AA as a substrate generates. Study confirmed that atherosclerosis, hypertension, diabetes and other serious diseases that affect human health have close relationship with the leukocyte-type 12-LO expression. Studies have shown that the key enzyme-12-LO gene and protein levels with arachidonic acid (AA) metabolism, not only increase the expression in glomerular mesangial cells, podocyte and diabetes model (type 1 and type 2) renal by high glucose-stimulated, but also its role are similar with TGF-βand Ang□, and thus that it is regarded as a new key factor closely related to the progress of DN. Studies have shown that 12-LO can also cause glomerular ECM accumulation. The above studies have shown that 12-LO play an important role in the promotion of the occurrence and development of DN in the pathology. However, Could 12-LO give rise to glomerular hypertrophy, there were no relevant reports.hypertrophy cells have the character of increasing in cyclin kinase inhibitor (CKI) expression and intracellular protein synthesis and so on. All causes of kidney damage can be expressed as cell proliferation, apoptosis and cell hypertrophy, and these changes are related to the regulation of cell cycle abnormalities. Study confirmed that in DN, there are some cells proliferation occurring such as renal interstitial cell, but most of kidney cells, especially mesangial cells and renal tubular epithelial cells cause cell hypertrophy in stagnation of the the late G1 after activation of the Go period, which is characteristic of early diabetic nephropathy change, and it is different from a variety of other primary or secondary renal diseases characterized by cell cycle. Studies have shown that mesangial cells (MC) cultured by high glucose showed a short-lived transient cell proliferation, and then G1/S cell cycle escaping.Its DNA synthesis was inhibited while the protein and RNA synthesis increased, the increase in intracellular protein content were out of proportion with the DNA synthesis, cell growth was arrested in the G1 phase, which caused the increase of cell volume, leading to the occurrence of cell hypertrophy. The cell cycle is mainly controlled by the positive and negative cell cycle regulatory proteins. Cell cycle positive regulated proteins include cyclin and CDK, activity can only be obtained when the two proteins are combined into compound. Experiments show that diabetic glomerular hypertrophy is closely related to the occurrence and CKI, but not with the level of cyclin and CDK. Cyclin kinase inhibitor (CKI) is a negative regulator of cell cycle proteins, presenting in the nucleus, and closely combined with Cyclin-CDK complexes, inhibiting CDK activity, affecting the function of the downstream target proteins, thereby preventing conversion of G1/S and G2/M, leading to cell cycle arrest, inhibiting cell cycle operation. If the cell cycle arrest in late G1,cell hypertrophy will occur. CKI is divided into two family of INK and CIP/KIP. Research has clearly showed that CIP/KIP family proteins such as p21waf1, p27kipl, p57kip2 can inhibit a variety of, CDK activity by combining with it, which is the key factors leading to cell cycle escape and causing cell hypertrophy. In diabetic conditions, increases in p21waf1, p27kipl expression are related to renal cell hypertrophy and glomerular hypertrophy. p27kipl is a non-specific negative control of cell cycle proteins, can inhibit a number of different CDK complexes, which cause that the cell can not pass G1 phase, leading to cell hypertrophy. In normal resting state, p27kipl in the three kinds of glomerular intrinsic cells has a very strong expression of the structural expression, it play an important role in cell cycle regulation of glomerular disease, and it is a major factor in inducing cell hypertrophy. The level of p27kipl protein vary in the cell cycle, it reach the highest in quiescent (Go) and G1 phase, while it begin to decline in the transition from G1 phase to S phase and then the cells start to divide and hyperplasia. p27kip1 is necessary for the maintaining the quiescent state of cells. The expression and regulation of p27kipl is multi-faceted.The study shows the regulation and expression of p27kipl mostly occurs in post-transcriptional levels, including protein translation, protein degradation and phosphorylation levels of modification, while not at the transcriptional level. The degradation of p27kipl achieve mainly through the ubiquitin/proteasome pathway. Positioning of p27kipl in the cell has also a certain effect on its function into full play and degradation. Because the function of inhibiting CDK and degradation of p27kipl in G1 phase were done in entering into the nucleus, the intracellular location of abnormalities can also affect the expression and physiological function of p27kipl.Recent studies have showed that p21wafl, p27kipl are not only closely related to cell hypertrophy, but also to cell aging. A common feature of diabetic nephropathy and aging kidney possess is renal cell hypertrophy. In vitro experiments show that p21wafl, p27kip1 increased the expression in the elderly mesangial cells comparied with the younger glomerular mesangial cells, but the in vivo experiments show that only the glomerular p27kipl expression increased in the elderly glomeruli compared with young, there is no change of p21wafl expression in glomeruli in the elderly, its mechanism is unclear. Thus, p27kipl is landmark proteins which associated both diabetes and aging of glomerular mesangial cells with glomerular hypertrophy in the CIP/KIP family proteins. p27kipl is not only a sign of cell hypertrophy and aging, but also main functions of proteins promoting cell hypertrophy and senescence.Research Methods 1) Observe the mesangial cell p27kipl protein expression under 12-LO product 12 (S)-HETE (10"7mmol/L) stimulation with or without p38 MAPK(p38 MAPK) inhibitor (SB203580, 1umol/L) and investigate whether 12-LO induce p27kipl protein expression through p38 MAPK pathway.2) Investigate the role of 12-LO in the glomerular hypertrophy using STZ-induced diabetic animal model. Male SD rats were divided into normal diet control group, normal diet+12-LO inhibitor (CDC,8mg/kg,3 times /week, subcutaneously) treatment group, high-fat diet low-dose streptozotocin (STZ 35mg/kg, intraperitoneal injection)-induced type 2 diabetes group, high-fat diet with low-dose STZ-induced type 2 diabetes+CDC treatment group, continuous subcutaneous injunction CDC for 1 months. The wild-type and 12-LO gene knockout C57BL/6 mice were randomly divided into wild-type control group,12-LO gene knockout group, wild-type STZ-induced type 1 diabetic group,12-LO gene knock-STZ-induced type 1 diabetic group, feeding for 16 weeks. Collect urine, blood and extract the kidney, and then glomeruli were isolated with the series of sifting after the end of the experiment. Detect the target protein changes by Western blot and immunohistochemistry.The results of this study are as follows:1) It is effective to block expression of p27kipl in mesangial cell induced by 12 (S)-HETE through inhibiting p38MAPK activity, the difference was statistically significant (P <0.01).2) CDC can decrease type 2 diabetic rats glomerular volume and reduce urinary albumin excretion(P<0.01).3) CDC can significantly decrease glomerular p38 MAPK activity and p27kip1 protein expression, compared with CDC untreated diabetic rats (P<0.01).4) Compared with 12-LO knock-out diabetic mice, p38 MAPK activity, p27kip1 protein expression and ECM accumulation increased in the glomeruli in wild-type diabetic mice, the difference was statistically significant (P<0.01).From this study,we can draw the following conclusions:1) 12-LO can induce p27kipl protein expression through p38 MAPK pathway.2) 12-LO inhibition not only reduce urinary albumin excretion and glomerular volume,but also decrease glomerular p38 MAPK activation and p27kipl protein expression under diabetic conditions.In summary,12-LO induces glomerular p27kipl expression through the p38 MAPK pathway in diabetic conditions. |
