| Diabetic nephropathy is one of common microvascular complications in diabetes mellitus, also contributed to increase the rate of cardiovascular events and mortality in diabetic patients. Approximately 30%-40% end-stage renal failure patients are the diabetic patients in foreign, and the domestic number of incidence is also increasing. Recently, DN is ranking to the important place as the because of the chronic kidney disease in China. DN occult begin, and rapid progress. Once the patient is diagnosed as DN, the patient is already in irreversible kidney damage and has entered the stage of rapid development. The clinical result of DN is not satisfactory, and poor prognosis becomes a major problem in clinical work. As we all know, the glomerular hypertrophy and increased extracellular matrix are the characteristic pathological changes in the early stage of DN. With the progress of the disease, the glomerular becomes sclerosis and interstitial fibrosis, and clinical manifestations express the chronic renal insufficiency. The early stage of DN can be reversed; therefore, current researches are focusing on how to know the possibility of DN for the diabetic patient as soon as possible, how to take effective intervention, and how to delay the progress of the DN?12-lipoxygenase (12-LO) is a kind of polyunsaturated fatty acid, in vivo 12-LO activation can produce the metabolism product such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] at the base of Arachidonic acid. A lot of study results show that,12-LO and its metabolism product 12(S)-HETE can cause a variety of diseases by oxidative stress and inflammatory response, such as hypertension, coronary heart disease, diabetes, atherosclerosis and so on. Therefore,12-LO and its metabolic pathway are emphasized by more and more researchers. Bleich D and his companions found the blood glucose did not increase in the 12-LO knock out C57BL/6 mice by injected with STZ for 5 days as early as 1999. In recent years, Kim YS and Kang found high glucose stimulation could increase the 12(S)-HETE levels in mesangial cells, and Reddy MA and his companion found the 12-LO could increase the expression of FN and Collagen by the P38MAPK pathway in diabetic environment. Therefore,12-LO has an important effect on the progress of the DN.The glomerular hypertrophy and increased extracellular matrix are the characteristic pathological of DN; moreover, glomerular hypertrophy is closely related to the CKI expression associated with cell cycle regulation. This study was to explore the interaction between 12-LO metabolic pathway and the expression of cyclin-dependent kinase inhibitor (CKI), glomerular hypertrophy; to observe the regulated effect of accumulating extracellular matrix (ECM) on 12-LO and CKI expressions and its molecular mechanism, and to know the effect of 12-LO on the glomerular hypertrophy and accumulating ECM, to further clarify the role of 12-LO in the progress of the DN and provide a new therapeutic target for clinical treatment. Glomerular mesangial cells, rat models injected 12(S)-HETE by osmotic mini-pump and high-fat diet with low-dose STZ-induced type 2 diabetes models were used in this study. We have do some experiments, as following:①To investigate the effect of 12 (S)-HETE injected by mini-pump on the expression of CKI, ECM;②To investigate the effect of CDC by injected subcutaneously on the glomerular hypertrophy and the expression of CKI, ECM;③To investigate the effect of 12(S)-HETE stimulated mesangial cells on the expression of CKI and its signaling pathway;④To investigate the effect of high glucose stimulated mesangial cells on the expression of CKI and the CDC effection in the whole progress;⑤To investigate the effect of ECM environment stimulated mesangial cells on the expression of CKI, the CDC effection in the whole progress and the signaling pathway.The main results in this study are as follows:1.12(S)-HETE injected by osmotic mini-pump increase P21,P16 and CollagenⅣexpressions in the normal rats glomerular (p<0.01), but there is no notably effect on the glucose.2. The kidney weight to body weight and the glomerular volume significantly rised in the DN rats compared with the normal rats (p<0.01). Treatment with CDC did not affect diabetic glucose levels, but significantly attenuated diabetes-induced increases in kidney weight to body weight ratio, glomerular hypertrophy (p<0.05).3. The 12 (S)-HETE levels significantly increased in the DN rats glomerulr, and the P21, P16 and CollagenⅣexpressions also increased in the DN (p<0.01), CDC could decreased the P21, P16, CollagenⅣexpressions and 12 (S)-HETE levels compared to DN without treatment with CDC (p<0.05). The change of the P38MAPK activity is consist of the 12 (S)-HETE levels.4. The P21 and P16 expressions significantly increased in the mesangial cells stimulated by 12(S)-HETE (p<0.01), and the P38MAPK inhibitor decreased P21, P16 expressions (p<0.05).5. High stimulation could increase 12(S)-HETE levels in mesangial cells (p＜0.01), and CDC could decrease 12(S)-HETE levels (p＜0.05). 6. High stimulation could increase P21, P16 expressions in mesangial cells (p<0.01), and CDC could decrease P21, P16 expressions (p<0.05).7. ECM environment could increase 12(S)-HETE levels in mesangial cells (p<0.01), and CDC could decrease 12(S)-HETE levels (p<0.05). The change of the P38MAPK activity is consist of the 12 (S)-HETE levels.8. ECM environment could increase P21, P16 expressions in mesangial cells (p<0.01), and CDC could decrease P21, P16 expressions (p<0.05).We draw some conclusions as follows:1. The 12-LO metabolic product 12(S)-HETE could up-regulate the CKI (P21, P16) expressions in the normal rats glomerular, and increase the ECM expression (Collagen IV).2. The P21, P16, Collagen IV expressions significantly increased in the DN rats compared with the normal rats. Treatment with CDC did not affect diabetic glucose levels, but significantly decreased diabetes-induced increases in The P21, P16, CollagenⅣexpressions3.12(S)-HETE stimulation could up-regulate the P21, P16 expresions, and P38MAPK inhibitor could decrease P21, P16 expressions in mesangial cells. These results above suggest the CKI expression is mediated at least in part by 12-LO-P38MAPK signaling pathway.4. High stimulation could increase the 12(S)-HETE levels, at same time up-regulate the CKI (P21, P16) expressions, CDC treatment could significantly reduce P21, P16 expressions, which suggest high glucose could participate in the progress of DN at least in part by increasing the activity of 12-LO.5. ECM environment could up-regulate P21, P16 expressions and increase the P38MAPK activity. After treatment with CDC, P21, P16 expressions and the P38MAPK activity significantly decreased. Moreover,12-LO could increase the ECM expression in the type 2 DN. Therefore, accumulated ECM could increase the activity 12-LO further to deteriorate the progress of DN. Above results could demonstrate that ECM could mediate the P21, P16 expression at least in part by 12-LO-P38MAPK signaling pathway.In summary, this study further illustrates the glomerular hypertrophy and accumulating ECM are not independent pathological changes in the DN, there may be promoted and cuase-effect relationships between two pathological changes. We not only investigate the effect of 12-LO on the diabetic glomerular hypertrophy, but also deeply investigate the signaling pathways. Simultaneously, we link the ECM with CKI and 12-LO to further investigate the 12-LO effect on the progress of DN by ECM stimulation in MCs, and to provide a theoretical basis for the future clinical trials. This study's innovation is to link two pathological changes together, while in vivo to invigate the 12-LO role on the both pathological by the artificial environment, and to futher improve the 12-LO role on the pathogenesis of the DN.
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