| Background and ObjectivesDiabetic nephropathy (DN) is one of the most common complications diabetes in mellitus (DM), which is a major cause of death of diabetes in patients. The major of its clinical manifestations is microalbuminuria, with the progress of the disease will appear continuous proteinuria, edema, hypertension, glomerular filtration rate descent of kidney and other clinical symptoms. It result in irreversible and eventually develop into end-stage renal disease pathological, which is the extracellular matrix (ECM) composition accumulation caused by glomerular hypertrophy, thickening of basement membrane, mesangial matrix broadening. Finally it development to glomerular sclerosis. Therefore, to better understand and explore the pathogenesis of DN and its pathophysiologic changes and better prevention and treatment of DN, which becomes an urgent problem. Previous experimental studies have shown that high glucose is a starting factor of chronic diabetic complications, and oxidative stress is a key factor development to and occurrence of diabetic nephropathy. Further more, it can interact with the inflammatory response and jointly promote the development of diabetic nephropathy occurs, but the specific mechanism is still not clarified. High glucose may induce oxidative stress (OS), and thus activate nuclear transcription factor NF-kappa B in the nucleus of cells and combine with DNA, then gene transcription regulation of inflammation. Finally the expression of tumor necrosis factor (TNF), monocyte chemotactic protein-1 (MCP-1), IL-6, IL-1, intercellular adhesion points-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), transforming growth factor β1(TGF-β1) and so on, leading to the extracellular matrix excess deposits and thus promoting the occurrence and development of diabetic nephropathy.Parthenolide (PTL) is a feverfew (tanacetum parthenium) purification of sesquiterpene lactone compounds, many studies show parthenolideis a special and strong effective inhibitor of nf-kappa B, which can prevent the nf-kappa B and (or) IKK complexes directly or indirectly, and then prevent the nf-kappa B into the nucleus and combine with the purpose gene. The treatment of migraine, rheumatic fever, anti-inflammatory, anti-oxidation and anti-tumor and other pharmacological effects with parthenolide. So far, there is few research of treatment of DN with parthenolide. Therefore, this study intends to using model of diabetic nephropathy with type 2 diabetes db/db mice, to explore the effect of parthenolide on the treatment of diabetic nephropathy in db/db mice.Contents and methods1. The animal group and interventionThe experimental animals were type 2 DM spontaneous db/db mice with 8 weeks, and which were divided into four groups. Group basis:the average weight and urine protein without abnormal individual, the remaining db/db mice were completely random divided by the SPSS statistical software. So in order to achieve no statistically difference between groups, each db/db mouse has a single number, and keeping a record at all time. Total of 12 db/db mice were divided into model group and the treatment group, each group were 6 db/db mice; Total of 12 db/m mice were divided into normal control group and the intervention group, each treatment group were given as stomach intervention with parthenolide 50 mg/(kg·d), and the normal control group and the db/db mice model group were given equal normal saline lavage.2. The general observation and biochemical index determination of db/m mice and db/db miceObservation with the state of mind, food, drinking water and health status and record of db/m mice and db/db mice. With a regular basis to determine the db/db mice and db/m mice body weight, the urinary ratio of albumin to creatinin, At the end of the experiment with continuous treatment of 12 weeks. We put all the mice to death, and according to weight, kidney weight, the urinary ratio of albumin to creatinin, to compare the difference of urinary albumin excretion, blood detection of serum creatinine, blood urea nitrogen and other biochemical indicators in db/m mice with that in db/db mice.3. The kidney pathology examination of db/m mice and db/db miceAt the end of the experiment with treatment of 12 weeks. All the mice were putting to death, HE and PAS staining of db/m and db/db mice kidney tissue were done and then we observe the pathological change of kidney tissue morphology.4. The damage and drug intervention mechanism research of db/m mice and db/db miceTo detect nuclear transcription factor (NF-κB), MCP-1 and TGF-p expression in db/db mice with tissue Enzyme-linked immunosorbent (Elisa), immunohistochemical technique, polyacrylamide gel electrophoresis (Western blotting) and quantitative real time PCR(qRT-PCR).StatisticsExpressed in X±S on the experimental data, the data analysis using SPSS 17.0 statistical analysis software, began to normal distribution data and f test. Comparison between the two groups by using two independent samples t-test; Comparison between groups:if accord with normal distribution (p>0.05) and variance qi values (p>0.05), using the single factor analysis of variance of LSD method are compared, and the multiple values (p<0.05) considered statistically significant; If not neat, do not accord with normal distribution or variance using nonparametric test K-ruskal Wallis H method, if K-ruskal Wallis H test results significantly (p<0.05), and then to convert data in order, after comparing between two more groups of two, value (p <0.05)was statistically significant.Result1. During the time of rearing, db/db mice showed more drinking, taking food, polyuria and fat comparede with db/m mice. At 24 weeks of the end of the experiment, the urinary ratio of albumin to creatinin of db/db mice was significantly higher than db/m mice (p<0.05). After the treatment with PTL 50 mg/(kg·d), the urinary ratio of albumin to creatinin of db/db mice was obviously decreasd (p<0.05).2. The general morphology observation. Morphological observation showed that db/db mice model group a significantly more drinking, taking food, polyuria and obese compared with normal db/m group. Further more, glomerular volume, broadening mesangial area, mesangial matrix of db/db mice increased significantly, and also part of the glomerular capillary expansion, endothelial cell swelling, degeneration, part of renal tubular epithelial cell vacuoles degeneration. PAS red dye area expanded value, the kidney tissues of of db/m mice didn’t change obviously. The pathological changes, matrix accumulation, sedimentary mitigation and mesangial cells was significantly reduced after treatment with parthenolide 50mg/(kg·d), but it has not yet returned to normal.3. Parthenolide 50 mg/(kg·d) can obviously reduce the expression of TGF-β in db/db mice and relieve renal fibrosis of db/db mice.4. The detection of polyacrylamide gel electrophoresis and Western blotting testing. We found that 8-hydroxy deoxyguanosine (8-OHdG) protein expression was significantly increased compared with normal control group (p<0.05). After treatment with parthenolide,8-OHdG protein expression significantly was decreased in mice kidney (p<0.05), but the 8-OHdG protein expression was increased compared with db/m group (p<0.05),8-OHdG protein expression in treatment group was almost with db/m group in mice kidney tissues (p>0.05).5. The detection of polyacrylamide gel electrophoresis and Western blotting testing. We found that NF-κB protein expression of db/db mice were significantly increased compared with db/m mice. After treatment with parthenolide 50 mg/(kg·d), the expression of NF-κB protein was significantly decreased.6. The result of ELISA showed that MCP-1 protein expression were significantly increased in db/db mice and reversed by parthenolide 50 mg/(kg·d) (p<0.05).7. The result of qRT-PCR showed that parthenolide with 50 mg/(kg·d) can decrease MCP-1 mRNA expression significantly in db/db mice kidney (p<0.05), these results were consistent with the protein level of MCP-1 in renal tissue of mice.ConclusionParthenolide can down regulate the gene expression of MCP-1mRNA and protein by inhitbiting NF-κB pathway, subsequently attenuate renal fibrosis and delay the development of DN. |
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