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Astragalus and Pueraria transdifferentiation of renal tubular epithelial cells of diabetic nephropathy

Author: LiYuJie
Tutor: WangQian
School: Beijing University of Traditional Chinese Medicine
Course: Chinese and Western medicine combined with the basis
Keywords: EMT Type 2 diabetes mellitus Puerarin Injection Astragalus injection KKA ~ y mice RIF Signaling pathway
CLC: R259
Type: Master's thesis
Year: 2011
Downloads: 110
Quote: 0
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Abstract


Diabetic nephropathy (DN) is one of the microvascular complications of diabetes mellitus (DM), has become the major cause of death of patients with diabetes. Interstitial fibrosis in the kidney lesions of diabetic nephropathy. Renal interstitial fibrosis (RIF) is the clinical variety of chronic progressive kidney disease progress to end-stage lead to renal failure common pathological changes, qualitative and quantitative changes in tubular basement membrane composition, stromal extracellular matrix (ECM) tubular atrophy and accumulation of myofibroblasts is characterized by change, the most important lesions appear by tubular epithelial cells transfected myofibroblast differentiation from (MyoF). TGF-β1 is a the main fibrogenic factor, tubular epithelial cell transdifferentiation (EMT) in central link is caused by a variety of factors, it can be caused by a variety of ways EMT, resulting in MyoF generation and RIF, including TGF-β1 / Smads pathway is the currently accepted tubular epithelial cell transdifferentiation process is the most important signal transduction pathways. Chinese medicine in the prevention and treatment DN shows good results, but the specific mechanism of action is not yet clear, so the depth to explore the mechanism of action of the Chinese medicine treatment of DN is of great significance for guiding clinical. Through literature review and preliminary studies show, Astragalus injection combined with puerarin injection the RIF good effect, but they are targets of the specific role of the RIF unclear. Therefore, we would like to Astragalus injection combined with puerarin injection on treatment of KKAy diabetic mice renal interstitial fibrosis and TGF-β1-induced tubular epithelial cells (HK-2) EMT process, to further explore traditional Chinese medicine Astragalus and Pueraria therapy to the mechanism of renal interstitial fibrosis and to provide experimental evidence for Chinese medicine prevention renal interstitial fibrosis. Objective: KKAy diabetic mice RIF and human renal tubular epithelial cell EMT process variation of TGF-β1 and its Smads signaling pathway, to further explore traditional Chinese medicine (Astragalus injection combined with puerarin injection) by blocking the EMT process to reach the mechanism of the treatment of renal interstitial fibrosis, to reveal Astragalus and Pueraria compatibility of the treatment of diabetes RIF role link to provide a scientific basis for the treatment of diabetes RIF Astragalus and Pueraria compatibility. Research methods: (a) the overall experiment (1) SPF level of type 2 diabetes model KKAy mice were divided into the model group and Astragalus injection combined with puerarin injection group (treatment group). Peers C57BL / J mice were used as the normal group. Intraperitoneal injection in mice treated with 14-week-old administration, the mice were sacrificed at 24 weeks of age, take kidney renal pathological morphological changes. (2) E-cadherin immunohistochemistry assay in mouse renal tissue of a-SMA, TGF-p, and TGFp-RI protein expression. (3) Western blot detection of mouse kidney tissue Smad3. Smad7 protein expression. (4) RT-PCR method, the detection of renal tissue in mice TGFp-RI and Smad3 mRNA expression. (B) in vitro experiments (1) In vitro cultured human renal tubular epithelial cells (HK-2), divided into normal group, model group, the treatment group. Model group to 10% of the cell culture medium containing TGF-β1 (8ng/ml) (DMEM/F12 medium containing 10% fetal bovine serum), co-cultured 48h; treatment group were treated with TGF-β1 in (8ng/ml) and Astragalus injection Jiage root injection of 10% of the total cell culture medium for 48h, and changes in cell morphology was observed under inverted phase contrast microscope. (2) immunohistochemical method to detect E-cadherin, a-SMA expression in HK-2 cells. (3) Real-time PCR assay HK-2 cells, E-cadherin, a-SMA-TGF-β1, TGFβ-RI, Smad3 and Smad7 mRNA expression. (4) Western blot to detect the protein expression of TGF-β1 in HK-2 cells. Results: (a) the whole experiment (1) KKAy model mice 24 weeks of age, renal pathological changes visible tubulointerstitial fibrosis, inflammatory cell infiltration, vacuolar degeneration of renal tubular epithelial cells, tubular dilatation; a-SMA, TGF-p1, TGFβ-RI and Smad3 protein expression increased E-cadherin and decreased expression of Smad7 expression of; the TGFP-R I and Smad3 mRNA expression increased. (2) Astragalus injection combined with puerarin injection after treatment, KKAy mice renal tissue injury alleviate kidney tissue a-SMA protein expression of Smad3, TGF-β1 and TGFP-R I reduce, E-cadherin and Smad7 The increase in protein expression. (3) Astragalus injection combined with puerarin injection KKAy mouse renal tissue decreased expression of TGFβ-RI and Smad3 mRNA. (B) in vitro experiments (1) HK-2 cells to give the TGF-β1 stimulation 48h, cell morphology changed significantly by the cobblestone into a fibroblast-like; intracellular a-SMA protein and mRNA levels expression increased significantly reduced expression of E-cadherinzai protein and mRNA levels of TGF-p1, TGFβ-RI and Smad3 mRNA expression was elevated, Smad7 mRNA expression decreased. (2) Astragalus injection combined with puerarin injection of a-SMA and TGF-β1 protein expression in HK-2 cells to reduce the increase in the expression of E-cadherin protein. (3) Astragalus injection combined with puerarin injection of a-SMA in HK-2 cells, TGF-β1, TGFβ-RI and Smad3 mRNA expression was inhibited E-cadherin and Smad7 mRNA expression increased. Research conclusions: (1) combining Astragalus injection puerarin injection can significantly alleviate the the KKAy mice renal tubular interstitial fibrosis, inhibit and reverse the process of EMT in the stimulation of TGF-β1 in HK-2 cells to restore epithelial cells phenotype. (2) Astragalus injection combined with puerarin injection against possible mechanism of renal interstitial fibrosis: by inhibiting and blocking the KKAy mouse renal interstitial fibrosis and TGF-β1 and its receptor in HK-2 cells EMT process gene and protein expression in the the Smads signaling pathways mediated improve Smad7 and expression of E-cadherin, blocked and reversed EMT process, reduce the renal interstitial fibroblasts and the expression of extracellular matrix components, recovery of renal tubular epithelial normal cell phenotype, delay the occurrence and development of renal interstitial fibrosis.

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