Dissertation > Excellent graduate degree dissertation topics show

Effect of JNK Signaling Transduction Pathway on Nephrotoxicity Mechanism of Cyclovirobuxine D

Author: HeMiao
Tutor: XuLi
School: Nanjing University of Traditional Chinese Medicine
Course: Pharmacology
Keywords: CVB-D renal toxicity apoptosis P-JNK HK-2 SP600125
CLC: R965
Type: Master's thesis
Year: 2013
Downloads: 11
Quote: 0
Read: Download Dissertation

Abstract

Objective:To observe the nephrotoxicity of cyclovirobuxine D (CVB-D) in rats by long intraperitoneal injection, and to explore the role of JNK signal transduction pathway in CVB-D induced kidney and HK-2cells apoptosis. Reveal the mechanism of CVB-D on nephrotoxicity to provides the reasonable reference for the clinical application.Methods:1.eighty rats were randomly divided into normal control group, the CVB-D2.5,5and10mg·kg-1group (n=20).The control group was given the same volume of solvent solution,every rat in each group is given ip eight weeks and observe the rates states daily regularly. On the forth and eighth weekend after giving drug, take10rats f rom each group.①carotid artery blood serum was separated,the detection of blood urea nitrogen (BUN),creatinine(Cr),sodium(NA+),potassium (K+), chlorine(Cl-);②collected the24h urine recording urine detecting N-acetyl-β-amino glucosidase(NAG), β2-micro globulin (beta2-MG), micro albumin (mAlb), immunoglobulin (IgG) and transferrin (TRF);βtake k idney tissues histopathology.④Using TUNEL method to observe apoptosis of kidney cells in each group;⑤Immunohistochemical method to detect Caspase-3expression in kidney;⑥Kit to detect Na+,K+-ATPase in renal cortex homogenate;⑦Western blot method to d etermine the changes in protein expression of JNK/P-JNK signal in renal cell.2.①Act CVB-D16.25、32.5、65、130、260、520μM on the HK-2cells in logarithmic phase respectively. After12,24and48hours, apply MTT method to detect the inhibitory action of CVB-D on the HK-2cells;②Using Hoechst staining method to observe the changes in nuclear morphology after CVB-D32.5、65、130μM acting on HK-2cells for24hours;③Using Annexin V/PI double staining method to observe the apoptotic rate changes after CVB-D32.5、65、130μ M acting on HK-2cells24h and48h;④Using Western blot method to detect protein expression changes of P-JNK、Smac/DIABLO and Caspase-3;⑤Take logarithmic growth phase HK-2cells, preproccess30minutes using SP600125, and add65μM CVB-D acting on HK-2cells for24hours. Using MTT method to detect cell inhibitory rate, Hoechst staining method to observe nuclear changes, and Western blot method to detect P-JNK changes in protein expression.Results:1. Compared with the reference group, after four weeks of injecting ip, in CVB-D medium-dose group and high-dose group, the BUN and CREA were significantly increased in rats serum, at the same time, the content of IgG and TRF in Urine were significantly increased(P<0.05, P<0.01); The content of NAG and β2-MG in urine is also increased in high-dose group(P<0.05), the content of (32-MG and IgG in urine is increased in low-dose group(P<0.05), HE detection shows the hydropic degeneration of kidney tubules; Continuous ip injection of CVB-D after8weeks, the weight of female rates in CVB-D medium-dose group and high-dose group was significantly decreased compared with the reference group(P<0.05, P<0.01); The content of BUN in rat serum was significantly increased in every CVB-D group, the CREA concentration in rats serum was significantly increased in CVB-D medium-dose group and high-dose group; At the same time, the amount of urine was significantly increased in CVB-D high-dose group; the content of β2-MG and IgG in urine were significantly increased in CVB-D low-dose group; The content of β2-MG and TF were significantly increased in CVB-D medium-dose group; The content of β2-MG、IgG and TRF were significantly increased in CVB-D high-dose group(P<0.05, P<0.01), HE detection showed tubular degeneration and renal interstitial inflammation generation, and with the increasing dose, the appearance proportion and symptoms getting worse, the TUNEL method results showed, CVB-D can cause apoptosis in rat kidney cells. CVB-D2.5mg·kg-1dose group has difference in apoptotic index compared with the reference group(P<0.05),5and10mg·kg-1dose group have significant difference with the reference group(P<0.01); Immunohistochemical results showed, after8weeks of giving drugs, Caspase-3expression increased in CVB-D group, and CVB-D10mg·kg-1dose group has increased significantly compared with the reference group(P<0.05);The activity of Na+-K+-ATPase in rats renal cortex was significantly reduced compared with the reference group, and showed a clear dose in accordance with patience. CVB-D5.0and10mg·kg-1dose group have obviously decreased compared with the reference group(P<0.05, P<0.01). Western blot analysis showed the JNK protein changes in each CVB-D dose group has no difference compared with the reference group. P-JNK expression was less in Blank group, but the P-JNK expression were significantly increased (P<0.05, P<0.01) in CVB-D5.0and10mg·kg-1dose-group, with a certain concentration by patience.2. MTT assay results showed that, CVB-D has growth inhibition in HK-2cells by dose-and time according to the patient; The Hochst staining showed, HK-2cells can be observed nuclear poly condensation and nuclear broken typical apoptotic phenomena under a fluorescence microscope after CVB-D treatment of24hours; Apoptosis was detected by Annexin V/PI double staining flow cytometry, CVB-D acting on the HK-2cells, apoptosis and apoptosis rate increased with the increase of the concentration of CVB-D, with a time-and dose-by patience; After CVB-D acts on HK-2cells24hours, Western blot method showed, CVB-D can promote P-JNK phosphorylation in a dose-dependent manner, and CVB-D can also increase Smac/DIABLO and Caspase-3expression; SP600125+CVB-D group compared with CVB-D, the apoptosis rate decreased significantly (P<0.01), Nucleus polycondensation apoptosis reduced and P-JNK expression was also reduced. Conclusion:1)Long-term intraperitoneal injection of CVB-D can produce renal toxicity in rats, and there is a certain amount of time, dose-dependent and mechanisms of toxicity may be associated with the damage of renal tubular and glomerular filtration membrane;2) CVB-D to kidney toxicity shows it induced renal cell apoptosis and inhibits the activity of the biological membrane, and apoptosis may be related to increase of Caspase-3expression and cause phosphorylation of JNK;3) Through the activation of JNK, CVB-D contributes to mitochondrial releasing of pro-apoptotic factors Smac/DIABLO, and increase Caspase-3expression, suggesting CVB-D may be involved in mitochondria-mediated HK-2cells apoptotic pathway.

Effect of JNK Signaling Transduction Pathway on Nephrotoxicity Mechanism of Cyclovirobuxine D

Abstract

Related Dissertations