Transcript
THE MOLECULAR MECHANISM OF LEPTIN SECRETION AND EXPRESSION INDUCES BY ARISTOLOCHIC ACID IN KIDNEY FIBROBLAST
Chung-shi yang; et al.
BY: ALEJANDRA ALVAREZ
SILVANA ZAPATA Estudiantes medicina UPB
III sermestre
OBJETIVE
The aim of this study is to explorethe effect of AA on leptinproduction and to dissect the AA-induced downstream signaling invitro.
INTRODUCTION
Leptin is a 16 kDa peptidehormone of cytokine familymainly secreted byadipocyte into blood streamand functions as a centralmediator that negativelyregulates satiety inhypothalamus1.
1: Zhang Y.; et al. 1994.
INTRODUCTION
Aristolochic acid (AA) is a famous botanical toxin which has been characterized to associate with the development of aristolochic acid nephropathy (AAN) 2
2: Vanherweghem JL, et al. (1993)
INTRODUCTION
A fibroblast is a type of cell that synthesizes the
extracellular matrix and collagen, the structural framework (stroma) for
animal tissues, and plays a critical role in wound
healing. Fibroblasts are the most common cells of
connective tissue in animals3, 4.3: Lan HY.;(2003) .
4: Roberts IS.; (1997).
INTRODUCTION
Leptin has been considered to playan important role in progressive renalfibrosis. Renal fibroblast, in particularunder the progression of fibrosisinduced by AA.
Findings imply the fibroblast-producedleptin maybe one of the factor whichpromotes the progression of renal fibrosisinduced by AA5.
5: Merabet E, et al. (1997)
MATERIALES Y METODOS
1. Cultivo celular :
Se obtuvieron fibroblastosNRK-49f a través de unacolección de cultivo tipoamericano .
2. La viabilidad celular :
Fue evaluada mediante la reducción dependiente de mitocondrias a purpura formazan y la incorporación de BrdU respectivamente .
3. Medición de la secreción de Leptina:
Fue determinada mediante el método de ELISA .
4. Western blot:
Fue utilizado para medir la expresión de la leptina inducida por AA.
• 5. RT-PCR y PCR en tiempo real:
La RT-PCR de RNAm a cDNA
PCR en tiempo real cuantificar concentración de RNAm.
6. Transfección:
Introducción de siRNA para bloquear la expresión del gen de la leptina.
7. Inmuno precipitación:
•Anticuerpo anti-Akt
RESULTADO 2.B
(B) Leptin expression was examined by Western Blot. NRK-49f kidney fibroblasts were treated without or with 12 mM AA for 24 and 48 h.
RESULTADO 2C
(C) Expression of leptin mRNA. Fibroblasts were treated with 0, 6 and 12 mM AA for 12and 24 h, the level of leptin mRNA was determined by RT-PCR
RESULTADO 3A
(A) Enhancement of C/EBP a DNA binding activity by AA. Fibroblasts were treated with 0, 6 and 12 mM AA for 3 h, nuclear extracts were isolated and C/EBP a DNA binding activity was analyzed by DAPA.
RESULTADO 3B
(B) Knockdown of C/EBP a expression by siRNA. NRK-49f cells were transfected with 0, 90 and 180 nM of C/EBP a siRNA or control scramble siRNA for 8 h, and then cells were treated with 12 mM AA for another 48 h, the expression level of C/EBP a was examined by immunoblotting (upper panel) and RTPCR (lower panel).
RESULTADO 4A
• (A) Examination of Akt activation. NRK-49f cells were incubated with 0 and 12 mM AA for 1, 3 and 6 h. The phosphorylated activation form of Akt was detected by immunoblotting
RESULTADO 4B
• (B) The levels of phosphorylated PDK1 and PI3K-p85 were evaluated by Western Blot at indicated periods of AA treatment.
RESULTADO 4C
• (C) The interaction of phosph-PDK1 with Akt. After 1 h of 0 and 12 mM AA treatments, cells were immunoprecipitated with anti-Akt antibody. The immunoprecipitated pellets were further immnoblotted with anti-phospho PDK1 or anti-Akt antibody. The input and mouse IgG were served as positive and negative control, respectively.
RESULTADO 5A
• (A) Knockdown of Akt by siRNA. Cells were transfected with 0, 60 and 120 nM of Akt siRNA or control scramble siRNA for 8 h, and then cells were treated with AA for another 48 h, the expression level of Akt was examined by immunoblotting (upper panel) and RT-PCR (lower panel).
RESULTADO 5C
• (C) Analysis of Akt activation. Cells were pretreated with 10 mMLY294002 (PI3K inhibitor) or 50 nM rapamycin (mTOR inhibitor) 1 h prior 12 mM AA addition. After 3 h incubation, level of Aktphosphorylation was determined by immunoblotting.
RESULTADO 5E
• (E) immunoblotting, respectively.
RESULTADO 5F
• (F) Expression of leptin mRNA. After the 1 h pretreatment of indicated inhibitors, cells were then treated with AA for another 24 h. RNA was isolated, and the level of leptin mRNA was assessed by RT-PCR.
RESULTADO 6A
• (A) DAPA
RESULTADO 6B
• (B) ChIP.
DISCUSION
Mapa silvana zapata
Mapa Alejandra Alvarez
CONCLUSION• Leptin is an important hormone in obesity, kidney
disease and has implications in different organs ofthe body.
• The Molecular Biology is very important in theinvestigation and diagnosis of different diseases.
• Leptin is part of the regulation of AA and this isinvolved in renal fibrosis.
• Genetic research to deliver various changes to findthe solution to many diseases.
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