[Enhance effect of Ox-LDL on NF-kappaB activity in glomerular mesangial cells from the Zucker rats].
OBJECTIVE
To study the effect of oxidized low-density lipoprotein (Ox-LDL) on nuclear NF-kappaB activity in cultured glomerular mesangial cells (GMCs) from the Zucker rats and the correlation between the change of NF-kappaB activity and ras's age and genotype.


METHODS
Four groups of GMCs (O(3m),O(10m), L(3m) and L(10m)) from the 3- and 10- month (fa/fa) and 3- and 10- month lean (Fa/Fa) Zucker rats were stimulated with Ox-LDL. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activity of NF-kappaB. Gel supershift assay(GSA) was used to detect the subunit of NF-kappaB dimer.


RESULTS
(1) NF-kappaB activity after induction with Ox-LDL in the 3 groups (O(3m),O(10m) and L(3m)) of GMCs were significantly higher than that in the control group (P<0.01). (2) With the increased of concentration and stimulation time of Ox-LDL, the NF-kappaB activity was increased correspondingly and reached the highest when GMCs were stimulated with 50 mg/L Ox-LDL for 4 hours. (3) Supershift assay demonstrated that Ox-LDL mainly activated the two subunits p65 and p50 in GMCs. (4) As the activity of NF-kappaB, O(3m) group vs O(10m) group, O(3m) group vs L(3m) group, and O(10m) group vs L(3m) group had all markedly differences (P<0.01), while the differences between L(3m) group vs L(10m) group was inferior to 3 groups above (P<0.05).


CONCLUSIONS
Ox-LDL could significantly activate NF-kappaB in GMCs from Zucker rats, in the time- and dose- dependence manner. There was a close relationship between the NF-kappaB activity in GMCs induced with Ox-LDL, the age and genotype of Zucker rats. NF-kappaB activity in GMCs of the Zucker rats induced with Ox-LDL plays an important role in the early renal lesion of diabetes mellitus.
Gan
Wei-hua
WH
Department of Pediatrics, the Second Affiliated Hospital, Nanjing 210011, China. weihuagan@yahoo.com
Din
Gui-xia
GX
Chen
Rong-hua
RH
D000375:Aging / Q000502:physiology; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005838:Genotype; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000008:administration & dosage / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit / Q000378:metabolism; D009765:Obesity / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011924:Rats, Zucker; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism
D008077:Lipoproteins, LDL; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D051996:Transcription Factor RelA; C099252:oxidized low density lipoprotein