Short interfering RNA targetting NF-kappa B induces apoptosis of hepatic stellate cells and attenuates extracellular matrix production.
BACKGROUND
Pharmacological inhibition of the NF-κB activity enhances hepatic stellate cell apoptosis and reverses experimental fibrosis. However, there is no report on the effects of NF-κB knockdown on apoptosis and extracellular matrix secretion in hepatic stellate cells. The aim of the present study is to explore the effects of siRNA targetting NF-κB on the apoptosis and extracellular matrix production in hepatic stellate cells.


METHODS
The immortalised hepatic stellate cell line HSC-T6 was transfected with siRNA; 72h later, cells were stimulated by LPS for 1h; these cells were collected for further use. Hepatic stellate cell apoptosis was determined by fluorescence activated cell sorter analysis, TUNEL assay and caspase-3 activity measurement. Matrix metalloproteinase 2 activity was evaluated with Gelatin zymography. The quantities of mRNA transcriptions of NF-κB p65, type I collagen, tissue inhibitor of metalloproteinases-1, α-smooth muscle actin and transforming growth factor beta 1 and anti-apoptotic protein A1 were evaluated with quantitative reverse transcriptase real-time polymerase chain reaction.


RESULTS
siRNA targetting NF-κB p65 effectively abrogated the expression of NF-κB p65 in hepatic stellate cells; decreased anti-apoptotic protein Bcl-2 and the mRNA transcription of hepatic type I collagen, α-smooth muscle actin, transforming growth factor beta 1, A1 and tissue inhibitor of metalloproteinases-1; increased matrix metalloproteinase 2 activity and promoted hepatic stellate cell apoptosis.


CONCLUSIONS
NF-κB knockdown enhances hepatic stellate cell apoptosis and attenuates extracellular matrix production.
Cui
Donglai
D
Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang Hebei 050000, China.
Zhang
Sui
S
Ma
Junji
J
Han
Jing
J
Jiang
Huiqing
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics; D002417:Cattle; D002461:Cell Line, Transformed; D024042:Collagen Type I / Q000235:genetics; D005109:Extracellular Matrix / Q000187:drug effects* / Q000473:pathology; D020868:Gene Silencing / Q000187:drug effects; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D008106:Liver Cirrhosis, Experimental / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D051996:Transcription Factor RelA / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D024042:Collagen Type I; D016328:NF-kappa B; D034741:RNA, Small Interfering; D019715:Tissue Inhibitor of Metalloproteinase-1; D051996:Transcription Factor RelA; D053773:Transforming Growth Factor beta1; D053148:Caspase 3