The Effect Of Short Hairpin RNA Of Nuclear Factor Kappa B On The Apoptosis Of Hepatic Stellate Cell

The chronic hepatic disease is one of the important cosmopolitan diseases that are severely harmful to the health of human. Hepatic fibrosis is the middle and key element of this process. Hepatic stellate cell(HSC) is the beginning of whole thing and plays an important role in the process of the hepatic fibrosis. Inducing the apoptosis of the activated hepatic stellate cell can reverse the process of hepatic fibrosis. The increasing research has indicated that the activation and proliferation of HSC were related with the inhibition of apoptosis. Nuclear factor-κB (NF-κB) has a close relation with apoptosis. NF-κB, a protein that binds specifically to an enhancerκB sequence ofκimmunoglobin light chain is generated via homodimeric and heterodimeric interactions of Rel subunits. Mammalian Rel proteins found by now include p65 (RelA,NF-κB3), p50 (NF-κB 1) ,Rel (c-Rel),RelB和p52 (NF-κB2), and p65/p50 are found earliest, distribute most widely, and are the NF-κB that play chief physiological functions. NF-κB can induce transcription and expression of multiple cellular cytokines by sequence specifically binding to their promoter or enhancer region, thus promotes inflammation, immunological response, and has been closely correlated with certain important pathological and physical process including cell proliferation, transformation and apoptosis. Recently the appearance of RNA interference opens a new way to inhibit the gene expression.RNA interference is a process that after double-stranded RNA, complementary to the endogenous mRNA, transfected into cells, the mRNA degrades specifically, so that the gene the mRNA codes can't express, and gene silences, showing us that we can silence the gene expression of NF-κB through the application of RNA interference, which can increase the apoptosis of the HSC and slow down the process of the hepatic fibrosis.Objective: Through RNA interference by designing short hairpin RNA on the NF-κBp65, the test investigates the effects on apoptosis of lipopolysaccharide (LPS) - stimulated HSC.Methods: Rat HSC was cultured in vitro and divided into 5 groups①blank control group;②LPS group: dealing with LPS;③negative group: dealing with negative siRNA and LPS;④positive group: dealing with NF-κBp65 shRNA and LPS;⑤lipidosome 2000 group: dealing with lipidosome 2000 and LPS. The expression levels of NF-κBp65, p50 and IκBαprotein were detected by western blot, the expression level of NF-κBp65 and procollagenⅠmRNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and the change of apoptosis of HSC was detected by flow cytometry method and the method of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL).Results: 1 SiRNA to monitor efficiency in HSC: A fluorescein-labeled non-special siRNA was used to monitor efficiency in HSC, demonstrating nealy 90% transfection efficiency after transfected with 33nmol/L siRNA at 72h.2 The expression of NF-κBp65 protein increased in the cell nucleus after LPS stimulated HSC: the result of the western blot displayed that NF-κBp65 protein expression in the cell nucleus increased after LPS stimulated HSC. The expression was highest at one hour after the stimulation, and the increase of the expression with 1000ng/ml stimulating(232.15%±5.28%) was higher than that with 100ng/ml stimulating(168.18%±3.39%). The result showed us that LPS could stimulate the expression of NF-κBp65 in HSC, the expression arrived at the peak at one hour after LPS stimulated HSC and then fell off to the normal, and the expression of NF-κBp65 increased while the concentration of LPS increased.3 NF-κBp65 protein and mRNA expression decreased in the cell nucleus after NF-κBp65 shRNA transfected into the HSC: the result of the western blot displayed that NF-κBp65 protein expression of the positive group in the cell nucleus decreased. The expression at 24 hours after the interference decreased by 72.41%±1.22% than the LPS group, the expression at 48 hours after the interference decreased by 77.45%±1.23% than the LPS group, and the expression at 72 hours after the interference decreased by 86.34%±1.01% than the LPS group, and the decrease was highest at 72 hours after the interference. The expression of the negative group and lipidosome 2000 group had not marked change.The result of the RT-PCR displayed that NF-κBp65 mRNA expression of the positive group in the cell decreased obviously. The expression of the negative group and lipidosome 2000 group had not marked change. The results showed us that the experiment constructed the short hairpin RNA which aimed at the 1543-1561 of the NF-κBp65 mRNA in rat HSC, the short hairpin RNA interfere effectively the expression of NF-κBp65 mRNA and protein after the short hairpin RNA was transfected into the HSC, and the efficiency of the interference arrived at the peak at 72 hours after the interference.4 The expression of NF-κBp50 and IκBαprotein after NF-κBp65 shRNA transfected into the HSC: the result of the western blot displayed that NF-κB p50 protein expression of the positive group in the cell nucleus decreased, and the expression at 72 hours after the interference decreased by 75.45%±0.93% than the LPS group. And the expression of the negative group and lipidosome 2000 group had not marked change. IκBαprotein expression of the positive group in the cell cytoplasm decreased, and the expression at 72 hours after the interference decreased 70.61%±2.19% than the LPS group. And the expression of the negative group and lipidosome 2000 group had not marked change. The result showed us that the expression of NF-κBp50 and IκBαprotein decreased while NF-κBp65 shRNA interfered the expression of mRNA and protein of NF-κBp65 after it transfected into HSC.5 The apotosis of HSC increased after interfering with the gene expression of NF-κBp65: the result of the flow cytometry method displayed that the apoptosis of HSC of the positive group increased, and the apoptosis at 72 hours after the interference was higher than the LPS group, the apoptosis ratio increased by 15.40%±0.72%. And the apoptosis of the negative group and lipidosome 2000 group had not marked change. The result of the TUNEL assay displayed that the apoptosis of HSC of the positive group increased after NF-κBp65 shRNA interfered the LPS-stimulated HSC, and the apoptosis at 72 hours after the interference was higher than the LPS group, the apoptosis ratio increased by 33.33%±1.06%. And the apoptosis of the negative group and lipidosome 2000 group had not marked change.6 ProcollagenⅠmRNA expression in the cell decreased after interfering with the gene expression of NF-κBp65: the result of the RT-PCR displayed that procollagenⅠmRNA expression of the positive group in the cell decreased by 56.84%±1.89%. The expression of negative group and lipidosome 2000 group had not marked change. The result showed us that mRNA expression of procollagenⅠdecreased after interfering with the gene expression of NF-κBp65.7 The avtivity of gelatinase increased after interfering with the gene expression of NF-κBp65: the result of the MMPs zymography showed that the avtivity of gelatinase of the positive group increased. The avtivity of negative group and lipidosome 2000 group had not marked change.Conclusion:1 The experiment constructed the short hairpin RNA which aimed at the 1543-1561 of the NF-κBp65 mRNA in rat HSC. And the short hairpin RNA interferes effectively the expression of NF-κBp65 mRNA and protein after the short hairpin RNA was transfected into the HSC. At the same time, the expression of NF-κB p50 and IκBαprotein decreased.2 The expression of NF-κBp65 protein increased after LPS stimulated HSC.3 The apoptosis of HSC increased after NF-κBp65 shRNA interfere effectively the expression of NF-κBp65 mRNA and protein, and the expression of ProcollagenⅠmRNA decreased and the activity of gelatinase increased, which can delay the process of hepatic fibrosis.