Mechanism Of Short Hairpin RNA Silence NF-κB Promotion Apoptosis Of Hepatic Stellate Cell

It is well known that the critical event of liver fibrosis is that hepatic stellate cells(HSCs)become active, the increasing of production and secretion of extracellular matrix(ECM)and the decreasing of degradation of them result in the accumulation of over ECM in the liver, especially Type[0]Ⅰprecollagen plays a key role in the deposition of ECM. In recovery period, the apoptosis of HSCs increase significantly, on the one hand it eliminates the origin of ECM, on the other hand it also relieves the inhibitory action of tissue inhibitor of metalloproteinase to matrix metalloproteinases, thus promotes the degeneration of ECM. So the apoptosis of HSCs plays a key role in the process of the resolution of hepatic fibrosis.Nuclear factor kappa B (NF-κB) is activated in many human cells, the NF-κB/Rel family can form various homo-or hetero-dimers. However, the most studied form is a heterodimer of the p50 and p65 subunits predominant in many kinds of cells. In most normal cells, NF-κB is sequestered in an inactive form through its tight association with the cytoplasmic inhibitor proteins, called inhibitors of NF-κB (IκB), which is catalyzed by an IκB kinase (IKK). A variety of extracellular stimulus factors such as TNF-α, IL-1, lipopolysaccharide (LPS) trigger a common signal transduction pathway based on the phosphorylation and degradation of IκB to freely active NF-κB. The released activated NF-κB is rapidly translocated to the nucleus and binds to the promoter region in the relevant downstream genes to touch off a series of transcriptional events.Constitutively activated NF-κB has been implicated in survival, adherence, transformation and proliferation of cell. NF-κB regulates the expression of many genes (COX2, cyclinD1, bcl-2) necessary for inhibition of apoptosis. And experiment discovered that the activation of NF-κB inhibited the initiation of caspase family by preventing the activiation of caspase-8, thus had suppressed the caspase-3 activation, further suppressed the apoptosis of cell. Investigation indicated that NF-κB is inactive in quiescent HSCs, but its activity sustains in actived HSCs. Thus, we have justification to make it believed that NF-κB is likely to make the number of HSCs steady by inducing its apoptosis, eventually leading to hepatic fibrosis. NF-κB signaling pathway has become a potential target for the therapeutic strategies due to its role. Nowadays, RNA interference (RNAi) has become a powerful strategy for knockdown and understanding gene function.Up to now, activation of NF-κB regulation the expression of many genes involve in inflammatory, immune, proliferative and apoptotic responses has been reported, but few studies have addressed NF-κB in proliferation and apoptosis of HSCs. Therefore, using in vitro cell culture techniques, activation NF-κB by LPS, the effects and possible mechanism of suppression NF-κB expression on apoptosis in HSC were investigated by using RNAi techniques specificity suppression the expression of NF-κB subunit p65, inhibiting hepatic fibrosis.Objective: NF-κB p65shRNA was used to transfect HSC in vitro, so as to evaluate the effects of NF-κB p65shRNA inhibited the NF-κB expression of HSC and the effect and possible mechanism on apoptosis in HSC.Methods: HSCs were cultured in vitro in DMEM medium supplemented with 2% Fetal Bovine Serum, 100 IU.mL-1penicillin, 100g·mL-1 streptomycin, 4 mmol·L-1 glutamine, and 1 mol·L-1 HEPES in a 37℃and 5% CO2 environment. HSC were transfected with p65shRNA to monitor the expression of p65 and a fluorescein-labeled non-special shRNA was used to monitor the best efficiency and the best transfection time in HSC; The protein levels of NF-κB and bcl-2 were analyzed by Western blotting; Flow cytometry (FCM),TUNEL staining and DNA ladder were given to detect the effects of p65shRNA on apoptosis in HSC; The activity of caspase-3 was detected by caspase-3 Activity Assay Kit; The activity of gelatinade was detected by SDS-PAGE enzymograph; in HSC was assayed by RT-PCR on mRNA level.Results:①Non-targeted shRNA to monitor efficiency in HSC: A fluorescein-labeled non-special shRNA was used to monitor efficiency in HSC, demonstrating nealy 90% transfection efficiency after transfected with 200pmol shRNA at 72h.②The expression of p65 and nuclear activation of the NF-κB were inhibited in HSC by p65shRNA: Western Blot shower that the expression of p65 protein was significantly highter in LPS group than that in control group(2.34±0.26 vs 0.62±0.11), increased by 227.42%, P<0.01;but there were no significant differences among LPS group,liposome group and negative group, P>0.05. the expression of p65 protein was significantly lower in p65shRNA group at 72h after transfection than that in negative group (0.27±0.09 vs 2.19±0.26), reduced by 87.67%, between which there was a statistic difference, P<0.01.③The effect of p65shRNA on the apoptosis of HSC: Flow cytometry (FCM) and TUNEL staining : There were no significant differences among LPS group,liposome group and negative group after LPS stimulation, P>0.05, but they were all lower than control group, P<0.05; while compared with control group,LPS group,liposome group and negative group, the apoptotic rate of HSC significantly increased after transfected with p65shRNA at 72h. This showed that the apoptotic rate of HSC was inhibited after LPS activating NF-κB, but effectively increased after the activity of NF-κB inhibited by p65 shRNA. In DNA ladder detection, there were ladder straps in p65ShRNA group, this showed that p65ShRNA group could facilitate the apoptosis of HSC at 72h.④p65shRNA down-regulated the expression of NF-κB-regulated gene products: Western Blot showed that the expression of bcl-2 protein was significantly higher in LPS group than that in control group (1.80±0.07 vs 0.59±0.11), increased by 205.08%, P<0.05; but there were no significant differences among LPS group,liposome group and negative group, P>0.05;the expression of bcl-2 protein was significantly lower in p65shRNA group at 72h after transfection than that in negative group (0.31±0.14 vs 1.71±0.06), reduced by 81.87%, between which there was a statistic difference, P<0.01. This showed that the expression of bcl-2 protein was increased after LPS activating NF-κB, but p65shRNA-mediated reduction in p65 levels down-regulated the expression of bcl-2 at 72h.⑤p65shRNA activated the activity of NF-κB-correlated caspase-3 expression: There were no significant differences among LPS group,liposome group and negative group after LPS stimulation, P>0.05;but they were all lower than control group, P<0.05; while compared with control group,LPS group,liposome group and negative group, the activity of Caspase-3 significantly increased after transfection with p65shRNA at 72h.This showed that the activity of Caspase-3 was inhibited after LPS activating NF-κB, but effectively increased after the activity of NF-κB inhibited by p65shRNA.⑥p65shRNA enhanced the activity of gelatinase: SDS-PAGE enzymograph: compared with control group,LPS group,liposome group and negative group, the activity of gelatinase significantly increased after transfection with p65shRNA at 72h, P<0.01. This showed that the activity of gelatinase effectively increased after the activity of NF-κB inhibited by p65shRNA.⑦p65shRNA down-regulated the expression of the levels of typeⅠprecollagen: The RT-PCR was used to determine the expression of typeⅠprecollagen and GAPDH mRNAs at 72h after p65shRNA transfection, the scan assay showed that compared with control group,LPS group,liposome group and negative group, the expression of typeⅠprecollagen significantly degraded after transfection with p65shRNA at 72h, P<0.01.Conclusion: RNA interference has been used as an experimental tool with which to suppress specific p65 expression and to study the function of NF-κB in HSC; p65shRNA inhibits the active NF-κB gene expression, down-regulates the expression of bcl-2 and activates the caspase path, which may be the mechanism of induction apoptosis in HSC.