The Effect Of Sodium Nitroprusside On Proliferation And Apoptosis Of Hepatic Stellate Cells

The activation and proliferation of Hepatic stellate cell ( HSC) are the key to liver fibrosis, which secrete a large number of type I procollagen (Precollagen I) and tissue inhibitors of matrix metalloproteinaseas (TIMP-1), the apoptosis of activated HSC plays a key role in reversing hepatic fibrosis. Therefore, the inducing apoptosis of activated HSC is an important strategy in anti-fibrosis therapy.Nuclear factor-κB (NF-κB) is a nuclear transcription regulation factor which widely exists in boby cells. NF-κB can mediate transcription expression of a variety of inflammatory mediators, it is also involved in the regulation of cell apoptosis. Recent Studies have indicated that NF-κB plays an important role in HSCs apoptosis and reversing liver fibrosis.In recent years, it is discovered that nitric oxide (NO) has a new NO biological effect on the regulation of gene expression. It is reported that NO may control NF-κB activity and induce HSCs apoptosis via the mitochondrial membrane pathway, while the mechanism which NO induces HSC apoptosis via NF-κB pathway has not yet reported. In this study, we demonstrate the mechanism which NO donor sodium nitroprusside (SNP) induces HSC apoptosis via NF-κB pathway, thus the drug provides a new hope and theoretical basis for treating liver fibrosis in the clinical application. The experiment mainly contains two parts as follows:Part 1:The effect of Sodium nitroprusside (SNP) on proliferation and apoptosis of activated Hepatic Stellate Cells in vitro and mechanisms involvedObjective: To investigate SNP on activated HSC in vitro proliferation, apoptosis and its mechanisms involved.Methods: The HSC-T6 was cultured in vitro, the proliferation of HSC-T6 was determined by MTT; The HSC apoptosis rate was determined by Propidium Iodide-labelled flow cytometry (FCM), while the HSC apoptosis morphological change was observed by Fluorescence microscope and Hoechst 33258 staining; The expressions of matrix metalloproteinase inhibitor-1 (TIMP-1), I-type pre-collagen (Precollagen I) mRNA were detected by real-time Polymerase Chain Reaction (PCR). Experimental groups are as follows:①Control group, Only 8% FBS-containing DMEM cell culture medium cultured cells;②tumor necrosis factorα(TNF-α) group, 100ng/ml TNF-α;③SNP group, the concentration is 1, 5, 10, 20mmol/L SNP respectively;④SNP+TNF-αgroup, 10mmol/L SNP before 100ng/ml of TNF-α.Results:①MTT showed that SNP of different concentrations obviously inhibited the proliferation of HSC-T6. With the ever-increasing SNP concentration, the proliferation inhibition rate was increased significantly. 1, 5, 10, 20 mmol/L SNP, compared with control group, respectively is (0.89±0.23, 0.50±0.13, 0.46±0.04, 0.36±0.09 )vs (1.24±0.22), the difference was statistically significant (P<0.05).②Propidium Iodide-labelled FCM showed the HSCs apoptosis rate in SNP group was increased significantly compared with that control group (20.78±5.91% vs 3.25±1.26%, P<0.05); The HSCs apoptosis rate in SNP group was increased significantly compared with that TNF-αgroup (20.78±5.91% vs 1.06±0.14%, P<0.05); The HSCs apoptosis rate in SNP+TNF-αgroup was increased significantly compared with that TNF-αgroup (15.36±5.97% vs 1.06±0.14%, P<0.05); While the HSCs apoptosis rate in control group was not changed significantly compared with that TNF-αgroup (P>0.05).③Hoechst 33258 fluorescent staining showed: the HSC nuclear in control group showed diffuse homogeneous fluorescence; The part of the HSC nuclear in SNP group showed dense nuclear stain block or granular fluorescence, indicating condensed nuclei, chromatin condensation, DNA fragmentation, cells were apoptotic.④The expressions of TIMP-1, Precollagen I were detected by real-time PCR. The TIMP-1, Precollagen I mRNA expression could be applied to the relative quantitative (2-△△Ct method). The mRNA expression in control group was assigned a reference value of 1. 1, 5, 10mmol/L SNP Precollagen I mRNA expression was (0.80±0.17, 0.56±0.16, 0.43±0.15) fold respectively, (P=0.004); 1, 5, 10mmol/L SNP TIMP -1 mRNA expression was (0.67±0.35, 0.50±0.28, 0.37±0.27) fold respectively, (P=0.003).With the ever-increasing SNP dose, Precollagen I, TIMP-1 mRNA expression was reduced, suggesting that SNP could inhibit Precollagen I, TIMP -1 mRNA expression.Conclusions: SNP can inhibit proliferation and induce apoptosis of HSC-T6, reduce Precollagen I, TIMP-1 mRNA expression, reducing the occurrence of liver fibrosis and development.Part 2:Induction of apoptosis with SNP in TNF-α-stimulated hepatic stellate cellsObjective: To explore the NO donor SNP-induced apoptosis in rat HSC-T6 and the relationship between NF-κB pathway.Methods: The activity of NF-κB P65 via the nuclear translocation was studied with laser scanning confocal microscopy fluorescent; The expressions of growth arrest and DNA damage-inducible protein (GADD45β) mRNA was detected by real-time PCR. Experimental groups are as follows:①Control group, Only 8% FBS-containing DMEM cell culture medium cultured cells;②tumor necrosis factorα(TNF-α) group, 100ng/ml TNF-α1h;③SNP group, 10mmol/L SNP;④SNP+TNF-αgroup, 10mmol/L SNP 1h before 100ng/ml of TNF-α1h.Results:①Laser scanning confocal microscope observated: HSC-T6 NF-κB P65 exists in the cytoplasm in resting state; The HSC NF-κB P65 was transferred from the cytoplasm to the nucleus when it was activated.The results showed that HSC NF-κB was activated in TNF-αgroup, while TNF-α-mediated activation of HSC NF-κB P65 was inhibited in SNP group.②The expression of GADD45βmRNA was detected by real-time PCR. The GADD45βmRNA expression could be applied to the relative quantitative (2-△△Ct method). The mRNA expression in control group was assigned a reference value of 1. 1, 5, 10mmol/L SNP GADD45βmRNA expression was (0.43±0.20, 0.36±0.18, 0.26±0.07)fold respectively, (P=0.001). With the ever-increasing SNP dose, GADD45βmRNA expression was reduced, suggesting that SNP could inhibit GADD45βmRNA expression.Conclusions: The mechanism which SNP induces HSCs apoptosis may be associated with inhibiting NF-κB activity, blocking the expression of downstream target gene GADD45β.