| Objective Nuclear transcription factors Kappa B (NF-κB) is found in the 1980s with a kind of DNA specific loci union, and the regulation of gene transcription protein complexes. This cytokine has a vital relationship with the transcription and translation, especially in the ischemia/reperfusion injury (I/RI) occurs at the early stage after liver transplantation. Ribosomal protein S3 (RPS3) was originally considered as a subunit of ribosome to participate in the protein translation. In recent years, it is found that RPS3 is essential function subunits of NF-κB complex, and has a key role in gene transcriptional process. This might become another breakthrough in signal transduction knowledge in NF-κB signal channel. In this paper, using the RNAi technique to silence donor liver RPS3, observe the influences to NF-κB and early I/RI after liver transplantation in rats.Methods Construct the adenovirus (Ad) particles which carrier RPS3 gene specificity short hairpin RNA (shRNA). The basic techniques of liver harvesting and orthotopic transplantation without hepatic arterial reconstruction derived from the method described by Kamada with a few modifications. Adult male Sprague Dawley (SD) rats were divided into three groups: (1) RPS3 gene specificity short hairpin RNA processing group (Ad-RPS3-shRNA), (2) blank adenovirus control group (Ad-blank-control), (3) saline control group (NS-control). Ad-shRNA-hRPS3, blank adenovirus vectors, and identical volume of 0.9% saline were injected through the portal vein to prospective donor animals under brief ether anesthesia, respectively. 48h later, the donor livers were harvested. The rats in the three groups were sacrificed at 3h and 12h after reperfusion, blood samples were collected from the precava, and the liver tissues were harvested for further studies. Automatic biochemistry analyzer test plasma ALT, AST level; liver tissues sections were stained with hematoxylin-eosin (H-E) and evaluated by light microscopy. Histopathological analysis, for the severity of liver I/RI, was based on the scoring system proposed by Suzuki; ELISA was used to test plasma inflammation factors TNF-α,IL-1βand ICAM-1 levels; qRT-PCR was used to detect TNF-α,IL- 1β,ICAM-1and RPS3 mRNA levels in liver tissues; Western blot was used to detect NF-κB level; EMSA was used to detect NF-κB protein activity.Results Liver function: 3h and 12h after reperfusion, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in Ad-RPS3-shRNA group (725.9±67.3 U/L, 1842.7±176.6 U/L; 863.7±72.7 U/L , 1513.4±187.6 U/L) significantly lower than those in the Ad-blank-control group (1736.5±173.7 U/L, 3232.6±273.1 U/L; 1874.6±163.7 U/L, 3149.1±282.6 U/L) and NS-control group (1472.2±178.4 U/L, 3347.6±293.5 U/L; 2109.3±188.7 U/L, 3662.4±269.6 U/L) (P < 0.01), the differences between Ad-blank-control group and NS-control group were not significant (P > 0.05). Histologic injury: 3h and 12h after reperfusion, in Ad-RPS3-shRNA group Suzuki score (3.72±0.31, 3.94±0.34) significantly lower than those in the Ad-blank-control group (7.93±0.63, 8.18±0.66) and NS-control group (8.22±0.61, 8.25±0.69) (P < 0.01), the differences between Ad-blank-control group and NS-control group were not significant (P > 0.05). Cytokines in Plasma, Cytokine mRNA levels in liver tissues, NF-κB protein levels and the activity tends to be consistent with that of Liver function and the differences between Ad-shRNA-hRPS3 group and the two control groups were significant (P < 0.01).Conclusion In This experiment, we proved the portal vein infusion of Ad-RPS3-shRNA, can achieve the purpose of effectively carrying and control the goal of gene expression; In liver transplantation, via the portal vein infusion Ad-RPS3-shRNA, can significantly reduce the NF-κB activity and express, reduce inflammation factor expression that reduces histologic injury after liver transplantation. In conclusion: Silencing RPS3 in Kupffer cells impairs ischemia-reperfusion injury following orthotopic liver transplantation in rats.
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