Proteomics to display tissue repair opposing injury response to LPS-induced liver injury

AIM: To examine the protein expression alterations in liver injury/repair network regulation as a response to gut-derived lipopolysaccharide (LPS) treatment, in order to anticipate the possible signal molecules or biomarkers in signaling LPS-related liver injury.METHODS: Male BALB/c mice were treated with intraperitoneal (i.p.) LPS (4 mg/kg) and sacrificed at 0, 6, 24 and 30 h to obtain livers. The livers were stained with hematoxylin and eosin for histopathologic analyses. Total liver protein was separated by two-dimensional gel electrophoresis (2-DE). The peptide mass of liver injury or repair related proteins were drawn up and the protein database was searched to identify the proteins.RESULTS: Observations were as follows: (1) TRAIL-R2 was down regulated in livers of LPS-treated mice. TNFAIP1 was significantly up regulated at 6 h, then down- regulated at 24, 30 h with silent expression during senescent stage.(2) The amount of metaxin 2 and mitochondria import inner membrane translocase subunit TIM8a (TIMM8A) was increased upon treatment with LPS, (3) P34 cdc2 kinase was significantly up-regulated 30 h after LPS administration with silent expression during senescent, 6, 24 h treated stage. (4) The amount of proteasome activator 28 alpha subunit (PA28), magnesium dependent protein phosphatase(MDPP) and lysophospholipase 2 was decreased 6 h after LPS treatment but recovered or up-regulated 24 and 30 h after LPS treatment.CONCLUSION: LPS-treated mouse liver displaying a timedependent liver injury can result in expression change of some liver injury or repair related proteins.

Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

AIM: To increase exogenous gene expression level bymodulating molecular conformations of targeting gene drugs.METHODS: The full length cDNAs of both P40 and P35subunits of human interleukin 12 were amplified throughpolymerase chain reaction (PCR) and cloned into eukaryoticexpressing vectors pcDNA3.1(±) to construct plasmids of P(+)/IL-12, P(+)/P40 and P(-)/P35. These plasmids werecombined with ASOR-PLL to form two targeting gene drugs[ASOR-PLL-P(+)/IL-12 and ASOR-PLL-P(+)/P40 + ASOR-PLL-P(-)/P35] in optimal ratios. The conformations of these twodrugs at various concentrations adjuvant were examined underelectron microscope (EM) and the drugs were transfected intoHepG2 (ASGr+) cells. Semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) was performed withtotal RNA extracted from the transfected cells to determinethe hiL12 mRNA transcript level. The hiL12 protein in thecultured supernatant was measured with enzyme-linkedimmunosorbent assay (ELISA) 48 hours after transfection.RESULTS: Targeting gene drugs, whose structures weregranular and circle-like and diameters ranged from 25 nmto 150 nm, had the highest hIL-12 expression level. ThehIL-12 expression level in the group co-transfected withASOR-PLL-P(+)/P4o and ASOR-PLL-P(-)/P35 was higher thanthat of ASOR-PLL-P(+)/IL-12 transfected group.CONCLUSION: The molecular conformations of targetinggene drugs play an important role in exogenous geneexpression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. Thesizes and linking styles of exogenous genes also have someeffects on their expression level.