Expression And Significance Of NF-κB P65in Ratsliver Tissue Affecting By IGFBPrP1Packaging In Adenovirus Vector

BackgroundLiver fibrosis is the common pathological basis for diverse liver injuries.The activation of hepatic stellate cells (HSCs) is initial factor. The information networks including series of cytokines and cellular signal transduction pathways make HSCs activation、 a-smooth muscle actin (a-SMA) expression and ECM synthesis.Nuclear transcription factor-κB(NF-KB) is a family of multi-directional transcriptional activation protein, presenting in many cells. It may participate in the development of liver fibrosis by regulation hepatocytes, HSCs and Kupffer cells proliferation、 activation.Liu et al discovered and confirmed IGFBPrPl was a new factor-induced hepatic fibrosis. Which intraperitoneal injection, capsules implanted osmotic pump and adenovirus vector selectively cause liver fibrosis.On the basis of these reviews， we inject IGFBPrP1mediated by adenovirus vector into rats’ caudal vein, observe the change of NF-κB p65in liver tissue, explore whether NF-κB signaling pathway is involved in IGFBPrPl induced liver fibrosis process.Objective To investigate the expression and significance of NF-κB p65in rat liver tissue which affected by IGFBPrPl mediated by adenovirus vector.Methods Thirty-two clean male6-week-old SD rats were set into four groups randomly(8in each). Normal control group (the same volume of normal saline was injected into caudal vein once), Negative control Ad-EGFP group(Ad-EGFP of4×109pfu was injected into caudal vein once), Ad-IGFBPrP12weeks group (Ad-IGFBPrP1of4×109pfu was injected into caudal vein once), Ad-IGFBPrP14weeks group (Ad-IGFBPrP1of4×10pfu was injected into caudal vein once).Two weeks and four weeks later, rats in each group narcotize with diethyl ether to collect and fix left hepatic lobe. Morphological changes of liver tissue observed by HE staining; Collagen fiber content in liver tissue were detected by picric acid-Sirius red staining; Distribution and dynamic expression of IGFBPrP1, a-SMA. NF-κB p65, Fn, in each group were detected by immunohistochemistry staining.Result1. Histomorphological changes in rats liver (HE staining and saturated picric acid-Sirius red staining)In normal control and negative control Ad-EGFP group, the hepatic lobular structural were integrity, hepatic cords were neat, plump cytoplasm of liver cells, without inflammation, necrosis et al pathological changes. Only thin collagen fibers were visible in the vascular wall; the liver in Ad-IGFBPrPI2weeks group were disorder with fatty degeneration of hepatocytes, inflammatory cell infiltration and rarely necrosis, apoptosis; Ad-IGFBPrPl4weeks group, the degeneration of hepatocyte steatosis, inflammatory cells and liver nuclei fragmentation, dissolution, disappearance increased, lobular structure and hepatic cord were disorder, fiber cord appeared. Collagen fibers in central venous and portal area significantly increased, collagen deposited in the liver sinusoid, fiber cord stretched between central veins.2. The results of immunohistochemical staining in liver tissueThe contents of IGFBPrP1, NF-κB p65, a-SMA, Fn were significantly increased in Ad-IGFBPrP1group of2,4weeks compared with normal control group and negative control Ad-EGFP group, which were gradually increased with process of fibrosis.(1) In normal control group and negative control Ad-EGFP group, IGFBPrPl expressed in the central vein endothelial, rarely hepatocytes and portal area, stained pale; in Ad-IGFBPrP12weeks group, the central venous wall, around the central vein of liver cells and perisinusoidal space distributed a lot of brown particles, deeply stained, four weeks group also observed deeply stained particles around fiber cord.(2) Normal control group and negative control Ad-EGFP group, NF-κB p65is only expressed in the minute quantity hepatic cytoplasm around the central vein, the coloring lighter; Ad-IGFBPrP12weeks group mainly positive particles in perisinusoidal space, degeneration of liver cells, fibrosis area, in addition, Ad-IGFBPrP14weeks group was also observed in the nucleus.(3) Only in the portal area could find lighter dyed particles in the normal control group and negative control Ad-EGFP group. Ad-IGFBPrPl group, a-SMA expressed abundantly in fibrous interval area, stained deeper. As time goes on, it became more and more strongger.(4) In Ad-IGFBPrP1group, around the central vein and fibrous interval area, Fn expressed larger and deeper colored particles,4weeks group was significantly increased more than2weeks group. Normal control group and negative control Ad-EGFP group were positive staining only in fewer liver cells.3. Saturated picric acid-Sirius red staining results of statistical analysisBy the arcsine square root transformation, collagen area percentage index respectively were Ad-IGFBPrP12weeks group6.36±1.64, Ad-IGFBPrP14weeks group8.02±1.61VS normal control group1.78±0.38, negative control Ad-EGFP group2.03±0.76, P<0.01. Ad-IGFBPrP14weeks group was significantly higher than2weeks group, P<0.01.4. The results of immunohistochemistry statisticalCompared with normal control group and negative control Ad-EGFP group, IGFBPrP1, NF-±B p65, a-SMA and Fn expression in Ad-IGFBPrPl group were significantly enhanced. IOD value of the statistical results are as follows.±IGFBPrP1:Ad-IGFBPrP12weeks group1.41±0.43,4week group4.93±0.18VS normal control group0.21±0.06, negative control Ad-EGFP group0.25±0.07, P<0.01.②NF-±B p65:Ad-IGFBPrP12weeks group3.66±1.59, Ad-IGFBPrP1group4weeks4.39±1.47VS normal control group0.02±0.02, negative control Ad-EGFP group0.16±0.13, P<0.01.③ct-SMA:Ad-IGFBPrPl2weeks group2.23±0.99, Ad-IGFBPrP14weeks group4.11±0.21VS normal control group0.11±0.07, negative control Ad-EGFP group0.12±0.06, P<0.01.④Fn:Ad-IGFBPrP12weeks group1.68±0.34, Ad-IGFBPrP14weeks group5.66±2.81VS normal control group0.002±0.001, negative control Ad-EGFP group0.04±0.06, P<0.01. In addition, the IOD of these four indicators in Ad-IGFBPrP14week group was significantly higher than Ad-IGFBPrP12weeks group, the difference was statistically significant (P<0.01).ConclusionAd-IGFBPrPl injected into caudal vein in rats can result in the high expression of NF-κBp65, correlated with that of a-SMA, Fn, suggesting that NF-κB signaling pathway may be involved in the formation and development of hepatic fibrosis.