| BackgoundAn increasing number of studies have shown that the liver secondaryto chronic inflammatory injury after liver fibrosis, inflammatory responseand development process plays a very important role in hepatic fibrosis.Alcohol, pathogenic microorganisms and other related inflammatoryresponse objects can also activate the immune system which inducedinflammatory response leading to the formation of liver fibrosis. Inautoimmune inflammation of the liver inflammatory response of acute andchronic phase, the liver cells, endothelial cells, hepatic stellate cells secretea variety of cytokines and chemokines which induced the inflammatorycells infiltration in the injury site, involved in mediating the acute phase theresponse of tissue repair and injury.Toll receptors can sense pathogens and directly make a defensivereaction of the innate immune receptor. Including MyD88and TRIF is twomajor signaling pathways. They have the important positive role in all TLRadapter-mediated intracellular signal transduction. Studies have shown thatsome can be inflammatory or pro-inflammatory factor regulation of TLRsexpressed in liver cells. Liver cell surface TLR expression in liver disease caused by liver cell injury also plays an important role. LPS together itsreceptor TLR4binding and activation of downstream signal transductionpathways which a series of immune responses in vivo, and therebycontributed to fibrosis. Thus, TLRs play an important role in the process ofhepatic fibrosis. However, current research is focused on the upstreamsignal, few reports on the characteristics and the innate immunedownstream signaling factor expressed in liver fibrosis.MyD88and TRIF as an important signaling pathway, but itsexpression changes in characteristics are still not clear. Thus, we want toobservate the the expression and role of the characteristics of tolldownstream signaling factor of MyD88and TRIF, to explore thepathogenesis of liver fibrosis and treatment.ObjectiveTo observe the characteristics of the innate immune signal factorMyD88expression in liver fibrosis, and investigating the occurrence anddevelopment of MyD88and liver fibrosis. Study innate immune signalingfactor, TRIF expression characteristic in liver fibrosis, gene and proteinexpression pattern, explore the relationship of the TOLL-like receptordownstream signal factor TRIF and development of the pathologicalmechanisms of liver fibrosis.Methods:1. Modeling and material:40SD rats were randomly divided into normal control group (n=10) and the experimental group (30). Experimentalgroup rats were given self-preparation of high-fat low protein diet(containing39.5%cornmeal+40%wheat flour+20%lard and0.5%cholesterol), while giving the experimental group, subcutaneous injectionof CCl4oil,2times a week, for a total of12weeks. The first week ofsubcutaneous dose are all0.5ml/100g weight after each injection dose0.3ml/100g weight, arrange a week2and week5, even10%alcohol perday as the only beverage. Normal control rats fed with normal diet. sterilepyrogen-free conditions from rat abdominal aorta blood injected in theheparin plus pyrogen-free glass test tubes, centrifuged to obtain plasmaplaced in a glass tube sealed cryopreservation. Take fresh liver left lobeimmediately fixed in4%formaldehyde for24h dehydration, transparent,embedded in paraffin; part of the placed to be stored at-80℃.2. HE staining: liver paraffin sections for HE staining to observe the liverlesions.3. Immunohistochemical detection: specimens routine dewaxing, gradientdehydration, over the hydrogen peroxide inactivation of endogenousperoxidase, citric acid and hot fixes. Goat serum (1%Triton X-100) for15minutes. Rabbit anti MyD88,1:50dilution,4°C overnight as the firstantibody, HRP-labeled antijbody,37°C incubated for60minutes, the PBSrinse, DAB stainning, dehydration, transparent Fengpian after microscopicexamination. 4. RT-PCR method to detect the expression of the live MyD88: extractionof rat liver total RNA, reverse transcription at42℃under20minutes, theproduct of cDNA. Amplification reaction and products electrophoresis. Gelimager mining, Quantityone the one image analysis software.5electron microscope specimen preparation and testing0.1mol/L PBS solution wash, cut into small pieces of tissue,10g/Lsodium pentobarbital anesthetized rats,50mg/kg, intraperitoneal injection,remove the liver into more than2%POM-2.5%glutaraldehyde pre-fixed.Cold buffer, rinsed15min×3times,1%osmium tetroxide at roomtemperature for2h in accordance with the routine embedding of theelectron microscope, sliced, uranyl acetate and lead citrate electronicstaining, Hitachi S-7500transmission electron microscope.6. Western blot analysis: cracking the liver tissue, the BCA Protein Assay,laminated plastic and separation gel electrophoresis, transferred to thenitrocellulose membrane, nonfat dry milk at room temperature closed,rabbit anti MyD88polyclonal antibody at4℃overnight PBST washed byadding horseradish conjugated goat anti-rabbit IgG, and room temperaturefor2h, chemiluminescent detection and software analysis.7. Rat behavioral observation: in the process of immunization, and closeobservation of the rats in each group behavioral changes, and determinewhether the abnormal performance.8Statistical analysis: SPSS17.0package for data analysis, data±s, among the groups first single factor homogeneity of variance, homogeneityof variance analysis of variance, heterogeneity of variance by rank-sumtest, P <0.05indicates statistical significance.Results1. Behavior Evaluation: normal control rats in good health, good mentalstate, normal weight, normal diet, and wool bright. Remove the liverspecimens of visible liver surface lubrication rosy, soft texture; and modelgroup, decreased activity, diet to reduce wool dull dull, poor spirit, weightdecreased. Remove the liver dark plot knot, the weight increase.2. Immunohistochemical detection results: the rules of normal ratshepatic lobules arranged orderly and no abnormal cells and other structures.In normal liver cells, liver parenchymal cells was weakly expressed,expression of MyD88and TRIF are mainly concentrated in the Kupffercells, endothelial cells, biliary epithelial cells and other non-substantivehepatocytes. Hepatic lobule structure of the model group was significantlydamaged, significantly reduced the number and structural disorder disorder,replaced by a large number of abnormal cell infiltration, fibrosis, and othercharacteristics. MyD88and TRIF expression increased significantly havestrongly expressed in endothelial cells, astrocytes, and mainly to thenucleus expression, positive and negative control, no positive expressionproducts.3. RT-PCR of detection MyD88mRNA expression: compared with normal control group, liver fibrosis group MyD88mRNA expression wassignificantly increased, the difference was significant (p <0.01).4. Western blot analysis MyD88and TRIF protein: compared withnormal control group, hepatic fibrosis MyD88and TRIF protein expressionwas significantly increased, the difference was significant (p <0.01).5TEM results of detectionNormal control rat liver cell membrane integrity, uniform cytoplasm,nucleus round and centered, prominent nucleoli. The sinusoidal week gapno fiber deposition, lipid droplets rare; compared with the normal controlgroup, liver fibrosis were observed in the high-density material, the livercells surrounded by a large number of collagen fiber depositionphenomenon. Perisinusoidal fibrosis, and dieldrin clearance dilated andvisible adipogenic cells; cytoplasm visible dissolution phenomena in thecavity of dieldrin, hepatic stellate cells in the nucleus dissolved, thesinusoidal endothelial cell cytoplasm, the nuclei are dissolved.Conclusion1. MyD88gene and protein expression of the liver fibrosis wassignificantly enhanced, in liver parenchymal cells and non-parenchymalcells have obvious expression distribution, MyD88may play an importantrole in fibrogenesis.2. TRIF protein expression of the liver fibrosis was significantlyenhanced, in liver parenchymal cells and non-parenchymal cells showed significant expression distribution, TRIF may play an important role infibrogenesis. |
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