The Study On Effect Of Qizhu Granule On P38Mapk Signaling Transduction Pathways In The Process Of Hepatic Fibrosis

1Theoretical investigationThrough the analysis collation of the relevant literature of the modem medical research progress and medicine for liver fibrosis research profile, combined with the basis of previous studies of our group, to further explore the specific mechanism of "collaterals blood-stasis dissolution"in the prevention and treatment of hepatic fibrosis,complete the theoretical study of the collateral disease on the treatment of hepatic fibrosis.According to the modern knowledge of anatomy, the hepatic portal vein, hepatic artery, hepatic vein are progressively branch.Sinusoidal as one of the largest permeability of the sinusoids, it is the terminal branches of the hepatic vessel,it can protection of the blood and liver cells material exchange histological basis, also can participatory exchange of material, energy exchange, information transfer function.According to traditional Chinese medicine theory, Collaterals have penetrate each other qi and xue,and other unique physiological functions. It is the bridge and hub of the blood nutrition in the organs and tissues in the meridians. Therefore, from their network structure and function of the characteristics, modern medicine sinusoidal capillarization in hepatic fibrosis change can be attributed an important part of the traditional Chinese medicine liver network and its lesions.2Experiment researchAIM:Dynamic observation the effect of Qizhu granule on sinusoid capillarity mice which caused by carbon tetrachloride.To further explain the mechanism of action, resistance of Qizhu granule hepatic fibrosis and improve the collateral disease theory of hepatic fibrosis, to provide experimental basis for the collaterals blood-stasis dissolution mechanism.METHODS:Early stages of hepatic fibrosis minces, induced by CCL4,were randomized into Normal control group, Experimental control group,Model control and Qizhu granule treatment group.And liver tissue samples were taken on1,2and4weeks. Cut at a predetermined point in time100mg (<1cm X1cm) fresh liver tissue at-80℃liquid nitrogen and save, mice liver by Western blot detection of VEGF-mediated p38MAPK, p-p38MAPK,ATF-2and p-ATF-2protein expression levels. Taken lmm3samples,fixed and watered prived by glutaraldehyde(4℃2.5%),osmic aeid andaeetone, and embedded by ethoxyline resin, were cut ins lices with colored by citric acid, The ultrastrueture of liver cell and hepatic sinusoid were observed by JEOLJEM-1400TEM.RESULTS:1. Expression of total p38MAPK protein of the rat liver tissueEach group of rat liver tissue in one,two and four weeks time point, a quantity of total p38MAPK protein expression, showing doctrinal. At one weeks,Model control group and Qizhu granule group shows different with the Normal Control group (1.75±0.25vs1.13±0.28,1.68±0.26vs1.13±0.28; p<0.05). At two weeks,Model control group and Qizhu granule group shows no different with the Normal Control group(1.56±0.44vs1.22±0.26,1.38±0.24vs1.22±0.26; p>0.05). At four weeks,Model control group shows significant different with the Normal Control group and Experimental group(1.83±0.26vs1.28±0.21,1.83±0.26vs1.38±0.22;p<0.01). At one and two weeks,There is no different between Qizhu granule group and Model control group (1.75±0.25vsl.68±0.26;1.56±0.44vs1.38±0.24; p>0.05) At four weeks, Model control group shows different with the Qizhu granule group.It have no different of total p38MAPK protein expression levels between one week、two weeks and four weeks in the four group (1.13±0.28vs1.22±0.26vsl.28±0.21;1.30±0.26vs1.18±0.31vsl.38±0.22;1.75±0.25vs1.56±0.44vs1.83±0.26;1.68±0.26vs1.38±0.24vs1.45±0.35; p>0.05)2. Expression of p-p38MAPK protein of the rat liver tissueEach group of rat liver tissue in one week and two weeks of the two time points, p-p38MAPK protein was weakly expressed in very light color. Apparent at4weeks p-p38MAPK protein expression, the color darker.At one week,Model group shows different with the normal control group and Experimental control group(0.36±0.11vs0.21±0.07,0.36±0.11vs0.24±0.07; p<0.05). At two weeks and four weeks, Model group shows no different with the normal control group and Experimental control group(0.40±0.12vs0.28±0.12vs0.31±0.10,1.51±0.41vs1.15±0.22vs1.22±0.13; p>0.05).At one week,two weeks and four weeks, here is no different between Model control group and Qizhu granule group (0.36±0.1lvs0.31±0.11,0.40±0.12vs0.37±0.14,1.51±0.41vs1.32±0.45; p>0.05).It have significant different of p-p38MAPK protein expression levels between one week、two weeks and four weeks in the four group(1.15±0.22vs0.21±0.07、1.15±0.22vs0.28±0.12;1.22±0.13vs0.24±0.07、1.22±0.13vs0.31±0.10;1.51±0.41vs0.36±0.11、1.51±0.41vs0.40±0.12,1.32±0.45vs0.31±0.11、1.32±0.45vs0.37±0.14, p<0.01).3. Expression of total ATF-2protein of the rat liver tissueEach group of rat liver tissue in one week and two weeks total ATF-2protein was weakly expressed in very light color. Apparent at4weeks total ATF-2pr otein expression, the color darker.At one week,Model control group and Qizhu granule group shows no different with the Normal Control group(0.85±0.21vs0.61±0.14,0.81±0.33vs0.61±0.14,p>0.05).There is no different between Qizhu gr anule group and Model control group(0.85±0.21vs0.81±0.33, p>0.05). At two weeks, Model group shows different with the normal control group and Experi mental control group(1.29±0.28vs0.75±0.22,1.29±0.28vs0.92±0.35; p<0.05), Th ere is no different between Qizhu granule group and Model control group(1.29±0.28vsl.13±0.26; p>0.05). At four weeks,Model control group and Qizhu gra nule group shows significant different with the Normal Control group(1.51±0.26vs1.12±0.21,1.44±0.36vs1.12±0.21; p<0.01). There is no different between Qi zhu granule group and Model control group(1.51±0.26vsl.44±0.36; p>0.05).It have significant different of total ATF-2protein expression levels between one week、two weeks and four weeks in the Normal Control group(1.12±0.21vs0.61±0.14,1.12±0.21vs0.75±0.22; p<0.01). It have significant different of p-p38MAPK protein expression levels between one week and four weeks in the Model control group and Qizhu granule group(1.44±0.36vs0.81±0.33,1.51±0.26vs0.85±0.21; p<0.01).4. Expression of p-ATF-2protein of the rat liver tissueEach group of rat liver tissue in one week and two weeks of the two time points, p-ATF-2protein was weakly expressed in very light color. Apparent at4weeks p-ATF-2protein expression, the color darker. At one week,Model grou p shows significantly different with the Normal control group(0.58±0.14vs0.38±0.14; p<0.01).Model group shows different with the Experimental control gro up(0.58±0.14vs0.38±0.04; p<0.05). There is no different between Qizhu granul e group and Model control group(0.58±0.14vs0.55±0.14; p>0.05).At two weeks, Model group shows significantly different with the Normal control group(1.11±0.38vs0.62±0.13; p<0.01).Model group shows different with the Experimental control group(1.11±0.38vs0.71±0.25; p<0.05). There is no different between Qi zhu granule group and Model control group(1.11±0.38vs0.85±0.35; p>0.05).At one week,Model group and Qizhu granule group shows different with the Nor mal control group and Experimental control group(1.77±0.41vs1.13±0.38,1.77±0.41vs1.29±0.31;1.38±0.40vs1.13±0.38,1.38±0.40vs1.29±0.31; p<0.05).There is no different between Qizhu granule group and Model control group(1.77±0.41vs1.38±0.40; p>0.05).It have significant different of p-ATF-2protein expression levels between o ne week、two weeks and four weeks in the four groups(1.13±0.38vs0.38±0.14,1.13±0.38vs0.62±0.13;1.29±0.31vs0.38±0.04,1.29±0.31vs0.71±0.25;1.77±0.41v s0.58±0.14,1.77±0.41vsl.11±0.38;1.38±0.40vs0.55±0.14,1.38±0.40vs0.85±0.35; p<0.01).Experimental control group and Model group shows different between one week and two weeks(0.38±0.04vs0.71±0.25,0.58±0.14vs1.11±0.38; p<0.05). 5. TEM RESULTNormal control group:Rat liver cells were round or oval, Cell boundaries are clear, Cell membrane integrity smooth, Visible bile duct, a large number of round mitochondria, Crest clear, No swelling, Rough endoplasmic reticulum arranged in neat rows, Surface attached to the ribosome particles, Nuclear membrane is clearly visible, complete a continuous, visible nucleoli, Liver SEC,have many fenestrae,There is a wealth of hepatocyte microvilli, Ito in the Disse gapbetween the liver cells sinus surface of the sinusoidal walls.Experimental control group:Liver ultrastructural changes is basically the same of the normal group.Model control group:Rat CCL4model of one week, varying sizes in lipid drops in the cytoplasm of liver cells, Some of the liver cells and vacuolization ofthe endoplasmic reticulum serious expansion, lots pf Round mitochondria,cristaeclear, glycogen granules than the normal group.A wealth of hepatocyte microvilli in the Disse gap,SEC have many fenestrae, Nothing outside the basement membrane structure. In Two weeks, Model group of liver cells with mild or severe damage,The Mild damage, Within the cytoplasm of liver cells of varying sizes in lipid drops, lots pf Round mitochondria, cristae clear, glycogen granules less than the normal group, The Severe damage, Hepatocyte vacuolization,Serious expansion of the endoplasmic reticulum,In the4weeks, Lipid droplets within the cytoplasm of liver cells fusion was large lipid droplets, Nucleus extrusion, some Nucleolus appeared pyknosis, The endoplasmic reticulum is a serious expansion induced liver cell vacuolization change, A lot of mitochondria within the chamber expansion, stromal thinning, Electron density decreased, Crest becomes shorter and less moved to the edge or broken, many Crest becomes shorter and less moved to the edge or broken.Qizhu granule group:Rat CCL4model of one week,varying sizes in lipid drops in the cytoplasm of liver cells, But less than the model group, No hepatocyte vacuolization change, lots of Round mitochondria, cristae clear, lots of glycogen granules, A wealth of hepatocyte microvilli in the Disse gap, SEC have many fenestrae, No significant difference compared with model group,Nothing outside the basement membrane structure.In the two weeks,Liver cells mild damage, The cytoplasm of varying sizes in lipid drops, But less than the model group, A large number of mitochondria, the crest is clear, more glycogen granules. A wealth of hepatocyte microvilli in the Disse gap, Many fenestrae in liver SEC, but no significant difference compared with model group, without the structure of the basement membrane in the endothelial cells.In the four weeks,T he cytoplasm of varying sizes in lipid drops, Part of the lipid droplet fusion were large lipid droplets, To the nucleus extrusion, No liver cell vacuolar change,a large number of mitochondria, clear ridge, A small amount of mitochondria, the electron density decreased, Visible glycogen granules and fibroblasts, Nearby Collagen fibers proliferation, SEC fenestrae less than two weeks, but see fenestrae than the model group, Nothing outside the basement membrane structure.CONCLUSIONS:Qizhu granule can regulate VEGF-mediated p38MAPK pathway, affecting the total p38MAPK and p-of p38MAPK, total ATF-2and p-ATF-2protein expression,thus improve the sinusoidal capillarization, and reversed the process of liver fibrosis.