| Carapax Trionycis derives from turtle back of Trionyx sinensis Wiegamann,it has been classified as a special type of Traditional Chinese Medicine both used for medicine and food., the< Shen Nong's Chinese Materia Medica> included in the products that it has such activities as replenishing'yin', restraining'yang', softening and resolving hard masses, reducing fever and removing steam. recorded that Carapax Trionycis is used for anti-fever,abdominal mass,windy syndrome agents in compound increase body resistance, cancer and a wide range of applications. Clinical experience shows that the Carapax Trionycis as the central medicine in the compound is good at combating chronic liver damage patients. And the modern pharmacological research shows that the turtle shell extract, especially the small molecule Oligonucleotide peptide played a good role in treating chronic liver damage model animals.This experiment is divided into two parts.The first part:the pharmacological studies of turtle shell oligonucleotide peptide I-C-f-6 resistance of acute liver damage in miceKunming mice, Male, weight (22±3)g, randomly divided into 4 group, normal group(n=10), model group(n=15), turtle shell oligonucleotide peptide I-C-f-6 low dose group(n=10) and high dose group(n=10), drug toxicity group(n=2).Normal group and model group breed conventionally. Low dose group and high dose group give I-C-f-6 by subcutaneous injection according to 0.17μg/g,0.34μg/g a week and record their weight. Establish mice acute liver damage model by intraperitoneal inject 0.15% CCl4 to model group, low dose group and high dose group. At the third week, collecting blood by remove eyeball, testing liver function, excising Liver, spleen and thymus, fixing liver, spleen and thymus by 10% formalin, or storing at-70 C.Experimental results1. Mice characterization changesAt the beginning of molding, fur color of model group mice are fade, confused and disordered, eat less, bleeding and blood cake appeared at tail. At the same time, normal group mice,low dose group and high dose group mice eat regularly, fur still burnished and orderly, no bleeding exist at tail.2. Mice viscera coefficient changesCompared to normal group, Liver and spleen weight of model group increased bemarkedly. Liver and spleen index increased significantly (P<0.01).Contrast with model group, liver and spleen index decreased significantly of each group of I-C-f-6(P<0.01)and presented dose dependent. The results show that turtle shell oligonucleotide peptide I-C-f-6 can obviously reduce live, spleen and thymus index which caused by CCl4 of liver damage.3. Mice liver function levelContrast with normal group, ALT and AST of model group increased obviously(P<0.01).Compare to model group, ALT and AST of each dose of turtle shell oligonucleotide peptide I-C-f-6 group decreased significantly(P<0.01).The results show that turtle shell oligonucleotide peptide I-C-f-6 can significantly suppress levels of ALT, AST which caused by CC14 of liver damage.4. Morphology test resultsThe normal group cells arranged like restis. Hepatic sinus linked with central vein and emit around. Central venous wall of model group are thicken, around are Debris shape necrosis of liver cells. Nuclei dissolved and disappeared, or turned into empty bubbles. Some perivascular fibrosis serious, false flocculus formed. low dose group Hepatic sinus expaned. steatosis more than normal group, but less than model group. steatosis appeared around central vein in high dose group, did not appeared large necrotic, lighter than model group. The results show that turtle shell oligonucleotide peptide I-C-f-6 can significantly suppress the changes of morphology.The second part:Studies on Activity of turtle shell oligonucleotide peptide I-C-f-6 to resist rat chronic liver damage induced by CCl4SD rats, male, weight(170±20)g, randomly divided into 4 groups:normal group(n=12), model group(n=12), turtle shell oligonucleotide peptide I-C-f-6 low dose group(n=12), turtle shell oligonucleotide peptide I-C-f-6 high dose group(n=12). Normal group were breed conventionally; Low dose group and high dose group were given I-C-f-6 by subcutaneous injection according to 0.12μg/g,0.24μg/g. The model was established by injecting 30%CCl4 intraperitoneally,2ml/kg, twice a week,7weeks, in the model group, the low dose group and the high dose group at the same time. At the end of the 7th week, blood was collected, and then the liver function was tested. Liver, spleen and thymus were incised and stored partly in 10% formalin and partly at-70℃. MDA,SOD,IL-4,IL-10,TNF-a were tested in serum or liver homogenate. Observe the changes of ibdv infected under light-microscopy dyed by HE, the expression changes of E-cadherin,VEGF,TGF-β1 in liver tissue stained by immunohistochemy.Experiment results1. Rats weight changesNormal group weight increased steadily. Model group weight increased stable in the late experiment. Low dose group was similar to normal group, but growed slowly. The high dose group weight increased between the model group and the low dose group. The results show that turtle shell oligonucleotide peptide compounds I-C-f-6 can significantly suppress the weight loss of chronic liver damage rats induced by CCI4.2.Rat organs coefficient changesContrast with normal group, liver weight of model group increased obviously. Liver and spleen index increased significantly(P<0.01).Compare to model group, Liver and spleen index of each dose of turtle shell oligonucleotide peptide I-C-f-6 group decreased significantly(P<0.01)and presented dose dependent. The results show that turtle shell oligonucleotide peptide I-C-f-6 can obviously reduce live, spleen and thymus index which caused by CCl4. of liver damage3. Rats liver function levelContrast with normal group, ALT and AST of model group increased obviously(P<0.01).Compare to model group, ALT and AST of each dose of turtle shell oligonucleotide peptide I-C-f-6 group decreased significantly(P<0.01).The results show that turtle shell oligonucleotide peptide I-C-f-6 can significantly suppress levels of ALT, AST which caused by CCl4 of liver damage.4. The content of MDA and SODContrast with normal group, SOD of model group decreased obviously(P<0.01), the content of MDA increased significantly. Low and high dose groups can enhance the activity of SOD in different levels, reducing the generation of MDA. Compare to model group, it is significant difference(P<0.05).The effect of low dose group were much better.5. The content of Cell factors (IL-4, IL-10, TNF-a)Contrast with normal group,thecontent of TNF-a in model group increased obviously, and the content of IL-4 and IL-10 decreased significantly. Low and high dose groups can reduce the content of TNF-a, enhancing that of IL-4 of IL-10.Compare to model group, it is significant different(P<0.05).6. HE dyeing resultsThe normal group cells arranged normally. Liver cell flake necrosis serious, false flocculus has formed in liver tissue of model group. Low dose group were similar to normal group, but a few of liver fat empty bubbles can be seen. High dose group of liver cells fat were serious, but have not form a flake necrosis. Model group are light. The results show that turtle shell oligonucleotide peptide I-C-f-6 can significantly suppress the changes of morphology which caused by CCl4 of liver damage.7. Immunohistochemical stains results(E-cadherin, VEGF, TGF-β1) E-cadherin, VEGF, TGF-β1, these three antibodies are without express or small amounts of expression in normal group. Model group is strong positive expression; Low dose group of expression in weak positive; High dose group of expression between low dose group and model group. The results show that turtle shell oligonucleotide peptide compounds I-C-f-6 is probably inhibit increase of E-cadherin, VEGF, TGF-β1 to suppress the chronic liver damage occurs which induced by CCl4. |
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