Mechanism Of Cod Skin Collagen Peptide On Liver Injury In Mice

Melanogrammus aeglefinus is known as the "North Atlantic cod",in the north of the Yellow Sea and the East Sea of china it has a rich production.In recent years,the study found that cod skin is rich in protein,the active peptide of the protein have antioxidant,anti-viral,anti-ulcer and other effects.Liver is one of the most important metabolic organ of body,while the liver is susceptible to damage and the drug treatment of liver disease will increase the metabolic burden of the liver,thus developing a safe and effective health food for liver disease patients has become a top priority of liver disease prevention and treatment.Preliminary experiments show that cod skin collagen peptide(CSCP)extracted from the skin of cod has good repairing function to the damage liver cell.For this,taking CSCP as raw materials and studying its' protecting function and mechanisms to the mice liver injury by establishing animal model of liver injury in mice and giving drugs to the mice through oral administration way.This study provides a theoretical basis for the development of code skin and the application of marine active substances on health food of liver disease patients.Research contentsThis paper selects the mice with acute and chronic liver injury as animal models,formulates the CSCP into various concentrations as raw materials to study the protecting effects and the mechanism of CSCP in curing the acute and chronic liver injury of mice from the following aspects.1.Giving mice different concentrations of CSCP through oral administration way and doing acute toxicity test by giving mice as large as possible dose of drugs to determine the maximum dosage of CSCP.2.Establishment of mice acute liver injury model: The experiment lasted for seven days;The first seven days giving the mice different concentrations of CSCP and positive drugs through oral administration way;After the last administration in the seventh day injecting the peanut oil solution contains CCl4 of 0.3% concentration in it into the peritoneal of mice;Prohibit eating but drinking after 24 hours dissecting and obtaining the mice' blood and livers.Establishment of mice chronic liver injury model: The experiment lasted for eight weeks;Injecting the peanut oil solution contains CCl4 of 25% concentration in it into the peritoneal of mice three times every week;Giving the mice different concentrations of CSCP and positive drugs through oral administration way every day from the fourth week;Prohibit eating but drinking after the last oral administration dissecting and obtaining the mice' blood and livers.3.The mice daily morphology and visceral were observed,which was giving CSCP before and after,the mice weight changes in each group during the experiment.4.Detecting the activity of AST,ALT,ALP,?-GT in the mice serum and SOD,GSH-Px,CAT in the mice liver homogenates and testing the contents of MDA through kit method.5.Observing the pathological changes of the paraformaldehyde solution immobilized fresh mice liver by HE staining and observing the ultrastructural changes of glutaraldehyde solution fixed mice liver by transmission electron microscopy.6.Determining the levels of TNF-? in liver through immunohistochemistry.7.Measuring the situation of Bax,Bcl-2,Cleavage Caspase-3,Caspase-9,Cytosolic Cyt c,TNF-?,JNK and P-JNK protein of mice liver tissue which effected by CSCP through Western blot.Research results1.Acute toxicity test results show that the maximum dose of CSCP reaches 8.0 g / kg,the mice have no abnormal behavior and no death;Dissecting the mice after feed and finding that the heart,liver,spleen and other organs are complete;It shows that CSCP is safety and non-toxic on mice.2.After modeling the activity of AST and ALT in mice serum are significantly increased and they have a significant difference;It shows that AST,ALT is released a lot through the cell membrane and the cells are damaged seriously.The activity of AST,ALT in the mice serum of CSCP treatment group decreased,the release of transaminases has been inhibited.The activity of ?-GT in the liver injured mice Serum increased;It shows that the small bile ducts or bile capillary within the liver suffered serious injuries,the activity of ?-GT of CSCP treatment group decreased,it achieves the effect of suppressing.ALP activity was significantly increased in the model group,it means that the ALP in the liver secretes a lot under the stimulation of CCl4 and then the activity of ALP increases;Compared to the model group the ALP activity of CSCP treatment group decreases significantly,it inhibits the activity of ALP in injured liver cell increasing;It means that CSCP can help recover the activity of transaminase in the liver injured mice serum.3.We observed the mice daily morphology,the normal group mice hair was brighter,the mental were in better state,which is appear to initiate feeding and eating tendencies;the model group hair tends to gray,spiritual became badly,appeared lethargy phenomenon and without influence change in environment;the CSCP group mice and the positive drug group mice gradually improved mental state compared with the model group.The mice visceral changes were observed,the normal group mice organs were good;the model group liver became larger and darken;the CSCP group mice and the positive drug group mice organs were good,liver color gradually compared became lighter;the low-dose group mices liver color were deep than the high-dose group,the high-dose groups mices liver size were less than the low-dose group.Through analysised the mices weight observation,we found that the mice weight changes were increase in each group during the experiment,the CSCP group mices weight increases were more than other group;compared with the model group,the positive drug group mices weight were obvious;the CSCP group mices were significantly increased in weight,the CSCP group were better than the positive drug group.4.After modeling the activity of SOD in mice liver homogenates is significantly decreased;Antioxidant capacity is blocked;At the same time the content of lipid oxidation products--MDA also increased significantly,it shows the liver cell of mice is injured seriously;In the CSCP administration group,the activity of antioxidant enzymes SOD is enhanced,the content of MDA is decreased,it indicates that CSCP have the ability to enhance the activity of antioxidant enzymes in liver injured mice;The GSH-Px and CAT have a lower activities in liver injury model,it shows that GSH-Px can't play its specific catalysis and can't reduce free radicals to water and hydroxyl radicals;In the CSCP administration group,the activity of GSH-Px and CAT is enhanced,it shows that CSCP can increase the activity of antioxidant enzymes to some extent and inhibit the content increase of MDA.5.After modeling,it is observed that the lobule in mice is incomplete,liver cord disorders,inflammatory cell infiltration and fatty degeneration through HE staining.It shows the liver cell of mice is damaged seriously;In the CSCP administration group,the mice lobule is much more complete,liver cord arrangement is clear,inflammatory cells is reduced and fatty degeneration is weaken.It indicates that CSCP can repair liver tissue effectively;Further observing the ultrastructure of mouse liver by transmission electron microscopy,it is found that the mitochondria in liver tissue swelling significantly,fatty degeneration and inflammatory cell infiltration after modeling.It shows the liver cell of mice is damaged seriously;After giving CSCP the damage of mice liver tissue is improved,the mitochondrial swelling,fatty degeneration and the inflammatory cell infiltration are reduced,the result of HE staining and transmission electron microscopy are consistent.6.Immunohistochemical staining shows that the expression of tumor necrosis factor TNF-? is apparent in the model group and its' important role in apoptosis has also been confirmed,it shows the liver cell of mice in model group is damaged seriously;The expression of tumor necrosis factor TNF-? is decreased in the CSCP administration group,it shows the degree of liver damage is reduced.7.By Western blotting further explore the mechanism of liver injury in mice,the results shows that the JNK phosphorylation of JNK pathway in mice liver protein is enhanced,the JNK phosphorylation may start a death receptor pathway,prompt the occurrence of apoptosis signal;The protein expression of pro-apoptotic gene Bax in Bcl-2 family is increased;The protein expression of antiapoptotic gene Bcl-2 is reduced;The expression of Bcl-2 and Bax is unbalanced and then accelerates apoptosis;A large number of cytochrome C Cytosolic Cyt c is released to the cytosol under the influence of Bcl-2 family or the regulation of the body's own;Then stimulating Caspase family to play its role and activating Caspase-9;Cleavage Caspase-3 executes apoptosis under the chain reaction.After giving CSCP the phosphorylation of JNK gene in mice liver protein is weakened;The expression of pro-apoptotic protein Bax gene in Bcl-2 family is decreased;The protein expression of antiapoptotic gene Bcl-2 is increased;The release of cytochrome C Cytosolic Cyt c in the cytosol is reduced;The protein expression of Caspase-9 activity is reduced;Cleavage Caspase-3 can't execute the apoptosis;The protein expression of tmor necrosis factor TNF-? is decreased,finally achieving the protective effect on liver injury in mice.ConclusionsThrough a series of experimental results of our study,we find that CSCP has a good protective function on acute and chronic liver injury models of mice.1.Acute toxicity test shows the CSCP reaction was safe and nontoxic with mice,which make sure in mices experiments well.2.After modeling,mice have a malaise mental state,loss appetite and become lethargy.The activity of AST,ALT,ALP,?-GT in the mice serum of model group is significantly increased;The activity of SOD,GSH-Px,CAT in liver tissue homogenates is significantly reduced;The content of MDA is increased a lot;Pathology observation finds that the liver of mice is damaged seriously,modeling is succeed.3.The mice daily morphology and visceral were observed,which was giving CSCP before and after,the mice weight changes in each group during the experiment.We can conclude that there is not only conducive to recovery of the mices mental in giving CSCP,but also is helpful in terms to restoring the damaged in organs,and CSCP could increase the weight of mice.4.CSCP can significantly reduce the activity of AST,ALT,ALP,?-GT in liver injured mice serum,significantly increase the activity of SOD,GSH-Px,CAT in liver homogenates and significantly reduce the content of MDA.5.The result of HE staining shows that CSCP can improve liver extent of disease,the hepatic lobule is almost complete,liver cord arranges regularly,the inflammatory cell infiltration and vacuoles are reduced,karyopyknosis is not obvious.Transmission electron microscope observations show that after CSCP effect on liver injured mice the liver ultrastructure of mice is significantly improved;Karyotheca boundaries become clear gradually;Organelles are intact;The number of mitochondria are increased;Heterochromatin edge dimerization is weakened;The expansion of rough endoplasmic reticulum is moderated.6.Immunohistochemical staining finds that CSCP can significantly reduce TNF-? protein expression in the liver and the extent of liver damage.7.Western blotting shows that after CSCP effect on liver injured mice the extent of phosphorylation of JNK gene in two model is decreased;The protein expression of tumor necrosis factor TNF-? is reduced;The protein expression of antiapoptotic gene Bcl-2 is increased;The protein expression of Pro-apoptotic gene Cleavage Caspase-3,Caspase-9 in Bax and Caspase family are significantly reduced;The protein expression of Cytochrome C Cytosolic Cyt is also significantly reduced.