| With the high development of aquaculture intensive farming,environmental pollution and the use of high protein,high fat or moldy feed,and the abuse of antibiotics and pesticides can cause damage to fish liver and the normal function of hepatocyte,leading to liver cell rupture,liver disease,liver function decline,eventually leading to the death of fish.The research confirmed that in the hepatocyte injury in various liver diseases,a variety of enzymes,free radicals and lipid peroxidation have changed a lot;at the same time,during the development process of liver injury,a series of cytokines have been activated,which can damage liver cells directly,cause toxic effects indirectly through inducing the inflammatory mediators,and play an important role in the disease process.At present,all kinds of antibiotics,pesticides and antiviral drugs could not effectively control the occurrence of fish liver disease.Therefore,it is important to use the liver injury model with biological activity to screen the hepatoprotective drug quickly with high throughput and explore its action principle.There are many ways to establish animal models,such as carbon tetrachloride(CCl4),xenogeneic protein,ethanol,dimethylnitrosamine and liver fibrosis model caused by complex factors.Wherein CCl4 is the earliest selective liver toxic substances,has been most widely used in the experimental liver injury animal model as its high modulus,good repeatability and easy to operate.In the late 1960s,traditional Chinese medicine has attracted attention as an immune enhancer.Studies have shown that traditional Chinese medicine can regulate a variety of cytokines,enhance the body's antioxidant system function,and has a good anti-virus,anti-tumor and anti-aging effect.During the past 10 years,Chinese herbal medicine has been widely used in aquatic animal disease prevention and control of their health,besides,its natural and toxic side effects are low and there is no residue,as the result it has remarkable effects in the fish enteritis control and immunity enhancement.At present,studies have found that Chinese herbal medicine also has a good effect in the prevention and treatment of liver damage with unique advantages,and is expected in the near future to become an efficient medicine widely used in the treatment of fish liver disease.In this study,CCl4-induced acute liver(cell)injury model of Cyprinus carpio.Jian was constructed.25 Chinese herbal medicine(extracts)were screened on this basis,and the liver protection effection and combined application of three Chinese herbs were studied.The research will lay a a theoretical foundation for the development of the formulation of the traditional Chinese medicine.1.The construction of liver injury model induced by CCl4To construct a model of acute liver injury in vitro and in vivo of Cyprinus carpio.Jian,CCl4 was used as a hepatotoxic agent.Methods:In vivo experiment,the intraperitoneal injection was used for modeling.Fish individuals were injected respectively with 0,6.25%,12.5%,15%,18.75%and 25%CCl4 olive oil(V/V)at 0.1 mL/10 g for one time,and blood was collected 24?144 h after modeling,then glutamic-pyruvic transaminase(GPT),glutamic oxalacetic transaminase(GOT),lactate dehydrogenase(LDH)and malondialdehyde(MDA)were determined in the serum of Cyprinus carpio.Jian.In vitro,CCl4 with 0,2,4,8,16,32 mM were applied respectively to the primary cultured o hepatocytes for 4 h,or CCl4 with 0,2,4,6,12,24 mM were applied respectively to precision-cut liver slices(PCLS)of Cyprinus carpio.Jian for 4h,MTT methodd was used to detect the survival status of hepatocytes or PCLS,and the supernatant or slice of cell culture was collected at the same time,then the activities of GPT,GOT,LDH,MDA,superoxide dismutase(SOD)and glutathione(GSH)were determined.Results:The result showed that 24 h after intraperitoneal injection with 12.5%and 15%CCl4-olive oil,the GOT,GPT and MDA in Cyprinus carpio.Jian serum were significantly increased(P<0.05 or P<0.01),48 h after modeling with 15%CCl4 solution,LDH level in serum could be significantly increased(P<0.05),the 4 indicators were recovered to normal level 96h after CCl4 treatment.4h after 8?32 mM CCl4 application to hepatocytes cultured in vitro,GPT,GOT,LDH and MDA content in the supernatant were significantly increased,activities of SOD and GSH and survival rate of hepatocytes were significantly declined and were 66.29%,54%and 37%of the control group,respectively with dosage dependence.4h after treatment with 4?24 mM CCl4,the contents of GOT,GPT,LDH and MDA in the cultured supernatant of the Cyprinus carpio.Jian PCLS were increased,and SOD and GSH levels were declined with significant difference comparing with the control group(P<0.05 or P<0.01).4h after treatment with 12mM CCl4,the activity of liver slices was maintained at more than 65%.Conclusion:Intraperitoneal injection with 15%CCl4-olive oil at 0.1 mL/10 g for 72h,treatment with 8 MM CCl4 on cultured hepatocytes in vitro for 4h and treatment with 12mM CCl4 on PCLS for 4h could construct in vivo and vitro acute liver injury model.This provided the possibility for high-efficiency and mass screening of hepatocellular traditional Chinese medicine.2.The preliminary screening of hepatocyte injury model of Cyprinus carpio Jian based on CCl4 induction on 25 Chinese herbal medicines(extracts)25 Chinese herbal medicines(extracts)were preliminary screened using the Cyprinus carpio Jian acute hepatocyte damage model constructed by CCl4 induction.Methods:8 mM CCl4 was applied to in vitro cultured hepatocytes for 4 h in order to construct model,L-15 mediums containing low,medium and high doses of Chinese herbal medicines(extracts)were used for pretreatment,pre-treatment and post-treatment,and post-treatment for 4h,respectively,and the liver protection effects of the 25 Chinese herbal medicines(extracts)were observed by determining the hepatocyte activity and biochemical indexes,such as LDH,GPT,GOT,SOD and GSH-PX in the supernatant of cell culture.Results:The protective effects of Chinese herbal medicine on the hepatocyte damage induced by CCl4 were similar.Traditional Chinese medicine could significantly inhibit the increase of the cell proliferation activity induced by CCl4(P<0.05 or P<0.01),significantly inhibit the increase of GPT,GOT and LDH activities in the supernatant of cell culture induced by CCl4(P<0.05or P<0.01),significantly recover the GSH-PX and SOD level in the supernatant(P<0.05or P<0.01),and inhibit the MDA production(P<0.05).The application sequence of the Chinese herbal medicine extracts and the damaging agents affect the protective effect of Chinese herbal medicine extracts on hepatocytes.The protective effect of pre-treatment and post-treatment group of the Chinese herbal medicine extracts on the damaged hepatocytes was significantly superior to the pre-treatment group and post-treatment group.The protective effect of Eucommia ulmoides extract was not obvious.Conclusion:Most of the traditional Chinese herbal medicines(extracts)have protective effect on the hepatocyte damage induced by CCl4 in Cyprinus carpio Jian,which is related to their scavenging free radicals and antioxidant ability.The preventive effect of Chinese herbal medicine extracts on liver injury is more important than that of therapeutic effect,3.Study on the liver protection effect of Glycyrrhetinic acidPrecision liver damage model induced by CCl4 was used to evaluate the effect of Glycyrrhetinic acid(GC)on the anti-oxidation and relieving inflammatory response of Cyprinus carpio Jian PCLS.Methods:2 mM CCl4 was used to damage PCLS for 4 h,L-15 medium containing GC(2.5,5 and 10 ?g/ml)and PDTC(4 ?g/ml)was used for preincubating for 6 h,liver slices and supernatants of cell culture were collected,and activity of liver slices,biochemical enzymes associated with liver damage in supernatants and liver tissue homogenate were respectively determined.The changes in mRNA expression and protein level of nuclear transcription factor NF-?B/C-Rel and cytokines iNOS,IL-1?,IL-6and IL,8 in liver tissue using real-time quantitative PCR and Western blots.Results:Pretreatment with GC on PCLS for 6 h could significantly inhibit the cytotoxicity of CCl4,GC at 5 and 10 ?g/ml could extreme significantly improve the activity of slices.GC could alleviate the oxidation and stress degree of PCLS induced by CCl4,GC at 5 and 10 ?g/ml could significantly promote the recovery of SOD and GSH activity in the homogenate(P<0.05)and extreme significantly inhibit the MDA production at the same time(P<0.01).GC at 5 and 10 ?g/ml),could significantly inhibit the increase of mRNA expression of NF-k·B/C-Rel,iNOS,IL-1?,IL-6,IL-8 and other inflammatory cytokines induced by CCl4,and significantly inhibit the protein level of C-Rel(P<0.05 or P<0.01).6 h after treatment of nuclear transcription factor NF-kB inhibitor,PDTC(4 ?g/ml)on PCLS,the NF-kB expression was significantly inhibited(P<0.01).The expression of iNOS,IL-1?,IL-6 and IL-8 in the down-stream was significantly down-regulated correspondingly,the activity of the slices was significantly increased(P<0.05 or P<0.01)and the GC result was consistent.Conclusion:GC could play a role in liver protection by inhibiting NF-kB signaling pathway to fight inflammation and reduce cytotoxicity.4.Study of the liver protective effect of CurcuminThe liver protective effect and mechanism of curcumin was studied in depth using in vivo liver damage model of Cyprinus carpio Jian.Methods:Feeds containing curcumin of 0.1%,0.5%and 1.0%were used to feed carp for 60 d,then 30%CCl4 mixture was intraperitoneal injected,then blood and liver tissue were collected,and liver indexes,comet assay,content of liver injury biochemical enzyme in serum and liver tissue homogenate were determined,respectively.In addition,pathological slices of liver tissue were observed,and changes in mRNA expression level of nuclear transcription factor NF-kB/C-Rel and cytokine IL-1?,TNF-? and IL-12 in tissue were determined using ng real-time quantitative PCR.Results:0.5%and 1.0%curcumin could significantly inhibit the increase of liver indexes caused by CCl4(P<0.05).Comet assay showed curcumin could effectively reduce DNA fragment generation of hepatic cells;tail moment,olive tail moment,tail length and tail DNA%were positively changed in fish pretreated with 0.5 and 1.0 g/kg curcumin.Curcumin could obviously reduce the extensive vacuolar degeneration,cell outline unclear,nuclear pyknosis and karyolysis and other histological lesions caused by CCl4.0.5%and 1.0%curcumin could significantly inhibit the increase of GOT content in serum caused by CCl4 and promote the recovery of SOD lever in liver tissue homogenates(P<0.05).1.0%curcumin could significantly decrease the GPT and LDH content(P<0.05)in carp serum,significantly increase T-AOC and GSH level(P<0.05)in homogenate(P<0.05),significantly inhibit MDA synthesis(P<0.05);0.5 and 1.0%curcumin could significantly inhibit the increase of expression of NF-kB/C-Rel,IL-1? and TNF-a induced by CCl4(P<0.05).While medicines with low,median and high concentration could significantly down-regulate the expression of IL-12(P<0.05).With the increase of curcumin concentration,liver indexes,enzyme indicators,pathological slices and mRNA expression of cytokines all showed dosage effect.Conclusion:curcumin may regulate its antioxidant capacity and secretion of related cytokines through improving the degree of hpatic cell DNA damage,enhancing the activity of carp antioxidant enzyme,inhibiting NF-KB activation and down-regulating the expression of cytokine IL-1?,TNF-a and IL-2 in liver tissue,thereby reducing the liver injury induced by CCl4.5.Effect of Lentinan on the activation of immunological cells in Cyprinus carpioThe head kidney(HK)macrophages and the peripheral blood leukocytes were isolated from the head kidney and blood of Cyprinus carpio respectively by percoll gradient density centrifugation,cells were cultured in vitro and exposed to different concentrations of Lentinan(LNT).Methods:to evaluate the immunostimulating effects of LNT,proliferation of the peripheral blood leukocytes,respiratory burst of macrophage and IL-1? mRNA expression of macrophages were investigated by MTT,NBT(nitroblue tetrazolium)reduction,Griess reaction and real time PCR,respectively.Results:proliferation of peripheral blood leukocytes after 24 h incubation by the induction of LNT at 100 and 1000 ?g/mL was markedly stimulated(P<0.01).LNT at 1,100 and 1000 ?g/mL could induce superoxide anion in macrophages markedly(P<0.05 or P<0.01).Reduction of nitric oxide of HK macrophages after 96 h incubation LNT showed it had no conspicuous effect on nitrogen burst activity of macrophages;moreover it inhibited the nitrogen burst activity at higher doses(P<0.01).The expression of IL-1? in the HK macrophages after 24 h stimulation by the polysaccharide showed LNT stimulated IL-1?expression in carp head kidney(P<0.01).Conclusion:LNT can modulate the immune response of C.carpio.6.Study of the liver protective effect of Curcumin combined with Glycyrrhizic acid and LentinanThe Cyprinus carpio Jian acute hepatocyte damage model constructed by CCl4 induction to evaluate the combined liver protective effect of curcumin,Glycyrrhizic acid and Lentinan.Methods:Feeds containing curcumin of 5 g/kg,Glycyrrhizic acid of 3 g/kg,Lentinan of 3 g/kg and combined groups containing low(1 g/kg curcumin +1 g/kg GC+1g/kg LNT),medium(2.5 g/kg curcumin +1.5 g/kg GC+1.5 g/kg LNT)and high(5 g/kg curcumin +3 g/kg GC+3 g/kg LNT)dose of extracts were used to feed carp for 60 d,then 30%CCl4 mixture was intraperitoneal injected.Then blood and liver tissue were collected,and growth indicators,content of liver injury biochemical enzyme in serum and liver tissue homogenate were determined,respectively.In addition,the changes in mRNA expression level of NF-kB/C-Rel and cytokine IL-1?,iNOS,TNF-?,IL-6 and IL-8 in tissue were determined using real-time quantitative PCR.Results:GC and high dose combined group could significantly increase RWR and SGR(P<0.05),reduce FCR(P<0.05).Curcumin,GC and LNT and combined group could significantly inhibit the increase of GOT,GPT and LDH content(P<0.01)in carp serum caused by CCl4,promote the recovery of GSH and SOD lever in liver tissue homogenates(P<0.01)and significantly inhibit MDA synthesis(P<0.05).Meanwhile,curcumin,GC and LNT and combined group could significantly inhibit the increase of expression of NF-kB/C-Rel,iNOS,IL-1?,TNF-?,IL-6 and IL-8 induced by CCl4(P<0.01).Comparison with single drug group,the preventive effect of combined groups were more significant(P<0.05 or P<0.01)with a dose effect.Conclusion:the combination of curcumin,GC and LNT could synergistically amplify reducing the liver injury induced by CCl4. |
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