| Ochratoxin A(OTA)exists in food,fodder and cereal,which is one of the most toxic,highest amount of enterotoxigenic and the most seriously pollutional ochratoxin of agricultural products.OTA has been shown to be nephrotoxic,hepatotoxic,teratogenic,genotoxic and embryotoxic.The toxicity of OTA is strong and it can cause liver injury mainly in poultry.However there are many reports about the pathological symptom of OTA induced liver injury,the mechanisms are not very clear.Therefore,it is necessary to ascertain the pathological changes of liver injury induced by OTA and the effects of hepatoprotective drugs in chicken.This paper dealt with the mechanisms of OTA induced hepatocytes injury in vitro and the hepatoprotective effects of compound ammonium glycyrrhizin(CAG),L-arginine(L-Arg),silymarin(Sil)and glucurolactone(GA)which are used commonly in cilinical.The main research contents and results are as follows:1 Screening the optimal dose of OTA induced chicken hepatocytes injury in vitroChicken hepatocytes were obtained from Hailan chicken(40-50 days old)by a modified half-step in situ perfusion which was used IV collagenase circularly.The hepatocytes grew against the bottom of the plate after culturing for 24h.The supernatant was removed and 200 μL of medium containing 0(the control group),0.25,0.5,1,2,4μg/mL of OTA(n=3)was added,the cells were incubated at 37℃ for 24 h.The cellular morphology was observed using an inverted optical microscope;cell viability and the content of ALT,AST in culture media were measured.The results showed that the cells in control group were attached the bottom of plate firmly,presented polygon,formed the shape of islands and the nucleus were round and bright.In the experimental groups,cell density was decreased with the increase of OTA concentration and the hepatocytes ruptured and floated in cell cultured supernatant.Cell viability was decreased and the content of ALT,AST in culture media was increased in dose-dependent manner.The results showed that the cell viability was 51.33 ± 4.27%and the content of ALT,AST was significantly increased(P<0.05)compaired to the control group when the cells treated with 1 μg/mL OTA.The cell viability and the contant of ALT,AST were more appropriate in the dose of 1μg/mL in relation to the other groups,so it was determined to be the most appropriate concentration which could induce chicken hepatocytes injury.2 The effects of the OTA induced oxidative stress and apoptosis Chicken hepatocytes were cultured for 24 h,the cells were divided into two groups: the control group,which was not exposed to OTA;the OTA group,which was administered OTA at the dose of 1 μg/mL for 24 h.The levels of SOD,GSH and MDA in hepatocytes were measured.Cell apoptosis rates were assayed by flow cytometry and mRNA expression levels of Caspase-3,Bcl-2,Bax were measured by real time PCR.The results showed that the activity of SOD and the amount of GSH were decreased and the content of MDA was increased meaningfully(P<0.01).It showed that OTA could induce oxidative stress response.The cell apoptosis rate reached up to 32.06 ± 2.11%in comparision with the control group(1.80 ± 0.45%),the mRNA expression of Caspase-3,Bax increased meaningfully(P<0.01)and Bcl-2 decreased significantly(P<0.05).It showed that OTA may trigger the mitochondria apoptosis pathway.3 Protective effects of CAG,L-Arg,Sil and GA in chicken hepatocytes injury induced by OTA3.1 Protective effects of CAG,L-Arg,Sil and GA on hepatocytes membrane.Chicken hepatocytes were cultured for 24 h,then the cells were divided into six groups(n=3):The control group,which was neither administered hepatoprotective drugs nor exposed to OTA;The OTA group which was given 1μg/mL OTA for 24 h;The prevention groups that the cells were treated with CAG,L-Arg,Sil and GA at concentrations of 0.1,1,10 μg/mL respectively for 24 h before the addition of 1μg/mL OTA.Each group was incubated at 3 7℃ for 24 h.Cell viability and the content of ALT,AST in culture media were measured.The results showed that CAG,L-Arg,Sil and GA could improve cell viability and inhibited the elevation of ALT(P<0.01).Arg,Sil and GA decrased the content of AST in supernatant but CAG had no effect.It showed that CAG,Arg,Sil and GA possess the effects of protecting the chicken hepatocytes membrane.3.2 Protective effects of CAG,L-Arg,Sil and GA against oxygen free radical damage.Chicken hepatocytes were cultured for 24 h,then the cells were divided into six groups(n=3):the control group;the OTA group;the prevention groups that the cells were treated with CAG,Arg,Sil and GA at concentrations of 1 μg/mL for 24 h respectively before the addition of 1μ/mL OTA.The activities of SOD and the content of GSH,MDA in hepatocytes were measured.CAG,Arg,Sil and GA could increase the level of SOD and GSH,decrease the content of MDA expressively,and Sil had a better effect especially.It showed that CAG,L-Arg,Sil and GA can inhibit the hepatocytes injury induced by OTA by antioxidant effects.3.3 Protective effects of CAG,L-Arg,Sil and GA against cell apoptosis.Chicken hepatocytes were cultured for 24 h,then the cells were divided into six groups(n=3):the control group;the OTA group;the cells were treated with CAG,L-Arg,Sil and GA at concentrations of 1 μg/mL for 24 h respectively before the addition of 1 μg/mL OTA.The cells apoptosis rates of six groups were tested by flow cytometry and the mRNA expression levels of Caspase-3,Bcl-2,Bax were measured by real time PCR.The cell apoptosis rates were decreased observably which were pretreated with CAG,L-Arg,Sil and GA respectively(P<0.05)in comparison with the OTA group.The four kinds of the hepatoproective drugs could decrease the mRNA expression of Caspase-3,increase Bcl-2 expression,but there were no differences between the groups of Arg and GA and the group of OTA in the mRNA expression of Bax.It showed that CAG and Sil may play a protective effect by inhibiting the mitochondria apoptosis pathway.L-Arg and GA could suppress cell apoptosis through Caspase-3 and Bcl-2,but it needs more research about the extensional mechanisms. |
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