| Aflatoxin B1(AFB1)is the most common carcinogenic pollutant in the feed of livestock and poultry.AFB1 poisoning in broilers occur frequently and can negatively affect the growth efficiency and the fecundity of broilers.AFB1 poisoning will inhibit the immune system function of broilers and further increase the susceptibility of broilers to infectious disease so that result in their morbility and mortality growth.The AFB1 in the feed of broilers and its metabolites will store up in the liver,kidney and muscle.The AFB1 and its metabolites which stored up in chicken liver,kidney and muscle will induce chronic poisoning and even cancer in human.Thus AFB1 poisoning in broilers is extremely harmful for animal husbandry,public health and human health.At present,the mechanism of AFB1 in inducing sub-chronic liver damage in broilers is still unknown.Curcumin is a diketone compound that extracted from the rhizome of ginger family and Araceae plants.Curcumin shows anti-tumor,anti-oxidant and anti-inflammatory functions in many diseases.Recent researches reported that curcumin plays hepatoprotective role in fatty liver,hepatitis,liver cirrhosis,and liver cancer.While,the effect of curcumin in protecting broilers from AFB1-induced sub-chronic liver damage and the mechanism are still not clear.Arbor Acres(AA)broiler has the largest market share in China.This research is conducted with 1 day old broilers;Establish AFB1-induced sub-chronic liver damage animal model and intervene with different concentrations of curcumin;Use DNA/RNA specific staining,transmission electron microscope(TEM)and advanced molecular biology techniques such as Western blot and Real Time-PCR;To investigate the influence of AFB1 sub-chronic exposure in chicken hepatocytes autophagy,apoptosis,inflammatory and proliferation related genes expression,and the role of TLR4/NF-?B pathway in this process;Clear whether curcumin intervenes AFB1-induced chicken sub-chronic liver injury via affecting autophagy,apoptosis,inflammatory and proliferation related genes expression;Explore the sustainability of curcumin intervention in AFB1-induced sub-chronic liver damage in broiler chickens.Morphology and ultramicro morphology examination: Broilers exposed in AFB1 for 28 days persistently,the TEM results showed that in hepatocytes of AFB1 exposed group(group A),autophagosome was rare,ridges of mitochondria was broken and the structure of mitochondria was disordered,the chromatin in nucleus was edged compared to the control group(group C).Curcumin intervention persistent for 28 days,the pathological changes mentioned above were not obviously improved in group intervened with low concentration of curcumin(group L),but the pathological changes were significantly improved in groups intervened with median and high concentrations of curcumin(group M and group H).In hepatocytes of group H,the autophagosome occurred,the chromatin distribution in nucleus was balanced,the structure of mitochondria was clear.In curcumin control group(group CC),the ultramicro morphology of hepatocytes was not changed.Chicken liver sections stained by DNA/RNA specific dyes and apoptosis,necrosis cells counting results showed that the apoptosis and necrosis cells counting increased significantly in group A(P<0.01).Curcumin intervention reduced the count of apoptosis and necrosis cells dose dependently(L?M and H groups,P<0.01).In group CC,there is no evidence of abnormal.Stop AFB1 exposure and curcumin intervention for 7 days,the TEM results showed that in group A the autophagosome was still rare,the chromatin in nucleus was gathered and edged,the ridges of mitochondria were disordered,the fiber-like materials occurred in cell spaces.In the other groups,lysosome activity was high,autophagy was obvious,chromatin distribution in nucleus was balanced,mitochondria structure was clear,except for slight chromatin gather in nucleus in group L.DNA/RNA specific staining and hepatocytes apoptosis,necrosis counting results reveled that apoptosis and necrosis cells count in group A was much higher than group C(P<0.01).Curcumin intervention dose-dependently reduced the numbers of apoptosis and necrosis hepatocytes in comparison with the group C(P<0.01).The apoptosis and necrosis cells count in group CC were similar to group C.These results indicated that curcumin effectively intervened the AFB1 sub-chronic exposure induced autophagy inhibition and apoptosis and necrosis increase in broilers livers.In addition,the intervention results of curcumin are continuable within 7 days after stoping curcumin administration.Autophagy involved proteins and genes expression: Continually exposed to AFB1 for 28 days,the hepatic LC3 a protein expression,LC3b-II/LC3b-I protein expression ratio and beclin-1 m RNA expression were obviously reduced(P<0.01),and the hepatic m TOR protein,mRNA and cytoplasmic P53 protein expressions were significantly enhanced(P<0.01)in chicken of group A compared to that in group C.Continually intervened with curcumin for 28 days,compared to group A,the LC3 a protein expression,LC3b-II/LC3b-I ratio and beclin-1 mRNA expression were significantly increased(P<0.05 or P<0.01),and the mTOR protein,mRNA and cytoplasmic P53 protein expressions were significantly decreased(P<0.05 or P<0.01)in the curcumin intervention groups.The LC3b-II/LC3b-I ratio and the beclin-1 m RNA expression level in group CC were higher than that in group C(P<0.01).Seven days after stopping the AFB1 exposure and curcumin intervention,changes of the indexes mentioned above were similar to the 28 th day,except for,there was no statistical difference between group CC and C.The results indicated that curcumin may increases LC3 a and LC3b-II/LC3b-I ratio levels via enhancing beclin-1 expression and impeding m TOR and cytoplasmic P53 protein expressions,and further intervene the AFB1 sub-chronic exposure induced hepatocytes autophagy inhibition.In addition,the intervention results of curcumin are continuable within 7 days after stoping curcumin administration.Apoptosis related genes and proteins expression detection: Broilers exposed in AFB1 for 28 days persistently,hepatic Caspase-3,Bax,nuclear P53 protein,p53 m RNA,bcl-2 mRNA expressions were significantly increased compared to the control group(P<0.01).Curcumin intervened persistently for 28 days,all the indexes were dos-dependently reduced(P>0.05 or P<0.05 or P<0.01),except for the dose-dependently increase of bcl-2 mRNA expression(P>0.05 or P<0.01)in chicken livers.Compared with group C,the bcl-2 mRNA expression was enhanced in group CC(P<0.01).Seven days after stopping AFB1 exposure and curcumin intervention,all the indexes changes were similar to the 28 th day(P<0.05 or P<0.01)except for the bcl-2 m RNA expression was decreased(P<0.01)in group A compared to group C.In comparison with group A,each index change was similar to the 28 th day in curcumin intervention groups(P<0.05 or P<0.01).In group CC,nuclear P53 protein,p53 m RNA,caspase-3 m RNA and Bax protein expression levels were obviously declined(P<0.01),and bcl-2 m RNA expression level was significantly elevated(P<0.01)in comparison with group C.These results implied that curcumin may intervene AFB1 sub-chronic exposure induced chicken hepatocytes apoptosis by inhibiting the expression of genes and proteins which contribute to cell apoptosis,and enhancing the expression of anti-apoptosis genes.The intervention results of curcumin are continuable within 7 days after stoping curcumin administration.Inflammatory and cell proliferation related genes expression and TLR4/NF-?B pathway activity detection: Chicken persistently exposed to AFB1 for 28 days,the TNF-?,cyclin D1,IL-10,HO-1 m RNA and cytoplasmic NF-?B p65 protein expressions were much lower in group A than that in group C(P<0.05 or P<0.01).While the TLR4 protein,nuclear NF-?B p65 protein,and TGF-?,IL-1?,IL-6,IL-8,TLR4,NF-?B p65 m RNA expressions in group A were much higher than that in group C(P<0.01).Persistently intervened with curcumin for 28 days,chicken hepatic TNF-?,cyclin D1,TGF-?,IL-10,HO-1 m RNA and cytoplasmic NF-?B p65 protein expressions were significantly enhanced in curcumin intervention groups in comparison with group A(P>0.05 or P<0.05 or P<0.01).Compared to the group A,the hepatic TLR4 protein,nuclear NF-?B p65 protein and IL-1?,IL-6,IL-8,TLR4,NF-?B p65 m RNA expressions were reduced in the groups with curcumin intervention(P<0.05 or P<0.01).In group CC,the TNF-?,cyclin D1,TGF-?,IL-10,HO-1 mRNA expressions were much higher than the control group(P<0.01),and the TLR4 protein and NF-?B p65 m RNA expressions were much lower than the control group(P<0.01).Stop AFB1 exposure and curcumin intervention for 7 days,compared to group C,the cyclin D1 and TGF-? m RNA expression levels were not changed and the variation trends of the other indexes were similar to the 28 th day(P<0.01).In comparison with group A,all of the indexes changes were similar to the 28 th day(P>0.05 or P<0.05 or P<0.01)except for there was no statistical change in TGF-? m RNA expression in groups with curcumin intervention.In group CC,the TNF-?,cyclin D1,IL-10 m RNA expressions were higher than group C(P<0.01),and the TLR4 protein,m RNA and nuclear NF-?B p65 protein expressions were lower than group C(P<0.01).These results indicated that curcumin may intervene the AFB1 sub-chronic exposure induced liver inflammatory and proliferation related genes expression inhibition via improving anti-inflammatory genes and proliferation genes expression,as well as impeding proinflammatory genes expression by inactivating TLR4/NF-?B pathway.The intervention results of curcumin are continuable within 7 days after stoping curcumin administration.The results of this study implied that the intervention results of curcumin in protecting broiler chicken from AFB1-induced sub-chronic liver injury are realized by accelerating hepatocytes autophagy and cell proliferation related genes expression,and inhibiting hepatocytes apoptosis,necrosis and inflammatory.The effect of curcumin in intervening AFB-induced sub-chronic liver damage in broiler chicken is continuable within 7 days after stoping curcumin administration. |
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