The Effects Of Gossypol Acetate On Immunity And Toxic Metabolic Enzyme Gene Expression Level In Cyprinus Carpio

In recent years,with the expansion of aquaculture scale,the demand for feed is increasing.In order to relieve the pressure of aquaculture cost from the hign price of fishmeal and soybean meal,the cottonseed meal with low prise and rich protein level has became one of the important protein sources for aquatic feed.However,the usage amount of cottonseed meal was limited due to the presence of cyclopropane fatty acids,free gossypol and other anti-nutritional factors,especially the excessive accumulation of free gossypol can cause toxicity to aquatic animals.At present,the researches about free gossypol mainly focused on growth performance,reproductive and hepatic function,while the immunotoxicity and detoxification of free gossypol is still unclear in aquatic animals.In this study,the immunotoxicity and detoxification mechanism of free gossypol were investigated after oral administration continuously with gossypol acetate in C.carpio.The important results are as follows:1.Effect of gossypol acetate on serum biochemical indices and hepatocyte antioxidant enzyme activities in C.carpioIn this section,we detected the activities of GPT,GOT,LZM,AKP,ACP,SOD,CAT,GSH-PX,the contents of MDA,H₂O₂ and the expressions of cytokines TNF-α,TGF-?,IL-6,IL-10,IL-1?in C.carpio at1st,3th and 7th day following oral administration of gossypol acetate at doses of 0,10,40 mg/kg body weight for 7 days.The results showed that the activities of GOT,GPT,AKP in serum,the content of MDA,H₂O₂ in serum and liver were significantly higher in 10,40 mg/kg gossypol acetate groups than in control group（P<0.05）,while the activities of LZM,ACP and the expression levels of cytokines TNF-α,TGF-?,IL-6,IL-10,IL-1?in serum was significantly lower in the 10,40 mg/kg gossypol acetate groups than in the control group（P<0.05）at 1st,3th,7th day after oral administration with gossypol acetate for 7 days.Regarding SOD activity in serum and GSH-px activity in liver,there were no significant differences between the control and 10 mg/kg gossypol acetate group at 1st,3th,7th day after oral administration of gossypol acetate.The CAT activity in serum and SOD activity in liver were significantly lower in 10 mg/kg gossypol acetate group than that in control group at 1st,3th,7th day after oral administration（P<0.05）.The GSH-px activity of serum and CAT activity of liver also were significantly lower in 10 mg/kg gossypol acetate group than that in control group at 3th,7th day after oral administration（P<0.05）.In addition,the activities of SOD,CAT,GSH-px in serum and liver were significantly lower in 10 mg/kg gossypol acetate group than in the control group at 1st,3th,7th day after oral administration（P<0.05）.The results indicated that the gossyphenol acetate of 10,40 mg/kg doses could cause an immune response in C.carpio.Particularly,the dose of 40 mg/kg body weight had a serious effect on the liver function,immune-related enzyme activities and cytokines gene expression levels.2.Effect of gossypol acetate on the indexs of spleen and head kidney,immune-related enzyme activities and gene expression levels in C.carpioIn this section,we analyzied the indexs of the spleen and head kidney the activities of immune-related enzymes LZM,AKP,ACP and gene expression levels of TLR1,TLR2,TLR3,My D88,TRAF6,NF-?b,TNF-α,TGF-?,IL-6,IL-8,IL-10,IL-1?of spleen and head kidney in C.carpio at 1st,3th and 7th day following oral administration of 0,10,40 mg/kg body weight gossypol acetate.The results showed that the indexs of spleen and head kidney were significantly higher at the gossypol acetate groups than that at the control group at 1st,3th,7th days after oral administration with gossypol acetate for 7 days（P<0.05）,and the index value in the high dose gossypol acetate group was significantly higher than low dose group（P<0.05）.The activities of LZM,ACP,AKP in spleen and head kidney were significant difference compared with control group at 1st,3th,7th day after oral administration with gossypol acetate（P<0.05）for 7 days.In the spleen,the expression levels of TLR1,My D88,TRAF6,NF-?b,LZ-mc and LZ-mg both in two gossypol acetate groups were significantly higher,and TNF-α,TGF-?,IL-6,IL-8,IL-10 and IL-1?in the 40 mg/kg group were significantly higher than that in the control group at 1st,3th,7th day after 7 days of oral administration with gossypol acetate（P<0.05）.In the head kidney,the expression levels of IL-8,LZ-mc and LZ-mg in the gossypol acetate groups and TLR1,TGF-?,IL-1?in the hign gossypol acetate dose groups were significantly higher compared with the control group at 1st,3th,7th day after oral administration with gossypol acetate（P<0.05）for 7 days,the expression levels of TLR1,TLR2,TLR3,My D88,TRAF6 in the low gossypol acetate group at 3th day after oral administration and My D88,TRAF6in the hign gossypol acetate group at 3th,7th days after oral administration were significantly higher than that in control group（P<0.05）.The findings implied that gossypol acetate may induce spleen inflammation in C.carpio by activating toll-like receptor signal pathway,while head kidney inflammation may be induced by gossypol acetate via other signal pathway in C.carpio.3.Effect of gossypol acetate on anti-disease ability of C.carpioIn this section,30 fish from each group were selected randomly and injected 200?L,1×10⁶ cfu/m L Edwardsiella tarda byintraperitoneal injection after oral administration of gossypol acetate at doses of 0,10,40 mg/kg body weight for 7 days.We analyzed the cumulative mortality rates at 24,48,72,96 h,the activities of immune-related enzymes LZM,AKP,ACP in spleen and head kidney and the expression levels of cytokines TNF-α,TGF-?,IL-6,IL-10,IL-1?in serum at 96 h after infection with E.tarda.The results showed that,there was no death in 0,10 mg/kg gossypol acetate groups,while the mortalities of C.capito increased with the infection time extension in 40 mg/kg gossypol acetate group,and the cumulative mortality reached 30%at 96 h after infection.The activities of LZM in serum,spleen,head kidney were not significantly different in 10 mg/kg gossypol acetate group compared with that in the control group,but the activities of AKP,ACP in serum,spleen and head kidney were significant higher compared with control group（P<0.05）.The expression level of IL-10 in serum was not significantly different between 10 mg/kg gossypol acetate group and control group,while the expression levels of TNF-α、TGF-?、IL-1?were significant higher compared with control group（P<0.05）,the expression levels of IL-6 were significantly lower compared with control group（P<0.05）.The activities of LZM,AKP and ACP increased significantly in serum,spleen and head kidney in 40 mg/kg gossypol acetate group（P<0.05）.The expression levels of TNF-α,TGF-?,IL-6,IL-10 in serum were significant lower in 40 mg/kg gossypol acetate group than that in 10 mg/kg gossypol acetate group（P<0.05）,while the expression level of IL-1?was significant higher compared with control group（P<0.05）.The results suggested that gossypol acetate increased the susceptibility in C.carpio to Edwardian tardiness and decreased its resistance to pathogenic bacteria.4.Effect of gossypol acetate on m RNA relative expression levels of CYP2K,CYP3A in C.carpioIn this section,the m RNA expression levels of CYP2K,CYP3A in liver,body kidney,gut of C.carpio were measured at 1st,3th and 7th day following oral administration of gossypol acetate at doses of 0,10,40 mg/kg body weight for 7 days.The results showed that the m RNA expression levels of CYP2K in foregut at 1st and 7th day,and CYP2K in midgut and hindgut at 7th day were significant higher than that in control group after oral administration with 10 mg/kg dose of gossypol acetate for 7 days（P<0.05）.The m RNA expression levels of CYP2K in liver,CYP3A in liver,midgut and foregut at 1st,3th day were significantly lower compared with control group after oral administration with 10 mg/kg dose of gossypol acetate for 7 days（P<0.05）.The m RNA expression levels of CYP2K in body kidney,and CYP3A in midgut were significant lower than that in control group at 1st,3th,7th day after oral administration with 10 mg/kg dose of gossypol acetate for 7 days（P<0.05）.The m RNA expression levels of CYP3A in hindgut at 1st,7th day were significantly lower than that in control group after oral administration with 10 mg/kg dose of gossypol acetate（P<0.05）.The m RNA expression levels of CYP2K in foregut and midgut were significant higher compared with control group at 1st,3th,7th day after oral administration with 40 mg/kg dose of gossypol acetate for 7 days（P<0.05）.The CYP2K m RNA expression level in hindgut were significant higher than in control group,while the CYP2K transcript levels in liver were significantly lower than in control group at 1st,3th day after oral administration with 40 mg/kg dose of gossypol acetate for 7 days（P<0.05）.The m RNA expression levels of CYP2K in body kidney,and CYP3A in foregut,midgut,hindgut,liver,body kidney were significantly lower than in control group at 1st,3th,7th day afte oral administration with 40 mg/kg dose of gossypol acetate for 7 days（P<0.05）.The results suggested that the expression of CYP2K in gut was induced,and CYP2K was inhibited in the liver and kidney by gossypol acetate,while CYP3A was inhibited in the liver and gut.CYP2K may play an important role in the metabolism of gossypol acetate.