Safety Of APO And Its Effects On Bone Marrow Hematopoiesis And Humoral Immune Function In Mice

Color is the sensory element of food color,aroma and taste.Adding safe pigments to food can increase appetite and willingness to buy,which is of great significance to the development of food industry.In recent years,driven by interests,the safety problems caused by unsafe and unreasonable pigments have attracted social and public attention.Adding APO to egg and broiler feed can effectively improve the skin color of egg and broiler,promote product sales and improve breeding efficiency.At present,the research of APO mainly focuses on its effect on production performance,but there are few detailed safety studies,especially on human function.In this study,mice were selected as the object of observation to investigate the safety of APO and its effects on bone marrow hematopoiesis and humoral immunity in mice,in order to provide theoretical reference for objectively evaluating the safety risk of APO.Methods:（1）According to the standard of GB/T21603-2008 acute toxicity test,the LD₅₀ of APO in mice was determined.（2）After intragastric administration of APO at the dose of 1.4 g/kg·BW,the chromaticity changes of abdominal subcutaneous,ascites,peritoneum,auricle,tail and soles of feet were observed by RYCF colorimetric fan method at 4 h,12 h,36 h,7 d and 14 d.To explore the coloring and discoloration rule of APO in mice.（3）After intragastric administration of APO at the dose of 1.4 g/kg·BW,blood samples were collected at 0 h,4 h,12 h,36 h,60 h,7 d and 14 d to explore the change trend of blood routine indexes such as white blood cells,lymphocytes and red blood cells.After continuous intragastric administration of 1.4 and 2.2 g/kg·BW APO for 21days,urine and serum were collected for urine routine index and biochemical index determination,liver and kidney tissue sections were stained with HE,and histological damage was observed under microscope.（4）The mice were intragastric at 1.4 and 2.2 g/kg·BW for 30 days.The bone marrow of femur was collected from mice.The bone marrow nucleated cells were counted with blood counting plate,and the bone marrow cells were stained with Wright-Giemsa staining.The number of erythroid cells,granulocytes,lymphoid cells in the bone marrow was determined,and the proportion of hematopoietic cells in each bone marrow line was calculated.The changes of granulocyte colony stimulating factor and erythropoietin in serum were detected by Elisa.To evaluate the effect of APO on bone marrow hematopoiesis.（5）The mice were intragastrically administered with APO at the dose of 1.4 and 2.2 g/kg·BW for 21 days.To observe the effect of APO on the organ coefficient of spleen and thymus.The contents of immunoglobulin Ig M,Ig G,SIg A and immune cytokines IL-1b,TNF-α,IL-10 and IFN-γin serum were determined by Elisa.The relative expression of positive immunoregulatory factor IL-1b gene and negative immunomodulatory factor CTLA-4 and PD-1 m RNA gene was detected by real-time fluorescence quantitative PCR.To investigate the effect of APO on humoral immune function.Results:（1）The LD₅₀ of APO in mice was 2.74 g/kg·BW.According to the WHO toxicity standard,the range of 501～5000 mg/kg·BW was low toxic grade substance.（2）After intragastric administration of APO at the dose of 1.4 g/kg·BW,the yellowness of subabdominal skin,ascites,peritoneum and auricle increased in mice.Rising speed:subcutaneous>auricular skin>tail and soles of feet,while elimination speed is opposite.The chromaticity of all parts reached the highest value at 4 h,in which the chromaticity of tail and foot,abdominal subcutaneous and auricular skin,ascites and peritoneum increased significantly in 4～12 h,4～36 h,4 h～7d（P<0.01）.The chromaticity of each part gradually faded after 4 hours,the chromaticity of the tail and soles of feet disappeared after 7 days,and the chromaticity of other parts disappeared after 14 days（P>0.05）.The results showed that the tissue of mice could be quickly stained in vivo and in vitro,but the chromaticity was eliminated quickly after a large dose of APO was given at one time.（3）The white blood cell count,lymphoid cell count,neutrophil count,red blood cell count and average erythrocyte hemoglobin content of mice were significantly higher than those of the control group within 4～36 hours after a single oral administration of APO according to1.4 g/kg·BW.The number and content of leukocyte,lymphocyte and neutrophil,erythrocyte and average erythrocyte hemoglobin reached the peak at 12 h,4 h and 36 h,respectively.Then it decreased gradually,and there was no significant difference after 60 hours（P>0.05）.On the 21st day after continuous gastric administration of APO at the dose of 1.4 and 2.2g/kg·BW.The weight gain and average daily gain decreased significantly（P<0.01）,the contents of leukocyte,protein,microalbumin and ketone body in urine increased significantly（P>0.05）,and the contents of alkaline phosphatase and urea in blood increased significantly（P<0.01）.Uric acid,glutamic oxaloacetic transaminase and creatinine decreased significantly（P>0.05）.The pathological section of the liver showed disorder of hepatic sinusoid and hepatic cord structure,swelling and necrosis of hepatocytes,enlargement of kidney yellow staining,damage of renal corpuscle structure,disappearance of vascular glomerulus fragmentation and diffuse hemorrhage of medulla.（4）The number of nucleated cells and the proportion of lymphocyte in bone marrow of mice were significantly increased by APO at 1.4 and 2.2 g/kg·BW for 30d（P<0.05）.The proportion of granuloskeletal cells in 2.2 g/kg·BW group was significantly increased（P<0.05）.Serum granulocyte colony-stimulating factor content was significantly increased in 2 dose groups（P<0.05）,but erythropoietin content was not significantly changed（P>0.05）.（5）The mice were intragastrically fed with APO at 1.4 and 2.2g/kg·BW for 21 days.The relative expression levels of CTLA-4 and PD-1m RNA were significantly increased in both doses（P<0.05）.2.2 g/kg·BW significantly increased the relative expression level of IL-1b m RNA in mice（P<0.01）.The spleen organ coefficient was significantly increased（P<0.05）,the contents of inflammatory factors TNF-αand IL-10 in serum were significantly increased（P<0.01）,IL-1b was significantly increased（P<0.01）,and immunoglobulin was decreased,among which Ig M and SIg A contents were significantly decreased（P<0.05）.Conclusion:APO is a low toxic substance,and it is relatively safe for livestock,poultry and human beings according to the national standards.However,high-dose APO can make the skin and viscera of mice stain quickly,and the color fades quickly,can quickly increase the number of blood cells,and cause damage to the tissue structure of liver and kidney of mice.APO can stimulate the proliferation of bone marrow nucleated cells,in which lymphatic and granulocytic hematopoietic cells proliferate significantly,and increase the content of granulocyte colony stimulating factor has a positive effect on bone marrow hematopoiesis.APO has a bi-directional regulatory effect on humoral immune function.Damage immune organs and cause excessive inflammatory response,weaken the body’s positive immune regulation function,enhance the expression of negative regulatory factors,and promote negative immune regulation function.The results of this study provide theoretical reference for evaluating the safety of APO and its effect on human hematopoiesis and immunomodulatory effect at high dose.