| ObjectivesWith increasing age,our gut flora changes.China and even the world are facing a huge medical challenge brought about by the aging problem.How to ensure the healthy life of the elderly is of vital importance.In this study,we investigated whether there was dose-age difference in the toxicity of long-term exposure to DEHP on multiple organ functions in young and aged mice,which provided valuable data for the study of DEHP and aging.Methods(1)Thirty 4-week-old SPF C57BL/6J male mice were purchased by Beijing Vitong Lihua Experimental Animal Technology Co.,Ltd.,and 21 SPF C57BL/6J male mice were purchased and raised to the age of 19 months.(2)Thirty 4-week-old male C57BL/6J rats were randomly divided into 3 groups with 10 rats in each group,which were young control group(0 mg/kg· bw DEHP),young low-dose group(50 mg/kg· bw DEHP)and young high-dose group(500mg/kg· bw DEHP),respectively.Twenty-one 19-month-old C57BL/6J male rats were randomly divided into 3 groups with 7 rats in each group,which were the elderly control group(0 mg/kg· bw DEHP),the elderly low-dose group(50 mg/kg· bw DEHP)and the elderly high-dose group(500 mg/kg· bw DEHP).(3)After 12 weeks of feeding,mice in each group were sacrificed.The general indexes of mice in each group and the gross view of liver,kidney,spleen,stomach and ileum were compared,and the weight of liver and spleen was weighed.(4)Serum diamine oxidase(DAO)content was determined by ELISA,and D-lactic acid content was determined by colorimetric method.(5)HE staining was used to observe and compare the changes of liver,spleen,kidney,stomach and ileum structure and inflammation in each group.(6)High-throughput sequencing of 16 S r DNA to analyze the intestinal microbiota of mouse feces.Results(1)Changes in general condition,body weight and 0-12 weeks body weight of mice: compared with the respective control group,the activity,hair gloss and mental state of mice in the young high-dose group and the old high-dose group decreased in the later period of feeding,and the changes were more obvious in the old group.Compared with the control group,there was no statistical significance in the body weight of the low-dose and high-dose groups.Compared with the young low-dose group,the initial body weight of the young low-dose group was lower than that of the young high-dose group(P< 0.05),and the other was not statistically significant.In young mice,the weight gain of high-dose group was significantly lower than that of control group(P< 0.01)and low-dose group(P< 0.05);In aged mice,the weight loss of high-dose group was higher than that of control group(P<0.05)and low-dose group(P< 0.01).(2)The appearance of the liver,spleen,kidney and stomach of mice: it can be seen that in the young and the aged mice,the color of the control group was reddish brown,and the dark red deepened with the increase of dose,and the color of the old group was slightly darker than that of the younger group.In aged mice,the spleen of low-dose group was slightly smaller than that of control group.There was no significant difference in the appearance of kidney and stomach among all groups.(3)Liver weight,spleen weight,liver index,spleen index: in young mice,compared with the control group,liver index of low-dose group was increased(P<0.05),liver weight,liver index and spleen index of high-dose group were significantly increased(P< 0.0001),liver index and spleen index were significantly increased(P< 0.05);Compared with the low-dose group,the liver weight and spleen weight of the high-dose group were significantly increased(P< 0.001),liver index was significantly increased(P< 0.0001)and spleen index was significantly increased(P< 0.01).Compared with the control group,the weight of spleen and spleen index in the low-dose group were significantly decreased(P< 0.01)and decreased(P< 0.001),while the weight of liver in the high-dose group was increased(P< 0.05)and liver index was increased(P< 0.05).(4)Mouse liver and kidney function: in young mice,compared with the control group,ALP increased(P< 0.05)and UREA decreased(P< 0.05)in the low-dose group,while ALT increased(P< 0.05),ALP increased(P< 0.001)and UREA decreased(P< 0.05)in the high-dose group;Compared with low-dose group,ALT in high-dose group increased(P< 0.01).Compared with the control group,ALP and UREA in high-dose group were increased(P< 0.05),decreased(P< 0.05),and CR increased(P< 0.05).Compared with low dose group,ALT in high dose group was increased(P< 0.05).In young and aged mice,there was no significant difference in AST,although there was an upward trend compared with the control group.(5)The intestinal mucosal barrier function of mice,DAO and D-lactic acid: the serum DAO level in the high-dose group decreased compared with the low-dose group in the old mice(P< 0.05),but there was no significant difference in other groups.(6)The pathological changes of liver,spleen,kidney and intestines of mice: in young mice,the liver plate boundary was not clear,the nucleus was slightly larger,inflammatory cells were infiltrated,and blood vessels were dilated and congested in the high-dose group.More inflammatory cells were infiltrated in the cortex,and the structure of lymphatic follicles was not clear.The glomerular hemorrhage,renal tubule arrangement disorder,irregular distortion,interstitial inflammatory cell infiltration;The lymphatic follicles in the cortical region of the spleen are less clear and more neutrophils are present.In the elderly mice,liver vascular congestion and inflammatory cell ring vascular infiltration were observed in the high-dose group.There were more macrophages in the spleen.The renal area is poorly structured with focal inflammatory cell infiltration.No obvious structural changes were observed in ileum and stomach.(7)Changes of intestinal microflora in mice: Alpha diversity analysis showed that in young mice,compared with the control group,Shannon index in low and high dose groups decreased(P< 0.05,P< 0.05),while Simpson index increased(P<0.05,P< 0.05),indicating that intestinal microflora diversity decreased with the increase of dose.In the elderly,compared with the control group,the diversity of bacteria in the low-dose and high-dose groups was not statistically significant.PCo A principal coordinate analysis showed that there were differences in microflora structure between the control group and the high-dose group in young and old mice.Species composition analysis showed that in young mice,the relative abundance of Firmicutes and Actinomyces increased in low-dose and high-dose groups compared with the control group,while the relative abundance of Bacteroidetes decreased.In aged mice,the relative abundance of Firmicutes decreased,while the relative abundance of Bacteroidetes and Actinomycetes increased.At the genus level,the relative abundance of Bifidobacteria increased in both young and aged mice.Conclusions1.Long-term exposure to DEHP lead to slow weight gain in young mice and sustained weight loss in aged mice.2.Long-term high-dose DEHP intragastric mice can cause increased liver weight,increased liver index and liver enzymes in mice,which has toxic effects on the liver,kidney and spleen of young and aged mice,and causes greater damage to aged mice.3.500 mg/kg·bw DEHP do not significantly change the intestinal permeability of young and aged mice.4.DEHP can change the diversity and composition of intestinal microbiota in C57 mice of different ages. |
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