Application And Research Of Intestinal Microbiota Analysis In The Safety Evaluation Of Drugs In Juvenile Animals

Intestinal microbiota has been considered as an important"functional organ"of the body,participating in the growth and development of the body and the metabolism of drugs in the body.The composition of the intestinal microbiota is closely related to the influence of drug metabolism on the body.As the carrier of non-clinical safety evaluation of pediatric drugs,the intestinal microbiota of juvenile animals is still in the stage of formation and development,which is closely related to drug toxicity.Therefore,the composition of intestinal microbiota is particularly important in drug safety evaluation.However,there are few studies on the intestinal micorbiota of juvenile animals,and there are not too many studies on the intestinal microbiota as a drug safety evaluation index.Aims:1.This study intends to analyze the intestinal microbiota of healthy juvenile animals(rats,monkeys)feces at different ages to explore the changes in the intestinal microbiota of normal juvenile and adult animals;2.Taking the wall-removed Ganoderma lucidum spore powder as an example,the change characteristics of the intestinal microbiota of juvenile animals after administration and their relationship with immune,behavioral and other related juvenile animal toxicological indicators were investigatedMethods:1.Comparative analysis of normal intestinal microbiota of juvenile and adult animals:collect feces of SD rats of different ages(postnatal day 45(Postnatal Day,PND45),PND65,PND92,half male and female,n=10-20),and Macaca fascicularis(1-1.5 years old,3-4years old,all male,n=8),16S rRNA assay was performed,and the results were analyzed to analyzed the dynamic rules of dominant bacteria and intestinal mimcrobiota in different age stages(from young age to adult age).2.Toxicity test and intestinal microbiota analysis of wall-removed Ganoderma lucidum spore powder in juvenile rats after 6 weeks of repeated administration:1)128 juvenile SD rats of PND24,half male and half,were divided into 4 groups by weight balance randomization method,namely vehicle control group,low-dose group,medium-dose group and high-dose group,each with 32 rats.At the beginning of PND24,pure water,0.8 g/kg,1.8 g/kg and 4.0 g/kg wall-removed Ganoderma lucidum spore powder were given orally and intragastric at a dosage volume of 10m L/kg,once a day,for 6consecutive weeks,and then resumed observation for 4 weeks after withdrawal.At the end of the drug withdrawal inspection and the recovery period,the effects of wall-removed Ganoderma lucidum spore powder on the development and toxicity of juvenile rats were investigated by testing various conventional juvenile animal toxicological indicators.The feces of juvenile rats in the drug withdrawal and at the end of recovery period were collected for 16S rRNA detection and analysis of intestinal microbiota to find different bacteria in different periods,and correlation analysis with routine immunotoxicity and behavioral-related toxicological indicators was carried out.2)Take another 60 PND21 juvenile SD rats,half male and half,and divide them into 3groups by weight balance randomization method,namely vehicle control group,low-dose group and high-dose group,20 in each group,same as the above test plan(vehicle control,low-dose and high-dose groups only)for administration and perform T-cell dependent antibody response(TDAR)test.That is,when the wall-removed Ganoderma lucidum spore powder was administered for 4 weeks and the drug was stopped for 2 weeks(5rats/sex/time point),juvenile rats in each group were injected with keyhole limpet hemocyanin(KLH)through the tail vein.1ml per mouse,at each time point,the serum KLH-Ig M and KLH-Ig G antibody titers were detected at 1 week and 2 weeks after KLH injection;and the feces of the corresponding period were collected for intestinal microbiota analysis,and analyzed the relationship between TDAR index and intestinal microbiota after administration.Result:1.Comparative analysis of intestinal flora of juvenile and adult healthy animals:(1)SD rats:Based on 16S rRNA sequencing,in the PCA and PCo A analysis of the intestinal bacteria of normal rats(from young to adolescents to adults),it was found that as the age of the rats increased,the structure of the intestinal microbiota changed significantly and gradually tended to stable.The dominant bacteria in PND45 juvenile rats are Firmicutes,Bacteroidia,Bacilli,and Bacteroidales,Muribaculaceae,Lactobacillaceae,norank＿f＿Muribaculaceae and Lactobacillus.The dominant bacteria of PND65 and PND92 rats are similar.The common dominant bacteria of the two are Firmicutes,Bacteroidetes,Bacteroidales,Muribaculaceae and norank＿f＿Muribaculaceae.(2)Macaca fascicularis:Based on 16S rRNA sequencing,PCA and PCo A analysis of the intestinal microbiota of 1-1.5-year-old monkeys and 3-4-year-old adult monkeys found that the composition of the intestinal microbiota between the two groups was similar,and its dominant bacteria at the level of phylum,class,order,family,and genus are the same,namely Firmicute,Bacteroidia Bacteroidales,Prevotellaceae and Prevotella.Suggesting that the distribution and diversity of intestinal microbiota of 1-1.5-year-old monkeys is close to the adult level.2.Toxicity test and intestinal microbiota analysis of wall-removed Ganoderma lucidum spore powder after 6 weeks of repeated administration in juvenile rats:(1)After 6 weeks of administration of Ganoderma lucidum spore powder,the immune index(CD3~+CD4~+%,TDAR test-KLH-Ig G antibody titer)and behavior index(Morris water maze-the percentage of time in the target quadrant)significantly increased,suggesting that the wall-removed Ganoderma lucidum spore powder can enhance and regulate the immune function and increase the learning and memory ability of juvenile rats.(2)Based on 16S rRNA sequencing analysis,it was found that the intestinal microbiota diversity of male juvenile rats during the drug withdrawal period changed significantly,but there were no significant changes at the end of recovery period. PCA and PCo A analysis showed that the composition of intestinal microbiota was similar between male and female juvenile rats in the two periods.The differential bacteria of male and female young rats in the two periods are different.Correlation analysis between differential bacteria and routine immunity(peripheral blood lymphocyte typing)and behavioral indicators found that there was no significant correlation between the differential bacteria of female and male juvenile rats and the immune and behavioral indicators when the drug withdrawal.At the end of recovery period,the decrease in relative abundance of Butyricimonas,Peptococcus and Marinifilaceae and the increase in relative abundance of Prevotellaceae may be related to the improvement of immune and behavioral indexes of juvenile male rats,respectively.The increase in the relative abundance of Burkholderiales,Parasutterella,Flavonifractor and Intestinimonas and the decrease of relative abundance of Frisingicoccus may be related to the improvement of immune indexes in juvenile female rats.The increase in relative abundance of Bacteroides＿pectinophilus＿group,Oscillospirales and Allobaculum may be related to the improvement of behavioral indicators of juvenile female rats.Among them,Allobaculum may be the key bacteria to improve the ability of learning and memory.(3)In the TDAR test,correlation analysis between the differential bacteria in groups of juvenile rats and the KLH antibody titers found that during the period of drug withdrawal,the relative abundance of Dubosiella,Bacilli,Clostridiaceae, Clostridiales,Faecalibaculum,UCG＿005,Candidatus＿Stoquefichus,Turicibacter, Clostridium＿sensu＿stricto＿1,Enterorhabdus,Bifidobacteriaceae,Bifidobacterium,Bifidobacteriales and Actinobacteria decreased and increase in relative abundance of Eubacterium＿ventriosum＿group,Frisingicoccus and Butyricicoccaceae may be related to the enhanced immune response of male juvenile rats after giving the wall-removed Ganoderma lucidum spore powder,Clostridium and Bifidobacteriaceae/genus may be the main related bacteria.Conclusion:1.This study analyzed the composition and dynamics of the intestinal microbiota of SD rats and Macaca fascicularis at different ages(from juvenile to adult).The results showed that the dominant bacteria of PND45 juvenile rats are different from the dominant bacteria of PND65 juvenile rats and PND92 adult rats.The latter two had similar dominant bacteria;the dominant bacteria and diversity of intestinal microbiota of 1-1.5-year-old monkeys and3-4-year-old adult monkeys are similar.2.In this study,the toxicological indicators and intestinal microbitoa analysis of juvenile rats after 6 weeks of administration of the wall-removed Ganoderma lucidum spore powder showed that the wall-removed Ganoderma lucidum spore powder enhanced the immunity and learning and memory ability of juvenile rats,and the composition of the intestinal microbiota has changed significantly;the changes in the abundance of some differential bacteria are closely related to the above-mentioned juvenile toxicological indicators,suggesting that the intestinal microbiota is related to the immune and nervous system development of juvenile animals,providing a certain scientific reference for the intestinal microbiota as a new non-clinical safety evaluation index of pediatric drugs.