| Objective:Based on the theory of "sweet flavour nourishing spleen" in Huangdi Neijing,Xiaojianzhong decoction has been used to treat spleen deficiency and diarrhea in pediatrics department of our hospital with satisfactory results.To explore the mechanism of Xiaojianzhong decoction in the treatment of spleen deficiency and diarrhea,the mice model of spleen deficiency and diarrhea was established by using the method of “letdown of bitter cold” so as to observe the changes of intestinal flora and intestinal mucosal immunity via comparing with the normal mice model.Analyzing the impact of Xiaojianzhong decoction on intestinal flora and intestinal mucosa function by intervening young mice with spleen deficiency and diarrhea can provide laboratory basis for Xiaojianzhong decoction in the treatment of spleen deficiency and diarrheaMethods: According to Random grouping principle,Forty-eight young male mice were divided into 12 normal group(group A)and 36 model group by establishing the model of spleen deficiency and diarrhea to simulate the letdown of bitter cold of Dahuang.Then,model group were randomly separated into 3 groups: spleen deficiency and diarrhea group(group B),Xiaojianzhong decoction high-dose group(group C)and Xiaojianzhong decoction low-dose group(group D),with 12 mice in each group.During the intervention period,normal group and diarrhea group were fed with saline while Xiaojianzhong decoction group was given Xiaojianzhong decoction for 10 days.The spirit,fur,diet,body weight,activity and stool characteristics of young mice were observed daily.After treatment,16 S r DNA high-throughput sequencing was used to detect the structural composition and richness of intestinal flora in the cecum of young mice and the intestinal tissues of young mice were taken for SIg A detection by enzyme-linked immunosorbent assay(ELISA).Results:1.This study successfully replicated the model of spleen deficiency and diarrhea in young mice,and found that in comparison with the normal group,the expression of thymus index,spleen gland index and intestinal mucosal tissue s Ig A in the spleen deficiency and diarrhea group significantly decreased(P<0.05).After the intervention of Xiaojianzhong decoction,compared with the spleen deficiency and diarrhea group,the general condition of the young mice in the high-dose and low-dose Xiaojianzhong decoction groups was ameliorated,and the body weight and thymus index were significantly increased(P<0.05).The spleen index in the high-dose Xiaojianzhong decoction group was significantly improved(P<0.05),but there was no difference in the low-dose group(P>0.05).The content of SIg A in intestinal mucosa of Xiaojianzhong decoction high-dose and low-dose groups was significantly increased(P<0.05),but the content of SIg A in high-dose group was much higher(P<0.05).2.High throughput sequencing results of 16 S r DNA(1)The perspective of composition of intestinal flora:At the phylum level,Bacteroidetes and Firmicutes accounted for the largest number of cecum species in the four groups of mice,which were the dominant flora(2)Comparison of different species between groups :Compared with normal mice,in spleen deficiency and diarrhea group,the levels of Bacteroidetes,Proteobacteria and Gemmatimonadetes were decreased(P<0.05),while the levels of Epsilonbacteraeota and Tenericutes were significantly increased(P<0.05).At the genus level,the levels ofBacteroides,Alistipes and Alloprevotella were significantly decreased while the levels of Helicobacter were significantly increased(P<0.05),and the numbers of Escherichia-Shigella were also increased,but not significantly(P>0.05).Compared with the spleen deficiency and diarrhea group,Xiaojianzhong decoction can promote the growth of Bacteroidetes,Proteobacteria,Acidobacteria,Actinobacteria and Gemmatimonadetes but inhibit the growth of Epsilonbacteraeota and Tenericutes(P<0.05).The effect of high dose ofXiaojianzhong decoction on promoting the growth of Gemmatimonadeteswas superior to low dose group(P<0.05).At the genus level,Xiaojianzh ong decoction could promote the growth of Bacteroides,Alistipes and Alloprevotella but inhibit the growth of Helicobacter and Escherichia-Shigell a(P<0.05).The effect of high dose of Xiaojianzhong decoction on promo ting the growth of Alloprevotella was better than that of low dose of Xiaojianzhong decoction(P<0.05).Compared with the normal group,Xiaojianzhong Decoction can promote the restoration of the disordered flora structure to normal at both the phylum and the genus level.And Xiaojianzhong Decoction can promote the significant growth of Acidobacteria,which is higher than the normal group(P<0.05).Conclusion:1.A young mouse model of spleen deficiency and diarrhea can be successfully established by intragastric administration of raw Dahuang water decoction.The level of SIg A secreted in intestinal mucosa in the mice model of spleen deficiency and diarrhea was decreased,the structure and composition of intestinal flora were changed and the diversity of intestinal flora was decreased.2.Xiaojianzhong decoction can improve spleen deficiency syndrome of young mice with spleen deficiency and diarrhea,promote weight gain,increase the atrophy of thymus and spleen,and up-regulate the secretion level of intestinal mucosa SIg A.3.Xiaojianzhong decoction can facilitate the growth of beneficial bacteria,restrain the proliferation of pathogenic bacteria,restore the disordered bacterial structure to the normal level as well as improve the intestinal mucosal immune function which may be one of the potential mechanisms in treating spleen deficiency and diarrhea.The dose of Zhongjing original formula(the eastern han dynasty)of Xiaojianzhong Decoction was better than the recommended dose of modern textbooks in improving intestinal mucosal immunity for relieving spleen deficiency.The dose of Zhongjing original formula(the eastern han dynasty)of Xiaojianzhong Decoction was superior to the dose recommended in modern textbooks in regulating Gemmatimonadetes and Alloprevotella. |
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