Effect Of Heifu Tablet On Immune Function And Intestinal Flora In Immunosuppressed Mice

Aconitum carmichaeli Debx.(Ranunculaceae)is widely distributed and cultivated in Sichuan Jiangyou and Shaanxi Hanzhong.The processed lateral roots of A.carmichaeli,which is known as ' Fu Zi' in traditional Chinese medicine,have been widely used in China to treat various symptoms such as cadianeuria,rheumatalgia,neuralgia,and inflammation.Previous studies have revealed that monoester type alkaloid and Polysaccharide are the main active constituents of this drug.However,it is also well known for its toxicity,cooking alone or concurrently with other herb such as Gancao is often used to alleviate its toxicity.As aconite can improve immunity,therefore,people in Shaanxi Hanzhong,Sichuan Jiangyou,Yunnan Lijiang and other aconite main producing areas have the habit that eat cooked aconite in winter.Basic science and clinical studies have clearly shown that the potential impact of the species and structure of human intestinal flora on human health and immunity.In this study,we investigated whether the water extract of aconite could improve the immunity of immunosuppressed mice and change their intestinal microbial composition,and further explore the relationship between them,to providing the reliable theory for the effect of aconite on the mechanism of ameliorating immunity.1.Establishment of immunosuppressive modelThe normal mice were used as control.This study established immunosuppression animal model by cyclophosphamide(CY)injection,including low dose group(75mg/kg)and high dose group(100mg/kg)of CY.The results showed that compared with the control group,the thymus index,spleen index,white blood cell count and lymphocyte absolute count of the mice injected with CY were significantly decreased,indicating that both doses of CY can lead to reduced immunity in mice.As a lot of the mice in high dose of CY group died,ultimately low dose(75mg/kg)of CY modeling was used in the following experiments.2.The study of exploring best therapeutic concentration of aconite decoctionThe mice were injected with low dose of CY for modeling.The experimental groups as follow:normal group,model group,and four different doses of aconite decoction groups(30,20,10,2g/kg).The drugs used in this paper accorded with the 2015 version of the Pharmacopoeia standard.Drugs were given gastric lavage for 7 consecutive days in all mice.The results showed that compared with model group,all doses of the aconitum decoction could improve the organ index,white blood cell count and lymphocyte number in immunosuppressed mice.The very low dose(2g/kg)of aconite decoction showed the best therapeutic effect,the postoperative immune function closely to the normal group.So,2g/kg dose of aconite decoction was chosen for the subsequent experimental study.3.Study on the immunoregulation effect of aconite decoction and aconite polysaccharides on miceThe mice were injected with low dose of CY for modeling and given 2g/kg aconite decoction or 15mg/kg aconite polysaccharide for treatment.The experimental groups as follow:normal group,aconite decoction normal group,model group,aconite decoction model group and aconite polysaccharide model group.The results indicated that aconite decoction and aconite polysaccharide could significantly increased the organ index,improve the spleen CD4+FOXP3+positive cells rate,increase the mRNA expression of IL-6,TNF-?,NF-KB in spleen,and decrease the mRNA expression of IFN-?,COX2,CXCL,CXCR.On the basis of the studies above,both of aconite decoction and aconite polysaccharides showed strong effect on immune function and inflammatory process through multiple levels and different pathways.4.Effects of aconite decoction and aconite polysaccharides on intestinal flora in miceThe results of intestinal flora analysis showed that all mice domain bacteria are same,including Helicobacter,Mucispirillum,Prevotella,Oscillospira,Lactobacillus,and Bacteroides,and there were a few differences in the community composition of intestinal flora in each group,but the abundance of species was significantly different.Compared with the model group,the abundance of Lactobacillus and Helicobacter were significantly increased,while the abundance of Bacteroides was significantly decreased.The alpha diversity analysis such as the Chaol index,Simpson index and Shannon index obviously demonstrated that the alpha dinersity of mice intestinal flora increased significantly after administer aconite decoction and aconite polysaccharide,which indicating that both of aconite decoction and polysaccharide could change the mice intestinal microbial diversity and community structure.5.Relationship between intestinal flora and immune system in miceThe Pearson correlation analysis between cytokines and bacterial diversity showed that the mRNA expression of IL-6 was positively correlated with the abundance of Prevotella and Blautia.The abundance of Candidatus Arthromitus was very significantly or significantly negative correlation with the mRNA expression of IFN-y,CXCL,CXCR,TNF-a,NF-KB,at the same time,it also positively correlated with the spleen index significantly.There was a significantly positive correlation of the abundance of Bacteroides with the mRNA expression of NF-KB,while with CXCL was negatively correlated.In addition,the spleen index and white blood cell count was positively correlated with the abundance of Lactobacillus,while it was significantly negatively correlated with the Faecalibacterium.Hower,the abundance of Coprococcus and Fusobacterium was only positively correlated with the thymus index.In summary,aconite decoction and aconite polysaccharide promote and maintain the health of body maybe is through improving the immunity and adjusting intestinal flora structure/abundance.