Study On The Immune Regulation And Anti-immune Aging Mechanism Of The Buzhong Yiqi Decoction With Hedysarum Radix Or Astragali Radix As The Monarch Drug And The Hedysari Polysaccharide

Background:Hedysarum Radix is dry root of Hedysarum polybotrys Hand.-Mazz..There are wild and cultivated varieties,mainly produced in Gansu province.Among the various active substances contained in Hedysarum Radix,the in-depth study on the anti-aging effect of Hedysari polysaccharide is a tool for us to deal with the challenges brought by aging in China.Among the many traditional Chinese medicine prescriptions for delaying senescence,the efficacy of Buzhong Yiqi Decoction takes Astragali Radix as the monarch drug,focuses on replenishing qi and regulating the acquired origin of the body.There are a lot of same characteristic efficacy both Hedysarum Radix and Astragali Radix such as replenishing qi and raising Yang,fixing the surface and stopping sweating...and so on.Hedysarum Radix is mainly sold in Guangdong,Fujian and other places and exported.Hedysarum Radix is often considered to be a high-quality variety of Astragali Radix.This paper hopes to study the difference of immune regulation effect of Buzhong Yiqi Decoction of Hedysarum Radix or Astragali Radix as the monarch drug on immune senescence.Through the research on the influence of Hedysari polysaccharide,which has anti-aging effect on immune senescence,this paper serves as a basis for the rational development of Hedysarum Radix and its clinical scientific use in Gansu.Purpose:We wanted to find out the mechanism of the immune function of Hedysarum Radix,so we had compared the regulatory effect of Buzhong Yiqi Decoction with Hedysarum Radix or Astragali Radix as the monarch drug on the immune function of SAMP8 mice,and we had studied the regulatory mechanism of Hedysari polysaccharide 3(HPS-3),on the immune function of SAMP8 mice,to provide a theoretical basis for the rational use of Hedysarum Radix.Methods:1.Replace the Astragali Radix in Buzhong Yiqi Decoction with an equal amount of Hedysarum Radix,and prepare Hedysarum Radix in Buzhong Yiqi Decoction(HR-BZYQ)and Astragali Radix in Buzhong Yiqi Decoction(AR-BZYQ)respectively.10KM mice were regarded as the younger model group,and 40 SAMP8 mice were randomly divided into aging model group,thymosin positive control group,HR-BZYQ group and AR-BZYQ group.After continuous intragastric administration for 14d,the changes of the spleen structure were observed by HE was staining.ELISA was used to detect Cytokines in serum.Flow cytometry was used to detect the changes of T lymphocyte subsets.Real-time fluorescence quantitative PCR was used to detect the expression of p38MAPK mRNA.Western Blot was used to detect the expression of p38MAPK protein in spleen lymphocytes.Immunohistochemical method was used to detect the protein expression of p38MAPK in mouse spleen.2.The spleen lymphocyte suspension of KM mice was prepared.After the intervention of different concentrations of HPS-3,the optimal drug concentration of HPS-3 on the proliferation of spleen lymphocytes in mice was determined by the MTT method.On this basis,ELISA was utilized to detect the effects of HPS-3 on cytokines in the supernatant of lymphocyte culture.Flow cytometry was utilized to detect the effect of HPS-3 on T lymphocyte subsets.The influence of HPS-3 on ultrastructure of spleen lymphocytes was observed by transmission electron microscope.3.SAMP8 mouse spleen lymphocyte suspension was prepared.After HPS-3intervention for 72h,total proteins in lymphocytes were extracted.Label Free unmarked quantitative mass spectrometry data were analyzed by the Database for Annotation,Visualization and Integrated Discovery;Gene Ontology and Kyoto Encyclopedia of Genes and Genomes to find the biological signaling pathways in which differential proteins content may be involved.Functional protein association networks platform analyzes the interaction network of differential proteins content.Western Blot was used to verify the accuracy of the results.Results:1.Compared with the younger model group,the proportion of white pulp in the spleen of the aging model group was reduced,the boundary between red pulp and white pulp was blurred.The levels of IL-2,IFN-?and TNF-?were lower than the younger model group(P<0.05).In the aging model group,the proportion of CD4~+T lymphocyte subsets was increased,the proportion of CD8~+T lymphocyte subsets was decreased,the proportion of CD28~+T lymphocyte subsets was decreased,and the proportion of CD28~+CD152~+T lymphocyte subsets was increased(P<0.05).The mRNA and protein expressions of p38MAPK were down-regulated(P<0.05).Compared with the aging model group,the spleen structure of SAMP8 mice in the HR-BZYQ group and the AR-BZYQ group improved,the proportion of white pulp increased.Contents of IL-2,IFN-?and TNF-?in serum were all increased,and the differences were statistically significant(P<0.05).Contents of IL-2 in the HR-BZYQ group were higher than the AR-BZYQ group(P<0.05).The proportion of CD8~+T lymphocyte subsets in spleen was increased in both the HR-BZYQ group and the AR-BZYQ group,and more in the AR-BZYQ group than in the HR-BZYQ group.The proportion of CD4~+T lymphocyte subsets were reduced in the HR-BZYQ group and the AR-BZYQ group.The proportion of CD28~+T lymphocyte subsets were increased,and the proportion of CD28~+CD152~+T lymphocyte subsets were decreased,(P<0.05).The expression of mRNA and protein of p38MAPK was up-regulated in both the HR-BZYQ group and the AR-BZYQ group(P<0.05).2.Different concentrations of HPS-3 were co-cultured with mouse spleen lymphocytes for 72 h,and the RGR was computed to determine that 100?g/mL was the optimal experimental concentration of HPS-3 to promote the proliferation of mouse spleen lymphocytes.Compared with the Blank group,IL-4 in splenic lymphocyte culture supernatant of mice interfered by HPS-3 and ConA were reduced(P<0.05).IL-2,IFN-?and TNF-?were increased(P<0.05).CD3~+T lymphocyte subsets were increased(P<0.05).The proportion of CD3~+CD8~+T lymphocyte subsets increased(P<0.05).Splenic lymphocytes interfered by HPS-3 and ConA showed more organelles,purer lymphocyte structure,increased mitochondrial number,and purer mitochondrial Cristal structure.Contents of IFN-?and TNF-?in HPS-3 group were increased(P<0.05).The content of CD3~+CD8~+T lymphocyte subsets in HPS-3group was greater than in ConA group(P<0.05).3.Label Free unmarked quantitative mass spectrometry detection results show that the HPS-3 training after 72 h,SAMP8 mice spleen lymphocytes had 194 proteins abundance show significant changes(CON group and the expression of the HPS group difference ratio R value more than 1.5 times:R value<0.7 or>1.5,P<0.05as differences in protein),including 172 proteins significantly increase,22 proteins significantly lower.Database for Annotation,Visualization and Integrated Discovery,Gene Ontology and Kyoto Encyclopedia of Genes and Genomes results showed that these differential proteins were enriched into 12 pathways,which were mainly related to the ubiquitin-Proteasome pathway and metabolic pathway of lymphocytes.Functional protein association networks platform on different proteins showed that HPS-3 could up-regulate the activity of"ubiquitin-proteasome pathway".It can up-regulate the activity of NF-Kappa B signaling pathway.Conclusion:1.Both HR-BZYQ Decoction and AR-BZYQ Decoction can improve the immune function imbalance caused by aging.IL-2,IFN-?and p38MAPKmRNA expression in spleen lymphocytes in the HR-BZYQ group were higher than the AR-BZYQ group.2.HPS-3 can promote proliferation of spleen lymphocytes,increase of Th1cytokine's IL-2,IFN-?and TNF-?,meanwhile,decrease of Th2 cytokines IL-4,increase of proportion of CD3~+T and CD3~+CD8~+T lymphocytes,and increase of intracellular organelles in lymphocytes.HPS-3 can promote cellular immune response.3.HPS-3 can up-regulate the activity of"ubiquitin-proteasome"in lymphocytes and up-regulate the activity of NF-Kappa B signaling pathway.