Fundamental Substance And Effect Mechanism Of Total Ginsenosides Of Wild Ginseng On Tonifying Vital Energy Through "Intestinal-immune" Axis

Objective:According to Traditional Chinese Medicine(TCM),the spleen,located in the middle jiao,is responsible for digestion and transformation,and is considered as the foundation of postnatal life and the source of qi and blood production.Spleen qi deficiency is a common clinical syndrome in TCM,with symptoms including shortness of breath,lack of energy to speak,emaciation,mental fatigue,loss of appetite,and abdominal distention.Supplementing qi is often the primary treatment method.Wild ginseng has the effect of reinforce vital energy,and its main active component is ginsenoside.Ginsenoside has pharmacological effects such as enhancing immunity,lowering blood sugar,anti-inflammatory,analgesic,and antioxidant properties.Our previous research has shown that total ginsenosides of wild ginseng can regulate gut microbiota and bile acid metabolism in rats with spleen qi deficiency,reducing intestinal permeability.However,it is still unclear whether these effects are mediated through the"gut barrier-immune"axis and the specific material basis of its pharmacological effects.This study aims to clarify the qi-supplementing mechanism and the material basis of the immunoregulatory effects of total ginsenosides of wild ginseng(TG),providing experimental evidence and data support for the clinical application of wild ginseng in TCM.Methods:1.To establish a rat model of spleen qi deficiency,reserpine was administered through subcutaneous injection.By combining fecal transplantation(FMT)experiments,the symptoms and body weight of rats were recorded daily,and the content of D-xylose in serum was measured.After TG and FMT interventions,the immune organ index was calculated,and the content of immune factors in serum was determined by enzyme-linked immunosorbent assay(ELISA).Tissue morphological analysis was used to explore the pathological changes in spleen structure,aiming to investigate the immunomodulatory effects of TG and FMT on rats with spleen qi deficiency.2.By examining the histopathological improvement of colon tissue through histomorphology,the contents of DAO,D-Lac,and LPS in serum were measured using ELISA.Immunofluorescence and Western blot(WB)experiments were conducted to determine the expression of intestinal physical and chemical barrier proteins.Flow cytometry was used to measure the proportion of T cell subtypes in colon tissue.Additionally,16S r RNA gene sequencing technology was employed to determine the colonization of gut microbiota affected by TG after FMT intervention,clarifying that TG exerts immunomodulatory effects based on the repair of intestinal barriers through gut microbiota.3.Using methods such as UPLC-Q-TOF-MS~Etargeted metabolomics analysis,unsupervised principal component analysis,and supervised orthogonal partial least squares-discriminant analysis,the effects of TG and FMT on bile acid metabolites in the feces and serum of rat models with spleen qi deficiency were determined,aiming to screen out the major bile acid metabolites that TG and FMT regulate in these models.4.Experimental validation was conducted on the major bile acid metabolite,Taurineursodeoxycholic acid(TUDCA),regulated by TG.Tissue morphological analysis,WB experiments,immunofluorescence,and flow cytometry were used to analyze the restorative effects of TUDCA on intestinal barrier function.Additionally,WB experiments were employed to determine the impact of TUDCA on the expression levels of bile acid receptors FXR and FGF15 proteins.q RT-PCR technology was applied to measure the expression levels of T-bet,GATA3,RORyt,and Foxp3 m RNA in colonic T cells,validating the mechanism of TG’s qi-tonifying effect mediated by gut microbiota and the bile acid metabolite TUDCA through the FXR/FGF15 signaling pathway.5.Ginsenoside components were analyzed in the intestinal contents of TG-intervened rats using UPLC-Q-TOF-MS~E,and the main 10 ginsenosides components were quantified.Pearson correlation analysis was performed between ginsenoside components and immune indicators in rats with spleen qi deficiency,bile acid metabolite TUDCA,and intestinal barrier proteins.This analysis aimed to screen the main active ingredients of TG that exert qi-tonifying effects through the"gut-immune"axis,revealing the pharmacological material basis for TG’s immunomodulatory effects.Results:1.After modeling with reserpine,the rats’body weight significantly decreased,while the traditional Chinese medicine syndrome score increased significantly,showing typical manifestations of spleen qi deficiency.The D-xylose test results showed that the rats’absorption capacity decreased,indicating a stable and reliable model.After intervention with TG and FMT,the syndrome scores of the rats decreased(p<0.05),body weight increased(p<0.05),D-xylose absorption increased(p<0.05),and the immune organ index significantly increased(p<0.05).Additionally,the immune indicators IL-17 and IL-1βdecreased(p<0.05),while TGF-β(p<0.05),IL10(p<0.05),and IL-22 increased,significantly improving the histopathological condition of the spleen tissue.These results indicate that TG and FMT can improve the immune function of rats with spleen qi deficiency.2.TG and FMT significantly improved the histopathological condition of the intestinal tissue,reduced the levels of DAO,LPS,and D-Lac in serum(p<0.05),and increased the intestinal physical barrier function(ZO-1,Occludin,and Claudin-1 protein expression)and chemical barrier function(Tff3,Muc-2,and Reg3g protein expression).The number of Treg cells in the intestinal immune barrier increased significantly(p<0.05),while the number of Th17,Th1,and Th2 cells decreased significantly(p<0.05).16S r RNA gene sequencing results validated that FMT enabled the Lactobacillus increased by TG to colonize in the intestinal tract of model rats.These results indicate that both TG and FMT can reduce intestinal permeability,enhance intestinal barrier function,and TG has a direct effect on the gut microbiota.3.The targeted metabolomics analysis results showed that TG and FMT significantly regulated bile acid metabolites in the feces and serum of rats.Specifically,TG and FMT jointly regulated TUDCA,exhibiting significant differences compared to the spleen qi deficiency model group(p<0.05).Further correlation analysis of 13 bile acid metabolites with intestinal barrier indicators revealed that TUDCA is likely the primary bile acid metabolite regulated by TG.These results suggest that TG may repair intestinal barrier function and exert immunomodulatory effects through the bile acid metabolite TUDCA.4.The validation experiment results for the major bile acid metabolite TUDCA showed that TUDCA was able to improve the phenotypic characteristics of rats(p<0.05),increase body weight and immune organ index(p<0.05),and significantly enhance the absorption of D-xylose(p<0.05),thereby improving the structure of the spleen and colon.It also significantly increased the expression of proteins related to the intestinal physical and chemical barriers(p<0.05),promoting the differentiation of intestinal T cells towards Treg cells.Western blot(WB)results indicated that TUDCA intervention significantly increased the expression of FXR and FGF15 proteins in the intestine and liver.q RT-PCR results showed that TUDCA significantly downregulated the expression levels of T-bet,GATA3,and RORyt m RNAs in T cells(p<0.05)and upregulated the expression level of Foxp3m RNA(p<0.05).These results suggest that TG exerts immunomodulatory effects by improving gut microbiota and the bile acid metabolite TUDCA,as well as activating the bile acid receptor FXR.5.A total of 28 ginsenoside components were identified in the intestinal contents.The results of Pearson correlation analysis showed that ginsenosides were correlated with immune indicators,TUDCA,and intestinal barrier proteins in rats with spleen qi deficiency models.Among them,ginsenosides such as Rb1 exhibited strong correlations,suggesting that they may be the key active ingredients of TG that play a role in immunomodulation by regulating gut microbiota and TUDCA.Conclusion:TG can increase Lactobacillus in the intestine and regulate the bile acid metabolite TUDCA,further acting on the FXR target to regulate the expression of downstream protein FGF15.This process increases the Foxp3 m RNA level and the secretion of cytokines such as IL-22 and IL-10 in colonic Treg cells,restoring intestinal barrier function and exerting immunomodulatory effects,thereby elucidating its qi-tonifying effects.Ginsenosides Rb1 in intestinal contents are likely the main active ingredients of TG that play a role in immunomodulation.Combining traditional Chinese medicine theory with modern analytical techniques,this study reveals the immunomodulatory mechanism of TG’s qi-tonifying effects based on the"gut-immune"axis,initially elucidating the pharmacological material basis of TG,and providing an effective methodological reference for the systematic analysis of the mechanism of action of traditional Chinese medicine.