| Aging refers to the process that leads to the gradual deterioration of various human functions,and it has currently become a forefront and hot topic in the field of life sciences worldwide.The"Jing-Qi-Shen theory" of Qi Luo doctrine in traditional Chinese medicine serves as a guiding system for anti-aging research.The brain is one of the most significant organs reflecting the state of aging.Studies have found that with brain aging,the structure and function of the intestine also undergo a series of changes.The homeostasis of the gut can effectively maintain the body’s balance,and its dysfunction has a significant impact on the accelerated progression of brain aging.Therefore,it is of great significance to explore the etiology,pathogenesis,and treatment methods for brain aging under the guidance of the "Jing-Qi-Shen theory"of Qi Luo doctrine,as well as to conduct research on the mechanism of the representative drug Bazi Bushen Capsule in preventing and treating this disease.Objective:This study includes two parts:theoretical research and experimental research under the guidance of the theory of " Jing-Qi-Shen theory" of Qi Luo doctrine to explore the regulation of intestinal function in preventing and treating brain aging.The first part explores the traditional Chinese medicine’s understanding of brain aging and the theoretical research guided by the theory of"Jing-Qi-Shen theory " of Qi Luo doctrine in the prevention and treatment of brain aging.The second part,through the establishment of two models,SAMP8 accelerated aging mice and stress-accelerated aging mice,explores the experimental research guided by the theory of"Jing-Qi-Shen theory" of Qi Luo doctrine on the representative anti-aging drug Bazhi Bushen Capsule by regulating intestinal function to prevent and treat brain aging,providing theoretical guidance and data support for the research of TCM anti-aging.Method:1.Theoretical InvestigationTraditional Chinese medicine(TCM)has accumulated a wealth of experience in understanding brain aging over thousands of years of development,conducting in-depth explorations of TCM anti-aging research in different historical periods,summarizing the etiology and pathogenesis of brain aging,and forming a basic understanding of brain aging.Based on the experience of predecessors,Academician Wu Yiling systematically constructed a guiding theory for anti-aging research—"Jing-Qi-Shen theory" of Qi Luo doctrine.Based on this theory,an in-depth exploration of the prevention and treatment of brain aging is carried out.2.Experimental ResearchExperiment I:Research on the effect of Bazi Bushen Capsule on improving cognitive function by intervening in age-related intestinal damageFour-month-old male SAMP8 mice were randomly divided into the model group,low-dose Bazi Bushen Capsule group,high-dose Bazi Bushen Capsule group,rapamycin group,with age-matched SAMR 1 mice serving as the control group.① Nissl staining and two-photon detection were used to assess the cognitive function of SAMP8 mice.②Hematoxylin and eosin(HE)staining and immunofluorescence staining were used to examine the intestinal barrier function of SAMP8 mice.③Reverse transcription quantitative PCR(RT-q PCR)technology and immunohistochemical staining techniques were used to detect the levels of SASP secretion and macrophage infiltration,as well as the proliferative and differentiative abilities of intestinal cells in SAMP8 mice.④ 16S rRNA sequencing technology was employed to analyze the gut microbial composition of SAMP8 mice.⑤RT-q PCR technology and Western Blot technology were used to measure the levels of inflammasome and pyroptosis in SAMP8 mice.Experiment Ⅱ:Study on the effect of Bazi Bushen Capsule on improving cognitive function by regulating intestinal homeostasis through Akkermansia.The SAMP8 mice were divided into groups based on age:2-month-old group,4-month-old group,6-month-old group,and 8-month-old group.Additionally,40 SAMP8 mice were randomly assigned to the model group,low-dose Bazi Bushen Capsule group,high-dose Bazi Bushen Capsule group,and rapamycin group,with 10 SAMR1 mice serving as the control group.C57BL/6J mice were randomly divided into the blank control group,model group,and Akkermansia+model group.①The Barnes maze test and HE staining method,WB experiment were used to evaluate the cognitive abilities of SAMP8 mice.②HE staining,PAS staining,and immunofluorescence staining techniques were used to assess the intestinal function of the mice.③16S rRNA technology was employed to detect the gut microbiota of the mice.④LC-MS was used to analyze the metabolomics profile of the mice.⑤Fecal microbiota transplantation(FMT)technique was used to verify the protective effects of characteristic bacterial strains on the mice’s gut.Experiment Ⅲ:Research on the effect of Bazi Bushen Capsule on improving cognitive function by intervening in stress-induced intestinal injuryEleven-month-old C57BL/J mice were divided into a natural aging control group,a stress model group,a low-dose Bazi Bushen Capsule group,a high-dose Bazi Bushen Capsule group,and an NMN(nicotinamide mononucleotide)positive drug group,with 25 mice in each group.A chronic mild stress model was used for the creation of the model.During the modeling process,the medication was administered for 12 months.① Survival curves were used to evaluate the survival ability of the mice in each group.② The frailty index was used to assess the level of frailty in each group of mice.③The Barnes maze and three-box social test were used to evaluate the cognitive abilities of the mice in each group.④HE(Hematoxylin and Eosin)staining was used to assess the pathological conditions of the intestines.⑤RT-q PCR technology was used to detect the aging levels in the brain and intestinal tissues of the mice in each group.⑥Immunofluorescence technology was used to detect the levels of immune infiltration in the brain and intestinal tissues of the mice in each group.Results:1.Theoretical Research:The "Jing-Qi-Shen theory" of Qi Luo doctrine is a theoretical system in traditional Chinese medicine,which regards the concepts of "Jing","Qi",and "Shen" as the fundamental elements of life activities.This theory is used to explain and guide the diagnosis and treatment practices of TCM in anti-aging,and it proposes that "Jing is the origin of life,Qi is the driving force of life,and Shen is the manifestation of life." It reveals the key pathogenesis of aging:the deficiency of kidney essence is the root of aging,the depletion of original Qi is the key to aging,and the consumption of form and spirit is the manifestation of aging.The longevity or brevity of life depends on the abundance or deficiency of Jing,Qi,and Shen.Under the guidance of"Jing-Qi-Shen theory" of Qi Luo doctrine,the deficiency of kidney essence is identified as the core pathogenesis of brain aging,and the disharmony between the brain and intestines is the key pathogenesis in the progression of brain aging.The innovative Chinese medicine Bazi Bushen Capsule,developed around the treatment principles of "nourishing kidney essence,harmonizing Yin and Yang,warming and supporting original Qi,and nourishing form and spirit," possesses the effects of replenishing essence to transform Qi,boosting Qi to invigorate Shen,and nourishing form and spirit to combat brain aging.2.Experimental Research:Experiment Ⅰ:①Compared to the control group,the model group showed a significant decrease in the number of neurons in the cerebral cortex and hippocampus,and an increase in blood-brain barrier leakage(P<0.001);treatment with Bazi Bushen Capsule increased the number of neurons in SAMP8 mice and reduced the exudation in the cerebral cortex(P<0.01).②Compared to the control group,the model group of SAMP8 mice had impaired intestinal barrier and a reduction in the number of intestinal villi(P<0.0001).Treatment with Bazi Bushen Capsule could alleviate the damage to intestinal barrier function,repair the structure of intestinal villi,and increase the expression levels of ZO-1 and Occludin proteins in the ileum tissue(P<0.01).③Compared to the control group,the model group had elevated transcription levels of P53.TNF-α,NF-κB,and IL-6,and decreased transcription levels of IL-4 and IL-10(P<0.05).After taking Bazi Bushen Capsule,these indicators returned to normal(P<0.05).Additionally,the expression of F4/80 in the model group was increased;after taking Bazi Bushen Capsule,the indicator tended to normalize(P<0.01).④ The experimental results showed that the model group had decreased levels of intestinal cell proliferation and intestinal stem cell markers;after taking Bazi Bushen Capsule,these parameters tended to normalize.⑤ There were significant differences in the composition and abundance of microbes between the control group,the model group,and the Bazi Bushen Capsule group.The relative abundance of the Firmicutes/Bacteroidetes ratio was significantly reduced in the model group,while it was increased in the Bazi Bushen Capsule group.⑥ Bazi Bushen Capsule inhibited the increase in NLRP3 and cleaved caspase-1 expression induced by LPS in a concentration-dependent manner(P<0.01),and also had an inhibitory effect on IL-1 β and IL-18.Experiment Ⅱ:①Limited by the duration of detection technology,although the total time in the Barnes maze for 8-month-old SAMP8 mice did not continue to increase compared to 6-month-old SAMP8 mice,the experimental results clearly showed that as the age progresses,the cognitive function of SAMP8 mice is continuously declining(P<0.01).②Results from various ages indicated that as the age progresses,the number of neurons in SAMP8 mice is continuously decreasing.Along with the worsening of cognitive damage,intestinal structural damage is also worsening(P<0.01).③As the age progresses,the intestinal barrier function of SAMP8 mice is continuously declining.④ A correlation analysis between the results of the Barnes maze and the data on intestinal barrier function showed that as the intestinal barrier function decreases,cognitive levels are also continuously declining.⑤ Compared to the control group,the model group had a decreased expression level of PSD-95(P<0.01).After administration of Bazi Bushen Capsule,the expression level of PSD-95 increased(P<0.01).⑥The model group showed abnormalities in the structure of colon tissue.However,after administration of Bazi Bushen Capsule,the thickness of the mucus and the number of goblet cells improved,playing a role in protecting the mucus barrier function.⑦ In the results of α-diversity analysis,the abundance of SAMP8 mice significantly decreased in the Simpson and Shannon-Wiener indices,with statistical significance(P<0.05).In the Shannon index and observed_otus index,there was a slight decrease in abundance compared to SAMR1 mice,but the difference was not statistically significant.Notably,Bazi Bushen Capsule was able to increase its abundance in all four indices,with statistical significance(P<0.01).In β-diversity,compared to SAMR1 mice,SAMP8 mice showed significant changes in the composition and abundance of the microbial community,and after administration of Bazi Bushen Capsule,the composition and abundance of the microbial community were restored to a state close to that of SAMR1 mice.Compared to the SAMR1 group,the Firmicutes decreased and Bacteroidetes increased in the SAMP8 group;after administration of Bazi Bushen Capsule,the Firmicutes increased and Bacteroidetes decreased.Akkermansia was also found to be a potential characteristic bacterial strain through which Bazi Bushen Capsule exerts its effects.⑧ Metabolic product distribution states of SAMR1 mice,SAMP8 mice,and those treated with Bazi Bushen Capsule were distinctly differentiated.SAMP8 mice had 1,338 significantly upregulated and 2,235 significantly downregulated differential metabolites compared to SAMR1 mice;after administration of Bazi Bushen Capsule,808 significantly upregulated and 775 significantly downregulated differential metabolites were selected compared to SAMP8 mice.Bazi Bushen Capsule acts on biological processes regulated by Akkermansia,involving the biosynthesis of pantothenic acid and CoA,glutathione metabolism,and the metabolism of D-glutamine and D-glutamate,among others.⑨Results of fecal microbiota transplantation with Akkermansia:The results showed that after the transplantation of Akk bacteria,the intestinal barrier function was effectively protected..Experiment III:① Compared to the control group,the survival curve of the model group mice was significantly decreased(P<0.01).Compared to the model group,the survival curves of the low-dose Bazi Bushen Capsule group,high-dose Bazi Bushen Capsule group,and NMN group were significantly increased(P<0.05).②Compared to the control group,the frailty index of the model group mice was significantly increased(P<0.01).Compared to the model group,the high-dose Bazi Bushen Capsule group showed a significant decrease(P<0.01).③Compared to the control,the total time in the Barnes maze for the model group was significantly increased(P<0.05).After administration of Bazi Bushen Capsule,the time spent in the maze was significantly reduced(P<0.01).④ Compared to the control group,the social interaction coefficient of the model group mice was significantly reduced(P<0.05).After administration of Bazi Bushen Capsule,the social interaction coefficient was significantly increased(P<0.01).⑤Compared to the control group,the colon length of the model group mice was shortened(P<0.01),and administration of Bazi Bushen Capsule increased its length(P<0.01).⑥Compared to the control group,the ileal villi structure of the model group mice was damaged,and administration of Bazi Bushen Capsule could repair the structural damage.⑦Compared to the control group,the level of SASP in the brain tissue of the model group mice was significantly increased,and administration of Bazi Bushen Capsule could significantly reduce its expression level.⑧Compared to the control group,the level of SASP in the ileal tissue of the model group mice was significantly increased,and administration of Bazi Bushen Capsule could significantly reduce its expression level.⑨Compared to the control group,the level of p16 in the brain tissue of the model group mice was significantly increased,and administration of Bazi Bushen Capsule could reduce its level.At the same time,Bazi Bushen Capsule could also reduce the infiltration of pro-inflammatory cells induced by accelerated aging,such as CD4-positive T cells,CD8-positive T cells,and microglia.⑩Compared to the control group,the level of p16 in the ileal tissue of the model group mice was significantly increased,and administration of Bazi Bushen Capsule could reduce its level.At the same time,Bazi Bushen Capsule could reduce the infiltration of pro-inflammatory cells induced by accelerated aging,such as CD4-positive T cells,CD8-positive T cells,and M1-type macrophages,while increasing the levels of anti-inflammatory cells like M2-type macrophages.Conclusion:1.Guided by "Jing-Qi-Shen theory",this study explores the etiology,pathogenesis,therapeutic methods,and medicinal prescriptions for the prevention and treatment of brain aging,providing theoretical guidance for the research of traditional Chinese medicine in anti-aging.It is believed that "the deficiency of kidney essence is the core pathogenesis of brain aging,and the abnormality in the intestinal transmission is the key pathogenesis in the development of brain aging."This study provides research support for the application of traditional Chinese medicine in regulating the intestines to delay brain aging,improve the quality of life,and prevent and treat diseases associated with aging.2.This research,based on the SAMP8 accelerated aging and stress-induced accelerated aging experiments,reveals that the disruption of the brain-gut axis has a significant impact on accelerated aging,and intestinal functional impairment can speed up the process of brain aging.It clarifies that the representative anti-aging drug,Bazhi Bushen Capsule,can effectively prevent and treat brain aging by improving intestinal function. |
