ObjectiveTo elucidate the correlation between alterations in digestion and absorption functions and hepatic deficiency states in aging rats based on the R-spondin1/Wnt3a signaling pathway， and reveal the intervention mechanism of Bugansan. MethodsForty-eight SPF-grade male SD rats were randomly assigned to six groups： blank control， model， low-， medium-， and high-dose （7.03， 14.06， 28.12 g·kg^-1， respectively） Bugansan， and vitamin E （suspension， 27 mg·kg^-1）， with 8 rats in each group. The rat model of aging was established by intraperitoneal injection of D-galactose （400 mg·kg^-1）， while the blank control group was injected with normal saline. Since the day of modeling， rats in intervention groups received corresponding agents by gavage， and those in blank control and model groups received an equal volume of normal saline （10 mL·kg^-1）. General biological features such as fur color， activity， body mass， water intake， and food intake were observed. Meanwhile， the content of malondialdehyde （MDA） and the activities of superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） in the serum were measured to assess aging. Grip strength and the content of total bile acids （TBA） and the activity of α-amylase （AMY） in the serum were measured to evaluate hepatic deficiency states. The activity of β-galactosidase （β-gal） in the duodenum was measured to evaluate intestinal senescence. The levels of glucagon-like peptide-1 （GLP-1）， vasoactive intestinal peptide （VIP）， and D-xylose in the serum were determined to assess digestion and absorption functions of the small intestine. Hematoxylin-eosin staining was conducted to observe pathological changes of the duodenum to assess the small intestine damage. Immunohistochemical staining was employed to visualize the expression of B-cell-specific Moloney murine leukemia virus integration site 1 （Bmi1） and leucine-rich repeat-containing G protein-coupled receptor 5 （Lgr5） in the duodenal tissue. Moreover， Real-time quantitative polymerase chain reaction （Real-time PCR） was utilized to quantify the mRNA levels of Ki67， Bmi1， and Lgr5 to assess proliferation and regeneration of the small intestine. Additionally， the mRNA levels of R-spondin1， Wnt3a， β-catenin， and glycogen synthase kinase-3β （GSK-3β） and the protein levels of R-spondin1， Wnt3a， β-catenin， and phosphorylated GSK-3β （p-GSK-3β） in the duodenum were determined by Real-time PCR and Western blot， respectively， to analyze the mechanisms of intestinal digestion and absorption dysfunction in aging rats and the regulatory characteristics of Bugansan. ResultsCompared with blank control group， the model group showed decreases in body mass， water intake， food intake， grip strength， activities of SOD， GSH-Px， and AMY in the serum and content of GLP-1， VIP and D-xylose in the serum （P<0.05）， increases in the content of MDA and TBA in the serum and β-gal activity in the duodenum （P<0.05）， reductions in villus length， villus width， crypt depth， and villi/crypt （V/C） value， down-regulated mRNA and protein levels of Ki67， Lgr5， Bmi1， R-spondin1， Wnt3a， β-catenin， and up-regulated level of GSK-3β， phosphorylation （p）-GSK-3β （P<0.05）. Compared with the model group， Bugansan increased the body mass， water intake， food intake， grip strength， and activities of SOD， GSH-Px， and AMY and levels of GLP-1， VIP and D-xylose in the serum （P<0.05）， while decreasing the content of MDA and TBA in the serum and β-gal activity in the duodenum （P<0.05）. Furthermore， Bugansan increased the villus length， villus width， crypt depth， and V/C value， up-regulated the mRNA and protein levels of Ki67， Lgr5， Bmi1， R-spondin1， Wnt3a， β-catenin， and down-regulated the level of GSK-3β and p-GSK-3β （P<0.05）. ConclusionAging rats exhibit obvious impairments in digestion and absorption functions， accompanied by a state of hepatic deficiency. The traditional Chinese medicine approach of tonifying liver Qi effectively ameliorates aging-related changes by modulating the R-spondin1/Wnt3a signaling pathway to mitigate intestinal senescence and enhance digestion and absorption functions， ultimately contributing to the delay of aging.

Based on commonly used acupoints in the clinical acupuncture treatment of functional dyspepsia （FD）， this study systematically analyzes the therapeutic differences and synergistic effects between local and distal point selection. It also examines the suitability of primary acupoint selection for different FD subtypes， postprandial distress syndrome （PDS） and epigastric pain syndrome （EPS）. The findings suggest that a combination of local and distal acupoints may be more appropriate as primary points for PDS， whereas local acupoints alone may be more suitable for EPS. Additionally， the study explores the impact of various factors， such as stimulation techniques， needling order， intensity or stimulation parameters， and depth， on the efficacy of acupuncture. It concludes that the intrinsic properties of acupoints are the primary determinants of therapeutic direction. Other factors mainly influence the magnitude rather than the direction of the effect. Future research may further investigate how different acupoint combinations， local versus distal， affect the treatment outcomes of FD subtypes， providing new insights for clinical acupuncture prescriptions.

ObjectiveTo elucidate the potential mechanism of modified Sinisan （MSNS） in alleviating anxiety-like behaviors induced by chronic restraint stress （CRS） in mice at the metabolic level based on serum untargeted metabolomics and identify key metabolites and metabolic pathways regulated by MSNS. MethodsSeventy-two male C57BL/6 mice were randomly assigned into six groups： control， model， high-dose （2.4 g·kg^-1） MSNS， medium-dose （1.2 g·kg^-1） MSNS， low-dose （0.6 g·kg^-1） MSNS， and positive control （fluoxetine， 2.6 mg·kg^-1）. Except the control group， the other groups were subjected to CRS for the modeling of anxiety. Mice were administrated with corresponding agents by gavage 2 h before daily restraint for 14 days. Anxiety-like behaviors were evaluated by the open field test （OFT）， elevated plus maze （EPM） test， and light/dark box （LDB） test. Serum levels of corticotropin-releasing hormone （CRH）， adrenocorticotrophic hormone （ACTH）， and corticosterone （CORT） were measured via ELISA to assess stress levels. Ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） was employed to detect 9 metabolites in the brain tissue and serum metabolites. Orthogonal partial least squares-discriminant analysis （OPLS-DA） was adopted to identify differential metabolites （VIP>1.0， P<0.05）. MetaboAnalyst 5.0 was used for metabolic pathway enrichment analysis of the differential metabolites. ResultsCompared with the control group， the model group showed reductions in the central activity time and central distance in the OFT （P<0.05）， the proportions of open-arm residence time and open-arm residence times in the EPM test （P<0.01）， and the proportions of open box activity time and open box activity distance in the LDB test （P<0.05）， which were increased in the medium- and high-dose MSNS groups compared with the model group （P<0.05）. Compared with the control group， the model group showed elevated levels of CRH， ACTH， and CORT in the serum （P<0.01）， and the elevations were diminished in the medium- and high-dose MSNS groups （P<0.05）. UPLC-MS results indicated that compared with the control group， the model group presented declined DA， GABA， 5-HIAA， 5-HT， and 5-HT/Trp levels （P<0.05， P<0.01） and raised Glu， NE， Kyn， and Kyn/Trp levels （P<0.05）. Compared with the model group， high-dose MSNS increased the GABA， 5-HIAA， and 5-HT/Trp levels （P<0.05） and lowered the Glu and Kyn/Trp levels （P<0.05）. Untargeted metabolomics identified that 16 CRS-induced metabolic disturbances were reversed by MSNS. KEGG pathway analysis indicated that MSNS primarily modulated eight core pathways including alanine/aspartate/glutamate metabolism， butyrate metabolism， arginine-proline metabolism， TCA cycle， unsaturated fatty acid biosynthesis， and tryptophan metabolism. The mechanisms involved multidimensional biological processes， including neurotransmitter homeostasis regulation， TCA cycle energy metabolism optimization， and inflammatory response suppression. ConclusionMSNS alleviates CRS-induced anxiety-like behaviors in mice by mitigating hypothalamic-pituitary-adrenal axis hyperactivity， improving hippocampal neurotransmitter and tryptophan metabolic pathways， and regulating alanine/aspartate/glutamate metabolism， butyrate metabolism， arginine-proline metabolism， and TCA cycle.

ObjectiveTo elucidate the potential mechanism of modified Sinisan （MSNS） in alleviating anxiety-like behaviors induced by chronic restraint stress （CRS） in mice at the metabolic level based on serum untargeted metabolomics and identify key metabolites and metabolic pathways regulated by MSNS. MethodsSeventy-two male C57BL/6 mice were randomly assigned into six groups： control， model， high-dose （2.4 g·kg^-1） MSNS， medium-dose （1.2 g·kg^-1） MSNS， low-dose （0.6 g·kg^-1） MSNS， and positive control （fluoxetine， 2.6 mg·kg^-1）. Except the control group， the other groups were subjected to CRS for the modeling of anxiety. Mice were administrated with corresponding agents by gavage 2 h before daily restraint for 14 days. Anxiety-like behaviors were evaluated by the open field test （OFT）， elevated plus maze （EPM） test， and light/dark box （LDB） test. Serum levels of corticotropin-releasing hormone （CRH）， adrenocorticotrophic hormone （ACTH）， and corticosterone （CORT） were measured via ELISA to assess stress levels. Ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） was employed to detect 9 metabolites in the brain tissue and serum metabolites. Orthogonal partial least squares-discriminant analysis （OPLS-DA） was adopted to identify differential metabolites （VIP>1.0， P<0.05）. MetaboAnalyst 5.0 was used for metabolic pathway enrichment analysis of the differential metabolites. ResultsCompared with the control group， the model group showed reductions in the central activity time and central distance in the OFT （P<0.05）， the proportions of open-arm residence time and open-arm residence times in the EPM test （P<0.01）， and the proportions of open box activity time and open box activity distance in the LDB test （P<0.05）， which were increased in the medium- and high-dose MSNS groups compared with the model group （P<0.05）. Compared with the control group， the model group showed elevated levels of CRH， ACTH， and CORT in the serum （P<0.01）， and the elevations were diminished in the medium- and high-dose MSNS groups （P<0.05）. UPLC-MS results indicated that compared with the control group， the model group presented declined DA， GABA， 5-HIAA， 5-HT， and 5-HT/Trp levels （P<0.05， P<0.01） and raised Glu， NE， Kyn， and Kyn/Trp levels （P<0.05）. Compared with the model group， high-dose MSNS increased the GABA， 5-HIAA， and 5-HT/Trp levels （P<0.05） and lowered the Glu and Kyn/Trp levels （P<0.05）. Untargeted metabolomics identified that 16 CRS-induced metabolic disturbances were reversed by MSNS. KEGG pathway analysis indicated that MSNS primarily modulated eight core pathways including alanine/aspartate/glutamate metabolism， butyrate metabolism， arginine-proline metabolism， TCA cycle， unsaturated fatty acid biosynthesis， and tryptophan metabolism. The mechanisms involved multidimensional biological processes， including neurotransmitter homeostasis regulation， TCA cycle energy metabolism optimization， and inflammatory response suppression. ConclusionMSNS alleviates CRS-induced anxiety-like behaviors in mice by mitigating hypothalamic-pituitary-adrenal axis hyperactivity， improving hippocampal neurotransmitter and tryptophan metabolic pathways， and regulating alanine/aspartate/glutamate metabolism， butyrate metabolism， arginine-proline metabolism， and TCA cycle.

ObjectiveTo multidimensionally analyze the clinical effects of Qianyang Yuyin granules on elderly hypertensive patients through an "energy-inflammation-aging" network. MethodsRelevant datasets were retrieved from the GEO database. Gene set enrichment analysis （GSEA） was performed on the gene expression profiles of peripheral blood cells from patients with essential hypertension in dataset GSE24752. The GSEA referenced "GO gene sets" and "KEGG gene sets" to identify significantly enriched gene sets. A clinical trial was conducted using a randomized controlled study design. A total of 40 patients meeting the inclusion criteria were enrolled. The control group received standard antihypertensive treatment with angiotensin receptor blockers （ARBs） or combined calcium channel blockers （CCBs）. In contrast，the treatment group received Qianyang Yuyin Granules in addition to the standard treatment for 12 weeks. Blood pressure levels and clinical efficacy were observed，and changes in energy metabolism indicators，DNA damage markers，and senescence-associated secretory phenotype （SASP） in blood were measured using ELISA before and after treatment. ResultsGSEA results indicated significant energy metabolism dysregulation in hypertensive patients. Clinical findings showed that both groups achieved blood pressure control without significant intergroup differences. In terms of clinical efficacy，the treatment group had a significantly higher effective rate compared to the control group （95% vs 65%，P0.05）. After treatment，the treatment group showed a significant increase in NAD⁺ levels （P0.01），with higher levels compared to the control group （P0.05）. The treatment group also exhibited a greater reduction in DNA damage marker 8-OHdG （P0.01） and cell adhesion factors ICAM-1 and VCAM-1 （P0.01） compared to the control group. Pro-inflammatory cytokines IL-1β and IL-6 were significantly reduced in the treatment group （P0.01），with greater reductions compared to the control group （P0.05，P0.01）. Anti-inflammatory cytokines IFN-α，IL-4，and IL-10 were significantly elevated in the treatment group （P0.01），with higher levels compared to the control group （P0.01）. No significant adverse reactions were reported in either group. ConclusionThe "energy- inflammation- aging" network plays an important role in the pathological mechanism of hypertension patients. Qianyang Yuyin granules may delay the aging process by increasing patients' energy metabolism levels，reducing DNA oxidative damage，and maintaining the balance of inflammatory factors.

OBJECTIVE: To explore the peripheral neural mechanism underlying representation along the distribution of stomach meridian induced by intestinal inflammatory reaction using diarrhea predominant-irritable bowel syndrome (IBS-D) mice. METHODS: Among 62 healthy male C57BL/6 mice of clean grade, 12 mice were randomly selected and divided into a control group and a model group, 6 mice in each group, additionally, 12 mice were randomly selected and divided into a Tianshu group, a Liangqiu group and a Zusanli group, 4 mice in each group. In the model group, citrobacter was administered orally to establish IBS-D model. In the control group and the model group, the visceral pain threshold was observed using fecal colorectal distension (fCRD) induced electromyography of external oblique muscle, the positive cell number of neutrophil in the colonic muscularis was detected by myeloperoxidase (MPO) staining, the number, location and distribution rule of Evans blue (EB) extravasation points were observed by injection of EB staining solution into the tail vein. In the Tianshu group, the Liangqiu group and the Zusanli group, fluorescent dye Dil was injected at bilateral "Tianshu" (ST25), "Liangqiu" (ST34) and "Zusanli" (ST36) respectively, to observe the dye-positive cell number in different dorsal root ganglion (DRG) segments. In the control group and the model group, the activation of satellite glial cells (SGCs) in different DRG segments was observed by immunofluorescence. RESULTS: Compared with the control group, in the model group, the area under curve of electromyography of external oblique muscle was increased at fCRD of 25, 50 and 75 μL distilled water (P<0.001, P<0.01); the MPO-positive cell number of neutrophil in the colonic muscularis was increased (P<0.01). Few EB extravasation points could be found in the control group, while there were much more EB extravasation points observed in the model group, which was specially distribution in the area of stomach meridian, from "Huaroumen" (ST24) to "Zusanli" (ST36), as well as the surface area dominated by L₂-L₅ segment of the spinal cord. The Dil-positive cells were mainly exhibited in the DRG of T₁₁, L₅ and L₄ segments in the Tianshu group, the Liangqiu group and the Zusanli group, respectively. Compared with the control group, the ratio of glial fibrillary acidic protein (GFAP)/glutamine synthetase (GS) co-expression was increased in the DRG of T₁₁, L₄ and L₅ segments in the model group (P<0.05, P<0.01). CONCLUSION The activation of SGCs within DRG of T₁₁, L₄ and L₅ segments may relate closely to the occurrence of the representation along the stomach meridian distribution in IBS-D mice.
Animals ; Male ; Mice ; Irritable Bowel Syndrome/therapy* ; Mice, Inbred C57BL ; Meridians ; Stomach/physiopathology* ; Humans ; Acupuncture Points ; Disease Models, Animal

Microbial-derived metabolites are important mediators of host-microbial interactions. In recent years, the role of intestinal microbial metabolites in colorectal cancer has attracted considerable attention. These metabolites, which can be derived from bacterial metabolism of dietary substrates, modification of host molecules such as bile acids, or directly from bacteria, strongly influence the progression of colitis-associated cancer (CAC) by regulating inflammation and immune response. Here, we review how microbiome metabolites short-chain fatty acids (SCFAs), secondary bile acids, polyamines, microbial tryptophan metabolites, and polyphenols are involved in the tumorigenesis and development of CAC through inflammation and immunity. Given the heated debate on the metabolites of microbiota in maintaining gut homeostasis, serving as tumor molecular markers, and affecting the efficacy of immune checkpoint inhibitors in recent years, strategies for the prevention and treatment of CAC by targeting intestinal microbial metabolites are also discussed in this review.
Humans ; Gastrointestinal Microbiome/physiology* ; Animals ; Carcinogenesis/metabolism* ; Colitis-Associated Neoplasms/microbiology* ; Fatty Acids, Volatile/metabolism* ; Bile Acids and Salts/metabolism* ; Colitis/microbiology*

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

This study aims to integrate data mining techniques of single cell transcriptomics and spatial transcriptomics, along with animal experiment validation, so as to systematically characterize the protective effects of Jianpi Tongluo Formula(JTF) on the cartilage in knee osteoarthritis(KOA) and elucidate the underlying molecular mechanisms. Single cell transcriptomics and spatial transcriptomics datasets(GSE254844 and GSE255460) of the cartilage tissue obtained from KOA patients were analyzed to map the single cell-spatial heterogeneity and identify key pathogenic factors. After that, a KOA rat model was established via knee joint injection of papain. The intervention effects of JTF on the expression features of these key factors were assessed through real-time quantitative polymerase chain reaction(PCR), Western blot, and immunohistochemical staining. As a result, the integrated single cell and spatial transcriptomics data identified distinct cell subsets with different pathological changes in different regions of the inflamed cartilage tissue in KOA, and their differentiation trajectories were closely related to the inflammatory fibrosis-like pathological changes of chondrocytes. Accordingly, the expression levels of the two key effect targets, namely nuclear receptor coactivator 4(NCOA4) and high mobility group box 1(HMGB1) were significantly reduced in the articular surface and superficial zone of the inflamed joints when JTF effectively alleviated various pathological changes in KOA rats, thus reversing the abnormal chondrocyte autophagy level, relieving the inflammatory responses and fibrosis-like pathological changes, and promoting the repair of chondrocyte function. Collectively, this study revealed the heterogeneous characteristics and dynamic changes of inflamed cartilage tissue in different regions and different cell subsets in KOA patients. It is worth noting that NCOA4 and HMGB1 were crucial in regulating chondrocyte autophagy and inflammatory reaction, while JTF could reverse the regulation of NCOA4 and HMGB1 and correct the abnormal molecular signal axis in the target cells of the inflamed joints. The research can provide a new research idea and scientific basis for developing a personalized therapeutic schedule targeting the spatiotemporal heterogeneity characteristics of KOA.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Osteoarthritis, Knee/pathology* ; Humans ; Male ; Cartilage, Articular/metabolism* ; Chondrocytes/metabolism* ; Rats, Sprague-Dawley ; Female ; Protective Agents/administration & dosage* ; Single-Cell Analysis ; Middle Aged ; HMGB1 Protein/metabolism*