As a distinctive resource of Chinese civilization， traditional Chinese medicine （TCM） technology transfer faces significant opportunities under the background of digital and intelligent transformation， while also being constrained by unique challenges such as the complexity of its theoretical system， lengthy industrial chains， and multidimensional policy restrictions， resulting in a "high-value-high-threshold" paradox. At present， TCM technology transfer is deeply trapped in a "threefold reluctance" dilemma， i.e.， unwillingness to transfer， inability to transfer， and lack of capacity to transfer. Specifically， the disconnection between scientific research evaluation systems and market demand leads to low conversion rates of research achievements， unclear ownership and compliance risks suppress innovation incentives， and the absence of professional services intensifies supply-demand mismatches. This article systematically analyzes the specific characteristics of TCM technology transfer and proposes a breakthrough pathway centered on full-chain digital and intelligent transformation. By integrating technologies such as intelligent sorting systems， blockchain-based traceability， and AI diagnostic models， the TCM ecosystem spanning "cultivation-production-service" can be reconstructed. In terms of standardization， promoting the progression from "experience-based data conversion" to "data standardization" and further to "intelligent standardization" is advocated to resolve quality control challenges. For example， a "three-no-one-full" certification system can strengthen quality trust. Policy coordination should focus on optimizing mechanisms for the transformation of scientific and technological achievements， while exploring intellectual property securitization and risk-sharing models to stimulate research momentum. In terms of internationalization， reliance on the Belt and Road Initiative platform to promote the export of geo-authentic medicinal material brands and standards is recommended to build a dual-driven model of "technology plus culture". Looking ahead， through the construction of national-level databases， the cultivation of interdisciplinary talent， and the mutual recognition of international standards， a new paradigm of "scientific intelligent manufacturing" can be formed， providing systematic solutions for the modernization of TCM and global health governance.

ObjectiveThis study aims to explore the potential of different orders of magnitude single-nucleotide polymorphism (SNP) locus combinations for predicting distant kinship relationships. A high-density SNP locus set was constructed, and a comprehensive assessment of its inference capability was conducted. MethodsFirstly, we selected three commercial chip panels, CGA (Chinese genotyping array, Illumina), GSA (Global screening array, Illumina), Affy (23MF_V2 high-density SNP array, Affymetrix) and merged them after quality control, forming a high-density SNP locus panel(1 180 k). Secondly, we selected 161 samples and collected their peripheral blood samples by using whole-genome sequencing technology. Within this sample population, the levels of kinship relationships fully covered the range from level 1 to level 9, and the number of kinship pairs at each level was consistently maintained at over 50 pairs. From 161 samples data of whole-genome sequencing, the 1 180 k locus set was extracted, which is referred to as the high-density SNP locus set in the following text. The kinship inference was conducted using the identity-by-descent (IBD) algorithm with the selected optimal parameters. To comprehensively evaluate the performance of the high-density SNP locus set in kinship inference, we compared it with the three commercial chip panels, the intersection of these three chip loci, and the control sets constructed by randomly reducing the number of the high-density SNP locus set. Based on the changes in the IBD lengths, as well as the dynamic trends in prediction accuracy, we conducted a scientific assessment of the kinship inference capability of the high-density SNP locus set. ResultsAfter screening, a set of 1 184 334 autosomal SNPs was obtained. During the process of screening the optimal IBD length threshold, the result revealed that 0 cM, 1 cM, and 2 cM all demonstrated good applicability. However, to avoid the issue of a large amount of redundant information caused by setting a too low IBD length threshold, this study ultimately selected 2 cM as the optimal threshold. Compared with the average results of three chip panels, the high-density SNP locus set increased the total IBD length and the average IBD length across levels 1-9; the accuracy of the confidence interval for level 8 was 70.97%, which represented a 3.50% improvement; the average confidence interval accuracy for levels 1-8 was 91.39%, representing a 1.00% increase; and the false negative rates at levels 8 and 9 were reduced by 2.42% and 6.76%, respectively. The system efficacy of the high-density SNP locus set for kinship inference of first to eighth degree relationships reached 98.91%. Through random reduction of the high-density SNP locus set results, it is found that increasing the number of SNPs with the panel, the detection efficiency of IBD length showed a significant upward trend. At the same time, the overall trend in the accuracy of kinship relationship prediction as well as the confidence interval accuracy also indicated that both metrics steadily increased with the addition of more loci. ConclusionThe results show that the high-density SNPs panel significantly enhances the efficacy of distant kinship inference, accurately covering kinship degrees, with the average confidence interval accuracy for first to eighth degree relationships stably above 90%. The study finds that increasing the number of SNPs panel can improve the ability to predict distant kinship.

ObjectiveSpinal cord injury (SCI) directly impairs the regulatory function of the autonomic nervous system, induces intestinal dysfunction, and significantly reduces patients’ quality of life. Preclinical studies have shown that electroacupuncture (EA) therapy can regulate the brain-gut axis and is used to treat central nervous system diseases such as major depressive disorder, Alzheimer’s disease and Parkinson’s disease. Recent research has established that fecal microbiota transplantation (FMT) from EA-treated SCI rats restored intestinal motility and colonic morphology. However, it remains unclear whether the regulation of gut microbiota by EA therapy directly contributes to neural repair after SCI. This study aims to explore whether gut microbiota mediates the neuroprotective effect of EA in the treatment of SCI and its possible mechanism. MethodsThe study employed RNA transcriptome analysis of spinal cord tissue to characterize gene expression profiles and to identify key signaling pathways following EA treatment for SCI. Hematoxylin-Eosin (HE) staining and Nissl staining were used to observe the morphological changes in spinal cord tissue. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) were applied to detect the effects of EA on the expression of proteins related to nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) -dependent pyroptosis. Using 16S rDNA sequencing, the study observed alterations in gut microbiota diversity and community composition in SCI rats. Prior to establishing SCI models, rats were pretreated with an antibiotic cocktail to induce gut dysbiosis, and the effects on intestinal function and spinal cord neural repair were evaluated. FMT was performed to investigate the regulatory effects of post-EA FMT on motor function, general status, liver and spleen indices, and NLRP3-mediated pyroptosis in SCI rats. ResultsEA improved motor function and reduced regulated neuronal cell death in SCI rats. Transcriptomic analysis demonstrated the activation of immune- and inflammation-related pathways post-SCI, including NOD-like receptors, nuclear factor-kappa B(NF-κB), and Toll-like receptor (TLR) pathways. EA primarily influenced intestinal inflammation and autoimmune functions. 16S rDNA sequencing illustrated that EA did not alter the diversity of gut microbiota. However, EA altered the gut microbiota composition in SCI rats, increasing Lactobacillus and Akkermansia genera while rebalancing the Firmicutes/Bacteroidetes ratio. Furthermore, depletion of gut microbiota by antibiotics disrupted the intestinal barrier, reduced the expression of intestinal barrier proteins Zonula Occludens-1 (ZO-1) and Occludin, elevated serum lipopolysaccharide-binding protein (LBP) levels, exacerbated spinal cord tissue damage, and hindered motor function recovery in SCI rats. FMT from donors treated with EA reduced LBP levels in the intestine, blood, and spinal cord of rats, inhibited the TLR4 myeloid differentiation primary response protein 88 (MyD88)-NF‑κB pathway and NLRP3-dependent pyroptosis, and improved motor function. On the other hand, FMT treatment resulted in decreased body weight and food intake, whereas FMT using EA-treated donors effectively alleviated these alterations. ConclusionEA effectively alleviated neuroinflammatory responses in rats with SCI, primarily through regulating the gut microbiota and suppressing the NLRP3-dependent pyroptosis signaling pathway.

ObjectiveSpinal cord injury (SCI) directly impairs the regulatory function of the autonomic nervous system, induces intestinal dysfunction, and significantly reduces patients’ quality of life. Preclinical studies have shown that electroacupuncture (EA) therapy can regulate the brain-gut axis and is used to treat central nervous system diseases such as major depressive disorder, Alzheimer’s disease and Parkinson’s disease. Recent research has established that fecal microbiota transplantation (FMT) from EA-treated SCI rats restored intestinal motility and colonic morphology. However, it remains unclear whether the regulation of gut microbiota by EA therapy directly contributes to neural repair after SCI. This study aims to explore whether gut microbiota mediates the neuroprotective effect of EA in the treatment of SCI and its possible mechanism. MethodsThe study employed RNA transcriptome analysis of spinal cord tissue to characterize gene expression profiles and to identify key signaling pathways following EA treatment for SCI. Hematoxylin-Eosin (HE) staining and Nissl staining were used to observe the morphological changes in spinal cord tissue. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) were applied to detect the effects of EA on the expression of proteins related to nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) -dependent pyroptosis. Using 16S rDNA sequencing, the study observed alterations in gut microbiota diversity and community composition in SCI rats. Prior to establishing SCI models, rats were pretreated with an antibiotic cocktail to induce gut dysbiosis, and the effects on intestinal function and spinal cord neural repair were evaluated. FMT was performed to investigate the regulatory effects of post-EA FMT on motor function, general status, liver and spleen indices, and NLRP3-mediated pyroptosis in SCI rats. ResultsEA improved motor function and reduced regulated neuronal cell death in SCI rats. Transcriptomic analysis demonstrated the activation of immune- and inflammation-related pathways post-SCI, including NOD-like receptors, nuclear factor-kappa B(NF-κB), and Toll-like receptor (TLR) pathways. EA primarily influenced intestinal inflammation and autoimmune functions. 16S rDNA sequencing illustrated that EA did not alter the diversity of gut microbiota. However, EA altered the gut microbiota composition in SCI rats, increasing Lactobacillus and Akkermansia genera while rebalancing the Firmicutes/Bacteroidetes ratio. Furthermore, depletion of gut microbiota by antibiotics disrupted the intestinal barrier, reduced the expression of intestinal barrier proteins Zonula Occludens-1 (ZO-1) and Occludin, elevated serum lipopolysaccharide-binding protein (LBP) levels, exacerbated spinal cord tissue damage, and hindered motor function recovery in SCI rats. FMT from donors treated with EA reduced LBP levels in the intestine, blood, and spinal cord of rats, inhibited the TLR4 myeloid differentiation primary response protein 88 (MyD88)-NF‑κB pathway and NLRP3-dependent pyroptosis, and improved motor function. On the other hand, FMT treatment resulted in decreased body weight and food intake, whereas FMT using EA-treated donors effectively alleviated these alterations. ConclusionEA effectively alleviated neuroinflammatory responses in rats with SCI, primarily through regulating the gut microbiota and suppressing the NLRP3-dependent pyroptosis signaling pathway.

ObjectiveType I interferon (IFN-I) signaling is essential for antiviral innate immunity, yet its sustained or excessive activation contributes to the pathogenesis of several autoimmune diseases and interferonopathies, such as systemic lupus erythematosus and Aicardi-Goutières syndrome. Current strategies targeting this pathway, exemplified by JAK inhibitors, act mainly on downstream signal transduction and provide limited direct control over upstream IFN-I production, while also carrying the risk of broad immunosuppression. Phyllanthus emblica L. has long been used in traditional medicine for inflammatory disorders, but the bioactive constituent responsible for its regulation of IFN-I signaling and the underlying molecular mechanism have not been clearly defined. This study aimed to identify the active anti-inflammatory component of P. emblica and to characterize its mechanism of action on the IFN-I pathway in macrophages. MethodsActive components ofP. emblica and their candidate targets were screened by network pharmacology using the TCMSP and DrugBank databases (oral bioavailability≥30%, drug-likeness≥0.18) and intersected with inflammation-related genes retrieved from public databases. The predicted interaction between EGCG and IFN-I pathway proteins (TBK1, IRF3, STAT1) was evaluated by molecular docking, with BX795 and GSK8612 used as reference TBK1 inhibitors. Mechanistic experiments were performed in THP-1-derived macrophages and primary bone marrow-derived macrophages (BMDM). Upstream signaling was activated by transfection of the nucleic acid analogs poly(I∶C) and poly(dA∶dT) or by lipopolysaccharide (LPS) stimulation, whereas downstream signaling was activated by exogenous IFN-β. An siRNA-mediated TREX1 knockdown model was used to mimic endogenous nucleic acid-driven interferonopathy. Expression of IFN-β1 and interferon-stimulated genes (ISGs) was measured by RT-qPCR, protein phosphorylation by Western blot, and IFN-β secretion by ELISA. Cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) were used to probe the interactionbetween EGCG and IRF3. ResultsNetwork pharmacology identified (-)-epigallocatechin-3-gallate (EGCG) as a candidate IFN-I-suppressive constituent of P. emblica, with predicted binding to TBK1, IRF3, and STAT1. Molecular docking yielded binding energies of -9.2, -7.2, and -8.2 kcal/mol for TBK1, IRF3, and STAT1, respectively, indicating an affinity for TBK1 comparable to that of the reference inhibitors BX795 (-5.7 kcal/mol) and GSK8612 (-6.4 kcal/mol). EGCG suppressed IFN-β1 and ISG mRNA expression under poly (I∶C), poly (dA∶dT), and LPS stimulation in both THP-1 macrophages and BMDM. At the protein level, EGCG reduced the phosphorylation of TBK1 and IRF3 without affecting the levels of the upstream sensors cGAS and RIG-I, and lowered IFN-β secretion in a concentration-dependent manner. CETSA and DARTS showed that EGCG did not enhance the thermal stability or protease resistance of IRF3, indicating that its effect on IRF3 is indirect. Following IFN-β stimulation, prolonged EGCG treatment reduced STAT1 phosphorylation in a time-dependent manner without an apparent change in IRF9, and partially attenuated ISG transcription; this effect was not monotonicly concentration-dependent, and CXCL10 showed the most consistent suppression. In TREX1-knockdown cells, the elevated mRNA levels of ISG15, ISG56, and CXCL10 were reduced by EGCG. ConclusionEGCG suppresses IFN-I responses by concurrently inhibiting TBK1-IRF3-dependent IFN‑β production and JAK-STAT1-mediated downstream transcription. These in vitro findings provide a mechanistic basis for the anti-inflammatory use of P. emblica in traditional medicine and identify EGCG as a candidate for further evaluation in interferon-driven autoimmune disease models.

This study investigated the effects and action mechanism of tannins from Galla chinensis cream(TGCC) on the skin wound of rat tail. Male Sprague Dawley(SD) rats were randomly divided into a control group, model group, model+low-dose TGCC(50 mg per rat) group, model+high-dose TGCC group(100 mg per rat), and model+TGC+FAK inhibitor(Y15) cream(100 mg+10 mg per rat) group, with 10 rats in each group. After the rat tail skin injury model was successfully constructed, in the treatment group, corresponding drugs were applied to the wound surface, while in the control and model groups, the same amount of cream base as the TGCC group was applied by the same method. Then, sterile gauze was wrapped around the wound edge, and these operations were performed three times a day for 28 consecutive days. The wound healing status at the third, seventh, eleventh, fourteenth, twenty-first, and twenty-eighth days was recorded, and the wound healing rate and healing time were calculated. On the day after the last dose of medication, rat serum and tail skin wound tissue were collected for analyzing the activities of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), creatinine(CREA), urea, reactive oxygen species(ROS), interferon gamma(IFN-γ), interleukin(IL)-1β, IL-6, IL-4, IL-10, tumor necrosis factor(TNF)-α, as well as catalase(CAT), glutathione(GSH), lactate dehydrogenase(LDH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC), platelet endothelial cell adhesion molecule-1(CD31), and leukocyte differentiation antigen 34(CD34) in the wound tissue of rat tail skin. Hematoxylin-eosin, Masson, and sirius red staining were used to observe the morphological changes in the wound tissue of rat tail skin. The thickness of the epidermis, the number of fibroblasts and blood vessels, and the contents of collagen fibers, typeⅠ collagen(COLⅠ), and COLⅢ were calculated. The mRNA expressions of keratin 10(KRT10), KRT14, vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF), epidermal growth factor(EGF), CD31, CD34, matrix metallopeptidase-2(MMP-2), MMP-9, COLⅠ, COLⅢ, desmin, fibroblast specific protein 1(FSP1), IFN-γ, IL-1β, TNF-α, IL-4, IL-6, and IL-10 in skin wound tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expressions of KRT10, KRT14, VEGF, FGF, EGF, MMP-2, MMP-9, COLⅠ, COLⅢ, desmin, FSP1, focal adhesion kinase(FAK), phosphorylated focal adhesion kinase(p-FAK), phosphatidylin-ositol-3-kinase(PI3K), phosphorylated phosphatidylin-ositol-3-kinase(p-PI3K), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results manifest that TGCC can dramatically elevate the healing rate of rat tail wounds and shorten wound healing time. Besides, it can reduce serum ROS levels, the contents of MDA, MPO, and LDH in the rat skin wound tissue, as well as the serum IFN-γ, IL-1β, IL-6, and TNF-α levels and the mRNA expression levels of IFN-γ, IL-1β, IL-6, and TNF-α in the skin wound tissue. It can elevate the activities of CAT, GSH, SOD, and T-AOC in wound tissue, the IL-4 and IL-10 contents in serum, and the mRNA expressions of IL-4 and IL-10 in the wound tissue. In addition, TGGC can inhibit inflammatory cell infiltration and increase the epidermal thickness, counts of fibroblasts and blood vessels, and contents of collagen fibers, COLⅠ, and COLⅢ. Besides, TGCC can elevate the mRNA and protein expressions of epidermal differentiation markers(KRT10 and KRT14), endothelial cell markers(CD31 and CD34), angiogenesis and fibroblast proliferation, differentiation markers(VEGF, FGF, EGF, COLⅠ, COLⅢ, desmin, and FSP1), reduce the mRNA and protein expressions of gelatinases(MMP-2 and MMP-9), and increase protein expressions of p-FAK, p-PI3K, p-Akt, p-mTOR, as well as ratios of p-FAK/FAK, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR. These results suggest that TGCC can significantly facilitate skin wound healing, and its mechanism may be related to the activation of the FAK/PI3K/Akt/mTOR signaling pathway, inhibition of inflammatory cell infiltration in skin wound tissue, elevation of epidermal thickness, counts of fibroblasts and vessels, and contents of collagen fiber, COLⅠ, and COLⅢ, and reduction of MMP-2 and MMP-9 expressions, thus accelerating wound healing.
Animals ; Male ; Wound Healing/drug effects* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Skin/metabolism* ; Proto-Oncogene Proteins c-akt/genetics* ; Tannins/pharmacology* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Focal Adhesion Kinase 1/genetics*

This study aims to explore the effects and mechanisms of the traditional Chinese medicine Cordyceps sinensis(CS) in ameliorating heart aging and injury in mice based on animal experiments and network pharmacology. A mouse model of heart aging was established by continuously subcutaneous injection of D-galactose(D-gal). Thirty mice were randomly assigned into a normal group, a model group, a low-dose CS(CS-L) group, a high-dose CS(CS-H) group, and a vitamin E(VE) group. Mice in these groups were administrated with normal saline, different doses of CS suspension, or VE suspension via gavage daily. After 60 days of treatment with D-gal and various drugs, all mice were euthanized, and blood and heart tissue samples were collected for determination of the indicators related to heart aging and injury in mice. Experimental results showed that both high and low doses of CS and VE ameliorated the aging phenotype, improved the heart index and myocardial enzyme spectrum, restored the expression levels of proteins associated with cell cycle arrest and senescence-associated secretory phenotypes(SASP), and alleviated the fibrosis and histopathological changes of the heart tissue in model mice. From the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),259 active ingredients of CS were retrieved. From Gene Cards and OMIM, 2 568 targets related to heart aging were identified, and 133common targets shared by CS and heart aging were obtained. The Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes( KEGG) pathway enrichment revealed that the pathways related to heart aging involved oxidative stress,apoptosis, inflammation-related signaling pathways, etc. The animal experiment results showed that both high and low doses of CS and VE ameliorated oxidative stress and apoptosis in the heart tissue to varying degrees in model mice. Additionally, CS-H and VE activated the nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathway and inhibited the expression of key proteins in the nuclear factor-κB(NF-κB) pathway in the heart tissue of model mice. In conclusion, this study demonstrated based on network pharmacology and animal experiments that CS may alleviate heart aging and injury in aging mice by reducing oxidative stress,apoptosis, and inflammation in the heart via the Nrf2/HO-1/NF-κB pathway.
Animals ; Cordyceps/chemistry* ; Mice ; NF-E2-Related Factor 2/genetics* ; NF-kappa B/genetics* ; Aging/genetics* ; Male ; Signal Transduction/drug effects* ; Network Pharmacology ; Drugs, Chinese Herbal/pharmacology* ; Heme Oxygenase-1/genetics* ; Heart/drug effects* ; Humans ; Myocardium/metabolism* ; Membrane Proteins/genetics*

The study was conducted to investigate the mechanism of Xinyang Tablets( XYP) in modulating the fat mass and obesity-associated protein(FTO)/N6-methyladenosine(m6A) signaling pathway to ameliorate ventricular remodeling in heart failure(HF). A mouse model of HF was established by transverse aortic constriction(TAC). Mice were randomized into sham, model, XYP(low, medium, and high doses), and positive control( perindopril) groups(n= 10). From day 3 post-surgery, mice were administrated with corresponding drugs by gavage for 6 consecutive weeks. Following the treatment, echocardiography was employed to evaluate the cardiac function, and RT-qPCR was employed to determine the relative m RNA levels of key markers, including atrial natriuretic peptide( ANP), B-type natriuretic peptide( BNP), β-myosin heavy chain(β-MHC), collagen type I alpha chain(Col1α), collagen type Ⅲ alpha chain(Col3α), alpha smooth muscle actin(α-SMA), and FTO. The cardiac tissue was stained with Masson's trichrome and wheat germ agglutinin(WGA) to reveal the pathological changes. Immunohistochemistry was employed to detect the expression levels of Col1α, Col3α, α-SMA, and FTO in the myocardial tissue. The m6A modification level in the myocardial tissue was measured by the m6A assay kit. An H9c2 cell model of cardiomyocyte injury was induced by angiotensin Ⅱ(AngⅡ), and small interfering RNA(siRNA) was employed to knock down FTO expression. RT-qPCR was conducted to assess the relative m RNA levels of FTO and other genes associated with cardiac remodeling. The m6A modification level was measured by the m6A assay kit, and Western blot was employed to determine the phosphorylated phosphatidylinositol 3-kinase(p-PI3K)/phosphatidylinositol 3-kinase(PI3K) and phosphorylated serine/threonine kinase(p-Akt)/serine/threonine kinase(Akt) ratios in cardiomyocytes. The results of animal experiments showed that the XYP treatment significantly improved the cardiac function, reduced fibrosis, up-regulated the m RNA and protein levels of FTO, and lowered the m6A modification level compared with the model group. The results of cell experiments showed that the XYP-containing serum markedly up-regulated the m RNA level of FTO while decreasing the m6A modification level and the p-PI3K/PI3K and p-Akt/Akt ratios in cardiomyocytes. Furthermore, FTO knockdown reversed the protective effects of XYP-containing serum on Ang Ⅱ-induced cardiomyocyte hypertrophy. In conclusion, XYP may ameliorate ventricular remodeling by regulating the FTO/m6A axis, thereby inhibiting the activation of the PI3K/Akt signaling pathway.
Animals ; Ventricular Remodeling/drug effects* ; Heart Failure/physiopathology* ; Signal Transduction/drug effects* ; Mice ; Male ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice, Inbred C57BL ; Humans ; Adenosine/analogs & derivatives* ; Myocytes, Cardiac/metabolism* ; Disease Models, Animal

This study aimed to investigate the effect of Dahuang Zhechong Pills(DHZCP) in delaying heart aging(HA) and explore the potential mechanism. Network pharmacology and molecular docking were employed to explore the targets and potential mechanisms of DHZCP in delaying HA. Furthermore, in vitro experiments were conducted with the DHZCP-containing serum to verify key targets and pathways in D-galactose(D-gal)-induced aging of cardiomyocytes. Active components of DHZCP were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCSMP), and relevant targets were predicted. HA-related targets were screened from the GeneCards, Online Mendelian Inheritance in Man(OMIM), and DisGeNET. The common targets shared by the active components of DHZCP and HA were used to construct a protein-protein interaction network in STRING 12.0, and core targets were screened based on degree in Cytoscape 3.9.1. Metaspace was used for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses of the core targets to predict the mechanisms. Molecular docking was performed in AutoDock Vina. The results indicated that a total of 774 targets of the active components of DHZCP and 4 520 targets related to HA were screened out, including 510 common targets. Core targets included B-cell lymphoma 2(BCL-2), serine/threonine kinase 1(AKT1), and hypoxia-inducible factor 1 subunit A(HIF1A). The GO and KEGG enrichment analyses suggested that DHZCP mainly exerted its effects via the phosphatidylinositol 3-kinase(PI3K)/AKT signaling pathway, HIF-1α signaling pathway, longevity signaling pathway, and apoptosis signaling pathway. Among the pathways predicted by GO and KEGG enrichment analyses, the PI3K/AKT/HIF-1α signaling pathway was selected for verification. The cell-counting kit 8(CCK-8) assay showed that D-gal significantly inhibited the proliferation of H9c2 cells, while DHZCP-containing serum increased the viability of H9c2 cells. SA-β-gal staining revealed a significant increase in the number of blue-green positive cells in the D-gal group, which was reduced by DHZCP-containing serum. TUNEL staining showed that DHZCP-containing serum decreased the number of apoptotic cells. After treatment with DHZCP-containing serum, the protein levels of Klotho, BCL-2, p-PI3K/PI3K, p-AKT1/AKT1, and HIF-1α were up-regulated, while those of P21, P16, BCL-2 associated X protein(Bax), and cleaved caspase-3 were down-regulated. The results indicated that DHZCP delayed HA via multiple components, targets, and pathways. Specifically, DHZCP may delay HA by reducing apoptosis via activating the PI3K/AKT/HIF-1α signaling pathway.
Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Apoptosis/drug effects* ; Myocytes, Cardiac/cytology* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Animals ; Rats ; Humans ; Molecular Docking Simulation ; Aging/metabolism* ; Protein Interaction Maps/drug effects* ; Heart/drug effects* ; Network Pharmacology

This study investigates the effect and underlying mechanism of Guizhi Tongluo Tablets(GZTL) in treating atherosclerosis(AS) in a mouse model. Apolipoprotein E-knockout(ApoE~(-/-)) mice were randomly assigned to the following groups: model, high-, medium-, and low-dose GZTL, and atorvastatin(ATV), and age-matched C57BL/6J mice were selected as the control group. ApoE~(-/-) mice in other groups except the control group were fed with a high-fat diet for the modeling of AS and administrated with corresponding drugs via gavage for 8 weeks. General conditions, signs of blood stasis, and body mass of mice were monitored. Aortic plaques and their stability were assessed by hematoxylin-eosin, Masson, and oil red O staining. Serum levels of total cholesterol(TC), triglycerides(TG), and low-density lipoprotein cholesterol(LDL-C) were measured by biochemical assays, and those of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) were determined via enzyme-linked immunosorbent assay. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL). Single-cell RNA sequencing(scRNA-seq) was employed to analyze the differential expression of CD72hi macrophages(CD72hi-Mφ) in the aortas of AS patients and mice. The immunofluorescence assay was employed to visualize CD72hi-Mφ expression in mouse aortic plaques, and real-time fluorescence quantitative PCR was utilized to determine the mRNA levels of IL-1β, TNF-α, and IL-6 in the aorta. The results demonstrated that compared with the control group, the model group exhibited significant increases in body mass, aortic plaque area proportion, necrotic core area proportion, and lipid deposition, a notable decrease in collagen fiber content, and an increase in apoptosis. Additionally, the model group showcased elevated serum levels of TC, TG, LDL-C, IL-1β, TNF-α, and IL-6, alongside marked upregulations in the mRNA levels of IL-1β, TNF-α, and IL-6 in the aorta. In comparison with the model group, the GZTL groups and the ATV group showed a reduction in body mass, and the medium-and high-dose GZTL groups and the ATV group demonstrated reductions in aortic plaque area proportion, necrotic core area proportion, and lipid deposition, an increase in collagen fiber content, and a decrease in apoptosis. Furthermore, the treatment goups showcased lowered serum levels of TC, TG, LDL-C, IL-1β, TNF-α, and IL-6. The data of scRNA-seq revealed significantly elevated CD72hi-Mφ signaling in carotid plaques of AS patients compared with that in the normal arterial tissue. Animal experiments confirmed that CD72hi-Mφ expression, along with several pro-inflammatory cytokines, was significantly upregulated in the aortas of AS mice, which were downregulated by GZTL treatment. In conclusion, GZTL may alleviate AS by inhibiting CD72hi-Mφ activity.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Atherosclerosis/immunology* ; Mice ; Mice, Inbred C57BL ; Macrophages/immunology* ; Male ; Humans ; Apolipoproteins E/genetics* ; Tablets ; Tumor Necrosis Factor-alpha/genetics* ; Apoptosis/drug effects* ; Interleukin-1beta/genetics* ; Interleukin-6/genetics* ; Disease Models, Animal ; Mice, Knockout