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

Astragalus membranaceus(A. membranaceus), a traditional tonic, contains flavonoids as one of its main bioactive components and key indicators for quality standard detection. These compounds predominantly exist in glycosylated forms after glycosylation modification within the plant. The catalytic products of flavonoid glycosyltransferases in A. membranaceus have been reported to be mostly monoglycosides, and only AmUGT28 catalyzes luteolin to form diglycosides. In this study, we cloned a glycosyltransferase gene, AmGT90, from A. membranaceus, with an ORF length of 1 335 bp, encoding 444 amino acids, and the protein had a relative molecular mass of 50.5 kDa. Phylogenetic tree analysis indicated that AmGT90 belongs to the UGT74 family. In vitro enzymatic reaction showed that AmGT90 had broad substrate specificity and could catalyze the glycosylation of various flavonoids, including isoflavones, flavones, flavanones, and chalcones. AmGT90 not only catalyzed the formation of monoglycosides but also diglycosides. In addition, the mechanism of AmGT90 catalyzing the formation of diglycosides from luteolin was preliminarily explored. The experimental results showed that AmGT90 may preferentially recognize C4'-OH of luteolin and then recognize C7-OH to form diglycosides. This study reported a glycosyltransferase from A. membranaceus capable of converting flavonoids into monoglycosides and diglycosides. This finding not only enhances our understanding of the biosynthetic pathways of flavonoid glycosides in A. membranaceus but also introduces a new component for glycoside production through synthetic biology.
Glycosyltransferases/chemistry* ; Flavonoids/chemistry* ; Astragalus propinquus/classification* ; Phylogeny ; Glycosylation ; Plant Proteins/chemistry* ; Substrate Specificity ; Cloning, Molecular ; Amino Acid Sequence

Ginsenoside Rg_2(GRg2) is a triterpenoid compound found in Panax notoginseng. This study explored its effects and mechanisms on diabetic retinopathy and angiogenesis. The study employed endothelial cell models induced by glucose or vascular endothelial growth factor(VEGF), the chorioallantoic membrane(CAM) model, the oxygen-induced retinopathy(OIR) mouse model, and the db/db mouse model to evaluate the therapeutic effects of GRg2 on diabetic retinopathy and angiogenesis. Transwell assays and endothelial tube formation experiments were conducted to assess cell migration and tube formation, while vascular area measurements were applied to detect angiogenesis. The impact of GRg2 on the retinal structure and function of db/db mice was evaluated through retinal thickness and electroretinogram(ERG) analyses. The study investigated the mechanisms of GRg2 by analyzing the activation of Yes-associated protein(YAP) and Toll-like receptors(TLRs) pathways. The results indicated that GRg2 significantly reduced cell migration numbers and tube formation lengths in vitro. In the CAM model, GRg2 exhibited a dose-dependent decrease in the vascular area ratio. In the OIR model, GRg2 notably decreased the avascular and neovascular areas, ameliorating retinal structural disarray. In the db/db mouse model, GRg2 increased the total retinal thickness and enhanced the amplitudes of the a-wave, b-wave, and oscillatory potentials(OPs) in the ERG, improving retinal structural disarray. Transcriptomic analysis revealed that the TLR signaling pathway was significantly down-regulated following YAP knockdown, with PCR results consistent with the transcriptome sequencing findings. Concurrently, GRg2 downregulated the expression of Toll-like receptor 4(TLR4), TNF receptor-associated factor 6(TRAF6), and nuclear factor-kappaB(NF-κB) proteins in high-glucose-induced endothelial cells. Collectively, GRg2 inhibits cell migration and tube formation and significantly reduces angiogenesis in CAM and OIR models, improving retinal structure and function in db/db mice, with its pharmacological mechanism likely involving the down-regulation of YAP expression.
Animals ; Ginsenosides/pharmacology* ; Diabetic Retinopathy/physiopathology* ; Mice ; YAP-Signaling Proteins ; Humans ; Male ; Signal Transduction/drug effects* ; Cell Movement/drug effects* ; Adaptor Proteins, Signal Transducing/genetics* ; Mice, Inbred C57BL ; Neovascularization, Pathologic/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Panax notoginseng/chemistry* ; Endothelial Cells/metabolism* ; Transcription Factors/genetics* ; Angiogenesis

This study explores the therapeutic differences and mechanisms of salt-processed Phellodendri Chinensis Cortex in improving insulin resistance(IR) based on network pharmacology, molecular docking, and cellular experiments. The components and intersection targets of Phellodendri Chinensis Cortex in improving IR were collected from databases, and a "drug-component-target-disease" network and protein-protein interaction(PPI) network were constructed to screen core components and targets. A total of 29 active components and 240 intersection targets were identified, of which 13 were core targets. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were used to identify key signaling pathways, and molecular docking was performed to validate the binding activity between core components and targets. An IR model in HepG2 cells was induced using insulin combined with high glucose, and the effects of Phellodendri Chinensis Cortex before and after salt-processing on cell glucose consumption were evaluated. The expression of proteins related to the mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT) signaling pathways was detected by Western blot. The cellular experimental results showed that, compared with the model group, glucose consumption in the drug-treated groups was significantly increased(P<0.01), the phosphorylation level of extracellular regulated protein kinase(ERK) was decreased(P<0.05), the phosphorylation levels of PI3K and AKT were increased, and the expression of glucose transporter 4(GLUT4) was also upregulated(P<0.05). Furthermore, the effect of salt-processed Phellodendri Chinensis Cortex was better than that of raw Phellodendri Chinensis Cortex. The study demonstrates that Phellodendri Chinensis Cortex, both before and after salt-processing, improves IR by regulating the expression of related proteins in the MAPK and PI3K-AKT signaling pathways, with enhanced effects after salt-processing.
Humans ; Network Pharmacology ; Phellodendron/chemistry* ; Insulin Resistance ; Drugs, Chinese Herbal/chemistry* ; Hep G2 Cells ; Signal Transduction/drug effects* ; Molecular Docking Simulation ; Protein Interaction Maps/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Glucose/metabolism*

This research investigated the impact of Ganoderma lucidum polysaccharides(GLP) on hepatoblastoma HepG2 and Huh6 cell models, as well as KM mouse model with in situ transplanted tumors, so as to provide a theoretical basis for the clinical application of GLP. Cell viability was assessed through the CCK-8 assay, whereas cell proliferation was evaluated by using the BeyoClick~(TM)EdU-488 test. Cell apoptosis was visualized via Hochest 33258 staining, and autophagy was detected through Mrfp-GFP-LC3 dual fluorescence staining. An in situ tumor transplantation model was created by using HepG2 cells in mice, and mice were treated with normal saline and GLP of 100, 200, and 300 mg·kg~(-1) for tumor count calculation and size assessment. Hematoxylin-eosin(HE) staining was used to observe pathological changes in tumor tissue and vital organs(liver, kidney, lung, spleen, and heart). Western blot analysis was conducted to measure the protein expressions of tumor protein P53(P53), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-caspase-3, Beclin-1, autophagy related protein-5(Atg-5), microtubule-associated protein-light chain-3Ⅰ(LC3Ⅰ)/LC3Ⅱ, autophagy adapter protein 62(P62), protein kinase B(Akt), p-Akt, mammalian target of rapamycin(mTOR), and p-mTOR. The in vitro experiment revealed that compared with the control group, after GLP treatment, tumor cell viability decreased significantly; apoptosis rate increased in a dose-dependent manner, and autophagic flux was inhibited. The in vivo experiments showed that compared with the model group, mice treated with GLP exhibited significantly fewer and smaller tumors. Western blot results showed that compared with the control group or model group, levels of P53, Bax, cleaved-caspase-3, Beclin-1, Atg-5, and LC3-Ⅱ/LC3-Ⅰ were significantly increased after GLP treatment, and the levels of Bcl-2, P62, p-Akt/Akt, and p-mTOR/mTOR were significantly decreased. These outcomes suggest that GLP promotes apoptosis and autophagy in hepatoblastoma cells by regulating the Akt/mTOR pathway.
Animals ; Humans ; Autophagy/drug effects* ; Reishi/chemistry* ; Mice ; Apoptosis/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Liver Neoplasms/genetics* ; Hepatoblastoma/genetics* ; Polysaccharides/pharmacology* ; Cell Line, Tumor ; Signal Transduction/drug effects* ; Male ; Cell Proliferation/drug effects* ; Hep G2 Cells

To investigate the mechanism of action of Syngnathus extract in treating knee osteoarthritis of rats, forty-eight male SD rats were randomly divided into the blank group, model group, positive drug group, as well as low-dose, medium-dose, and high-dose groups of Syngnathus extract. The rat model of knee osteoarthritis was constructed by intra-articular injection of sodium iodoacetate. After successful modeling, celecoxib(18 mg·kg~(-1)·d~(-1)) and Syngnathus extract(0.4, 0.8, and 1.6 g·kg~(-1)·d~(-1)) were given in different groups by gavage intervention for two weeks. Hematoxylin-eosin(HE) staining was used to observe the histopathological changes of cartilage in knee joints, and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of inflammatory factors in serum. Real-time fluorescence quantitative PCR, Western blot, and immunohistochemistry were used to detect the levels of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target protein of rapamycin(mTOR) pathway-related mRNA and protein expression. The results showed that, comparied with the blank group, the cartilage surface of the knee joints of rats in the model group was uneven, with disorganized levels and defective cartilage tissue. The serum levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) and the mRNA levels of PI3K, Akt, and mTOR in cartilage tissue, as well as the protein expression levels of phosphorylated PI3K(p-PI3K)/PI3K, phosphorylated Akt(p-Akt)/Akt, phosphorylated mTOR(p-mTOR)/mTOR, and P62 were significantly increased. Beclin1 protein expression was decreased. Comparied with the model group, the number of chondrocytes in the knee joint of rats in each group of Syngnathus extract increased, and the arrangement of chondrocytes was relatively neat. The cartilage layer was restored, and the serum levels of IL-1β, IL-6, and TNF-α, as well as the mRNA expression levels of PI3K, Akt, and mTOR in cartilage tissue were significantly reduced. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, and P62 were significantly reduced in the rats in the middle-dose and high-dose groups of Syngnathus extract, and the Beclin1 protein expression was significantly increased. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, and P62 in rats in the low-dose group of Syngnathus extract were significantly reduced. In summary, Syngnathus extract may be used to treat knee osteoarthritis by inhibiting the expression of PI3K/Akt/mTOR signaling pathway, so as to alleviate the inflammatory response in the organism, enhance the autophagy activity of chondrocytes, and reduce the apoptosis of chondrocytes.
Animals ; TOR Serine-Threonine Kinases/genetics* ; Male ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Rats ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Phosphatidylinositol 3-Kinases/genetics* ; Humans

This study explores the basic characteristics and potential functions of the terpene synthase(TPS) gene family members in Lonicera japonica. The L. japonica TPS(LjTPS) gene family was identified and functionally analyzed using bioinformatics methods. The results showed that a total of 70 members of the LjTPS gene family were identified in L. japonica, with protein lengths ranging from 130 to 1 437 amino acids. Most of these proteins were hydrophilic, and they were unevenly distributed across nine chromosomes. Phylogenetic analysis showed that the LjTPS gene family members were divided into six subfamilies, mainly consisting of members from the TPS-a, TPS-b, and TPS-e subfamilies. Promoter cis-acting element analysis showed that LjTPS members contained a large number of stress-responsive cis-acting elements. Aphid inoculation experiments showed that key enzyme genes in the MVA pathway for terpenoid backbone synthesis in L. japonica, such as HMGS, HMGR, MK, MPD, and the key enzyme gene in the DXP pathway, DXS, exhibited an initial increase followed by a decrease under aphid stress. The qRT-PCR analysis showed that the expression levels of the α-farnesene synthase genes LjTPS34 and LjTPS39 were down-regulated, while the expression levels of(E)-β-caryophyllene synthase genes LjTPS15 and LjTPS17 were up-regulated 12 h before aphid feeding, then began to decline. Farnesyl pyrophosphate synthase(FPS), which interacted with these genes, also displayed a pattern of increasing followed by decreasing expression. The expression of linalool synthase genes LjTPS12 and LjTPS33 was significantly up-regulated after 72 h of aphid feeding(P<0.000 1), reaching 24.39 and 22.64 times the initial expression, respectively. This pattern was in close alignment with the trend of linalool content in L. japonica. This study provides a theoretical foundation for future research on the interaction between L. japonica and pests, as well as on the functional roles of the LjTPS gene family.
Animals ; Aphids/physiology* ; Alkyl and Aryl Transferases/chemistry* ; Lonicera/parasitology* ; Phylogeny ; Plant Proteins/chemistry* ; Gene Expression Regulation, Plant ; Multigene Family ; Terpenes/metabolism*

This study aims to investigate the optimal ratio of Astragali Radix-Curcumae Rhizoma(AC) for inhibiting the proliferation of 143B osteosarcoma cells, and to investigate the mechanism by which AC inhibits osteosarcoma growth and metastasis through angiogenesis and cell adhesion mediated by the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/hypoxia inducible factor-1α(HIF-1α) pathway. A subcutaneous 143B tumor-bearing nude mouse model was successfully established and randomly divided into the model group, and the AC 1∶1, 2∶1, and 4∶1 groups. Body weight, tumor volume, and tumor weight were recorded. Real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot were used to detect the mRNA and protein expression levels of PI3K, Akt, phosphorylated Akt(p-Akt), HIF-1α, vascular endothelial growth factor A(VEGFA), transforming growth factor-β1(TGF-β1), epithelial cadherin(E-cadherin), neural cadherin(N-cadherin), vimentin, matrix metalloproteinase 2(MMP2), matrix metalloproteinase 9(MMP9), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and caspase-3 in the hypoxic core region of the tumor tissue. A cell hypoxia model was established, and the effects of AC-medicated serum(model group, AC 1∶1, 2∶1, and 4∶1 groups) on angiogenesis, proliferation, adhesion, invasion, and migration of 143B osteosarcoma cells were examined through CCK-8, flow cytometry, Transwell assay, cell adhesion assay, and HUVEC tube formation assay. The results showed that compared with the model group, the tumor weight and volume were smallest in the 2∶1 group. The expression levels of PI3K, Akt, p-Akt, HIF-1α, VEGFA, and TGF-β1 were significantly decreased, and the protein expression of E-cadherin was significantly increased, while the protein expression of N-cadherin, vimentin, MMP2, and MMP9 was significantly decreased. Additionally, the protein expression of Bax and caspase-3 was significantly increased, and Bcl-2 protein expression was significantly decreased. In vitro experiments showed that after intervention with AC-medicated serum at a 2∶1 ratio, the cell activity, adhesion, invasion, and migration of 143B cells were significantly reduced, apoptosis was significantly increased, and HUVEC tube formation was significantly decreased. In conclusion, the 2∶1 ratio of AC showed the most effective inhibition of 143B cell growth. AC can inhibit the growth and metastasis of osteosarcoma 143B cells by regulating the PI3K/Akt/HIF-1α signaling pathway, inhibiting angiogenesis and reducing cell adhesion, invasion, and migration.
Osteosarcoma/pathology* ; Animals ; Proto-Oncogene Proteins c-akt/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Humans ; Mice ; Cell Adhesion/drug effects* ; Cell Proliferation/drug effects* ; Neovascularization, Pathologic/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Phosphatidylinositol 3-Kinases/genetics* ; Cell Line, Tumor ; Mice, Nude ; Signal Transduction/drug effects* ; Astragalus Plant/chemistry* ; Bone Neoplasms/physiopathology* ; Male ; Rhizome/chemistry* ; Mice, Inbred BALB C ; Angiogenesis

Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
Nicotiana/metabolism* ; Genetic Vectors/metabolism* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering/methods* ; Green Fluorescent Proteins/metabolism* ; Gene Expression

To study the differences in transcript levels among different organs of Spatholobus suberectus and to explore the genes encoding enzymes related to the catechin biosynthesis pathway, this study utilized the genome and full-length transcriptome data of S. suberectus as references. Transcriptome sequencing and bioinformatics analysis were performed on five different organs of S. suberectus-roots, stems, leaves, flowers, and fruits-using the Illumina NovaSeq 6000 platform. A total of 115.28 Gb of clean data were obtained, with GC content values ranging from 45.19% to 47.54%, Q20 bases at 94.17% and above, and an overall comparison rate with the reference genome around 90%. In comparisons between the stem and root, stem and leaf, stem and flower, and stem and fruit, 10 666, 9 674, 9 320, and 5 896 differentially expressed genes(DEGs) were identified, respectively. The lowest number of DEGs was found in the stem and root comparison group. KEGG enrichment analysis revealed that the DEGs were mainly concentrated in the pathways of phytohormone signaling, phenylalanine biosynthesis, etc. A total of 39 genes were annotated in the catechin biosynthesis pathway, with at least one highly expressed gene found in all organs. Among these, PAL1, PAL2, C4H1, C4H3, 4CL1, 4CL2, and DFR2 showed high expression in the stems, suggesting that they may play important roles in the biosynthesis of flavonoids in S. suberectus. This study aims to provide important information for the in-depth exploration of the regulation of catechin biosynthesis in S. suberectus through transcriptome analysis of its different organs and to provide a reference for the further realization of S. suberectus varietal improvement and molecular breeding.
Catechin/biosynthesis* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Fabaceae/metabolism* ; Transcriptome ; Flowers/metabolism* ; Plant Stems/metabolism* ; Plant Leaves/metabolism* ; Plant Roots/metabolism* ; Fruit/metabolism*