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Real-time quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for gene expression studies. However, choosing the appropriate reference gene is essential to obtain reliable results for RT-qPCR assays. In the present work, the expression of eight candidate reference genes, (elongation factor 1- ), (glyceraldehyde 3-phosphate dehydrogenase), (ubiquitin-conjugating enzyme), (polyubiquitin), (actin), ( -tubulin), (adenine phosphoribosyltransferase 1), and (18S ribosomal RNA), was evaluated in samples using two algorithms, geNorm and NormFinder. The samples were classified into groups according to developmental stages, various tissues, stresses (cold, heat, drought, NaCl), and hormone treatments (MeJA, IBA, SA). Suitable combination of reference genes for RT-qPCR normalization should be applied according to different experimental conditions. In this study, , , and genes were verified as the suitable reference genes across all tested samples. To validate the suitability of the reference genes, we evaluated the relative expression of , which is a gene that may be involved in phytosterol synthesis. Our results provide the foundation for gene expression analysis in and other species of Amaranthaceae.

Achyranthes bidentata Blume (ABB; Chinese name: Huai Niuxi) and Cyathula officinalis K.C.Kuan (COK; Chinese name: Chuan Niuxi), two botanical drugs collectively termed “Niuxi” in traditional Chinese medicine (TCM), are widely used for rheumatoid arthritis (RA) management. This review comprehensively summarized the pharmacological mechanisms and therapeutic potential of the metabolites of ABB and COK on RA, while addressing limitations of current evidence. Of the 314 and 185 metabolites contained in ABB and COK, respectively, 22 metabolites (including Chikusetsusaponin V and chikusetsusaponin Ⅳa), showed multiple anti-RA activities. The mechanisms underlying the effects of ABB and COK with respect to the occurrence and development of RA (including inflammatory processes, immunoregulation, fibroblast-like synoviocytes, angiogenesis, oxidative stress, cartilage degradation, and bone destruction) were evaluated (Graphical Abstract). Numerous signaling pathways, such as the nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), are involved in RA. The metabolites contained in ABB and COK have significant medicinal value and potential in the treatment of RA, while in-depth mechanism studies and clinical research are warranted to support the clinical application of these metabolites.

The aim of this study was to investigate whether polysaccharides (ABPSs) alleviate hypoxic renal injury (HRI) and the possible mechanism. The HRI rat model was established using a hypobaric hypoxia chamber. Rats were divided into a control group, a hypoxia group, low-dose ABPS (ABPL) group, a high-dose ABPS (ABPH) group, a DNase I-positive control group, and an NLRP3 agonist nigericin sodium salt (NSS) group. Blood serum components relevant to neutrophil extracellular traps (NETs), including cell-free DNA (cf-DNA), myeloperoxidase-DNA (MPO-DNA), neutrophil elastase-DNA (NE-DNA), citrullinated histone 3 (cit-H3), blood urea nitrogen (BUN), serum creatinine (Scr), cystatin C (CysC), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1), were analyzed. NOD-like receptor protein 3 (NLRP3) pathway proteins, MPO, NE, cit-H3, and reactive oxygen species (ROS) in renal tissues were analyzed by multiplex fluorescence immunohistochemistry. MPO and cit-H3 in renal tissues were analyzed via Western blot. Oxidative stress markers such as malondialdehyde (MDA) and superoxide dismutase (SOD) were analyzed using the thiobarbituric acid (TBA) assay. The results demonstrated that ABPSs exerted protective effects against hypoxic renal injury. First, ABPSs significantly reduced the levels of cf-DNA, MPO-DNA, and cit-H3, with efficacy comparable to that of DNase I. Second, ABPSs markedly suppressed the activation of the NLRP3 inflammasome pathway by degrading NETs, as evidenced by reduced protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1, accompanied by significant decreases in interleukin 1β (IL-1β) and IL-18. Furthermore, ABPSs effectively alleviated oxidative stress by reducing MDA, enhancing SOD activity, and attenuating ROS. Finally, these molecular and cellular improvements translated into functional recovery as high-dose ABPS treatment restored renal function to near-normal levels, including a 58.1% reduction in BUN, a 34.5% reduction in Scr, a 23.6% reduction in NGAL, a 29.6% reduction in KIM-1, and a 32.2% reduction in CysC. Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining and quantitative scoring analysis of kidney injury revealed severe tubular necrosis and glomerular damage in rats in the hypoxia group, which were significantly attenuated in both the ABPL and ABPH groups ( < 0.05). ABPS mitigates hypoxic renal injury by reducing NETs and synergistically regulating oxidative stress. ABPS shows potential as a multi-target, low-toxicity candidate for renal protection.

Temporomandibular joint osteoarthritis (TMJOA) is an inflammatory disorder with limited treatment options. Given the key role of oxidative stress in TMJOA progression, targeting this pathway may offer therapeutic benefits. In this study, we isolated and characterized exosome-like nanovesicles from Achyranthes bidentata (ABNVs), a traditional Chinese herb known for its antioxidant properties. ABNVs exhibited an average size of 134.7 nm and a negatively charged surface (-30.32 mV), and were enriched with antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD). In a rat TMJOA model, ABNVs (1 µg/mL) effectively reduced cartilage degradation, bone damage, and synovial inflammation while maintaining high biocompatibility. Immunohistochemical analysis revealed that ABNVs suppressed M1 macrophage polarization. In vitro, ABNVs did not impair chondrogenic differentiation but mitigated chondrocyte inflammation. Mechanistically, ABNVs scavenged reactive oxygen species (ROS) and reduced macrophage inflammation through CAT/SOD-mediated ROS clearance and modulation of the JNK/FOXO1 pathway. Additionally, ABNVs indirectly enhanced chondrogenic differentiation by inhibiting M1 macrophage activation. Our findings demonstrate that ABNVs hold promise as a therapeutic strategy for TMJOA by targeting oxidative stress and inflammation via ROS scavenging and JNK/FOXO1 regulation. Graphical Abstract Scheme 1: The mechanism of Achyranthes bidentata-derived nanovesicles (ABNVs) ameliorates TMJOA by ROS scavenging and modulation of inflammatory levels in M1-type macrophages. This results in reduced cartilage matrix degradation and enhanced matrix synthesis in the TMJOA animal model

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion. Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion, but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear. Therefore, in the current study, we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons. Hippocampal neurons were treated with Mg2+-free extracellular solution containing glutamate (300 µM) for 3 hours as a model of glutamate-mediated excitotoxicity (glutamate group). In the normal group, hippocampal neurons were incubated in Mg2+-free extracellular solution. In the Achyranthes bidentata polypeptide group, hippocampal neurons were incubated in Mg2+-free extracellular solution containing glutamate (300 µM) and Achyranthes bidentata polypeptide at different concentrations. At 24 hours after exposure to the agents, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear morphology, respectively. Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay, respectively. At various time points after glutamate treatment, reactive oxygen species in cells were detected by H2DCF-DA, and mitochondrial membrane potential was detected by rhodamine 123 staining. To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors, electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons. Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate. In addition, Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current. Furthermore, the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment. These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway. All animal studies were approved by the Animal Care and Use Committee, Nantong University, China (approval No. 20120216-001) on February 16, 2012.

ABPPk, the active ingredient separated from polypeptides, is a traditional Chinese medicine with multiple pharmaceutical properties. In this study, we investigated the molecular mechanisms of ABPPk in protecting Schwann cells (SCs) from H O -induced cell apoptosis. The viability of SCs pretreated with ABPPk was elevated significantly by MTT assay estimation. Meanwhile, the apoptosis of SCs was reduced which was showed in flow cytometry and transferase-mediated dUTP nick end labeling analysis. Furthermore, the addition of ABPPk also increased the activities of SOD and GSH accompanied with a decrease in MDA and LDH activities. According to Western blot analysis, the upregulation of Bcl-2, also downregulation of Bax and cleaved caspase-3 were demonstrated in SCs which was ABPPk pretreated. Further research showed that PI3K/AKT and ERK1/2 pathways in SCs have been activated after pretreatment of ABPPk. Collectively, results in our study suggested that ABPPk protected SCs from H O -induced oxidative damage by reducing the expression of apoptotic molecules and enhancing the activities of antioxidant enzymes, which inhibited the apoptosis of SCs modulated by PI3K/AKT and ERK1/2 signaling pathways. In our perspectives, ABPPk as an active factor with its antioxidative activities has potential and promising therapeutic effects in the prevention of neurologic disorders.

Achyranthes bidentata, a traditional Chinese medicinal herb, has garnered significant attentions due to its diverse bioactivities and substantial market potential. Recent advancements in phytochemical and pharmacological research have identified polysaccharides as some of its primary bioactive constituents. These polysaccharides demonstrate a wide range of biological effects both in vitro and in vivo, such as antioxidant, immunomodulatory, antitumor, anti-inflammatory, anti-osteoporotic, and gut microbiota-regulating properties. This review comprehensively examines the extraction and purification techniques, structural features, modifications, pharmacological effects, and structure–activity relationships of A. bidentata polysaccharides (ABPs) over the past three decades. By highlighting the multifaceted biological roles and structure–activity correlations of ABPs, this review aims to enhance the understanding of their potential applications and foster further innovation in bioactive research and development.

is a popular perennial medicine herb used for 1000s of years in China to treat various diseases. Although this herb has multiple pharmaceutical purposes in China, no transcriptomic information has been reported for this species. In addition, the understanding of several key pathways and enzymes involved in the biosynthesis of oleanolic acid and ecdysterone, two pharmacologically active classes of metabolites and major chemical constituents of root extracts, is limited. The aim of the present study was to characterize the transcriptome profile of the roots and leaves of to uncover the biosynthetic and transport mechanisms of the active components. In this study, we identified 100,987 transcripts, with an average length of 1146.8 base pairs. A total of 31,634 (31.33%) unigenes were annotated, and 12,762 unigenes were mapped to 303 pathways according to the Kyoto Encyclopedia of Genes and Genomes pathway database. Moreover, we identified a total of 260 oleanolic acid and ecdysterone genes encoding biosynthetic enzymes. Furthermore, the key enzymes involved in the oleanolic acid and ecdysterone synthesis pathways were analyzed using quantitative real-time polymerase chain reaction, revealing that the roots expressed these enzymes to a greater extent than the leaves. In addition, we identified 85 ATP-binding cassette transporters, some of which might be involved in the translocation of secondary metabolites.

The root of Achyranthes bidentata Blume (AB) is a well-known traditional Chinese medicine for treating osteoporosis. Plenty of studies focused on the pharmacological mechanism of the whole extract; however, the contribution of different components to the anti-osteoporosis effect remains unknown. The aim of this study is to explore the anti-osteoporosis mechanism of different components of crude and salt-processed AB under the guidance of network pharmacology, metabolomics, and microbiomics. First, network pharmacology analysis was applied to constructing the compound-target-disease network of AB to provide a holistic view. Second, the anti-osteoporosis effects of the four components were evaluated in female Wistar rats. The subjects were divided into a normal group, a model group, a 17α-estradiol (E2)-treated group, a polysaccharide-component-treated groups, and a polysaccharide-knockout-component-treated groups. All the serum, urine, and feces samples of the six groups were collected after 16 weeks of treatment. Biochemical and microcomputed tomography (μCT) parameters were also acquired. Coupled with orthogonal partial least-squares discrimination analysis, one dimensional nuclear magnetic resonance (NMR) was used to monitor serum metabolic alterations. A total of twenty-two biomarkers, including lipids, amino acids, polyunsaturated fatty acids, glucose, and so on were identified for the different components-treated groups. Through pathway analysis, it is indicated that glyoxylate and dicarboxylate metabolism, glycine, serine, and threonine metabolism, alanine, aspartate, and glutamate metabolism, d-glutamine, and d-glutamate metabolism were the major intervened pathways. Levels of these biomarkers shifted away from the model group and were restored to normal after treatment with the four components. In addition, 16S rDNA sequencing demonstrated that the abundance of Anaerofilum, Rothia, and Turicibacter bacteria was positively correlated with an anti-osteoporosis effect, whereas the abundance of Oscillospira was negatively correlated. The osteoprotective effect of the polysaccharide components of crude and salt-processed AB is related to the regulation of the abundance of these gut microbiota.

Key message The transcriptome profiling in replanting roots revealed that expression pattern changes of key genes promoted important metabolism pathways, antioxidant and pathogen defense systems, adjusted phytohormone signaling and inhibited lignin biosynthesis. The yield of the medicinal plant Achyranthes bidentata could be significantly increased when replanted into a field cultivated previously for the same crop, but the biological basis of this so-called “replanting benefit” is unknown. Here, the RNA-seq technique was used to identify candidate genes responsible for the benefit. The analysis of RNA-seq libraries prepared from mRNA extracted from the roots of first year planting (normal growth, NG) and second year replanting (consecutive monoculture, CM) yielded about 40.22 GB sequencing data. After de novo assembly, 87,256 unigenes were generated with an average length of 1060 bp. Among these unigenes, 55,604 were annotated with public databases, and 52,346 encoding sequences and 2881 transcription factors were identified. A contrast between the NG and CM libraries resulted in a set of 3899 differentially transcribed genes (DTGs). The DTGs related to the replanting benefit and their expression profiles were further analyzed by bioinformatics and qRT-PCR approaches. The major differences between the NG and CM transcriptomes included genes encoding products involved in glycolysis/gluconeogenesis, glutathione metabolism and antioxidant defense, in aspects of the plant/pathogen interaction, phytohormone signaling and phenylpropanoid biosynthesis. The indication was that replanting material enjoyed a stronger level of defense systems, a balance regulation of hormone signals and a suppression of lignin formation, thereby promoting root growth and development. The study provides considerable significant insights for a better understanding of the molecular mechanism of the replanting benefit and suggests their possible application in developing methods to reinforce the effects in medicinal plants.