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In traditional Chinese medicine, Angelica dahurica is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for A. dahurica gene expression studies. Real-time quantitative PCR (RT-qPCR) is widely employed as the predominant method for investigating gene expression. In order to ensure the precise determination of target gene expression outcomes in RT-qPCR analysis, it is imperative to employ stable reference genes. In this study, a total of 11 candidate reference genes including SAND family protein (SAND), polypyrimidine tract-binding protein (PTBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), TIP41-like protein (TIP41), cyclophilin 2 (CYP2), elongation factor 1 α (EF1α), ubiquitin-protein ligase 9 (UBC9), tubulin β-6 (TUB6), thioredoxin-like protein YLS8 (YLS8), and tubulin-α (TUBA) were selected from the transcriptome of A. dahurica. Subsequently, three statistical algorithms (geNorm, NormFinder, and BestKeeper) were employed to assess the stability of their expression patterns across seven distinct stimulus treatments. The outcomes obtained from these analyses were subsequently amalgamated into a comprehensive ranking using RefFinder. Additionally, one target gene, phenylalanine ammonia-lyase (PAL), was used to confirm the effectiveness of the selected reference genes. According to the findings of this study, the two most stable reference genes for normalizing the expression of genes in A. dahurica are TIP41 and UBC9. Overall, our research has determined the appropriate reference genes for RT-qPCR in A. dahurica and provides a crucial foundation for gene screening and identifying genes associated with the biosynthesis of active ingredients in A. dahurica. [ABSTRACT FROM AUTHOR]

Background: Wenshen Jianpi recipe (WSJPR), a blended traditional Chinese medicine, is considered to have the possible beneficial effect on the progression of diabetic nephropathy (DN). This present study was designed to elucidate this protective activity in a rat model with streptozotocin (STZ)-induced DN and to explore the possible underlying mechanism. Methods: Adult Sprague Dawley (SD) rats were induced to develop DN through intraperitoneal injection of STZ (60 mg/kg). Animals were orally administered saline, WSJPR at 7.5, 15, 30 g/kg, and valsartan (25 mg/kg) daily for 8 weeks. Blood and 24-h urine samples of each rat were collected for biochemical examination at 2-week intervals. Microcirculatory blood flow in the renal cortex and hemorheology index were also measured. At the end of 8 weeks, all rats were sacrificed to obtain the kidney tissues for histological examination and reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the transcriptional levels of nephrin and podocin genes. Results: WSJPR could improve serum total protein (TP) and albumin (ALB), reduce the excretion rates of urine-TP (U-TP), urine-ALB (U-ALB) and urine urea nitrogen (UUN) (P < 0.05), although it did not significantly alter the hyperglycemia. In addition, treatment with WSJPR could strongly reduce blood flow, erythrocyte aggregation index, and ameliorate microcirculation. In histological measurement, WSJPR-treated rats showed a significant amelioration in glomerular hypertrophy and mesangial expansion. By RT-PCR, we found WSJPR up-regulated the nephrin and podocin expression at mRNA levels. Conclusion: This study suggested that WSJPR could effectively relieve renal damage and improve renal function of DN rats by ameliorating metabolism disorder and increasing the gene expression of nephrin and podocin, which might be a useful approach for the treatment of DN. [ABSTRACT FROM AUTHOR]

Celastrus orbiculatus Thunb. is an ancient traditional Chinese herb with a long history of medicinal use. The ethyl acetate extract of Celastrus orbiculatus Thunb. (COE) has been shown to have anti-tumor effects in various preclinical studies. However, the anti-invasive and metastatic efficacy of COE in non-small cell lung cancer (NSCLC) and the mechanism by which COE regulates cellular oxidation levels are yet to be elucidated. To study the anti-dissemination effect of COE on NSCLC and to elucidate the molecular mechanism of COE in regulating cellular oxidation levels and its effect on lung cancer invasion and metastasis. CCK-8 assay was used to detect the toxic effects of COE on NSCLC. Transwell assay and high-content imaging was used to detect the Motility of NSCLC. Transmission electron microscopy and three-dimensional (3D) imaging of mitochondrial fluorescence were employed to detect the number and structure of mitochondria. JC-1 probe was used to detect the level of mitochondrial membrane potential. Firefly luciferase assay was used to detect the level of total intracellular ATP. MitoSox probe and DCFH-DA probe were applied to detect the level of reactive oxygen species (ROS) inside the mitochondria and the total intracellular ROS, respectively. Immunohistochemistry was used to detect protein expression in xenograft tumors. COE inhibited motility and induced DJ-1 downregulation in NSCLC at low toxic concentrations, and the antiseptic effect of COE was reduced significantly after the overexpression of DJ-1. COE induced structural disruption of mitochondria in NSCLC and accumulation of superoxide compounds, decreased the volume of membrane potential depolarization, and impaired energy production, ultimately leading to a large accumulation of ROS at the cellular level. The antioxidant acetylcysteine (NAC) significantly reversed the antiseptic capacity of COE. In a xenograft tumor model, protein expression of DJ-1, E-cadherin, N-cadherin, and MMP-2 in COE group was significantly changed compared to the model group. In the present study, COE inhibited NSCLC invasion and metastasis and was associated with the downregulation of DJ-1 and elevated ROS. COE-mediated downregulation of DJ-1 may be the primary cause of mitochondrial structural and functional dysfunction in NSCLC, eventually leading to ROS accumulation. [Display omitted] [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Cerebrovascular diseases (CBVDs), characterized by striking morbidity and mortality, have become the most common life-threatening diseases. The existing drugs of CBVDs target one or a few of pathogenic factors, the efficacy of which is limited because of the complexity of CBVDs. Traditional Chinese medicine (TCM), featured by multi-component and multi-target endows the great effectiveness in CBVDs treatment. For instance, Erigeron breviscapus (vant.) Hand. Mazz. （Erigeron breviscapus) has been used to treat CBVDs for a long time and the efficacy has been verified through years’ of practice. Nevertheless, the mechanisms of Erigeron breviscapus for treating CBVDs are still unclear. The aim of the study Systematically decipher the mechanisms of Erigeron breviscapus for treating CBVDs. Materials and methods The systems pharmacology approach is utilized by integrating ADME pharmacokinetic screening, target fishing, protein-protein interaction (PPI), network analysis and in vitro experiments verification. Results First, 14 potentially active molecules were screened out through in silico ADME pharmacokinetic evaluation, most of which have been reported with excellent biological activities. Then 169 targets of active molecules were read out using our in-house softwares, systems drug targeting (sysDT) and Weighted Ensemble Similarity(WES). We found that the targets of the active compounds were significantly enriched to the CBVDs therapeutic targets by analyzing their biological processes and protein-protein interactions (PPIs). A multi-layer network analysis including compound-target network, target-pathway network and “CBVDs pathway” indicated that the Erigeron breviscapus exerts a protective effect on CBVDs via regulating multiple pathways and hitting on multiple targets. Meanwhile in vitro experiments confirmed that the stigmasterol, scutellarein, and daucosterol from Erigeron breviscapus increased the MEK and PLCγ proteins levels, and decreased the expression of Bax, PI3K, and eNOS, which led to the cell survival, proliferation and contraction. Conclusion The approach used in this work offers a new exemplification for systematically understanding the mechanisms of herbal medicines, which will give an impulse to the CBVDs drug development. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance For many years, Guangzhou University of Chinese Medicine has been successfully using the empirical Wenyang Huoxue Jiedu formula (WHJF) to treat coronary heart disease. Modern theories of acute coronary syndrome mainly focus on rupture of thin-cap fibroatheromas (TCFAs), which is closely related to the release of vascular endothelial growth factor and its receptor (VEGF/VEGFR). Aim of study We investigated the effects of WHJF on the formation of TCFA plaques and the potential mechanism (VEGF/VEGFR signaling pathway). Materials and methods For the in vivo experiments, WHJF was administered to ApoE -/- mice, as a model of TCFA plaque formation. Aortic sections of the mice were obtained, and the vulnerability index and new vessel density of plaques were calculated by the Movat staining assay and immunohistochemistry kit, respectively. Protein and mRNA expression levels of VEGF/VEGFR in aortas were assayed by capillary electrophoresis immunoassay and quantitative real-time polymerase chain reaction analyses. In vitro , WHJF serum was produced in rats on the fourth day 2 h after the first administration of different concentrations of WHJF. Proliferation, migration, and lumen formation ability of human umbilical vein endothelial cells (HUVECs) treated with sera from these rats were assayed by the CKK-8 kit, Transwell plates, and Matrigel assay, respectively. Protein and mRNA expression levels of signaling molecules in the VEGF/VEGFR pathways were also examined. Results In vivo , the vulnerability index and new vessel density of plaques in the WHJF group were lower than those values in the blank control group ( P  < 0.05). No differences were found between the groups in the expression levels of VEGF/VEGFR ( P  > 0.05). In vitro , the proliferation, migration, and tube formation of HUVECs in the high-dose WHJF group were reduced compared to the control group ( P  < 0.05). This finding was in agreement with the downregulation of VEGFR-2 and pERK ( P  < 0.05). The mRNA expression of signaling molecules showed no difference between the groups ( P  > 0.05). Conclusions WHJF inhibits TCFA formation by influencing the VEGF/VEGFR signaling pathway. [ABSTRACT FROM AUTHOR]