Late-Stage Skeletal Muscle Transcriptome in Duchenne muscular dystrophy shows a BMP4-Induced Molecular Signature.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease due to loss-of-function mutations in the DYSTROPHIN gene. DMD-related skeletal muscle wasting is typified by an aberrant immune response involving upregulation of TGFß family of cytokines. We previously demonstrated that bone morphogenetic protein 4 (BMP4) is increased in DMD and BMP4 stimulation induces a 20-fold upregulation of Smad8 transcription. However, the role of BMP4 in severely affected DMD skeletal muscle is unknown. We hypothesized that transcriptomic signatures in severely affected human DMD skeletal muscle are driven by BMP4 signaling. Transcriptomes from skeletal muscle biopsies of late-stage DMD vs. non-DMD controls and C2C12 muscle cells with or without BMP4 stimulation were generated by RNA-Seq and analyzed for single transcript differential expression as well as by Ingenuity Pathway Analysis and weighted gene co-expression network analyses. A total of 2,328 and 5,291 transcripts in the human muscle and C2C12 muscle cells, respectively, were differentially expressed. We identified an overlapping molecular signature of 1,027 genes dysregulated in DMD muscle that were induced in BMP4-stimulated C2C12 muscle cells. Highly upregulated DMD transcripts that overlapped with BMP4-stimulated C2C12 muscle cells included ADAMTS3, HCAR2, SERPING1, SMAD8 , and UNC13C. The DMD transcriptome was characterized by dysregulation of pathways involving immune function, extracellular matrix remodeling, and metabolic/mitochondrial function. In summary, we define a late-stage DMD skeletal muscle transcriptome that substantially overlaps with the BMP4-induced molecular signature in C2C12 muscle cells. This supports BMP4 as a disease-driving regulator of transcriptomic changes in late-stage DMD skeletal muscle and expands our understanding of the evolution of dystrophic signaling pathways and their associated gene networks that could be explored for therapeutic development.

Tandem Vinylogous Aldol and Intramolecular [2 + 2] Cycloaddition toward Benzocyclobutenes by UV Light Photocatalysis.

A facile and efficient radical tandem vinylogous aldol and intramolecular [2 + 2] cycloaddition reaction for direct synthesis of cyclobutane-containing benzocyclobutenes (BCBs) under extremely mild conditions without using any photocatalysts is reported. This approach exhibited definite compatibility with functional groups and afforded new BCBs with excellent regioselectivity and high yields. Moreover, detailed mechanism studies were carried out both experimentally and theoretically. The readily accessible, low-cost, and ecofriendly nature of the developed strategy will endow it with attractive applications in organic and medicinal chemistry.

Influence of microbial inoculation site on trichloroethylene degradation in electrokinetic-enhanced bioremediation of low-permeability soils.

The integration of electrokinetic and bioremediation (EK-BIO) represents an innovative approach for addressing trichloroethylene (TCE) contamination in low-permeability soil. However, there remains a knowledge gap in the impact of the inoculation approach on TCE dechlorination and the microbial response with the presence of co-existing substances. In this study, four 1-dimensional columns were constructed with different inoculation treatments. Monitoring the operation conditions revealed that a stabilization period (∼40 days) was required to reduce voltage fluctuation. The group with inoculation into the soil middle (Group B) exhibited the highest TCE dechlorination efficiency, achieving a TCE removal rate of 84%, which was 1.1-3.2 fold higher compared to the others. Among degraded products in Group B, 39% was ethylene. The physicochemical properties of the post-soil at different regions illustrated that dechlorination coincided with the Fe(III) and SO42- reduction, meaning that the EK-BIO system promoted the formation of a reducing environment. Microbial community analysis demonstrated that Dehalococcoides was only detected in the treatment of injection at soil middle or near the cathode, with abundance enriched by 2.1%-7.2%. The principal components analysis indicated that the inoculation approach significantly affected the evolution of functional bacteria. Quantitative polymerase chain reaction (qPCR) analysis demonstrated that Group B exhibited at least 2.8 and 4.2-fold higher copies of functional genes (tceA, vcrA) than those of other groups. In conclusion, this study contributes to the development of effective strategies for enhancing TCE biodechlorination in the EK-BIO system, which is particularly beneficial for the remediation of low-permeability soils.

Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency.

OBJECTIVE: Glucose-6-phosphate isomerase (GPI) deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants. This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics, often posing challenges for precise diagnoses using conventional methods. To this end, this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family. METHODS: The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis. Novel compound heterozygous variants of the GPI gene, c.174C>A (p.Asn58Lys) and c.1538G>T (p.Trp513Leu), were identified using whole-exome and Sanger sequencing. The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure. RESULTS: By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study, we found that most variants were located in exons 3, 4, 12, and 18, with a few localized in exons 8, 9, and 14. This study identified novel compound heterozygous variants associated with GPI deficiency. These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids. CONCLUSION: Early family-based sequencing analyses, especially for patients with congenital anemia, can help increase diagnostic accuracy for GPI deficiency, improve child healthcare, and enable genetic counseling.

Uridine diphosphate glucuronosyltransferase 1A1 prevents the progression of liver injury.

BACKGROUND: Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) plays a crucial role in metabolizing and detoxifying endogenous and exogenous substances. However, its contribution to the progression of liver damage remains unclear. AIM: To determine the role and mechanism of UGT1A1 in liver damage progression. METHODS: We investigated the relationship between UGT1A1 expression and liver injury through clinical research. Additionally, the impact and mechanism of UGT1A1 on the progression of liver injury was analyzed through a mouse model study. RESULTS: Patients with UGT1A1 gene mutations showed varying degrees of liver damage, while patients with acute-on-chronic liver failure (ACLF) exhibited relatively reduced levels of UGT1A1 protein in the liver as compared to patients with chronic hepatitis. This suggests that low UGT1A1 levels may be associated with the progression of liver damage. In mouse models of liver injury induced by carbon tetrachloride (CCl4) and concanavalin A (ConA), the hepatic levels of UGT1A1 protein were found to be increased. In mice with lipopolysaccharide or liver steatosis-mediated liver-injury progression, the hepatic protein levels of UGT1A1 were decreased, which is consistent with the observations in patients with ACLF. UGT1A1 knockout exacerbated CCl4- and ConA-induced liver injury, hepatocyte apoptosis and necroptosis in mice, intensified hepatocyte endoplasmic reticulum (ER) stress and oxidative stress, and disrupted lipid metabolism. CONCLUSION: UGT1A1 is upregulated as a compensatory response during liver injury, and interference with this upregulation process may worsen liver injury. UGT1A1 reduces ER stress, oxidative stress, and lipid metabolism disorder, thereby mitigating hepatocyte apoptosis and necroptosis.

An optimized metastructure switchable between ultra-wideband angle-insensitive absorption and transmissive polarization conversion: a theoretical study.

An optimized metastructure (MS) switchable between ultra-wideband (UWB) angle-insensitive absorption, and transmissive linear-to-circular (LTC) polarization conversion (PC), is proposed, which is a theoretical study. The structural parameters of this MS are optimized by the thermal exchange optimization algorithm. By modulating the chemical potential (µc) of the graphene-based hyperbolic metamaterial embedded in the MS, the MS can achieve UWB absorption in the absorption state and LTC PC in the transmission state. At normal incidence, in the absorption state, the MS exhibits absorptivity exceeding 0.9 within 7-15.45 THz, with a relative bandwidth (RBW) of 75.28%. By elevating µc, an UWB LTC PC is realized, with a RBW of 118.8%, achieving transmittance above 0.9 and the axial ratio below 3 dB. When prioritizing the angular stability, in the absorption state, the MS secures the angular stability of 75° for TE waves and 65° for TM ones. In the transmission state, the angular stability of PC reaches 60°, with RBW = 100.7%. Moreover, by manipulating µc, the tunability of UWB absorption is realized. The optimized MS provides a reference for designing multifunctional intelligent terahertz modulators, with promising application potential in domains like electromagnetic shielding, communication systems, and THz modulation.

Erratum: [Corrigendum] Chloroquine potentiates the anticancer effect of sunitinib on renal cell carcinoma by inhibiting autophagy and inducing apoptosis.

[This corrects the article DOI: 10.3892/ol.2017.7635.].

Targeting the chromatin structural changes of antitumor immunity.

Epigenomic imbalance drives abnormal transcriptional processes, promoting the onset and progression of cancer. Although defective gene regulation generally affects carcinogenesis and tumor suppression networks, tumor immunogenicity and immune cells involved in antitumor responses may also be affected by epigenomic changes, which may have significant implications for the development and application of epigenetic therapy, cancer immunotherapy, and their combinations. Herein, we focus on the impact of epigenetic regulation on tumor immune cell function and the role of key abnormal epigenetic processes, DNA methylation, histone post-translational modification, and chromatin structure in tumor immunogenicity, and introduce these epigenetic research methods. We emphasize the value of small-molecule inhibitors of epigenetic modulators in enhancing antitumor immune responses and discuss the challenges of developing treatment plans that combine epigenetic therapy and immunotherapy through the complex interaction between cancer epigenetics and cancer immunology.

Increased Cyclic Adenosine Monophosphate Responsive Element is Closely Associated with the Pathogenesis of Drug-resistant Epilepsy.

BACKGROUND: Drug-resistant epilepsy (DRE) is a refractory neurological disorder. There is ample evidence that suggest that γ-aminobutyric acid-a (GABAA) receptors could be one of the mechanisms responsible for the development of drug resistance in epilepsy. It is also known that the cAMP response element binding protein (CREB) plays a possible key role in the transcriptional regulation of GABAA. OBJECTIVE: This study explores the role of CREB in the development of DRE and the effect of CREB on GABA-related receptors in DRE. METHODS: The CREB expression was increased or decreased in the hippocampus of normal rats by lentiviral transfection, who then underwent the lithium-pilocarpine-induced epilepsy model. Phenobarbital (PB) sodium and carbamazepine (CBZ) were used to select a drug-resistant epileptic model. The expression levels of GABAA receptor α1, ß2, and γ2 subunits and CREB protein were measured in the rat hippocampus by western blot and fluorescent quantitative PCR. RESULTS: The frequency and duration of seizures increased in the overexpression group compared to that in the control group. In addition, the severity, frequency, and duration of seizures decreased in the group with decreased expression. The hippocampus analysis of the expression levels of the CREB protein and CREB mRNA yielded similar findings. Altering the CREB protein expression in the rat hippocampus could negatively regulate the expression and transcript levels of GABAA receptors α1, ß2, and γ2, suggesting that CREB may serve as a potential target for the development of treatment protocols and drugs for epilepsy. CONCLUSION: Our study shows that enhanced CREB expression promotes the development of DRE and negatively regulates GABAA receptor levels and that the inhibition of CREB expression may reduce the incidence of DRE.

The azole biocide climbazole induces oxidative stress, inflammation, and apoptosis in fish gut.

Climbazole is an azole biocide that has been widely used in formulations of personal care products. Climbazole can cause developmental toxicity and endocrine disruption as well as gut disturbance in aquatic organisms. However, the mechanisms behind gut toxicity induced by climbazole still remain largely unclear in fish. Here, we evaluate the gut effects by exposing grass carp (Ctenopharyngodon idella) to climbazole at levels ranging from 0.2 to 20 µg/L for 42 days by evaluating gene transcription and expression, biochemical analyses, correlation network analysis, and molecular docking. Results showed that climbazole exposure increased cyp1a mRNA expression and ROS level in the three treatment groups. Climbazole also inhibited Nrf2 and Keap1 transcripts as well as proteins, and suppressed the transcript levels of their subordinate antioxidant molecules (cat, sod, and ho-1), increasing oxidative stress. Additionally, climbazole enhanced NF-κB and iκBα transcripts and proteins, and the transcripts of NF-κB downstream pro-inflammatory factors (tnfα, and il-1ß/6/8), leading to inflammation. Climbazole increased pro-apoptosis-related genes (fadd, bad1, and caspase3), and decreased anti-apoptosis-associated genes (bcl2, and bcl-xl), suggesting a direct reaction to apoptosis. The molecular docking data showed that climbazole could form stable hydrogen bonds with CYP1A. Mechanistically, our findings suggested that climbazole can induce inflammation and oxidative stress through CYP450s/ROS/Nrf2/NF-κB pathways, resulting in cell apoptosis in the gut of grass carp.

Motor neurons generate pose-targeted movements via proprioceptive sculpting.

Motor neurons are the final common pathway1 through which the brain controls movement of the body, forming the basic elements from which all movement is composed. Yet how a single motor neuron contributes to control during natural movement remains unclear. Here we anatomically and functionally characterize the individual roles of the motor neurons that control head movement in the fly, Drosophila melanogaster. Counterintuitively, we find that activity in a single motor neuron rotates the head in different directions, depending on the starting posture of the head, such that the head converges towards a pose determined by the identity of the stimulated motor neuron. A feedback model predicts that this convergent behaviour results from motor neuron drive interacting with proprioceptive feedback. We identify and genetically2 suppress a single class of proprioceptive neuron3 that changes the motor neuron-induced convergence as predicted by the feedback model. These data suggest a framework for how the brain controls movements: instead of directly generating movement in a given direction by activating a fixed set of motor neurons, the brain controls movements by adding bias to a continuing proprioceptive-motor loop.

Accurate preoperative prediction of nodal metastasis in papillary thyroid microcarcinoma: Towards optimal management of patients.

OBJECTIVE: To enhance the accuracy in predicting lymph node metastasis (LNM) preoperatively in patients with papillary thyroid microcarcinoma (PTMC), refining the "low-risk" classification for tailored treatment strategies. METHODS: This study involves the development and validation of a predictive model using a cohort of 1004 patients with PTMC undergoing thyroidectomy along with central neck dissection. The data was divided into a training cohort (n = 702) and a validation cohort (n = 302). Multivariate logistic regression identified independent LNM predictors in PTMC, leading to the construction of a predictive nomogram model. The model's performance was assessed through ROC analysis, calibration curve analysis, and decision curve analysis. RESULTS: Identified LNM predictors in PTMC included age, tumor maximum diameter, nodule-capsule distance, capsular contact length, bilateral suspicious lesions, absence of the lymphatic hilum, microcalcification, and sex. Especially, tumors larger than 7 mm, nodules closer to the capsule (less than 3 mm), and longer capsular contact lengths (more than 1 mm) showed higher LNM rates. The model exhibited AUCs of 0.733 and 0.771 in the training and validation cohorts respectively, alongside superior calibration and clinical utility. CONCLUSION: This study proposes and substantiates a preoperative predictive model for LNM in patients with PTMC, honing the precision of "low-risk" categorization. This model furnishes clinicians with an invaluable tool for individualized treatment approach, ensuring better management of patients who might be proposed observation or ablative options in the absence of such predictive information.

Limitations and modifications in the clinical application of calcium sulfate.

Calcium sulfate and calcium sulfate-based biomaterials have been widely used in non-load-bearing bone defects for hundreds of years due to their superior biocompatibility, biodegradability, and non-toxicity. However, lower compressive strength and rapid degradation rate are the main limitations in clinical applications. Excessive absorption causes a sharp increase in sulfate ion and calcium ion concentrations around the bone defect site, resulting in delayed wound healing and hypercalcemia. In addition, the space between calcium sulfate and the host bone, resulting from excessively rapid absorption, has adverse effects on bone healing or fusion techniques. This issue has been recognized and addressed. The lack of sufficient mechanical strength makes it challenging to use calcium sulfate and calcium sulfate-based biomaterials in load-bearing areas. To overcome these defects, the introduction of various inorganic additives, such as calcium carbonate, calcium phosphate, and calcium silicate, into calcium sulfate is an effective measure. Inorganic materials with different physical and chemical properties can greatly improve the properties of calcium sulfate composites. For example, the hydrolysis products of calcium carbonate are alkaline substances that can buffer the acidic environment caused by the degradation of calcium sulfate; calcium phosphate has poor degradation, which can effectively avoid the excessive absorption of calcium sulfate; and calcium silicate can promote the compressive strength and stimulate new bone formation. The purpose of this review is to review the poor properties of calcium sulfate and its complications in clinical application and to explore the effect of various inorganic additives on the physicochemical properties and biological properties of calcium sulfate.

Numb cheek syndrome in breast cancer: a case report.

Background: Numb cheek syndrome, a rare corollary of numb chin syndrome, is due to infra-orbital neuropathy. It can occur in association with an underlying malignancy, which can cause neuropathy by direct malignant nerve infiltration or via a paraneoplastic mechanism. Although numb cheek syndrome has been reported in association with a variety of cancers, it has previously not been reported in association with breast cancer. We report a case of left breast cancer presenting with left numb cheek syndrome. Case presentation: A 65-year-old woman presented to the Neurology clinic with a 7-month history of left cheek numbness and occasional cheek tenderness. Examination revealed slightly diminished pin-prick sensation in the left cheek and a vaguely palpable left breast lump. A magnetic resonance imaging scan of the brain showed abnormal enhancement of the left maxillary nerve at the foramen rotundum, but cerebrospinal fluid analysis was normal. Mammography, ultrasound scans, and core biopsy of the left breast confirmed the diagnosis of invasive left breast carcinoma (estrogen and progesterone receptor negative, c-erb-B2 equivocal, fluorescence in-situ hybridization negative). There was no evidence of distant metastases on computed tomography and bone scintigraphy scans. The patient underwent neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, followed by 4 cycles of paclitaxel and carboplatin), and left breast wide excision and sentinel lymph node biopsy, and a repeat magnetic resonance imaging scan performed 2 months after surgical resection showed resolution of the left maxillary nerve enhancement. The patient's left numb cheek symptoms improved over a course of 5 months after cancer resection but did not completely resolve. Conclusions: Our case represents the first reported left numb cheek syndrome in association with breast cancer, due to maxillary neuropathy without any discrete mass or compressive cause. To avoid delays in diagnosing malignancy, physicians and surgeons should be aware that numb cheek syndrome can occur in association with an underlying malignancy, and that breast cancer should be counted amongst the possibilities.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Combined aerobic and strength exercise training on biological ageing in Singaporean breast cancer patients: protocol for the Breast Cancer Exercise Intervention (BREXINT) Pilot Study.

Breast cancer (BC) is the most prominent cancer amongst women, but fortunately, early diagnosis and advances in multimodality treatments have improved patient survivability. Cancer survivors, however, experience increased biological ageing which may accelerate other co-morbidities. Exercise intervention is a promising clinical adjuvant approach to improve BC patients' physiological function, recovery from treatment, and quality of life. However, the effects of combined aerobic and strength exercise training on biological ageing in BC patients have not been studied. The Breast Cancer Exercise Intervention (BREXINT) Pilot Study will evaluate the effects of a 24-week combined aerobic and strength exercise intervention against usual care in 50 BC patients' post-treatment randomised to either group. The primary outcomes include changes in cardiorespiratory fitness, muscle strength, cancer-related symptoms, and rate of biological ageing following exercise intervention period. The secondary outcomes include habitual physical activity measured with tri-axial accelerometery and supporting questionnaires, including physical activity, food diary, and quality of life questionnaires. This study will identify the effects of combined aerobic exercise strength training on biological ageing in BC patients from Singapore. Results from this study could further support the implementation of regular exercise programmes as routine care for cancer patients.

Insight into the relationship between metabolic enzymes and oxadiazon degradation in Oryza sativa for reducing environmental risks.

Oxadiazon (ODZ) is extensively utilized in agricultural fields for weed control owing to its strong effectiveness. However, excessive loading of ODZ in water bodies and agricultural soils can lead to various environmental concerns. Therefore, it is crucial to understand the ODZ metabolic process and associated mechanisms in crops to assess the likelihood of ODZ contamination in the environment. This study aimed to assess the effects of ODZ on the growth and toxicological responses of rice (Oryza sativa). The growth of rice tissues was notably compromised with the increase in ODZ concentrations. RNA sequencing in combination with liquid chromatography-quadrupole-time-of-flight-high-resolution mass spectrometry/mass spectrometry (LC-Q-TOF-HRMS/MS) analysis allowed for the identification of numerous transcriptional components associated with ODZ metabolism. Four libraries comprising rice roots and shoots exposed to ODZ were RNA-sequenced in triplicate. The application of environmentally realistic ODZ concentrations upregulated the expression of 844 genes in shoots and 1476 genes in roots. Gene enrichment analysis revealed the presence of multiple enzymes involved in ODZ metabolism and detoxification. These enzymes play a critical role in mitigating environmental stress and facilitating xenobiotic metabolism. Notably, among differentially expressed genes, several key enzymes were identified, including cytochrome P450s, protein kinases, aminotransferases, and ATP-binding cassette transporters involved in the metabolic process. Using LC-Q-TOF-HRMS/MS, 3 metabolites and 13 conjugates were identified in multiple metabolic pathways involving oxidation, hydrolysis, glycosylation, acetylation, and methylation. This study successfully established a potential link between the specific metabolic products of ODZ and increased activities of their corresponding enzymes. Moreover, this study considerably elucidates the detailed pathways and mechanisms involved in ODZ metabolism. The study findings provide valuable insights into the development of genotypes for reducing ODZ residues in paddy fields and minimizing their accumulation in rice crops.

Effects of New Psychoactive Substance Esketamine on Behaviors and Transcription of Genes in Dopamine and GABA Pathways in Zebrafish Larvae.

Esketamine (ESK) is the S-enantiomer of ketamine racemate (a new psychoactive substance) that can result in illusions, and alter hearing, vision, and proprioception in human and mouse. Up to now, the neurotoxicity caused by ESK at environmental level in fish is still unclear. This work studied the effects of ESK on behaviors and transcriptions of genes in dopamine and GABA pathways in zebrafish larvae at ranging from 12.4 ng L- 1 to 11141.1 ng L- 1 for 7 days post fertilization (dpf). The results showed that ESK at 12.4 ng L- 1 significantly reduced the touch response of the larvae at 48 hpf. ESK at 12.4 ng L- 1 also reduced the time and distance of larvae swimming at the outer zone during light period, which implied that ESK might potentially decrease the anxiety level of larvae. In addition, ESK increased the transcription of th, ddc, drd1a, drd3 and drd4a in dopamine pathway. Similarly, ESK raised the transcription of slc6a1b, slc6a13 and slc12a2 in GABA pathway. This study suggested that ESK could affect the heart rate and behaviors accompanying with transcriptional alterations of genes in DA and GABA pathways at early-staged zebrafish, which resulted in neurotoxicity in zebrafish larvae.

Effective substances and molecular mechanisms guided by network pharmacology: An example study of Scrophulariae Radix treatment of hyperthyroidism and thyroid hormone-induced liver and kidney injuries.

ETHNOPHARMACOLOGICAL RELEVANCE: Scrophulariae Radix (Xuanshen [XS]) has been used for several years to treat hyperthyroidism. However, its effective substances and pharmacological mechanisms in the treatment of hyperthyroidism and thyroid hormone-induced liver and kidney injuries have not yet been elucidated. AIM OF THE STUDY: This study aimed to explore the pharmacological material basis and potential mechanism of XS therapy for hyperthyroidism and thyroid hormone-induced liver and kidney injuries based on network pharmacology prediction and experimental validation. MATERIALS AND METHODS: Based on 31 in vivo XS compounds identified using ultra-performance liquid chromatography tandem quadruple exactive orbitrap high-resolution accurate-mass spectrometry (UPLC-QE-HRMS), a network pharmacology approach was used for mechanism prediction. Systematic networks were constructed to identify the potential molecular targets, biological processes (BP), and signaling pathways. A component-target-pathway network was established. Mice were administered levothyroxine sodium through gavage for 30 d and then treated with different doses of XS extract with or without propylthiouracil (PTU) for 30 d. Blood, liver, and kidney samples were analyzed using an enzyme-linked immunosorbent assay (ELISA) and western blotting. RESULTS: A total of 31 prototypes, 60 Phase I metabolites, and 23 Phase II metabolites were tentatively identified in the plasma of rats following the oral administration of XS extract. Ninety-six potential common targets between the 31 in vivo compounds and the diseases were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that Bcl-2, BAD, JNK, p38, and ERK1/2 were the top targets. XS extract with or without PTU had the following effects: inhibition of T3/T4/fT3/fT4 caused by levothyroxine; increase of TSH levels in serum; restoration of thyroid structure; improvement of liver and kidney structure and function by elevating the activities of anti-oxidant enzymes catalase (CAT),superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px); activation anti-apoptotic proteins Bcl-2; inhibition the apoptotic protein p-BAD; downregulation inflammation-related proteins p-ERK1/2, p-JNK, and p-p38; and inhibition of the aggregation of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6, as well as immune cells in the liver. CONCLUSION: XS can be used to treat hyperthyroidism and liver and kidney injuries caused by thyroid hormones through its anti-oxidant, anti-inflammatory, and anti-apoptotic properties. In addition, serum pharmacochemical analysis revealed that five active compounds, namely 4-methylcatechol, sugiol, eugenol, acetovanillone, and oleic acid, have diverse metabolic pathways in vivo and exhibit potential as effective therapeutic agents.