ObjectiveThis study aims to explore the mechanism by which Yishen Huoxue Tongqiao Formula improves metabolism-neuroplasticity and treats unilateral vestibular labyrinth destruction by regulating the metabolic balance of glutamate （Glu）/γ-aminobutyric acid （GABA）. Methods48 Sprague-Dawley （SD） adult rats were randomly divided into the sham operation group， model group， Yishen Huoxue Tongqiao Formula groups with low， medium， and high doses （9.20， 18.39， 36.78 g·kg^-1）， and betahistine group （1.62 mg·kg^-1）. A unilateral vestibular labyrinth destruction （vestibular dysfunction） model was established by intratympanic injection of chloroform into the right ear， while the control group received intratympanic injection of normal saline. Drugs were administered once daily for seven consecutive days. During the period， behavioral tests were performed to evaluate the behaviors of rats after unilateral vestibular labyrinth destruction. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the neuronal morphology in the medial vestibular nucleus. Golgi staining was employed to assess the number of dendritic spines of neurons in the medial vestibular nucleus. Ultra-performance liquid chromatography-tandem mass spectrometry （LC-ESI-MS/MS） was utilized to detect Glu/GABA. Immunofluorescence and immunohistochemistry were used to detect the expressions of neuronal nuclei （NeuN）， growth-associated protein 43 （GAP-43）， and glial fibrillary acidic protein （GFAP）. Western blot and real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） were applied to determine the expressions of glutamate-immunoreactive （Glu-IR）， GABA， GFAP， postsynaptic density protein 95 （PSD-95）， and GAP-43. ResultsCompared with the sham operation group， the model group presented with head deviation， balance disorder， increased tail suspension score， nuclear consolidation of medial vestibular nerve neurons， and decreased Nissl bodies （P<0.01）. The number of dendritic spines in neurons and NeuN-positive cells decreased. The content of Glu decreased. The content of GABA increased （Glu/GABA decreased）. The expression of GAP-43 was down-regulated， and GFAP was up-regulated （P<0.05， P<0.01）. The expressions of Glu-IR， PSD-95， and GAP-43 proteins， as well as Glu-IR mRNA decreased， while the expressions of GABA and GFAP proteins and mRNA increased （P<0.05， P<0.01）. Compared with those in the model group， the head deviation， imbalanced behavior， and tail suspension scores in each treatment group decreased， with alleviated neuronal injury and recovered Nissl bodies （P<0.01）. The number of dendritic spines of neurons increased， and the number of NeuN-positive cells rebounded. The content of Glu increased， and the content of GABA decreased （Glu/GABA increased）. GFAP was down-regulated， and GAP-43 was up-regulated （P<0.05， P<0.01）. The expressions of Glu-IR， PMD-95， and GAP-43 proteins， as well as Glu-IR mRNA increased， while the expressions of GABA and GFAP proteins and mRNA decreased. The effect was more significant in the high-dose group （P<0.01）. ConclusionThe Yishen Huoxue Tongqiao Formula can alleviate vestibular dysfunction， and its mechanism may be associated with regulating the metabolic balance of Glu/GABA， mitigating neural damage， improving synaptic plasticity （promoting GAP-43 expression and inhibiting GFAP expression）， and facilitating vestibular compensation.

Objective To explore the potential targets and mechanism of Gufang Granules in treating osteoporosis through network pharmacology,molecular dynamics simulations,and in vitro experiment validation.Methods The active components of Gufang Granules were obtained from the TCMSP database and literature,and their related targets were predicted using SwissTargetPrediction database.Core drug targets were selected through protein-protein interaction(PPI)network analysis and machine learning models,and the predictive performance of the models was assessed by drawing receiver operating characteristic(ROC)curves on independent validation datasets.Gene Set Enrichment Analysis(GSEA)was used to analyze the expression and pathways of core targets.Molecular dynamics(MD)simulations were applied to evaluate the structural stability and interactions of the compound-target complexes.Non-cytotoxic concentrations of Gufang Granules containing serum were determined by the CCK-8 assay.RAW264.7 cells were treated with low,medium,and high concentrations of drug containing serum,respectively.The number of osteoclasts was quantified using TRAP staining.The expression levels of relevant genes and proteins were analyzed through qRT-PCR and Western blot methods.Results A total of 251 potential active components and 1 078 related targets of Gufang Granules were identified.The high expressions of core targets SRC and TNF were mainly associated with osteoclast differentiation,MAPK signaling pathway and PI3K/Akt signaling pathway.MD simulations showed that the core active component Glabridin exhibited strong stability and interaction with the SRC and TNF target proteins.The number of TRAP positive cells in all concentration groups of Gufang Granules was significantly reduced compared to the RANKL group(P<0.01,P<0.001).The serum containing Gufang Granules significantly reduced the mRNA expression of NFATc1,CTSK,SRC and TNF-α,and also downregulated the protein expression of NFATc1,CTSK,p-SRC and TNF-α(P<0.05,P<0.01,P<0.001).Conclusion Gufang Granules may inhibit osteoclast differentiation by downregulating the expression of NFATc1,CTSK,p-SRC and TNF-α,thereby slowing the pathological progression of osteoporosis.

Objective To explore the potential targets and mechanism of Gufang Granules in treating osteoporosis through network pharmacology,molecular dynamics simulations,and in vitro experiment validation.Methods The active components of Gufang Granules were obtained from the TCMSP database and literature,and their related targets were predicted using SwissTargetPrediction database.Core drug targets were selected through protein-protein interaction(PPI)network analysis and machine learning models,and the predictive performance of the models was assessed by drawing receiver operating characteristic(ROC)curves on independent validation datasets.Gene Set Enrichment Analysis(GSEA)was used to analyze the expression and pathways of core targets.Molecular dynamics(MD)simulations were applied to evaluate the structural stability and interactions of the compound-target complexes.Non-cytotoxic concentrations of Gufang Granules containing serum were determined by the CCK-8 assay.RAW264.7 cells were treated with low,medium,and high concentrations of drug containing serum,respectively.The number of osteoclasts was quantified using TRAP staining.The expression levels of relevant genes and proteins were analyzed through qRT-PCR and Western blot methods.Results A total of 251 potential active components and 1 078 related targets of Gufang Granules were identified.The high expressions of core targets SRC and TNF were mainly associated with osteoclast differentiation,MAPK signaling pathway and PI3K/Akt signaling pathway.MD simulations showed that the core active component Glabridin exhibited strong stability and interaction with the SRC and TNF target proteins.The number of TRAP positive cells in all concentration groups of Gufang Granules was significantly reduced compared to the RANKL group(P<0.01,P<0.001).The serum containing Gufang Granules significantly reduced the mRNA expression of NFATc1,CTSK,SRC and TNF-α,and also downregulated the protein expression of NFATc1,CTSK,p-SRC and TNF-α(P<0.05,P<0.01,P<0.001).Conclusion Gufang Granules may inhibit osteoclast differentiation by downregulating the expression of NFATc1,CTSK,p-SRC and TNF-α,thereby slowing the pathological progression of osteoporosis.

ObjectiveTo evaluate the pharmacological effect of Buzhong Yiqitang on brain development in offspring of rats with subclinical hypothyroidism （SCH） during pregnancy and explore its potential mechanism. MethodsForty-eight SPF female SD rats were divided into sham operation group （n=8） and model group （n=40）. The rat model of subclinical hypothyroidism （SCH） was constructed by total thyroidectomy combined with postoperative subcutaneous injection of levothyroxine （L-T₄）. The modeled rats were randomly allocated into model， low-， medium-， and high-dose （5.58， 11.16， 22.32 g∙kg^-1， respectively） Buzhong Yiqitang， and euthyrox （4.5×10^-6 g∙kg^-1） groups， with 8 rats in each group. These rats were co-housed with normal male rats for mating. Drug administration started 2 weeks before pregnancy and continued until delivery. Hematoxylin-eosin staining and Golgi-cox staining were used to observe pathological changes in the hippocampal tissue of offspring rats. Western blot was employed to detect the effects of Buzhong Yiqitang on the protein levels of cytochrome C oxidase subunitⅠ （COX）Ⅰ and COXⅣ in the hippocampal tissue of offspring rats. A colorimetric method was used to measure the mitochondrial adenosine triphosphate （ATP） content in the hippocampal tissue of offspring rats. For in vitro experiments， a hydrogen peroxide （H₂O₂）-induced oxidative damage model was established with rat pheochromocytoma cells （PC12）. Interventions included the DNA methyltransferase inhibitor （SGI-1027）， Buzhong Yiqitang-medicated serum， and euthyrox-medicated serum. The cell counting kit-8 （CCK-8） assay was used to examine the effect of Buzhong Yiqitang on cell proliferation. Immunofluorescence staining was performed to evaluate the effect on tubulin beta 3 class Ⅲ （TUBB3） in PC12 cells. Western blot was employed to assess the effects on the protein levels of DNA methyltransferases （TETs and DNMTs） in PC12 cells. The fluorescent probe 2′，7′-dichlorodihydrofluorescein diacetate （DCFH-DA）， luciferase assay， and JC-1 staining were employed to assess the effects of Buzhong Yiqitang on the levels of reactive oxygen species （ROS） and ATP and the mitochondrial membrane potential in PC12 cells. ResultsCompared with the sham group， the model group showed a reduction in the number of hippocampal neurons， incomplete pyramidal cell bodies， loose arrangement， shortened average dendrite length， decreased dendritic complexity and dendritic spine density， and reduced expression levels of COXⅠ and COXⅣ and content of ATP in the brain tissue （P<0.05， P<0.01）. Compared with the model group， after administration of Buzhong Yiqitang and euthyrox， hippocampal neurons exhibited regular arrangement， complete morphology， extended dendrite， increased dendritic complexity and dendritic spine density， and restored expression levels of COXⅠ and COXⅣ and content of ATP （P<0.05， P<0.01）， with the medium-dose Buzhong Yiqitang group showing the best therapeutic effect. In the PC12 cell model of oxidative damage， Buzhong Yiqitang increased the cell viability （P<0.01）， enhanced neuronal differentiation， down-regulated the expression levels of DNMTs （P<0.05）， up-regulated the expression levels of TETs （P<0.05）， decreased the ROS content （P<0.01）， and restored the ATP content and mitochondrial membrane potential （P<0.01）. ConclusionBuzhong Yiqitang protects brain development in offspring of pregnant rats with SCH. It mainly acts on the oxidative stress and mitochondrial dysfunction resulted from abnormal mtDNA methylation， with DNMTs and TETs as the key proteins for its effects.

To develop biocontrol agents for the control of kiwifruit bacterial canker, we isolated a strain CXG2-5 with inhibitory activity against Pseudomonas syringae pv. actinidiae (Psa), the pathogen of kiwifruit bacterial canker, from the rhizosphere soil of kiwifruit by the plate confrontation test. The strain was identified by morphological observation, physiological and biochemical tests, and molecular biological methods. The indoor control efficacy of the strain was determined by the inoculation of the strain into detached branches with wounds and into leaf discs by vacuum infiltration. The ability of the strain to expand and colonize leaf veins was determined by fluorescent labeling and scanning electron microscopy. Subsequently, the strain was prepared into microcapsules, the field control efficacy of which was evaluated. The strain CXG2-5 was identified as Pseudomonas benzenivorans. It demonstrated good antagonistic activity against Psa, with an inhibition zone diameter of 22 mm and an inhibition rate of 72.7%. The preventive effects of the strain on kiwifruit bacterial canker were better than the therapeutic effects on both detached branches and leaves, with the preventive effects reaching 65% and 92.4%, respectively. The control effect of microcapsules of this strain in the field reached 60.89%, which was slightly lower than that of 20% kasugamycin and higher than that of Bacillus subtilis wettable powder. In conclusion, strain CXG2-5 serves as a candidate for the control of kiwifruit bacterial canker, and the prepared microcapsules have good value for development and application.
Actinidia/microbiology* ; Plant Diseases/prevention & control* ; Pseudomonas syringae ; Pseudomonas/isolation & purification* ; Capsules ; Antibiosis ; Biological Control Agents ; Pest Control, Biological/methods*

Objective:To explore the prescription ideas of treating phlegm-drinking disease in Wang Xugao Lin Zheng Yi An; To analyze the medication law of Wang Xugao's clinical treatment of phlegm-drinking disease. Methods:The database was established based on the medical records of the chapter of phlegm, fluid retention and liver wind and phlegm fire contained in Wang Xugao Lin Zheng Yi An. Excel 2017 software was used to analyze the frequency, taste and meridian tropism of all Chinese materia medica. For Chinese materia medica with frequency≥10, IBM SPSS Modeler 18 software was used to analyze the association rules based on Apriori algorithm, and SPSS 25.0 software was used for cluster analysis based on Ochiai algorithm. Results:A total of 80 medical cases were included, involving 114 prescriptions, including 191 flavors of Chinese materia medica . High-frequency Chinese materia medica mainly included Poria, Pinelliae Rhizoma, Citri Reticulatae Pericarpium, Atractylodis Macrocephalae Rhizoma and Armeniacae Semen Amarum, etc. The main properties in Wang Xugao's medication for the treatment of phlegm-drink disease were warm, followed by cold and mild. The main tastes were sweet, bitter and pungent. Drugs mainly belong to the lung meridian and spleen, stomach, liver, kidney meridians; several core medicinal pairs were obtained, such as Farfarae Flos - Armeniacae Semen Amarum, Pinelliae Rhizoma - Zingiberis Rhizoma, Uncariae Ramulus cum Uncis - Haliotidis Concha, etc. Eight groups of core drug combinations could be sorted out by clustering analysis.Conclusions:In the treatment of phlegm-drinking disease, Wang Xugao paid attention to the simultaneous treatment of multiple viscera to coordinate the balance between the viscera, emphasized the complex etiology of phlegm-drinking disease combined with cold, fire and dampness, attached importance to the treatment of healthy qi to retreat pathogens, the regulation of three-energizer to regulate qi flow. The treatment of three-energizer, promoting yang and reducing phlegm, clearing liver and dispelling wind are the main methods. Medication mainly chooses properties of sweet and warm, with bitter and pungent.

The fiber type transition of skeletal muscle is an intricate and essential physiological process in the body, significantly influencing both the function and metabolism of skeletal muscle. This phenomenon is not only affected by external environmental changes but also intricately regulated by internal physiological mechanisms. Therefore, exploring the physiological process of muscle fiber type transition holds considerable significance for the treatment of human neuromuscular disorders and the improvement of meat quality in livestock and poultry. It has been discovered that the cytokines secreted by skeletal muscle, i.e., myokines, play a role in the fiber type transition of skeletal muscle. Myokines mainly act on skeletal muscle in autocrine and paracrine forms to participate in signal transduction and regulate the fiber type transition of skeletal muscle. This paper reviews the functional differences among various muscle fiber types, expounds the effects and mechanisms of myokines in regulating the transition processes of these fiber types, and prospects the future research directions in this field. This review is expected to provide theoretical support for enhancing the meat quality of livestock and poultry and treating skeletal muscle-related diseases.
Humans ; Animals ; Cytokines/metabolism* ; Muscle Fibers, Skeletal/metabolism* ; Muscle, Skeletal/metabolism* ; Signal Transduction ; Muscle Fibers, Slow-Twitch/metabolism* ; Muscle Fibers, Fast-Twitch/metabolism* ; Myostatin/metabolism* ; Myokines

Rhodotorula toruloides is a non-conventional red yeast that can synthesize various carotenoids and lipids. It can utilize a variety of cost-effective raw materials, tolerate and assimilate toxic inhibitors in lignocellulosic hydrolysate. At present, it is widely investigated for the production of microbial lipids, terpenes, high-value enzymes, sugar alcohols and polyketides. Given its broad industrial application prospects, researchers have carried out multi-dimensional theoretical and technological exploration, including research on genomics, transcriptomics, proteomics and genetic operation platform. Here we review the recent progress in metabolic engineering and natural product synthesis of R. toruloides, and prospect the challenges and possible solutions in the construction of R. toruloides cell factory.
Gene Editing ; Metabolic Engineering ; Rhodotorula/metabolism* ; Lipids

Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N⁶-methyladenosine (m⁶A), an epigenetic modification prevalent in mRNAs, is implicated in the transcriptional regulation during spermatogenesis. However, the dynamics of m⁶A modification in non-rodent mammalian species remains unclear. Here, we systematically investigated the profile and role of m⁶A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m⁶A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m⁶A pattern between porcine and murine genes with spermatogenic function. We found that m⁶A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m⁶A modification. Our data show that m⁶A plays the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point are three genes (SETDB1, FOXO1, and FOXO3) that are crucial to the determination of the fate of SSCs. To the best of our knowledge, this study for the first time uncovers the expression profile and role of m⁶A during spermatogenesis in large animals and provides insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.
Animals ; Male ; Mice ; Cell Differentiation/genetics* ; Mammals/metabolism* ; Methylation ; RNA, Messenger/metabolism* ; Spermatogenesis/genetics* ; Spermatozoa/metabolism* ; Swine/genetics* ; Testis/metabolism* ; Transcriptome ; RNA Methylation/genetics*

Rapid development of high-throughput technologies has permitted the identification of an increasing number of disease-associated genes (DAGs), which are important for understanding disease initiation and developing precision therapeutics. However, DAGs often contain large amounts of redundant or false positive information, leading to difficulties in quantifying and prioritizing potential relationships between these DAGs and human diseases. In this study, a network-oriented gene entropy approach (NOGEA) is proposed for accurately inferring master genes that contribute to specific diseases by quantitatively calculating their perturbation abilities on directed disease-specific gene networks. In addition, we confirmed that the master genes identified by NOGEA have a high reliability for predicting disease-specific initiation events and progression risk. Master genes may also be used to extract the underlying information of different diseases, thus revealing mechanisms of disease comorbidity. More importantly, approved therapeutic targets are topologically localized in a small neighborhood of master genes in the interactome network, which provides a new way for predicting drug-disease associations. Through this method, 11 old drugs were newly identified and predicted to be effective for treating pancreatic cancer and then validated by in vitro experiments. Collectively, the NOGEA was useful for identifying master genes that control disease initiation and co-occurrence, thus providing a valuable strategy for drug efficacy screening and re-positioning. NOGEA codes are publicly available at https://github.com/guozihuaa/NOGEA.