Potassium dehydroandrographolide succinate regulates the MyD88/CDH13 signaling pathway to enhance vascular injury-induced pathological vascular remodeling / 中国天然药物

Pathological vascular remodeling is a hallmark of various vascular diseases. Previous research has established the significance of andrographolide in maintaining gastric vascular homeostasis and its pivotal role in modulating endothelial barrier dysfunction, which leads to pathological vascular remodeling. Potassium dehydroandrographolide succinate (PDA), a derivative of andrographolide, has been clinically utilized in the treatment of inflammatory diseases precipitated by viral infections. This study investigates the potential of PDA in regulating pathological vascular remodeling. The effect of PDA on vascular remodeling was assessed through the complete ligation of the carotid artery in C57BL/6 mice. Experimental approaches, including rat aortic primary smooth muscle cell culture, flow cytometry, bromodeoxyuridine (BrdU) incorporation assay, Boyden chamber cell migration assay, spheroid sprouting assay, and Matrigel-based tube formation assay, were employed to evaluate the influence of PDA on the proliferation and motility of smooth muscle cells (SMCs). Molecular docking simulations and co-immunoprecipitation assays were conducted to examine protein interactions. The results revealed that PDA exacerbates vascular injury-induced pathological remodeling, as evidenced by enhanced neointima formation. PDA treatment significantly increased the proliferation and migration of SMCs. Further mechanistic studies disclosed that PDA upregulated myeloid differentiation factor 88 (MyD88) expression in SMCs and interacted with T-cadherin (CDH13). This interaction augmented proliferation, migration, and extracellular matrix deposition, culminating in pathological vascular remodeling. Our findings underscore the critical role of PDA in the regulation of pathological vascular remodeling, mediated through the MyD88/CDH13 signaling pathway.

Artemisia argyi extract subfraction exerts an antifungal effect against dermatophytes by disrupting mitochondrial morphology and function / 中国天然药物

Artemisia argyi (A. argyi), a plant with a longstanding history as a raw material for traditional medicine and functional diets in Asia, has been used traditionally to bathe and soak feet for its disinfectant and itch-relieving properties. Despite its widespread use, scientific evidence validating the antifungal efficacy of A. argyi water extract (AAWE) against dermatophytes, particularly Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum, remains limited. This study aimed to substantiate the scientific basis of the folkloric use of A. argyi by evaluating the antifungal effects and the underlying molecular mechanisms of its active subfraction against dermatophytes. The results indicated that AAWE exhibited excellent antifungal effects against the three aforementioned dermatophyte species. The subfraction AAWE6, isolated using D101 macroporous resin, emerged as the most potent subfraction. The minimum inhibitory concentrations (MICs) of AAWE6 against T. rubrum, M. gypseum, and T. mentagrophytes were 312.5, 312.5, and 625 μg·mL-1, respectively. Transmission electron microscopy (TEM) results and assays of enzymes linked to cell wall integrity and cell membrane function indicated that AAWE6 could penetrate the external protective barrier of T. rubrum, creating breaches ("small holes"), and disrupt the internal mitochondrial structure ("granary"). Furthermore, transcriptome data, quantitative real-time PCR (RT-qPCR), and biochemical assays corroborated the severe disruption of mitochondrial function, evidenced by inhibited tricarboxylic acid (TCA) cycle and energy metabolism. Additionally, chemical characterization and molecular docking analyses identified flavonoids, primarily eupatilin (131.16 ± 4.52 mg·g-1) and jaceosidin (4.17 ± 0.18 mg·g-1), as the active components of AAWE6. In conclusion, the subfraction AAWE6 from A. argyi exerts antifungal effects against dermatophytes by disrupting mitochondrial morphology and function. This research validates the traditional use of A. argyi and provides scientific support for its anti-dermatophytic applications, as recognized in the Chinese patent (No. ZL202111161301.9).

Estrogen receptor beta suppresses the androgen receptor oncogenic effects in triple-negative breast cancer / 中华医学杂志(英文版)

BACKGROUND@#Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer associated with poor prognosis and limited treatment options. The androgen receptor (AR) has emerged as a potential therapeutic target for luminal androgen receptor (LAR) TNBC. However, multiple studies have claimed that anti-androgen therapy for AR-positive TNBC only has limited clinical benefits. This study aimed to investigate the role of AR in TNBC and its detailed mechanism.@*METHODS@#Immunohistochemistry and TNBC tissue sections were applied to investigate AR and nectin cell adhesion molecule 4 (NECTIN4) expression in TNBC tissues. Then, in vitro and in vivo assays were used to explore the function of AR and estrogen receptor beta (ERβ) in TNBC. Chromatin immunoprecipitation sequencing (ChIP-seq), co-immunoprecipitation (co-IP), molecular docking method, and luciferase reporter assay were performed to identify key molecules that affect the function of AR.@*RESULTS@#Based on the TNBC tissue array analysis, we revealed that ERβ and AR were positive in 21.92% (32/146) and 24.66% (36/146) of 146 TNBC samples, respectively, and about 13.70% (20/146) of TNBC patients were ERβ positive and AR positive. We further demonstrated the pro-tumoral effects of AR on TNBC cells, however, the oncogenic biology was significantly suppressed when ERβ transfection in LAR TNBC cell lines but not in AR-negative TNBC. Mechanistically, we identified that NECTIN4 promoter -42 bp to -28 bp was an AR response element, and that ERβ interacted with AR thus impeding the AR-mediated NECTIN4 transcription which promoted epithelial-mesenchymal transition in tumor progression.@*CONCLUSIONS@#This study suggests that ERβ functions as a suppressor mediating the effect of AR in TNBC prognosis and cell proliferation. Therefore, our current research facilitates a better understanding of the role and mechanisms of AR in TNBC carcinogenesis.

Monotropein Induced Apoptosis and Suppressed Cell Cycle Progression in Colorectal Cancer Cells / 中国结合医学杂志

OBJECTIVE@#To determine whether monotropein has an anticancer effect and explore its potential mechanisms against colorectal cancer (CRC) through network pharmacology and molecular docking combined with experimental verification.@*METHODS@#Network pharmacology and molecular docking were used to predict potential targets of monotropein against CRC. Cell counting kit assay, plate monoclonal assay and microscopic observation were used to investigate the antiproliferative effects of monotropein on CRC cells HCT116, HT29 and LoVo. Flow cytometry and scratch assay were used to analyze apoptosis and cell cycle, as well as cell migration, respectively in HCT116, HT29, and LoVo cells. Western blotting was used to detect the expression of proteins related to apoptosis, cell cycle, and cell migration, and the expression of proteins key to the Akt pathway.@*RESULTS@#The Gene Ontology and Reactome enrichment analyses indicated that the anticancer potential of monotropein against CRC might be involved in multiple cancer-related signaling pathways. Among these pathways, RAC-beta serine/threonine-protein kinase (Akt1, Akt2), cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase-9 (MMP9), epidermal growth factor receptor (EGFR), cell division control protein 42 homolog (CDC42) were shown as the potential anticancer targets of monotropein against CRC. Molecular docking suggested that monotropein may interact with the 6 targets (Akt1, Akt2, CDK6, MMP9, EGFR, CDC42). Subsequently, cell activity of HCT116, HT29 and LoVo cell lines were significantly suppressed by monotropein (P<0.05). Furthermore, our research revealed that monotropein induced cell apoptosis by inhibiting Bcl-2 and increasing Bax, induced G1-S cycle arrest in colorectal cancer by decreasing the expressions of CyclinD1, CDK4 and CDK6, inhibited cell migration by suppressing the expressions of CDC42 and MMP9 (P<0.05), and might play an anticancer role through Akt signaling pathway.@*CONCLUSION@#Monotropein exerts its antitumor effects primarily by arresting the cell cycle, causing cell apoptosis, and inhibiting cell migration. This indicates a high potential for developing novel medication for treating CRC.

Application of PolyPRep tools on HIV protease polyproteins using molecular docking / Aplicação da ferramenta PolyPRep em poliproteínas de protease de HIV usando docking molecular

Abstract In recent years, the development of high-throughput technologies for obtaining sequence data leveraged the possibility of analysis of protein data in silico. However, when it comes to viral polyprotein interaction studies, there is a gap in the representation of those proteins, given their size and length. The prepare for studies using state-of-the-art techniques such as Machine Learning, a good representation of such proteins is a must. We present an alternative to this problem, implementing a fragmentation and modeling protocol to prepare those polyproteins in the form of peptide fragments. Such procedure is made by several scripts, implemented together on the workflow we call PolyPRep, a tool written in Python script and available in GitHub. This software is freely available only for noncommercial users.

Resumo Nos últimos anos, o desenvolvimento de tecnologias de alto rendimento para obtenção de dados sequenciais potencializou a possibilidade de análise de dados proteicos in silico. No entanto, quando se trata de estudos de interação de poliproteínas virais, existe uma lacuna na representação dessas proteínas, devido ao seu tamanho e comprimento. Para estudos utilizando técnicas de ponta como o Aprendizado de Máquina, uma boa representação dessas proteínas é imprescindível. Apresentamos uma alternativa para este problema, implementando um protocolo de fragmentação e modelagem para preparar essas poliproteínas na forma de fragmentos de peptídeos. Tal procedimento é feito por diversos scripts, implementados em conjunto no workflow que chamamos de PolyPRep, uma ferramenta escrita em script Python e disponível no GitHub. Este software está disponível gratuitamente apenas para usuários não comerciais.

Potencial inibitório do ácido elágico sobre a formação do complexo Keap1-Nrf2: implicações terapêuticas em doenças crônico-degenerativas / Inhibitory potential of ellagic acid on the formation of the keap1-Nrf2 complex: Therapeutical implications in chronic-degenerative diseases

Objetivos ­ Avaliar o potencial inibitório do ácido elágico sobre as interações do complexo Keap1-Nrf2, com o intuito de esclarecer um dos eventuais mecanismos associado à atividade antioxidante do ácido elágico. Métodos ­ Foram empregadas simulações de docagem molecular para prever o modo de ligação do ácido elágico no sítio ligante da proteína Keap1, o qual foi comparado com o modo de ligação obtido experimentalmente e descrito na literatura para o ligante natural, a proteína Nrf2, e um potente inibidor monoácido do complexo Keap1-Nrf2. Resultados ­ As simulações de docagem revelaram que o ácido elágico apresenta potencial para realizar uma rede de ligações de hidrogênio com resíduos de aminoácidos da proteína Keap1 considerados importantes para o reconhecimento do Nrf2, se assemelhando ao perfil observado para inibidores do complexo Keap1-Nrf2 descritos na literatura. Conclusão ­ O ácido elágico apresenta características químicas e espaciais favoráveis para a inibição do complexo Keap1-Nrf2 e a elucidação do seu modo de ligação pode auxiliar na identificação de novos produtos naturais com propriedades antioxidantes e potencializar o desenvolvimento de fármacos contra doenças crônico-degenerativas.

Mannich bases derivatives of 2-Phenyl-5-Benzimidazole sulfonic acid; Synthesis, Characterization, Computational studies and Biological evaluation

Abstract A new series of N-Mannich bases of 2-Phenyl-5-benzimidazole sulfonic acid have been synthesized through amino methylation reaction with secondary amines. The two moieties were held together through a methylene bridge, which comes from formaldehyde (Formalin Solution 37%) used in the reaction. Chemical structures of the newly synthesized compounds have been confirmed using FT-IR, 1HNMR and 13CNMR. Different in vitro assays including Anti-oxidant, Enzyme inhibition, Anti-microbial and Cytotoxicity assay were performed to evaluate the biological potential with reference to the standard drug. Among the synthesized library, compound 3a shows maximum alpha-glucosidase inhibition with an IC50 value of 66.66 µg/ml, compound 3d was found most toxic with LC50 value of 10.17 µg/ml. ADME evaluation studies were performed with the help of Molinspiration online software. Docking calculations were also performed. Given the importance of the nucleus involved, the synthesized compound might find extensive medicinal applications as reported in the literature.

Fosfatase Cdc25B: Interações transientes intramoleculares e complexação com pequenos ligantes / Cdc25B phosphatase: Transient intramolecular interactions and small molecules complexation

Proteínas tirosina-fosfatase (PTPs) possuem papel fundamental na regulação da transdução de sinais e estão envolvidas em diversos processos fundamentais do ciclo celular. As Cdc25 (Cell Division Cycle 25) são fosfatases duais encontradas em todos os organismos eucarióticos e atuam em checkpoints do ciclo celular, permitindo ou inibindo o prosseguimento deste. Este grupo de proteínas pertence à classe de PTPs com atividade baseada em cisteína, apresenta domínio catalítico altamente conservado assim como o motivo catalítico, P-loop. Devido sua função, as Cdc25 são consideradas possíveis alvos terapêuticos para tratamento de câncer e sua interação com pequenas moléculas e inibidores tem sido investigada de forma que análises estruturais e de ligação das Cdc25 com inibidores podem elucidar aspectos importantes do mecanismo de ação destes além de direcionar para o desenho racional de fármacos. Interações cátion-π são interações intra ou intermoleculares não-covalentes que ocorrem entre uma espécie química catiônica, como o grupo guanidino de argininas, e uma das faces de um sistema π rico em elétrons, como dos anéis indólicos de triptofanos. Apesar de pouco discutidas na literatura, quando em comparação às interações não-covalentes mais convencionais, do ponto de vista energético as interações cátion-π são tão importantes na estruturação de proteínas quanto às ligações de hidrogênio ou pontes salinas. De fato estas interações são observadas com frequência em estruturas proteicas resolvidas. O domínio catalítico da Cdc25B possui diversas argininas expostas em sua superfície e um único resíduo de triptofano localizado na região C-terminal flexível, muito próximo do sítio catalítico da proteína. A flexibilidade de proteínas ou de regiões proteicas apresenta importante papel no reconhecimento entre biomoléculas participantes de vias de sinalização e tem sido muito estudada atualmente. Aqui, simulações de dinâmica molecular, experimentos de 1H-15N HSQC RMN, ensaios de cinética de inibição e de ancoragem molecular, evidenciam a existência de contatos cátion-π transientes na superfície de um importante membro da família das Cdc25, a Cdc25B, e de sítios de interação entre inibidores testados e a proteína com destaque a sítios na proximidades do P-loop, região próxima ao C-terminal desordenado, onde se demonstra estabilidade da interação com os pequenos ligantes

Protein tyrosine phosphatase (PTPs) play a fundamental role in the regulation of signal transduction and are involved in several fundamental processes of the cell cycle. Cdc25 (Cell Division Cycle 25) are dual phosphatases found in all eukaryotic organisms and act at checkpoints of the cell cycle, allowing or inhibiting its progression. This group of proteins belongs to the class of PTPs with cysteine-based activity, presenting a highly conserved catalytic domain as well as the catalytic motif, P-loop. Due to their function, Cdc25 are considered possible therapeutic targets for cancer treatment and their interaction with small molecules and inhibitors has been investigated so that structural and binding analyzes of Cdc25 with inhibitors can elucidate important aspects of their mechanism of action besides directing to rational drug design. Cation-π interactions are non-covalent intra- or intermolecular interactions that occur between a cationic chemical species, such as the guanidino group of arginines, and one of the faces of an electron-rich system, such as the indole rings of tryptophans. Although little discussed in the literature, when compared to more conventional non-covalent interactions, from the energetic point of view, cation-π interactions are as important in the structuring of proteins as hydrogen bonds or salt bridges. In fact, these interactions are frequently observed in solved protein structures. The catalytic domain of Cdc25B has several arginines exposed on its surface and a single tryptophan residue located in the flexible C-terminal region, very close to the catalytic site of the protein. The flexibility of proteins or protein regions plays an important role in the recognition between biomolecules participating in signaling pathways and has been extensively studied today. Here, molecular dynamics simulations, 1H-15N HSQC NMR experiments, inhibition kinetics and molecular anchoring assays, evidence the existence of transient cation-π contacts on the surface of an important member of the Cdc25 family, Cdc25B, and of sites of interaction between tested inhibitors and the protein, with emphasis on sites in the vicinity of the P-loop, a region close to the disordered C-terminus, where stability of the interaction with the small ligands is demonstrated

Expression of β-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance / 生物工程学报

The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

Improving the position specificity of Themomyces lanuginosus lipase based on semi-rational design / 生物工程学报

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.

Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis / 生物工程学报

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

Screening of small molecule inhibitors of IL-15Rα using molecular docking and surface plasmon resonance technology / 生理学报

The study aims to explore the active molecules of traditional Chinese medicine that specifically bind to interleukin-15 receptor α (IL-15Rα) using molecular docking and surface plasmon resonance (SPR) technology. AutoDock molecular docking software was used to perform simulated docking of more than 3 000 compounds from 48 traditional Chinese medicines at IL-15Rα and screen the specific binding compounds. Then Biocore T200 biomolecular interaction analysis system of SPR was used to confirm the binding specificity of the selected target compounds. Finally, the biological effects of the target compounds on IL-15Rα were verified by cell biological experiments. The results showed that neoprzewaquinone A (Neo) possessed the highest specific binding affinity among the active molecules from traditional Chinese medicine, and the dissociation constant (KD) value was (0.62 ± 0.20) µmol/L. The results of cell experiment showed that Neo significantly inhibited the proliferation of Mo7e cells induced by IL-15, and the IC50 was 1.075 µmol/L, approximately 1/120 of the IC50 of Cefazolin (IL-15 specific antagonist). These results suggest that Neo is a specific inhibitor of IL-15Rα and may be a potential active drug for the treatment of diseases related to the dysfunction of the IL-15Rα signaling.

Plasma components of Danzhi Xiaoyao Formula and its mechanism of action in treating perimenopausal depression based on UPLC-Q-TOF-MS~E integrated with network pharmacology / 中国中药杂志

In this study, ultra-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS~E) was used to analyze the plasma components of Danzhi Xiaoyao Formula after oral administration. Forty-nine plasma components were found in the serum of rats by comparing the compound extract, drug-containing serum, and blank serum. Components, such as 6-hydroxycoumarin, poricoic acid F, deoxoglabrolide, 30-norhederagenin, kanzonol R, 3',6'-di-O-galloylpaeoniflorin, 16α-hydroxytrametenolic acid, 16-deoxyporicoic acid B, 3-O-acetyl-16α-hydroxytrametenolic acid, and 16α,25-dihydroxydehydroeburiconic acid, were first found in rat serum. Behavioral tests, including the tail suspension test, novel object recognition test, and novelty-suppressed feeding test, were conducted for behavioral analysis. It was confirmed that this formula had therapeutic effects on perimenopausal depression. Furthermore, in combination with the network pharmacology method, 53 core targets including MAPK1, HRAS, AKT1, EGFR, and ESR1 were screened, and these targets participated in 165 signaling pathways, including PI3K-AKT, AMPK, VEGFA, MAPK, and HIF-1. In summary, the potential effects of Danzhi Xiaoyao Formula in treating perimenopausal depression are associated with mechanisms in accelerating inflammation repair, improving neuroplasticity, affecting neurotransmitters, regulating estrogen levels, and promoting new blood vessel formation.

Mechanism of anti-hyperplasia of mammary glands of Xihuang Pills blood-entering component based on UPLC-Q-TOF-MS and network pharmacology / 中国中药杂志

In this study, based on network pharmacology and molecular docking method, the mechanism of anti-hyperplasia of mammary glands of Xihuang Pills blood-entering components was explored, and the efficacy and key targets of Xihuang Pills blood-entering components were experimentally verified by MCF-10A proliferation model of human mammary epithelial cells. In order to clarify the material basis and mechanism of Xihuang Pills in realizing anti-hyperplasia of mammary glands, the blood-entering components of Xihuang Pills were qualitatively analyzed by UPLC-Q-TOF-MS, and 22 blood-entering components were identified. By taking the blood-entering components as the research object, the network pharmacology prediction and molecular docking verification were carried out, and finally, three key targets were screened out, namely JAK1, SRC, and CDK1. In vitro experiments show that Xihuang Pills can inhibit the proliferation of MCF-10A cells, promote the apoptosis of MCF-10A cells, and reduce the expression of JAK1, SRC, and CDK1 targets in cells. To sum up, Xihuang Pills can promote the apoptosis of mammary epithelial cells by regulating the expression of JAK1, SRC, and CDK1 and then play an anti-hyperplasia role, which provides an experimental basis for clarifying the material basis of Xihuang Pills for anti-hyperplasia effect.

Advancements in virtual screening techniques for study of enzyme inhibitor compounds / 中国中药杂志

Enzymes are closely associated with the onset and progression of numerous diseases, making enzymes a primary target in innovative drug development. However, the challenge remains in identifying compounds that exhibit potent inhibitory effects on the target enzymes. With the continuous expansion of the total number of natural products and increasing difficulty in isolating and enriching new compounds, traditional high-throughput screening methods are finding it increasingly challenging to meet the demands of new drug development. Virtual screening, characterized by its high efficiency and low cost, has gradually become an indispensable technology in drug development. It represents a prominent example of the integration of artificial intelligence with biopharmaceuticals and is an inevitable trend in the rapid development of innovative drug screening in the future. Therefore, this article primarily focused on systematically reviewing the recent applications of virtual screening technology in the development of enzyme inhibitors and explored the prospects and advantages of using this technology in developing new drugs, aiming to provide essential theoretical insights and references for the application of related technologies in the field of new drug development.

Comprehensive identification of metabolites and metabolic characteristics of luteolin and kaempferol in Simiao Yong'an Decoction in rats by UHPLC-LTQ-Orbitrap MS/MS / 中国中药杂志

Simiao Yong'an Decoction is a classic prescription for treating gangrene. Modern medical evidence has proven that Si-miao Yong'an Decoction has therapeutic effects on atherosclerosis(AS), vascular occlusion angeitides, and hypertension, while its pharmacodynamic mechanism remains unclear. The evidence of network pharmacology, molecular docking, literature review, and our previous study suggests that luteolin and kaempferol are two major flavonoids in Simiao Yong'an Decoction and can inhibit macrophage inflammation and exert anti-AS effects. However, due to lack of the metabolism studies in vivo, little is known about the metabolic characteristics of luteolin and kaempferol. This study employed ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS/MS) and relevant software to identify the metabolites and metabolic pathways of luteolin and kaempferol in rat plasma, urine, and feces, after oral administration of luteolin and kaempferol, respectively. After the administration of luteolin, 10, 11, and 3 metabolites of luteolin were detected in the plasma, urine, and feces, respectively. After the administration of kaempferol, 9, 3, and 1 metabolites of kaempferol were detected in the plasma, urine, and feces, respectively. The metabolic pathways mainly involved methylation, glucuronidation, and sulfation. This study enriches the knowledge about the pharmacological mechanism of luteolin and kaempferol and supplies a reference for revealing the metabolic process of other flavonoids in Simiao Yong'an Decoction, which is of great significance for elucidating the pharmacological effects and effective substances of this decoction in vivo.

Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells / 中国中药杂志

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Mechanism of Zhongfeng Xingnao Decoction in improving microcirculatory disorders in cerebral hemorrhage based on network pharmacology and molecular docking techniques / 中国中药杂志

This study aimed to explore the mechanism of Zhongfeng Xingnao Decoction(ZFXN) in intervening microcirculatory di-sorders in cerebral hemorrhage by network pharmacology and molecular docking techniques. The information on the components of ZFXN was obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and the predicted targets of chemical components were obtained from PubChem and SwissTargetPrediction. The relevant targets of cerebral hemorrhage and microcirculatory disorders were collected from the GeneCards database, and the common targets of the components and diseases were analyzed by the Database for Annotation, Visualization, and Integrated Discovery(DAVID) for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. Visualization of the correlation network was carried out using Cytoscape software to further screen important chemical components for molecular docking prediction with disease targets. The animal experiment validation was performed using modified neurological severity score(mNSS), enzyme-linked immunosorbent assay(ELISA), quantitative real-time polymerase chain reaction(qRT-PCR), immunofluorescence, and Western blot to detect the effects of ZFXN intervention in mice with cerebral hemorrhage. The results showed that there were 31 chemical components and 856 targets in the four drugs contained in ZFXN, 173 targets for microcirculatory disorders in cerebral hemorrhage, and 57 common targets for diseases and components. The enrichment analysis showed that common targets were mainly involved in biological processes, such as cell proliferation and apoptosis, and signaling pathways, such as tumor pathway, viral infection, phosphoinositide-3-kinase/protein kinase B(PI3K/AKT) signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway. Molecular docking results revealed that the common components β-sitosterol of Rhei Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Ginseng Radix et Rhizoma Rubra showed good docking with proto-oncogene tyrosine-protein kinase(SRC), signal transducer and activator of transcription 3(STAT3), phosphoinositide-3-kinase catalytic alpha polypeptide gene(PIK3CA), recombinant protein tyrosine phosphatase non receptor type 11(PTPN11), AKT1, epidermal growth factor receptor(EGFR), calcium adhesion-associated protein beta 1(CTNNB1), vascular endothelial growth factor A(VEGFA), and tumor protein p53(TP53). Moreover, sennoside E of Rhei Radix et Rhizoma showed good docking with MAPK1. The results revealed that the ZFXN relieved the neural injury in mice with cerebral hemorrhage, decreased the expression of S100 calcium-binding protein B(S100β), neuron specific enolase(NSE), matrix metalloproteinase 9(MMP9), tumor necrosis factor α(TNF-α), interleukin 1β(IL-1β), SRC, EGFR, CTNNB1, VEGFA, TP53, glial fibrillary acidic protein(GFAP), and leukocyte differentiation antigen 86(CD86), and increased the expression of p-PI3K, p-AKT, and zona occludens 1(ZO-1). The results indicate that ZFXN may inhibit neuronal apoptosis and inflammatory response through PI3K/AKT/p53 pathway to protect the blood-brain barrier, thereby slowing down microcirculatory impairment in cerebral hemorrhage.

Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway / 中国中药杂志

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

Mechanism of Gegen Qinlian Decoction in improving glucose metabolism in vitro and in vivo by alleviating hepatic endoplasmic reticulum stress / 中国中药杂志

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.