To establish the cells stably co-expressing NOD1 receptor and enhanced green fluorescent protein (EGFP)-tagged nuclear factor of activated T cells 2 nuclear factor (NFAT2) (EGFP-NFAT2) in U2OS cell, the NOD1 (NM_006092) pcDNA3.1-3×Flag-hygro recombinant plasmid was transfected into U2OS-EGFP-NFAT2 cells, which were screened by pressure of hygromycin B and then incubated with NOD1 agonist lipopolysaccharides (LPS) for 30 min, and the green fluorescence intensity in the nucleus of the cells was detected by the high content screening assay. There were 46 cell strains expressing NOD1 in U2OS-EGFP-NFAT2 cells by EGFP-NFAT2 nuclear translocation assay. Among these cells, cells No 4 had the highest nuclear translocation function. Therefore, it was selected as the U2OS-EGFP-NFAT2-NOD1 cell for functional validation. The expression levels of NOD1 mRNA and protein in the selected U2OS-EGFP-NFAT2-NOD1 cells and the control cell U2OS-EGFP-NFAT2 were examined by real-time quantitative PCR (RT-qPCR) and Western blot. The results showed that NOD1 mRNA was stably expressed in this stably transfected cell line for 5-20 generations, and NOD1 protein was expressed in U2OS-EGFP-NFAT2-NOD1 stably transfected cell line, whereas no NOD1 protein was expressed in the control cell U2OS-EGFP-NFAT2. U2OS-EGFP-NFAT2-NOD1 cells were treated with histones or LPS for 30 min, and the EGFP-NFAT2 nuclear translocation was detected by the high content screening assay. Histones were found to significantly increase the EGFP-NFAT2 nuclear translocation in U2OS-EGFP-NFAT2-NOD1 stably transfected cells over a range of concentrations. The U2OS-EGFP-NFAT2-NOD1 cells were divided into the solvent control group, NOD1 receptor antagonist nodinitib-1+histone group, and histone group. The drug incubation time was 30 min, and the specificity of the NOD1 cells was verified by observing the EGFP-NFAT2 nuclear translocation through the high content screening assay. Compared with the histones group, the nodinitib-1+histones group significantly decreased EGFP-NFAT2 nuclear translocation in U2OSEGFP-NFAT2-NOD1 cells (P < 0.05). In conclusion, U2OS-EGFP-NFAT2-NOD1 cells stably co-expressing NOD1 and EGFP-NFAT2 are established, which can be used for screening antagonistic compounds targeting NOD1 pathogenic microorganisms with mechanism study.

Indigo naturalis [Baphicacanthus cusia (Nees) Bremek., QD], as a traditional Chinese medicine, has exhibited efficacy in the ulcerative colitis (UC). Cu Dian (CD), the current form of Indigo naturalis, has been regarded as the mainstream form of medicine today. Dian Hua (DH) is the traditional purified form of QD, in which the content of indigo and indirubin is higher than that of CD. The study evaluated the efficacy of DH and CD in UC and explored their mechanism. Male BALB/c mice were subjected to an 8-day regimen of 3% dextran sodium sulphate (DSS) drinking water to induce UC. The experiment was approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (approval number: 2024075). Concurrently, the mice received intragastric administration of CD (400, 200, and 100 mg·kg^-1) and DH (400, 200, 100, and 50 mg·kg^-1) for the same duration. The anti-inflammatory properties of CD and DH were evaluated by quantifying levels of myeloperoxidase (MPO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-18 in colon tissue. Western blot and immunofluorescence assays were employed to assess the protein levels of zonula occludens 1 (ZO-1) and occludin. Additionally, Western blot and RT-qPCR were utilized to analyze the protein and gene expression levels of AMP-activated protein kinase (AMPK), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), and related factors in colon tissue. CD and DH demonstrated a capacity to alleviate inflammatory responses in mice with UC. The protective impact of both CD and DH on the intestinal mucosal barrier was associated with an elevation of ZO-1 and occludin. Furthermore, the anti-inflammatory effects of CD and DH were attributed to the inhibition of the NLRP3 inflammasome through the activation of the AMPK/silent information regulator of transcription 1 (SIRT1) pathway. Notably, DH exhibited a more pronounced improvement in UC compared to CD, particularly at the dosage of DH-M (200 mg·kg^-1). Our investigation substantiates the effectiveness of CD and DH in mitigating DSS-induced UC in mice. They demonstrated a capacity to diminish the production of inflammatory cytokines and safeguard the integrity of the intestinal epithelial barrier, notably by elevating level of tight junctions. The anti-colonic inflammatory effects of CD and DH were elucidated through the inhibition of both the formation and activation of the NLRP3 inflammasome, mediated by the AMPK/SIRT1 pathway.

Conjugated estrogens (CE) are widely used in menopausal hormone therapy (MHT) for the relief of menopause-related symptoms (e.g., vasodilatory symptoms, neuropsychiatric symptoms, genitourinary atrophy symptoms, etc.) and the prevention of postmenopausal osteoporosis. This article reviews the characteristics, pharmacological effects, and practical applications of different dosage forms of CE (tablets, creams, and injections) in clinical practice. It has been shown that tablet CE effectively regulates the body's temperature center through systemic treatment, reduces hot flashes and night sweats, promotes bone formation, and increases bone density. Cream CE is used for local treatment to improve the genitourinary syndrome of menopause (GSM). Injectable CE is used for the treatment of abnormal uterine bleeding caused by hormonal imbalance without organ pathology. CE administered intravenously is indicated only for short-term use to rapidly and temporarily increase estrogen levels. Additionally, CE plays an important role in metabolism by improving insulin clearance and sensitivity and potentially protecting the nervous and cardiovascular systems, demonstrating a wide range of therapeutic potential. This paper provides new ideas for the design and development of drug dosage forms by exploring the formulation and application of CE.

Autophagy is an important physiological process that can degrade cellular components and maintain cellular homeostasis. In the process of cancer development, autophagy plays a dual role in promoting or inhibiting autophagy, and targeting autophagy is considered to be an important means of cancer treatment. According to Chinese medicine theory, autophagy has the function of regulating the balance of Yin and Yang, and the balance of good and evil in the organism, and based on the theory of "supporting the positive and dispelling the evil", the use of active ingredients of traditional Chinese medicine (TCM) to target autophagy has been proven to be effective in the treatment of cancer. In addition, autophagic cell death, as a type Ⅱ programmed cell death, is often accompanied by autophagic features, and the regulation of autophagic cell death is an important way for autophagy to achieve anti-tumor effects. In recent years, more and more studies have found that the active ingredients of TCM have good effects in cancer treatment, among which, targeting autophagic cell death is an important way for TCM active ingredients to achieve anti-tumor effects. This paper outlines the understanding of cancer and autophagy in Chinese medicine theory, and summarizes the current Chinese medicine small molecule compounds targeting autophagic cell death and their mechanisms of action based on the classification of natural medicines. Finally, the development of Chinese medicine-derived compounds targeting autophagic cell death for the treatment of diseases is summarized and prospected, which hopefully can provide clues for subsequent exploration and research.

Resiquimod (R848) belongs to the class of weak base organic compounds that are derivatives of imidazole quinoline and is a potent agonist of Toll-like receptor (TLR) 7/8. R848 can be considered an effective immune adjuvant as it has the ability to activate different immune cells and regulate innate and adaptive immunity. A number of researches done recently have reported R848 potent anti-tumor capacity, especially when combined with other cytotoxic therapies. Nonetheless, some hurdles remain with the clinical use of R848 such as its limited ability to reach the tumor and likelihood of inflammation and autoimmune responses which might result from repeated delivery of high dosages of the drug. Nanoscale drug delivery systems may overcome some of these challenges. Various nanoformulations, including liposomes, polymeric nanoparticles, biocarriers, inclusion compound, metal-organic frameworks and inorganic nanoparticles, as well as several R848 prodrugs or derivatives-baesd nanoparticles, have been incorporated into present R848 delivery platforms. This article reviews the physicochemical properties, immune regulatory mechanisms and nano drug delivery systems of R848, in order to provide reference and support for anti-tumor research and new drug development.

Tumor immunotherapy represented by blocking programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) has gained better therapeutic effect in lung cancer treatment, and the development of small molecule drugs to block PD-1/PD-L1 provides a new strategy for lung cancer immunotherapy. In this study, we applied an in vitro cell model to investigate the regulation of PD-L1 expression and the mechanism of action of baicalin on A549 lung cancer cells. The effects of baicalin on the viability of A549 cells were detected by CCK8 assay; the expression of PD-L1 protein in A549 cells was detected by Western blot; the mechanism of baicalin cytotoxicity and its correlation with PD-L1 protein expression were investigated by using small-molecule inhibitors of apoptosis and autophagy; the formation of autophagic vesicles in A549 cells treated with baicalin was observed under transmission electron microscope. And the alterations of acid autophagic vesicles and autophagic lysosomes were observed under fluorescence microscope. The results of CCK8 experiments showed that baicalin (12.5-400 μmol·L^-1) inhibited the proliferation of A549 cells in a dose-dependent manner; at the same time, the elevation of PD-L1 protein expression after interferon-γ (IFN-γ) interference could be reduced with the increase of baicalin concentration; on the other hand, treatment with autophagy inhibitors, wortmannin, and chloroquine, could call back the baicalin-induced cytotoxicity, transmission electron microscopy and fluorescence microscopy showed that the number of autophagic vesicles increased after baicalin treatment of A549 cells, and Western blot results showed that baicalin promoted the expression of autophagy-related proteins LC3-Ⅱ/Ⅰ and beclin-1; the number of baicalin-induced acidic autophagic vesicles in A549 cells was attenuated after the intervention of wortmannin, and at the same time the LC3-Ⅱ/Ⅰ expression was inhibited and the inhibitory effect on PD-L1 was regulated. The above results suggest that baicalin may exert antitumor effects by activating the autophagy protein LC3 in A549 cells to reduce the expression of PD-L1. This study lays the foundation for baicalin to have the ability to be developed as a small molecule blocker of the PD-1/PD-L1 signaling pathway.

This study aimed to clarify the mechanism and active components of Buyang Huanwu Decoction (BYHWD) in alleviating cerebral ischemia reperfusion injury (CIRI) by inhibiting pyroptosis. The key components and targets of BYHWD for CIRI were identified via network pharmacological analysis, followed by molecular docking performed with Autodock and Pymol software. The effects of BYHWD and its active components were validated in vivo and in vitro. A middle cerebral artery occlusion (MCAO) model was established in mouse to assess neural function alterations in mice under various conditions. Concurrently, an oxygen-glucose deprivation/reperfusion (OGD/R) model was developed utilizing mouse brain tissue astrocytes in vitro. Molecular biology experiments were used to verify the predicted key targets. We have determined that the principal components of BYHWD are baicalein and β-sitosterol. By analyzing genes associated with CIRI pathology alongside those linked to pyroptosis, 20 intersecting genes were identified. In conjunction with molecular docking binding energy assessment, TP53 and TNF emerged as pivotal core targets for subsequent validation. Molecular biology experiments confirmed that BYHWD effectively alleviates injury while reducing the expression level of P53. These findings indicate that the primary bioactive constituents of BYHWD were baicalein and β-sitosterol. In addition, BYHWD may inhibit pyroptosis via TNF and TP53 in protecting CIRI. The experiment has been approved by the Experimental Animal Welfare Ethics Committee of Zhejiang Academy of Traditional Chinese Medicine, approval number (KTSC2020037, KTSC2023030).

Non-small cell lung cancer (NSCLC) is the primary pathological type of lung cancer. Osimertinib, as a third-generation EGFR-TKI (epidermal growth factor receptor-tyrosine kinase inhibitor) targeted drug, effectively prolong the progression-free survival of patients with EGFR-mutated NSCLC. However, drug resistance limits the efficacy of osimertinib. Traditional Chinese medicine can effectively delay the resistance to EGFR-TKIs. In this study, Cucurbitacin B (CuB) was investigated to analyze its pharmacological effects in osimertinib-resistant NSCLC cells through cell viability, migration, and invasion experiments. Public databases were used to screen potential targets of CuB in osimertinib-resistant NSCLC cells, and the interactions between CuB and potential targets were verified through molecular docking, cellular thermal shift assay (CETSA), and microscale thermophoresis (MST). Western blot was used to detect the effects of CuB on downstream pathways of the targets. The results showed that CuB significantly reduced the proliferation activity of osimertinib-resistant NSCLC cells and inhibited the migration and invasion abilities of tumor cells. Mechanistically, CuB inhibited the expression of ERK and AKT molecules by binding to the tyrosine kinase receptor AXL. In summary, CuB exhibits resistance to osimertinib-resistant NSCLC by inhibiting the migration and invasion of resistant cells through regulating the abnormal activation of the AXL-ERK/AKT axis. This study provides a basis for the pre-clinical in vitro efficacy of CuB in the treatment of osimertinib targeted resistance in NSCLC and theoretical support for the development of clinical drug combinations.

Prenylated aromatic natural products (PANPs) are widely distributed in nature. PANPs exhibit a great structural diversity due to their various core scaffolds (coumarin, lignan, benzoic acid/benzyl alcohol, flavonoid, xanthone, anthraquinone, and aromatic alkaloid, etc.) and the distinct types and substitution sites of isoprenoid moieties which may possess either linear or cyclic structures. The structural diversity of PANPs endow them with various bioactivities including anti-bacterial, anti-oxidation, anti-cancer, anti-inflammatory and analgesic effects, which makes them a group of highly promising molecules for drug development. Notably, isoprenoid moieties are often the indispensable pharmacophores in these active PANPs. Aromatic prenyltransferases (aPTs) are responsible for prenylation in the biosynthesis of PANPs. aPTs can be divided into three classes according to their evolutionary relationships and structural features, i.e. membrane-bound aPTs (UbiA type), soluble aPTs with a PT barrel structure (ABBA type and DMATS type) and terpene synthase-like aPTs. Herein, we summarize 94 aPTs belonging to the different classes which were characterized in the past ten years, in particular introduce their substrate selectivity/tolerance, regioselectivity, evolutionary relationships and structural features. This would provide cues for discovery and engineering of new aPTs, and modification and bio-production of active PANPs.

The study investigates the therapeutic effects and mechanisms of Buyang Huanwu Decoction (BHD) in treating ischemic stroke (IS). Using a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model, we evaluated the neuroprotective effects of BHD, demonstrating significant improvements in neurological function scores, prolonged rotarod retention time, and reductions in both infarct volume and brain water content. An unsupervised clustering algorithm was employed to identify active components of BHD by clustering them with FDA-approved drugs for ischemic stroke treatment. Combined with network pharmacology analysis, the mechanisms of these active components were predicted to be associated with anti-inflammatory pathways. Further validation using a lipopolysaccharide (LPS)-induced BV-2 cell model demonstrated the anti-inflammatory efficacy of seven key active components, with their effects on anti-inflammatory activity and cell viability assessed via the Griess and MTT assays. Additionally, the content of these active components in BHD was quantified using liquid chromatography-mass spectrometry (LC-MS). In conclusion, this study elucidates the critical active components of BHD and their potential pharmacological mechanisms, providing valuable insights for the modernization of traditional Chinese medicine and its application in ischemic stroke therapy. All animal experiments were approved by the Animal and Medical Ethics Committee of Northeastern University (approval No.: NEU-EC-2023A052S).