Acute kidney injury (AKI) is a prevalent clinical syndrome characterized by a rapid deterioration in renal function. Naringin, a flavonoid abundant in the Rutaceae family, has been reported to provide protective effects against kidney injury. However, the mechanisms responsible for these effects remain inadequately elucidated. In the present study, the AKI mouse model was established in vivo through a single intraperitoneal injection of 20 mg·kg^-1 of cisplatin, and human kidney-2 (HK-2) cell injury was induced by cisplatin in vitro. Blood urea nitrogen (BUN) and creatinine (CRE) levels were measured using an ELISA kit. Hematoxylin and eosin (H&E) staining, along with periodic acid-Schiff (PAS) staining, were employed to evaluate changes in renal histopathology. Serum concentrations of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the mice were assessed using ELISA, and the levels of these cytokines were further analyzed. Morphological alterations in HK-2 cells were examined microscopically. The levels of reactive oxygen species (ROS) in HK-2 cells were quantified using an ELISA kit. The protein expression levels of p-MAPK/MAPK, p-JNK/JNK, p-ERK/ERK, p-NF-κB/NF-κB, p-IκBα/IκBα, IL-6, and TNF-α in renal tissue and HK-2 cells were evaluated through Western blot. The results showed that the serum BUN, Cre, IL-6, and TNF-α levels in the model group mice increased and the renal tissue structure was damaged when compared to the control group, whereas naringin could significantly ameliorate the above pathological changes. Moreover, naringin significantly reduced cisplatin-induced ROS production in HK-2 cells. Western blot results confirmed that naringin notably attenuated the expression of p-MAPK, p-JNK, p-ERK, p-NF-κB, p-IκBα, IL-6, and TNF-α proteins in vitro and in vivo. In conclusion, naringin may ameliorate cisplatin-induced AKI by inhibiting the activation of the MAPK/NF-κB pathway and reducing the production of ROS. All animal experiments were approved by the Institutional Animal Care and Use Committee of Hubei University of Chinese Medicine (approval No: HUCMS202204001).

To explore the standardization of the in vitro Nb2-11 cell proliferation bioassay for recombinant human growth hormone (rhGH), we first tested the biological activity of 2 batches of rhGH drug substances, 3 batches of rhGH for injection, and 24 batches of rhGH injections, based on which we proposed the experimental effective criteria. Furthermore, we conducted methodological validation in accordance with General Rule 9401 of the 2020 edition of the Pharmacopoeia of the People's Republic of China. Subsequently, the inter-laboratory consistency, intra-laboratory and inter-laboratory precision of seven labs were studied by determining the in vitro bioactivity of six batches of rhGH products and two batches of rhGH drug substances from four different manufacturers using this method. The consistency of the in vitro and in vivo bioassays was assessed by evaluating 28 batches of rhGH drug substances with both methods. Finally, multiple batches of rhGH samples with different expiration dates were tested to determine the standard limits of the bioassay. The results of this collaborative study indicate that the Nb2-11 cell bioassay exhibits good intra- and inter-laboratory reproducibility, with a 100% pass rate for the experimental effective criteria. The Nb2-11 cell bioassay had high consistency with in vivo animal bioassay. Animal experiments were approved by the Experimental Animal Welfare Ethics Committee of China National Institute of Food and Drug Control (approval number: NIFDC (Fu) No. 2024 (B) 004). This method is applicable to rhGH drug substances and products from different manufacturers. Collectively, the Nb2-11 cell bioassay can be used as a standardized method for determining the biological activity of rhGH products, replacing in vivo animal methods for quality control and release testing.

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).

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.

The HIV-1 capsid protein (CA) is an effective target for antiviral drug discovery. Targeting the capsid assembly inhibitor peptide (CAI) binding pocket in the C-terminal domain of HIV-1 CA (CA CTD) can be used to screen for CA protein inhibitors. In this study, we found that the small molecule suramin targets HIV-1 CA by binding to this pocket, with an IC₅₀ value of 2.1 μmol·L^-1 for inhibiting the CA CTD-CAI interaction, based on homogeneous time-resolved fluorescence (HTRF) technology. Bio-layer interferometry (BLI) experiments demonstrated that suramin binds directly to CA, with a binding affinity to CA hexamer (K_D = 248 nmol·L^-1) higher than to CA monomer (K_D = 227 μmol·L^-1). In addition, in vitro CA protein assembly assays showed that suramin promotes disordered multimerization of CA protein. Binding model analysis revealed that suramin binds to the interface between adjacent CA monomers in the CA hexamer through the N57, Q63, Q67 and Y169 residues, thereby enhancing CA multimerization. In summary, suramin is a CA protein inhibitor that targets the CA hexamer and can serve as a starting point for the discovery of new CA inhibitors.

A detection method using UHPLC-MS/MS was established for the determination of 53 prohibited veterinary drug residues in goat horns, including 16 tranquilizers, 14 β-agonists, chloramphenicol, 4 fluoroquinolones, 7 nitroimidazoles, 3 quinoxalines, and 8 other compounds. Samples were extracted using salting-out assisted liquid-liquid extraction (SALLE). A Zorbax Eclipse Plus C18 column (1.8 μm, 3.0 mm × 150 mm, Agilent) was used with 0.1% formic acid solution-acetonitrile as the mobile phase for gradient elution. Detection was performed in positive and negative electrospray ionization modes using multiple reaction monitoring (MRM). All 53 analytes showed good linearity within their respective concentration ranges, with correlation coefficients above 0.99. The average recoveries at three spiked levels ranged from 70.4% to 118.7%, with relative standard deviations (RSDs) ranging from 0.69% to 12.07%. The limits of detection (LODs) ranged from 0.1 to 50 μg·kg^-1, and the limits of quantitation (LOQs) ranged from 0.2 to 100 μg·kg^-1. This method has been applied to the determination of real samples.

The purpose of this study is not only to investigate the effects of Angelica sinensis polysaccharide (ASP) as a potential vaccine adjuvant on immune activation and cytokine release in RAW264.7 macrophages, but also to elucidate its underlying involved signaling mechanisms. Cell viability was evaluated by the CCK-8 assay. Flow cytometry was used to analyze the influence of ASP at five distinct concentration gradients on the expression of cluster of differentiation (CD) 80, CD86, and major histocompatibility complex Ⅱ (MHC Ⅱ) on RAW264.7 cell surfaces. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA) method. Molecular techniques, including quantitative polymerase chain reaction (qPCR) were utilized to assess the mRNA expression levels of Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-TNF receptor associated factor 6 (TRAF6)-nuclear factor kappa-B (NF-κB) signaling pathway. The levels of TRAF6, and the phosphorylation levels of IκB kinase (IKK) and p65 proteins were detected by Western blot. The results show that ASP at varying concentrations promote the proliferation of RAW264.7 cells without cytotoxicity. Surface molecules CD80, CD86, and MHC Ⅱ on RAW264.7 cells showed statistically significant up-regulation in response to ASP compared to the blank control (P < 0.05), with a dose-dependent effect within an optimal range. Furthermore, ASP also elevated cytokines IL-6 and TNF-α secretion levels by RAW264.7 cells compared to the normal control (P < 0.05), exhibiting a dose-response relationship within a specific concentration span. The qPCR results indicated that ASP groups at different concentrations all led to upregulation of mRNA expression levels of TLR4, MyD88, TRAF6, and NF-κB signaling pathway. The expression levels of TRAF6, p-IKK and p-p65 were increased by different concentrations of ASP. The TLR4 inhibitor TAK-242 significantly reduced the secretion of cytokines induced by APS (P < 0.05). This study highlights the immunostimulatory properties of ASP, emphasizing its potential as a vaccine adjuvant. By significantly enhancing the expression of co-stimulatory molecules and cytokines via the TLR4-MyD88-TRAF6-NF-κB signaling pathway, ASP offers a promising approach for modulating immune responses.

In this thesis, we propose to establish a phage display method for screening adalimumab (ADM)-specific binding peptide to provide a particular element of recognition for clinical studies of ADM, and to successfully establish an indirect enzyme-linked immunosorbent assay (i-ELISA) based on this peptide for clinical detection of ADM. With ADM as the target molecule, five rounds of peptide elution were performed using phage display technology by putting it into the M13 linear dodecapeptide library. By measuring the titer of each round of eluate, it was found that the recovery rate increased from 7.28×10^-6 to 1.55×10^-3, indicating that the peptides binding to ADM in the eluate were continuously enriched, and the enrichment effect was better; the fifth round of screening amplicons were sequenced, and it was found that five peptide sequences appeared with high frequency, which were considered to be potentially able to bind specifically to ADM and were synthesised; the specific binding ability of the five peptide sequences to ADM was verified and identified by surface plasmon resonance (SPR) and ELISA experiments, respectively, and the results showed that the first peptide sequence (Pep1) and the second peptide sequence (Pep2) showed specific binding to ADM, with the affinity constants (K_D) of 7.91×10^-5 mol·L^-1 and 1.67×10^-5 mol·L^-1, respectively, and of which the affinity constant of Pep1 with ADM isotype antibody IgG was 1.35×10^-4 mol·L^-1, which was one order of magnitude lower than that with ADM, and thus Pep1 was determined to be a specific binding peptide for ADM. Based on Pep1, an indirect ELISA method for the analysis of the antibody-drug ADM was successfully established. With the increasing concentration of ADM the OD₄₅₀ value showed a regular change, and the method can be used for clinical ADM blood concentration monitoring. In this study, we obtained a peptide that can specifically bind to ADM, and this peptide sequence can be used as a specific recognition element for ADM, which not only enables the monitoring of ADM blood concentration, but also provides an effective tool for the accurate qualitative and quantitative quantification of ADM in vivo and ex vivo, and at the same time provides a new strategy and idea for the discovery of recognition elements of other monoclonal antibody drugs.