OBJECTIVE: To investigate the biological activity and antitumor effect of pegylated recombinant human interleukin 2 (PEG-rhIL-2) obtained by site-specific conjugation of polyethylene glycol (PEG) with non-natural amino acids, and to explore its antitumor mechanism. METHODS: The binding activities of PEG-rhIL-2 at three different sites (T41, Y45, and V91) to human interleukin 2 receptors α (IL-2R_α) and β (IL-2R_β) and were detected by surface plasmon resonance (SPR) technology. Western blot was used to detect the levels of the Janus kinase-signal transducer and activator of transcription 5 (JAK-STAT5) signaling pathway activated by different doses of rhIL-2 and PEG-rhIL-2 in CTTL-2 and YT cells. Blood was collected after a single administration in mice to detect the drug concentration at different time points and evaluate the pharmacokinetic parameters of Y45-PEG-rhIL-2. Mouse hepatoma cell line Hepa1-6, pancreatic cancer cell line Pan-02, and colon cancer cell line MC-38 were selected. Tumor models were constructed in C57BL/6 mice. Different doses of Y45-PEG-rhIL-2 and excipient control were administrated respectively to evaluate the tumor suppression effect of the drug. In the MC-38 colon cancer model, the tumor suppression effect of Y45-PEG-rhIL-2 combined with anti-programmed death-1 (PD-1) monoclonal antibody was evaluated. Hepa1-6 mouse tumor models were constructed and rhIL-2, Y45-rhIL-2 and Y45-PEG-rhIL-2 were administrated respectively. The proportion of tumor-infiltrating lymphocytes was analyzed by flow cytometry. RESULTS: The SPR detection results showed that the binding activities of PEG-rhIL-2 to IL-2R_α/IL-2R_β were both reduced. The affinity of Y45-PEG-rhIL-2 to IL-2R_α was reduced to approximately 1/250, and its affinity to IL-2R_β was reduced to 1/3. Western blot results showed that the activity of Y45-PEG-rhIL-2 in stimulating JAK-STAT5 signaling in CTLL-2 cells expressing heterotrimeric IL-2 receptor complex IL-2R_αβγwas reduced to approximately 1/300, while its activity in YT cells expressing heterodimeric IL-2 receptor complex IL-2R_βγwas reduced to approximately 1/3. The pharmacokinetic evaluation after a single dose in the mice showed that the elimination half-life of Y45-PEG-rhIL-2 was 17.7 h. Y45-PEG-rhIL-2 has pharmacokinetic characteristics superior to those of rhIL-2. Y45-PEG-rhIL-2 showed dose-dependent tumor suppression activity, and the combination of Y45-PEG-rhIL-2 and anti-PD-1 antibody had a better tumor-inhibiting effect than the single use of Y45-PEG-rhIL-2 or anti-PD-1 antibody. Flow cytometry analysis demonstrated that 72 h after the administration of Y45-PEG-rhIL-2, the proportion of tumor-infiltrating cytotoxic T lymphocytes (CD8⁺T cells) increased by 86.84%. At 120 h after administration, the ratio of CD8⁺T cells to regulatory T cells (Treg) increased by 75.10%. CONCLUSION Y45-PEG-rhIL-2 obtained by site-specific conjugation via non-natural amino acids changed its receptor binding activity and inhibited tumor growth in dose-dependent manner in multiple tumor models by regulating CD8⁺T cells.
Interleukin-2/pharmacokinetics* ; Animals ; Mice ; Humans ; Recombinant Proteins/pharmacology* ; Polyethylene Glycols/chemistry* ; Cell Line, Tumor ; Antineoplastic Agents/pharmacokinetics* ; Signal Transduction/drug effects* ; STAT5 Transcription Factor/metabolism* ; Interleukin-2 Receptor alpha Subunit/metabolism* ; Interleukin-2 Receptor beta Subunit/metabolism*

OBJECTIVE: To investigate the anti-inflammatory effect of rutaecarpine (RUT) on monosodium urate crystal (MSU)-induced murine peritonitis in mice and further explored the underlying mechanism of RUT in lipopolysaccharide (LPS)/MSU-induced gout model in vitro. METHODS: In MSU-induced mice, 36 male C57BL/6 mice were randomly divided into 6 groups of 8 mice each group, including the control group, model group, RUT low-, medium-, and high-doses groups, and prednisone acetate group. The mice in each group were orally administered the corresponding drugs or vehicle once a day for 7 consecutive days. The gout inflammation model was established by intraperitoneal injection of MSU to evaluate the anti-gout inflammatory effects of RUT. Then the proinflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and the proportions of infiltrating neutrophils cytokines were detected by flow cytometry. In LPS/MSU-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and proinflammatory cytokines were measured by ELISA. The percentage of pyroptotic cells were detected by flow cytometry. Respectively, the mRNA and protein levels were measured by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot, the nuclear translocation of nuclear factor κB (NF-κB) p65 was observed by laser confocal imaging. Additionally, surface plasmon resonance (SPR) and molecular docking were applied to validate the binding ability of RUT components to tumor necrosis factor α (TNF-α) targets. RESULTS: RUT reduced the levels of infiltrating neutrophils and monocytes and decreased the levels of the proinflammatory cytokines interleukin 1β (IL-1β) and interleukin 6 (IL-6, all P<0.01). In vitro, RUT reduced the production of IL-1β, IL-6 and TNF-α. In addition, RT-PCR revealed the inhibitory effects of RUT on the mRNA levels of IL-1β, IL-6, cyclooxygenase-2 and TNF-α (P<0.05 or P<0.01). Mechanistically, RUT markedly reduced protein expressions of tumor necrosis factor receptor (TNFR), phospho-mitogen-activated protein kinase (p-MAPK), phospho-extracellular signal-regulated kinase, phospho-c-Jun N-terminal kinase, phospho-NF-κB, phospho-kinase α/β, NOD-like receptor thermal protein domain associated protein 3 (NLRPS), cleaved-cysteinyl aspartate specific proteinase-1 and cleaved-gasdermin D in macrophages (P<0.05 or P<0.01). Molecularly, SPR revealed that RUT bound to TNF-α with a calculated equilibrium dissociation constant of 31.7 µmol/L. Molecular docking further confirmed that RUT could interact directly with the TNF-α protein via hydrogen bonding, van der Waals interactions, and carbon-hydrogen bonding. CONCLUSION RUT alleviated MSU-induced peritonitis and inhibited the TNFR1-MAPK/NF-κB and NLRP3 inflammasome signaling pathway to attenuate gouty inflammation induced by LPS/MSU in THP-1 macrophages, suggesting that RUT could be a potential therapeutic candidate for gout.
Animals ; NF-kappa B/metabolism* ; Male ; Indole Alkaloids/therapeutic use* ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Inflammation/complications* ; Uric Acid ; Quinazolines/therapeutic use* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Humans ; Gout/chemically induced* ; Inflammasomes/metabolism* ; Cytokines/metabolism* ; THP-1 Cells ; Mitogen-Activated Protein Kinases/metabolism* ; Mice ; Molecular Docking Simulation ; Lipopolysaccharides ; Quinazolinones

Sonodynamic therapy (SDT) can potentially induce immunogenic cell death in tumor cells, leading to the release of ATP, and facilitating the initiation of an immune response. Nevertheless, the enzymes CD39 and CD73 can swiftly convert ATP into immunosuppressive adenosine (ADO), resulting in an immunosuppressive tumor microenvironment (TME). This study introduced a nanomedicine (QD/POM1@NP@M) engineered to reprogram TME by modulating the CD39/CD73/ADO pathway. The nanomedicine encapsulated sonosensitizers silver sulfide quantum dots, and the CD39 inhibitor POM1, while also incorporating homologous tumor cell membranes to enhance targeting capabilities. This integrated approach, on the one hand, stimulates the release of ATP via SDT, thereby initiating the immune response. In addition, it reduced the accumulation of ADO by inhibiting CD39 activity, which ameliorated the immunosuppressive TME. Upon administration, the nanomedicine demonstrated substantial anti-tumor efficacy by facilitating the infiltration of anti-tumor immune cells, while reducing the immunosuppressive cells. This modulation effectively transformed the TME from an immunologically "cold" state to a "hot" state. Furthermore, combined with the checkpoint inhibitor α-PDL1, the nanomedicine augmented systemic anti-tumor immunity and promoted the establishment of long-term immune memory. This study provides an innovative strategy for combining non-invasive SDT and ATP-driven immunotherapy, offering new ideas for future cancer treatment.

Ergopeptines or their derivatives are widely used for treating neurodegenerative and cerebrovascular diseases. The nonribosomal peptide synthetase-d-lysergyl peptide synthetase A (LPSA) determines ergopeptine formation but the detailed mechanism remains to be elucidated. Here, we characterized two LPSAs from Claviceps purpurea Cp-1 strain through heterologous expression in Aspergillus nidulans feeding with d-lysergic acid. We proved that Cp-LPSA1 catalyzed the formation of ergocornine, α-ergocryptine, and β-ergocryptine, precisely controlled by the substrate specificity of its three modules. Cp-LPSA2 was initially inactive but could be restored to catalyze α-ergosine formation. Using this platform, we validated that P1-LPSA1 and P1-LPSA2 from the reported C. purpurea P1 strain catalyzed ergotamine and α-ergocryptine formation, respectively. Typically, the non-ribosomal peptide codes implicated in every module of the LPSAs were defined and elucidated, in which certain key residues could play a switched role for substrate specificity and product interconversion. By constructing chimeric LPSAs through module assembly, the production of the desired ergopeptines was achieved. Notably, 1.46 mg/L of α-ergocryptine and 1.09 mg/L of ergotamine were produced respectively by mixed-culture of C. paspali No. 24 (fermentation supernatant) and the recombinants of A. nidulans. Our findings provide insights into the biosynthetic mechanism of ergopeptines and lay a foundation for directed ergopeptine biosynthesis.

Natural products are important sources of drug discovery. However, the traditional methods of extraction and isolation, as well as chemical synthesis for obtaining natural products are associated with issues such as operational complexity, high costs, low efficiency, and environmental pollution. Constructing microbial cell factories through synthetic biology methods to produce medicinal natural products has the advantages of high efficiency, low cost, and environmental protection. Nevertheless, the scope and yield improvement of the products are limited by the limitations of enzymes in microbial cell factories. Protein engineering is considered one of the most effective approaches to overcome these limitations. This article introduces commonly used methods of protein engineering technology and summarizes its specific applications in improving enzyme performance, modifying the enzymatic environment, and promoting the development of synthetic biology tools in the field of pharmaceutical natural product synthesis. Furthermore, it analyzes the current bottlenecks and challenges in protein engineering and looks forward to its future application prospects, offering insights for the development and practical use of protein engineering technology.

Nicotinic acetylcholine receptors (nAChRs) belong to ligand-gated ion channel receptors, of which α7 nAChR subtype is widely distributed in the cerebral cortex, thalamus, hippocampus, and also identified in microglia, macrophages, bone marrow cells, etc. Previous studies revealed that α7 nAChR is closely related to the function of the cholinergic anti-inflammatory pathway, and is a vital target for drug development of Alzheimer's disease and schizophrenia. The establishment of a stable α7 nAChR in vitro drug screening system is crucial for the efficient screening of novel drugs targeting this target. Recombinant expression of different subtypes of nAChRs on Xenopus laevis oocyte membranes and current detected by two-electrode voltage clamp (TEVC) is an advanced and complex model for novel drug screening. Molecular chaperones can assist the assembly of some nAChR subunits to form functional receptors, providing a stable expression model for the screening of compounds targeting this receptor. In this study, a molecular chaperone gene of α7 nAChR, transmembrane protein 35A (Tmem35a), was isolated and cloned from rats. We constructed the recombinant expression vector and obtained the cRNA of Tmem35a by in vitro transcription technique. Two cRNAs (Tmem35a and α7) were mixed and injected into X. laevis oocytes for expression. Then, the effects of this molecular chaperone on the current expression and pharmacological properties of α7 nAChR were evaluated by the TEVC. The results revealed that TMEM35A, also known as novel acetylcholine receptor chaperone (NACHO) could effectively increase the expression of α7 nAChR protein on oocyte membranes, and the amount of α7 nAChR protein was increased about 1-fold. The peak current induced by agonist acetylcholine (ACh) was increased about 10-fold. After injection of Tmem35a cRNA, the median effect concentration (EC₅₀) value of α7 nAChR to agonist ACh is 228.5 μmol·L^-1, which shows almost no difference from native α7 nAChR (EC₅₀: 223.3 μmol·L^-1), indicating the preservation of the normal properties of α7 nAChR. The results of this investigation indicate that the molecular chaperone NACHO effectively assists the heterologous expression of α7 nAChR in X. laevis oocytes, which provides a model for screening the potency of lead compounds targeting α7 nAChR. All animal experiments in this study were reviewed and approved by the Ethics Committee of Guangxi University (approval number: GXU-2023-0249).

N-type voltage-gated calcium (Ca²⁺) channels (N-type VGCC, Ca_V2.2) mediate Ca²⁺ influx in response to action potential at the presynaptic terminal, and play an important role in synaptogenesis, neurotransmitter release and nociceptive signal transduction. It is a new target for the development of drugs for the treatment of neuralgia (chronic pain) and other major diseases. Due to the difficulty of calcium channel expression in vitro and the detection of channel current, there is a great lack of new drug screening models. In this study, we established and optimized the electrophysiological drug screening model using Xenopus laevis oocytes for the recombinant expression of Ca_V2.2 in vitro (this study were reviewed and approved by the Ethics Committee of Guangxi University, approval number: GXU-2023-0249). Firstly, the linear plasmids encoding cDNA of major subunit α1B and auxiliary subunits α2δ1 and β3 of rat Ca_V2.2 were used as templates for in vitro transcription to generate their related mRNA (cRNA), after which three kinds of cRNA were injected into Xenopus laevis oocytes at the mass ratio of 2∶1∶1 for expression. The two-electrode voltage clamp (TEVC) technique was used to detect the inward current produced by Ca_V2.2. At the same time, the expression conditions of Ca_V2.2 were optimized, and its gating function was characterized from the aspects of channel activation and inactivation. The results showed that 3-5 days after cRNA microinjection, stable Ca_V2.2-mediated barium ion (Ba²⁺) currents were successfully detected. The interference of endogenous potassium channels and Ca²⁺-activated chloride channels can be eliminated by tetraethylammonium hydroxide (TEAOH) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (BAPTA-AM) treatment. The maximum potential for Ca_V2.2 activation is 0 mV, and the current reverses to be outward when the membrane potential is greater than +50 mV. By fitting the steady-state activation and inactivation curves, the half-maximal activation potential and half-maximal inactivation potential of Ca_V2.2 are identified as -15.9 and -60.2 mV. In this study, a stable Ca_V2.2 expression system was established based on Xenopus laevis oocytes. The in vitro expression system can provide a new way for the screening of Ca_V2.2 active compounds or lead drugs.

Objective To study the bioequivalence and safety of two olmesartan medoxomil and hydrochlorothiazide tablets in Chinese healthy subjects.Methods A total of 24 healthy subjects underwent fasting and postprandial tests in a single-center,randomized,open-label,single-dose,two-formulation,two-sequence,two-period,self-cross-over controlled design.The subjects were administered a single oral dose of the test formulation and reference formulation(each containingolmesartan medoxomil 20 mg and hydrochlorothiazide 12.5 mg)in a random cross-over fashion.The plasma concentrations of olmesartan and hydrochlorothiazide were determined by LC-MS/MS.The non-compartmental model analysis of olmesartan and hydrochlorothiazide was conducted using WinNonlin 7.0 software to calculate pharmacokinetic parameters and assess bioequivalence.Results In the fasting test,the pharmacokinetic parameters of olmesartan of test and reference were as follows:Cmax were(798.35±206.78)and(664.52±168.25)ng·mL-1,AUC0-t were(4 430.71±1 294.87)and(3 976.67±1 083.54)h·ng·mL-1,AUC0-∞ were(4 551.67±1 303.06)and(4 090.37±1 103.97)h·ng·mL-1.The pharmacokinetic parameters of hydrochlorothiazide of test and reference were as follows:Cmax were(92.39±35.96)and(96.15±38.76)ng·mL-1,AUC0_t were(548.69±217.11)and(564.41±208.68)h·ng·mL-1,AUC0-∞ were(603.04±228.59)and(619.26±223.27)h·ng·mL-1.In the fed test,the pharmacokinetic parameters of olmesartan of T and R were as follows:Cmax were(583.15±149.48)and(550.57±104.76)ng·mL-1,AUC0-t were(3 585.18±952.72)and(3 292.19±904.58)h·ng·mL-1,AUC0-∞ were(3 696.05±996.55)and(3 396.30±923.41)h·ng·mL-1.The pharmacokinetic parameters of hydrochlorothiazide of test and reference were as follows:Cmax were(70.30±17.88)and(74.70±21.65)ng·mL-1,AUC0-t were(476.60±119.39)and(492.91±144.81)h·ng·mL-1,AUC0-∞ were(523.37±132.67)and(535.81±151.92)h·ng·mL-1.In fasting and fed condition,the 90％confidence interval(90％CI)of Cmax,AUC0-t and AUC0-∞ of olmesartan and hydrochlorothiazide were in 80.00％-125.00％.Conclusion The two olmesartan medoxomil and hydrochlorothiazide tablets were bioequivalent under fasting and fed conditions,and good security.

ObjectiveTo investigate the protective effect of the extract of Liuwei Dihuangwan （LW） on mitochondrial damage in the Alzheimer's disease （AD） model of Caenorhabditis elegans （C. elegans）. MethodC. elegans transfected with human β-amyloid protein （Aβ） _1-42 gene was used as an AD model. The rats were divided into blank group， model group， metformin group （50 mmol·L^-1）， and low， medium， and high dose （1.04， 2.08， 4.16 g·kg^-1） LW groups. Behavioral methods were used to observe the sensitivity of 5-hydroxytryptamine （5-HT） in nematodes. Western blot was used to detect the expression of Aβ in nematodes. Total ATP content in nematodes was detected by the adenine nucleoside triphosphate （ATP） kit， and mitochondrial membrane potential was detected by the JC-1 method. In addition， the mRNA expression of Aβ expression gene （Amy-1）， superoxide dismutase-1 （SOD-1）， mitochondrial transcription factor A homologous gene-5 （HMG-5）， mitochondrial power-associated protein 1 （DRP1）， and mitochondrial mitoprotein 1 （FIS1） was detected by real-time fluorescence quantitative polymerase chain reaction （RT-PCR）. ResultThe extract of LW could reduce the hypersensitivity of the AD model of nematodes to exogenous 5-HT （P<0.05） and delay the AD-like pathological characteristics of hypersensitivity to exogenous 5-HT caused by toxicity from overexpression of Aβ in neurons of the AD model of nematodes. Compared with the blank group， in the model group， the mRNA expression of Aβ protein and Amy-1 increased （P<0.01）， and the mRNA expression of SOD-1 and HMG-5 decreased （P<0.01）. The mRNA expression of DRP1 and FIS1 increased （P<0.01）， and the level of mitochondrial membrane potential decreased （P<0.05）. The content of ATP decreased （P<0.01）. Compared with the model group， in the positive medicine group and medium and high dose LW groups， the mRNA expression of Aβ protein and Amy-1 decreased （P<0.05，P<0.01）， and the mRNA expression of SOD-1 and HMG-5 increased （P<0.01）. The mRNA expression of DRP1 decreased （P<0.05，P<0.01）， and that of FIS1 decreased （P<0.01）. The level of mitochondrial membrane potential increased （P<0.01）， and the content of ATP increased （P<0.05，P<0.01）. ConclusionThe extract of LW may enhance the antioxidant ability of mitochondria， protect mitochondrial DNA， reduce the fragmentation of mitochondrial division， repair the damaged mitochondria， adjust the mitochondrial membrane potential， restore the level of neuronal ATP， and reduce the neuronal damage caused by Aβ deposition.

In recent years, polysaccharides have received much attention because of their high safety and good immunological activity. The study of polysaccharide in vivo process is a key scientific problem that needs to be solved for polysaccharide drug development. Some progress has been made in the field of polysaccharide pharmacokinetics and immunomodulation. However, due to the lack of both chromogenic and light-absorbing groups and the complex molecular structure of polysaccharides, the in vivo processes and immunomodulatory mechanisms of polysaccharides have been slow to be investigated. The effective combination of multiple techniques can break the bottleneck of difficult tracing and unknown immunomodulatory mechanism of polysaccharides in vivo, and promote the development and utilization of polysaccharides. In this paper, we systematically summarize the key techniques in the study of polysaccharide in vivo processes and immunomodulatory mechanisms in order to provide technical references and research ideas for the study of polysaccharide in vivo processes and immunomodulatory mechanisms.