Inteins are unique protein insertion sequences capable of self-excision, enabling the covalent ligation of flanking extein peptides via amide bond formation. This process proceeds spontaneously without requiring external enzymes, cofactors, or chemical reagents, granting inteins exceptional biocompatibility and traceless performance in protein engineering applications. Split inteins represent a specialized and versatile subclass whose splicing domains are encoded by two separate gene fragments rather than a single continuous open reading frame. These fragments, known as the N-terminal (IntN) and C-terminal (IntC) split inteins, associate through non-covalent interactions including hydrophobic forces, hydrogen bonds, and van der Waals forces to assemble into an active three-dimensional structure, which then drives efficient extein ligation and enables protein trans-splicing. Protein trans-splicing mediated by split inteins has become a cornerstone for traceless protein ligation owing to its high specificity and irreversibility, fundamentally reshaping strategies for protein modification, assembly, and functional regulation. Compared with traditional chemical ligation methods, split intein systems require no complex chemical derivatization of peptide fragments and can operate efficiently at micromolar concentrations under physiological conditions, thus avoiding structural and functional damage caused by organic reagents. In contrast to enzymatic ligation tools such as sortase, split inteins eliminate the need for additional enzymes or cofactors, simplifying reaction systems, reducing costs, and minimizing non-specific side products. These distinctive advantages render split inteins highly promising for applications in chemical biology, synthetic biology, and biopharmaceutical development. In recent years, deepened mechanistic understanding has established structure-guided rational design as the primary approach to overcoming key limitations of split inteins, including intrinsic aggregation propensity, strict extein sequence dependence, and limited splicing efficiency. Bioinformatic tools have been used to identify aggregation-prone regions in the IntN fragment, and site-directed mutagenesis of hydrophobic residues, relocation of split sites, or removal of misfolding-prone sequences has substantially reduced in vitro aggregation and improved soluble expression and assembly activity. Rational engineering of catalytic residues and adjacent flexible loops has relaxed strict amino acid preferences at extein junctions, enhancing sequence tolerance and reducing the risk of functional impairment in target proteins. Consensus design based on multiple sequence alignments has yielded ultra-fast splicing variants such as Cfa DnaE and Cat-TerL, which exhibit significantly accelerated kinetics and improved tolerance to denaturing conditions. Meanwhile, advances in structural biology have further clarified the conformational dynamics and catalytic mechanisms of splicing, supporting the precise design of high-performance intein modules. On this basis, electrostatic interaction tuning and metagenomic screening have yielded multiple mutually orthogonal split intein pairs, enabling selective multi-fragment protein ligation and providing new routes for the efficient synthesis of large multi-domain functional proteins. With these engineered split inteins offering continuously improved performance and expanded applicability, protein trans-splicing has been widely applied in numerous cutting-edge areas of protein research and biomedicine. In gene delivery, split intein-based systems overcome the packaging limit of adeno-associated viral vectors, enabling the accurate reconstitution of large therapeutic proteins and base editors in target cells, thereby enhancing the efficacy and scope of gene therapy for genetic diseases. In internal protein sequence editing, split inteins mediate precise sequence replacement and modification in flexible regions or loops of target proteins, without the need for complex multi-step ligation and protein refolding involved in traditional protein semisynthesis. In protein-protein interaction studies, intein-mediated splicing covalently captures transient and weak intracellular complexes, enabling sensitive, high-throughput interaction detection and drug screening. In synthetic biology, conditionally controllable splicing systems support the construction of diverse intracellular and cell-surface biological logic gates for the precise regulation of cellular behavior. In mechanistic biochemical research, split inteins enable photocatalytic proximity labeling and site-specific tagging, allowing the preparation of homogeneous protein samples carrying precise post-translational modifications such as ubiquitination and polyglutamylation for chromatin interactome analysis and epigenetic studies. Moreover, covalent trapping strategies using split inteins stabilize transient enzymatic intermediates, providing unprecedented insights into molecular mechanisms such as nucleosome ubiquitination that are difficult to elucidate using conventional methods. This review systematically summarizes key technological advances in split inteins over the past decade, highlighting engineering strategies, mechanistic insights, and the development of orthogonal components. It comprehensively surveys emerging applications at the frontiers of protein research, analyzes current core challenges, and proposes future directions, particularly emphasizing artificial intelligence-driven de novo design and novel splicing pathways to break existing technical bottlenecks. By enabling traceless, efficient, and versatile protein manipulation, split inteins continue to serve as indispensable tools that drive innovation in protein engineering and fundamental life science research.

Body weight abnormalities, including overweight, obesity, and underweight, have become a dual public health challenge in Chinese adults: overweight and obesity lead to a variety of chronic complications, while underweight increases the risks of malnutrition, sarcopenia, and organ dysfunction. To systematically address these issues, multidisciplinary experts in endocrinology, sports science, nutrition, and psychiatry from various regions have held multiple weight management seminars. Based on the latest epidemiological data and clinical evidence, they expanded the guideline to include assessment and intervention strategies for underweight, in addition to the core content of obesity management. This guideline outlines the etiological mechanisms, evaluation methods, and multidimensional management strategies for overweight and obesity, covering key areas such as diagnosis and assessment, medical nutrition therapy, exercise prescription, pharmacological intervention, and psychological support. It is intended to provide a scientific and standardized approach to weight management across the adult population, aiming to curb the rising prevalence of obesity, mitigate complications associated with abnormal body weight, and improve nutritional status and overall quality of life.

OBJECTIVE: To evaluate the anti-hepatocellular carcinoma (HCC) activity of total alkaloids from Gelsemium elegans Benth. (TAG) in vivo and in vitro and to elucidate their potential mechanisms of action through transcriptomic analysis. METHODS: TAG extraction was conducted, and the primary components were quantified using high-performance liquid chromatography (HPLC). The effects of TAG (100, 150, and 200 µg/mL) on various tumor cells, including SMMC-7721, HepG2, H22, CAL27, MCF7, HT29, and HCT116, were assessed. Effects of TAG on HCC proliferation and apoptosis were detected by colony formation assays and cell stainings. Caspase-3, Bcl-2, and Bax protein levels were detected by Western blotting. In vivo, a tumor xenograft model was developed using H22 cells. Totally 40 Kunming mice were randomly assigned to model, cyclophosphamide (20 mg/kg), TAG low-dose (TAG-L, 0.5 mg/kg), and TAG high-dose (TAG-H, 1 mg/kg) groups, with 10 mice in each group. Tumor volume, body weight, and tumor weight were recorded and compared during 14-day treatment. Immune organ index were calculated. Tissue changes were oberseved by hematoxylin and eosin staining and immunohistochemistry. Additionally, transcriptomic and metabolomic analyses, as well as quatitative real-time polymerase chain reaction (RT-qPCR), were performed to detect mRNA and metabolite expressions. RESULTS: HPLC successfully identified the components of TAG extraction. Live cell imaging and analysis, along with cell viability assays, demonstrated that TAG inhibited the proliferation of SMMC-7721, HepG2, H22, CAL27, MCF7, HT29, and HCT116 cells. Colony formation assays, Hoechst 33258 staining, Rhodamine 123 staining, and Western blotting revealed that TAG not only inhibited HCC proliferation but also promoted apoptosis (P<0.05). In vivo experiments showed that TAG inhibited the growth of solid tumors in HCC in mice (P<0.05). Transcriptomic analysis and RT-qPCR indicated that the inhibition of HCC by TAG was associated with the regulation of the key gene CXCL13. CONCLUSION TAG inhibits HCC both in vivo and in vitro, with its inhibitory effect linked to the regulation of the key gene CXCL13.
Animals ; Apoptosis/drug effects* ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/genetics* ; Humans ; Alkaloids/therapeutic use* ; Gelsemium/chemistry* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Mice ; Xenograft Model Antitumor Assays

OBJECTIVE: To explore the therapeutic effects and underlying mechanisms of Dendrobium officinale (Tiepi Shihu) extract (DOE) on insomnia. METHODS: Forty-two male Sprague-Dawley rats were randomly divided into 6 groups (n=7 per group): normal control, model control, melatonin (MT, 40 mg/kg), and 3-dose DOE (0.25, 0.50, and 1.00 g/kg) groups. Rats were raised in a strong-light (10,000 LUX) and -noise (>80 db) environment (12 h/d) for 16 weeks to induce insomnia, and from week 10 to week 16, MT and DOE were correspondingly administered to rats. The behavior tests including sodium pentobarbital-induced sleep experiment, sucrose preference test, and autonomous activity test were used to evaluate changes in sleep and emotions of rats. The metabolic-related indicators such as blood pressure, blood viscosity, blood glucose, and uric acid in rats were measured. The pathological changes in the cornu ammonis 1 (CA1) region of rat brain were evaluated using hematoxylin and eosin staining and Nissl staining. Additionally, the sleep-related factors gamma-aminobutyric acid (GABA), glutamate (GA), 5-hydroxytryptamine (5-HT), and interleukin-6 (IL-6) were measured using enzyme linked immunosorbent assay. Finally, we screened potential sleep-improving receptors of DOE using polymerase chain reaction (PCR) array and validated the results with quantitative PCR and immunohistochemistry. RESULTS: DOE significantly improved rats' sleep and mood, increased the sodium pentobarbital-induced sleep time and sucrose preference index, and reduced autonomic activity times (P<0.05 or P<0.01). DOE also had a good effect on metabolic abnormalities, significantly reducing triglyceride, blood glucose, blood pressure, and blood viscosity indicators (P<0.05 or P<0.01). DOE significantly increased the GABA content in hippocampus and reduced the GA/GABA ratio and IL-6 level (P<0.05 or P<0.01). In addition, DOE improved the pathological changes such as the disorder of cell arrangement in the hippocampus and the decrease of Nissel bodies. Seven differential genes were screened by PCR array, and the GABA_A receptors (Gabra5, Gabra6, Gabrq) were selected for verification. The results showed that DOE could up-regulate their expressions (P<0.05 or P<0.01). CONCLUSION DOE demonstrated remarkable potential for improving insomnia, which may be through regulating GABA_A receptors expressions and GA/GABA ratio.
Animals ; Dendrobium/chemistry* ; Rats, Sprague-Dawley ; Male ; Sleep Initiation and Maintenance Disorders/blood* ; Plant Extracts/therapeutic use* ; Receptors, GABA-A/metabolism* ; Noise/adverse effects* ; Light/adverse effects* ; gamma-Aminobutyric Acid/metabolism* ; Sleep/drug effects* ; Rats ; Receptors, GABA/metabolism*

Functional dyspepsia (FD), characterized by persistent or recurrent dyspeptic symptoms without identifiable organic, systemic or metabolic causes, is an increasingly recognized global health issue. The objective of this guideline is to equip clinicians and nursing professionals with evidence-based strategies for the management and treatment of adult patients with FD using traditional Chinese medicine (TCM). The Guideline Development Group consulted existing TCM consensus documents on FD and convened a panel of 35 clinicians to generate initial clinical queries. To address these queries, a systematic literature search was conducted across PubMed, EMBASE, the Cochrane Library, China National Knowledge Infrastructure (CNKI), VIP Database, China Biology Medicine (SinoMed) Database, Wanfang Database, Traditional Medicine Research Data Expanded (TMRDE), and the Traditional Chinese Medical Literature Analysis and Retrieval System (TCMLARS). The evidence from the literature was critically appraised using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. The strength of the recommendations was ascertained through a consensus-building process involving TCM and allopathic medicine experts, methodologists, pharmacologists, nursing specialists, and health economists, leveraging their collective expertise and empirical knowledge. The guideline comprises a total of 43 evidence-informed recommendations that span a range of clinical aspects, including the pathogenesis according to TCM, diagnostic approaches, therapeutic interventions, efficacy assessments, and prognostic considerations. Please cite this article as: Zhang SS, Zhao LQ, Hou XH, Bian ZX, Zheng JH, Tian HH, Yang GH, Hong WS, He YY, Liu L, Shen H, Li YP, Xie S, Shu J, Zeng BF, Li JX, Liu Z, Xiao ZH, Xiao JD, Zheng PY, Huang SG, Chen SL, Fei GJ. International clinical practice guideline on the use of traditional Chinese medicine for functional dyspepsia (2025). J Integr Med. 2025; 23(5):502-518.
Dyspepsia/drug therapy* ; Humans ; Medicine, Chinese Traditional/methods* ; Practice Guidelines as Topic ; Drugs, Chinese Herbal/therapeutic use*

Objective To establish a rapid,sensitive,and specific multiplex PCR detection method for the simultaneous detection of Cryptosporidium,Eimeria,and bovine parvovirus.Methods Specific primers targeting the SSU rRNA genes of Cryptosporidium and Eimeria,as well as the VP2 gene of bovine parvovirus were designed and the corresponding recombinant plasmid standards were constructed.To establish the multiplex PCR method,the reaction conditions were optimized using temperature gradient PCR and single-variable control methods.The sensitivity,specificity,reproducibility,and clinical application of the protocol were evaluated.Results The optimal annealing temperature was found to be 60.5℃,and the forward and reverse primer concentrations were determined to be 0.2 μmol/L for Eimeria,and 0.4 μmol/L for Cryptosporidium and bovine parvovirus.The assay demonstrated high sensitivity,with detection limits of 243,260,and 3 110 copies for the recombinant plasmid standards of Cryptosporidium,Eimeria,and bovine parvovirus,respectively.Specificity testing showed no cross-reactivity with ten common bovine pathogens,including Salmonella,bovine viral diarrhea virus,and bovine rotavirus.Consistent intra-and inter-batch results confirmed the strong reproducibility of the method.Clinical application to 81 diarrhea samples from various regions in the Ganzi Prefecture,Sichuan,revealed positivity rates of 18.52%(15/81)for Cryptosporidium,34.57%(28/81)for Eimeria,and 18.52%(15/81)forbovineparvovirus,withamixedinfectionrateof3.7%(3/81).Conclusions Themultiplex PCR method established in this study offers a reliable tool for differential diagnosis and epidemiological investigation of the three common diarrheal pathogens in yaks.

This study was aimed at determining the molecular characteristics of Yersinia pestis in the natural plague foci around Qinghai Lake through single nucleotide polymorphism technology,to lay a foundation for molecular epidemiological and source-tracing analysis of Y.pestis in this area.Using the whole genome sequencing technology,we obtained the whole genome sequences of 84 representative Y.pestis strains.Using the sequences of Y.pestis and Yersinia pseudotuberculosis IP32953 from the NCBI database as references,we compared and analyzed the 2 298 SNP loci of these strains.From 1957 to 2020,84 representative strains of Y.pestis from the natural plague foci around Qinghai Lake were divided into two clades:1.IN2 and 3.ANT1.The 1.IN2 clade was the characteristic population of Y.pestis throughout all epidemic years in this area.Additionally,analysis of the SNP distribution and hosts in the region indicated that the 1.IN2 clade was located in five counties except Wulan,whereas the 3.ANT1 clade was isolated from Himalayan marmot and dog in two counties.In conclusion,the population structure of SNP of Y.pestis in the natural plague foci around Qinghai Lake is relatively simple,and SNP analysis of Y.pestis provided a scientific basis for tracing plague epidemic sources and formulating plague prevention and control measures in this area.

Objective To investigate the relationship between prolactin and mild cognitive impairment(MCI)in postmenopausal women with type 2 diabetes mellitus(T2DM).Methods A total of 319 postmenopausal women with T2DM who were hospitalized in the Department of Endocrinology,Drum Tower Hospital,Affiliated Hospital of Medical School,Nanjing University were enrolled in this study from August 2016 to October 2023.All the patients were divided into two groups according whether they had MCI:T2DM group(n=161)and MCI group(n=158).Differences in clinical characteristics were compared between the two groups.Pearson correlation was used to analyze the correlation between sex hormones and cognitive domains,and Logistic regression analysis was used to evaluate the influencing factors for MCI development.Results Serum prolactin levels were significantly lower in the MCI group than in the T2DM group[(5.5±2.1)vs(7.2±2.9)μg/L,P<0.05].Serum prolactin level was positively correlated with mini-mental state examination score,Montreal cognitive assessment score,immediate memory score,visuopatial constructional score,attention score and hippocampal volume(P<0.05),and negatively correlated with processing speed test(time)and executive function test(time)(P<0.05).Logistic regression analysis demonstrated that serum prolactin level was an influencing factor for the risk of MCI in postmenopausal women with T2DM(OR 0.715,95%CI 0.605～0.845,P<0.01).Conclusions The decrease of serum prolactin level is associated with an increased risk of MCI in postmenopausal women with T2DM.

This study aims to investigate the ameliorating effect of Corni Fructus(CF) on the myocardial ischemic injury and the pharmacokinetic properties of characteristic components of CF. The mouse model of isoproterenol-induced myocardial ischemia was established and administrated with the aqueous extract of CF. The general efficacy of CF in ameliorating the myocardial ischemic injury was evaluated based on the cardiac histopathology and the levels of myocardial injury markers: creatine kinase isoenzyme(CK-MB) and cardiac troponin I(cTn-I). The metabolomics analysis was carried out for the heart and serum samples of mice to screen the biomarkers of CF in ameliorating the myocardial ischemic injury and then the predicted biomarkers were submitted to metabolic pathway enrichment. The pharmacokinetic analysis was performed for morroniside, loganin, and cornuside Ⅰ in mouse heart and serum samples to obtain the pharmacokinetic parameters of these components. The pharmacokinetic parameters were then integrated on the basis of self-defined weighting coefficients to simulate an integrated pharmacokinetic profile of CF iridoid glycosides in the heart and serum of the mouse model of myocardial ischemia. The results indicated that CF reduced the pathological damage to cardiac cells and tissue(hematoxylin-eosin staining) and lowered the levels of CK-MB and cTn-I in the serum of the mouse model of myocardial ischemia(P<0.01). Metabolomics analysis screed out 31 endogenous metabolites in the heart and 35 in the serum as biomarkers of CF in ameliorating the myocardial ischemic injury. These biomarkers were altered by modeling and restored by CF. Six metabolic pathways in the heart and 5 in the serum were enriched based on these metabolic markers. The main integrated pharmacokinetic parameters of CF iridoid glycosides were T_(max)=1 h, t_(1/2)=(1.52±0.05) h in the heart and T_(max)=1 h, t_(1/2)=(1.56±0.50) h in the serum. Both concentration-time curves showed a double-peak phenomenon. In conclusion, CF demonstrated the cardioprotective effect by regulating metabolic pathways such as taurine and hypotaurine metabolism, and pantothenic acid and coenzyme A biosynthesis. The integrated pharmacokinetics reflect the general pharmacokinetic properties of characteristic components in CF.
Animals ; Cornus/chemistry* ; Mice ; Metabolomics ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Myocardial Ischemia/metabolism* ; Humans ; Troponin I/metabolism* ; Myocardium/pathology* ; Disease Models, Animal ; Biomarkers/metabolism* ; Creatine Kinase, MB Form/metabolism*

Grounded in the theory of "all marrows dominated by brain", this study explored the therapeutic mechanism of Zuogui Pills in modulating the oxytocin(OXT)/oxytocin receptor(OXTR) feed-forward loop in the treatment of postmenopausal osteoporosis(PMOP). A PMOP rat model was established using ovariectomy, and 70 Sprague-Dawley female rats were randomly divided into the following groups: sham operation group, model group, estradiol group(17β-estradiol, 0.05 mg·kg~(-1)·d~(-1)), Zuogui Pills low, medium, and high dose groups(0.2, 0.4, 0.8 g·kg~(-1)·d~(-1), respectively), and an antagonist group(atosiban 0.9 mg·kg~(-1)·d~(-1) + 17β-estradiol 0.05 mg·kg~(-1)·d~(-1) + Zuogui Pills 0.4 g·kg~(-1)·d~(-1)). After 12 weeks of model establishment, treatment was administered by gavage once daily for another 12 weeks, followed by sample collection. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of estrogen(E_2), OXT, tartrate-resistant acid phosphatase(TRACP-5b), and bone alkaline phosphatase(BALP). Histopathological changes in the left distal femur were observed through hematoxylin and eosin(HE) staining. Micro-computed tomography(micro-CT) was used to analyze the microstructure of the right distal femur. Western blot was employed to detect the expression levels of OXTR, small GTP-binding protein Ras, Raf1 proto-oncogene(Raf1), mitogen-activated protein kinase kinase 1/2(MEK1/2), and extracellular signal-regulated kinase 1/2(ERK1/2), and their phosphorylated forms in tibial tissues. Compared with the model group, the Zuogui Pills medium and high dose groups showed significantly increased levels of E_2, OXT, and BALP, with a notable decrease in TRACP-5b levels. Morphologically, the trabeculae in the left distal femur were more tightly arranged. The fibrous structure in the right distal femur was significantly improved in the Zuogui Pills high dose group. Additionally, the expression of OXTR, Ras, p-Raf1, p-MEK1/2, and p-ERK1/2 proteins in tibial tissues was significantly increased. The therapeutic effect of the Zuogui Pills high dose group was partially inhibited when an OXTR antagonist was administered. These findings suggest that Zuogui Pills can regulate the OXT/OXTR feed-forward loop, activate the phosphorylation of the downstream Ras/Raf1/MEK/ERK signaling pathway, and ultimately improve bone mineral density, thereby exerting therapeutic effects in PMOP.
Animals ; Rats, Sprague-Dawley ; Rats ; Female ; Drugs, Chinese Herbal/administration & dosage* ; Oxytocin/genetics* ; Receptors, Oxytocin/genetics* ; Humans ; Osteoporosis, Postmenopausal/genetics* ; Bone and Bones/drug effects* ; Brain/drug effects* ; Bone Marrow/drug effects*