ObjectiveThe widespread adoption of portable fundus cameras for primary care and community screening is hindered by limitations in current autofocus(AF) technologies. Image-based methods relying on sharpness evaluation require iterative searches, resulting in slow convergence, while projection-based techniques are susceptible to optical artifacts and calibration errors. To address these challenges, this study introduces a novel AF system based on direct wavefront sensing, designed to deliver simultaneous high speed, high precision, and operational robustness within the compact form factor essential for portable ophthalmic devices. MethodsOur approach fundamentally reimagines the AF process by directly measuring the ocular wavefront aberration. We developed a custom portable fundus camera integrating a miniaturized Shack-Hartmann wavefront sensor (SHWS) into the optical path. An 850 nm laser diode projects a point source onto the retina via oblique illumination to minimize corneal reflections. Light scattered from this spot carries the eye’s refractive error through the imaging optics and is directed to the SHWS, positioned at a plane optically conjugate to the primary color CMOS imaging sensor. A microlens array within the SHWS samples the incident wavefront, generating a pattern of focal spots on a CCD. Real-time centroid analysis of these spots provides a map of local wavefront slopes. These measurements are processed through a singular value decomposition (SVD) algorithm to fit a Zernike polynomial basis set, enabling real-time reconstruction of the wavefront phase. The defocus component (S) is extracted from the second-order Zernike coefficients, providing a direct, quantitative measure of the refractive error in diopters. This value serves as a precise error signal in a closed-loop control system, which commands a voice-coil actuated focusing lens to its null position in a single, deterministic step, eliminating the need for iterative search algorithms. ResultsComprehensive evaluation demonstrated the system’s high performance. Testing on a calibrated model eye (OEMI-7) established a highly linear relationship between the computed defocus S and the focusing lens position across a ±20 Diopter (D) compensation range, achievable within a 5 mm mechanical travel. The system achieved a focusing precision of 0.08 D, corresponding to an 18-fold improvement over a conventional projection spot-size method tested under identical conditions. The total focus acquisition time, encompassing wavefront measurement, computation, and lens actuation, averaged under 0.5 s. Clinical validation with 25 human volunteers (50 eyes, refractive range -15 D to +10 D) confirmed practical efficacy. The wavefront-sensing AF succeeded in 92% of attempts with a mean time of 0.5 s, substantially outperforming a projection-based benchmark which achieved only a 32% success rate with an average time of 4.25 s. The system provided instantaneous directional guidance and maintained stability during minor ocular movements. Objective assessment of image quality, via amplitude contrast of retinal vasculature, showed consistent and significant enhancement following AF correction across the entire tested diopter range. ConclusionThis work successfully implements and validates a direct wavefront-sensing autofocus paradigm for portable fundus cameras. By directly quantifying and compensating for the optical defocus aberration, this method bypasses the fundamental limitations of image-processing and projection-based techniques, enabling rapid, precise, and deterministic diopter compensation. The developed system delivers an exceptional combination of a wide operational range (±20 D), high accuracy (0.08 D), fast convergence (0.5 s), and a compact physical footprint. This technology provides a practical and high-performance focusing solution capable of enhancing the reliability, throughput, and diagnostic utility of portable retinal imaging in large-scale screening applications. Future efforts will be directed towards system cost optimization and performance adaptation for diverse ocular conditions.

ObjectiveTo explore the mechanism by which the Shenfu Xiongze prescription regulates autophagy in rats with myocardial remodeling through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsA rat model of myocardial remodeling induced by isoprenaline （ISO） was established. Rats were divided into the blank group，the model group，the low-，medium-， and high-dose groups of Shenfu Xiongze prescription，and the captopril group， 6 rats in each group. Except for the blank group，the rat model of myocardial remodeling was established in the other groups by intraperitoneal injection of 2.5 mg·kg^-1 ISO for 3 consecutive weeks. At the same time of modeling， the low-，medium-， and high-dose groups of Shenfu Xiongze prescription were administered the corresponding doses of Shenfu Xiongze prescription solution （8.4，16.8，and 33.6 g·kg^-1），and the captopril group was administered captopril solution （25 mg·kg^-1）. As for the blank group and the model group， the same volume of normal saline was given. The treatment was continued for 3 weeks. Echocardiography was used to observe the cardiac structure and function，and the heart weight index was detected. Masson staining and hematoxylin-eosin （HE） staining were used to observe the pathological morphology changes of myocardial tissue. The levels of interleukin-6 （IL-6） and B-type natriuretic peptide （BNP） in serum were detected by enzyme-linked immunosorbent assay （ELISA）. The expression of type Ⅰ collagen （Collagen Ⅰ），type Ⅲ collagen （Collagen Ⅲ），and microtubule-associated protein 1 light chain 3 （LC3） proteins in myocardial tissue was determined by immunohistochemistry. Autophagy was observed by transmission electron microscopy. The mRNA expression of Collagen Ⅰ，Collagen Ⅲ，α-smooth muscle actin （α-SMA），LC3，yeast Atg6 homolog protein （Beclin-1），AMPK，and mTOR in myocardial tissue was detected by quantitative real-time polymerase chain reaction （real-time PCR）. The protein expression of Collagen Ⅰ，α-SMA，transforming growth factor-β₁ （TGF-β₁），LC3，Beclin-1，p62， phosphorylation（p）-AMPK，p-mTOR，AMPK，and mTOR was detected by Western blot. ResultsCompared with the normal group，rats in the model group exhibited significantly decreased values of ejection fraction （EF） and left ventricular fractional shortening （FS） （P<0.01）， significantly increased values of left ventricular end-diastolic diameter （LVIDd） and left ventricular end-systolic diameter （LVIDs） （P<0.01）. Additionally， the model group also showed increased degrees of inflammatory infiltration and fibrosis of myocardial tissue， significantly elevated levels of serum IL-6 and BNP （P<0.01）， significantly increased mRNA and protein levels of Collagen Ⅰ，Collagen Ⅲ，α-SMA，and mTOR （P<0.01），and markedly decreased mRNA and protein levels of LC3，Beclin-1，and AMPK （P<0.05，P<0.01）. Compared with the model group， the low-，medium-， and high-dose groups of Shenfu Xiongze prescription presented significantly elevated EF and FS values （P<0.01） and lowered LVIDd and LVIDs （P<0.05）. In these groups， the inflammation and fibrosis were alleviated significantly. They also exhibited decreased serum levels of IL-6 and BNP （P<0.01）， significantly reduced protein expression of Collagen Ⅰ， α-SMA， TGF-β₁， p62， and p-mTOR （P<0.01）， significantly decreased mRNA expression of Collagen Ⅰ， Collagen Ⅲ， α-SMA， and mTOR （P<0.01）， and significantly increased mRNA and protein levels of LC3， Beclin-1， and AMPK （P<0.05，P<0.01）. ConclusionThe Shenfu Xiongze prescription can improve the myocardial remodeling induced by ISO in rats by regulating the autophagy level，enhance cardiac function，and reduce inflammatory and fibrotic levels. This effect may be achieved through the AMPK/mTOR signaling pathway.

ObjectiveThe essence of syndrome manifestation recognition in traditional Chinese medicine (TCM) is to infer the body’s latent pathogenesis state from clinical observational information, rather than to perform simple label matching. However, previous studies have largely modeled this task as syndrome pattern classification within a fixed label space, which does not adequately reflect the cognition process of TCM syndrome differentiation centered on pathogenesis reasoning, and is also insufficient to capture the openness, semantic variability, and cross-disease reusability of syndrome manifestation expression. This study aimed to investigate whether introducing pathogenesis reasoning chain-of-thought (PR-CoT) supervision into large language models (LLMs) could improve the quality and cognitive consistency of syndrome manifestation recognition and support cross-disease transfer. MethodsSyndrome manifestation recognition was formulated as a conditional generation task under the framework of clinical observational information (X)→pathogenesis structure (Z)→syndrome pattern output (Y), where Z serves as an explicit intermediate structural variable linking the clinical evidence and syndrome judgment. Within this framework, a PR-CoT-supervised dataset for syndrome manifestation recognition was constructed based on medical case records of spleen-stomach disorders. After preprocessing, information extraction, manual proofreading, and data cleaning, the dataset comprised 4 800 training cases, 400 development cases, and 400 test cases. Each sample was annotated with a structured PR-CoT consisting of three progressive levels: clinical information summarization, comprehensive pathogenesis analysis, and syndrome pattern output. Supervised fine-tuning was conducted on open-source LLMs, with an end-to-end model serving as the baseline. Qwen3-32B was used as the primary experimental model, and Qwen3-14B as the scale comparison model. A progressive multidimensional evaluation framework was further established, comprising a structural parsing level, a semantic similarity level, and an expert blind review level. At the structural parsing level, syndrome pattern expressions were decomposed into structural elements and evaluated using Precision, Recall, F1 score, and Jaccard similarity. At the semantic similarity level, independent LLMs scored the theoretical proximity between predicted and reference syndrome patterns. At the expert blind review level, three TCM experts independently evaluated model outputs on two dimensions: syndrome differentiation consistency and terminology standardization of syndrome patterns. In addition, zero-shot cross-disease transfer evaluation was conducted on gynecological and heart-system disorder test sets. ResultsAt the structural parsing level, PR-CoT supervision did not lead to a stable improvement in the element-wise overlap of syndrome pattern structural components. Compared with the corresponding baselines, neither Qwen3-32B nor Qwen3-14B showed consistent advantages in structural matching metrics after the introduction of PR-CoT supervision. In contrast, at the semantic similarity level, PR-CoT supervision produced stable positive gains across different model scales and evaluation systems. The average semantic score of Qwen3-32B increased from 6.425 8 in the baseline model to 6.585 0 after PR-CoT supervision, and that of Qwen3-14B increased from 5.870 0 to 5.964 2. At the expert blind review level, the overall score of Qwen3-32B (PR-CoT) was 7.026 0±0.107 7, higher than 6.416 3±0.288 9 for its baseline. In zero-shot cross-disease testing, the PR-CoT model still showed advantages in semantic evaluation and expert evaluation on both gynecological and heart-system disorder test sets, indicating a certain degree of transferability. ConclusionThe benefits of PR-CoT supervision are mainly reflected in TCM semantic consistency and clinical plausibility, rather than in improved hard matching of structural elements. These findings support understanding syndrome manifestation recognition as a process of generating and expressing latent pathogenesis structures, rather than as a classification task within a traditional fixed label space. By introducing pathogenesis reasoning as an explicit intermediate structure into the modeling process and combining it with a progressive multidimensional evaluation framework, this study provides a methodological pathway for intelligent TCM syndrome differentiation that integrates theoretical alignment, interpretability, and multi-level evaluation.

ObjectiveThe essence of syndrome manifestation recognition in traditional Chinese medicine (TCM) is to infer the body’s latent pathogenesis state from clinical observational information, rather than to perform simple label matching. However, previous studies have largely modeled this task as syndrome pattern classification within a fixed label space, which does not adequately reflect the cognition process of TCM syndrome differentiation centered on pathogenesis reasoning, and is also insufficient to capture the openness, semantic variability, and cross-disease reusability of syndrome manifestation expression. This study aimed to investigate whether introducing pathogenesis reasoning chain-of-thought (PR-CoT) supervision into large language models (LLMs) could improve the quality and cognitive consistency of syndrome manifestation recognition and support cross-disease transfer. MethodsSyndrome manifestation recognition was formulated as a conditional generation task under the framework of clinical observational information (X)→pathogenesis structure (Z)→syndrome pattern output (Y), where Z serves as an explicit intermediate structural variable linking the clinical evidence and syndrome judgment. Within this framework, a PR-CoT-supervised dataset for syndrome manifestation recognition was constructed based on medical case records of spleen-stomach disorders. After preprocessing, information extraction, manual proofreading, and data cleaning, the dataset comprised 4 800 training cases, 400 development cases, and 400 test cases. Each sample was annotated with a structured PR-CoT consisting of three progressive levels: clinical information summarization, comprehensive pathogenesis analysis, and syndrome pattern output. Supervised fine-tuning was conducted on open-source LLMs, with an end-to-end model serving as the baseline. Qwen3-32B was used as the primary experimental model, and Qwen3-14B as the scale comparison model. A progressive multidimensional evaluation framework was further established, comprising a structural parsing level, a semantic similarity level, and an expert blind review level. At the structural parsing level, syndrome pattern expressions were decomposed into structural elements and evaluated using Precision, Recall, F1 score, and Jaccard similarity. At the semantic similarity level, independent LLMs scored the theoretical proximity between predicted and reference syndrome patterns. At the expert blind review level, three TCM experts independently evaluated model outputs on two dimensions: syndrome differentiation consistency and terminology standardization of syndrome patterns. In addition, zero-shot cross-disease transfer evaluation was conducted on gynecological and heart-system disorder test sets. ResultsAt the structural parsing level, PR-CoT supervision did not lead to a stable improvement in the element-wise overlap of syndrome pattern structural components. Compared with the corresponding baselines, neither Qwen3-32B nor Qwen3-14B showed consistent advantages in structural matching metrics after the introduction of PR-CoT supervision. In contrast, at the semantic similarity level, PR-CoT supervision produced stable positive gains across different model scales and evaluation systems. The average semantic score of Qwen3-32B increased from 6.425 8 in the baseline model to 6.585 0 after PR-CoT supervision, and that of Qwen3-14B increased from 5.870 0 to 5.964 2. At the expert blind review level, the overall score of Qwen3-32B (PR-CoT) was 7.026 0±0.107 7, higher than 6.416 3±0.288 9 for its baseline. In zero-shot cross-disease testing, the PR-CoT model still showed advantages in semantic evaluation and expert evaluation on both gynecological and heart-system disorder test sets, indicating a certain degree of transferability. ConclusionThe benefits of PR-CoT supervision are mainly reflected in TCM semantic consistency and clinical plausibility, rather than in improved hard matching of structural elements. These findings support understanding syndrome manifestation recognition as a process of generating and expressing latent pathogenesis structures, rather than as a classification task within a traditional fixed label space. By introducing pathogenesis reasoning as an explicit intermediate structure into the modeling process and combining it with a progressive multidimensional evaluation framework, this study provides a methodological pathway for intelligent TCM syndrome differentiation that integrates theoretical alignment, interpretability, and multi-level evaluation.

Objective To explore the application value of computed tomography (CT) three-dimensional reconstruction in total knee arthroplasty (TKA). Methods A total of 102 patients who underwent TKA in our hospital between April 2022 and April 2025 were enrolled and divided using a random number table into a traditional group (51 cases, routine preoperative evaluation) and a study group (51 cases, CT three-dimensional reconstruction-assisted surgery). The therapeutic efficacy at 3 months postoperatively and perioperative indicators were compared between the two groups. The hip-knee-ankle (HKA) angle was measured preoperatively and at 3 months postoperatively. The Hospital for Special Surgery (HSS) knee score and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score were recorded preoperatively and at 3 and 6 months postoperatively. Complications were documented. Results The excellent/good rate in the study group (92.16%) was higher than that in the traditional group (74.51%) (P<0.05). The operation time, intraoperative blood loss, postoperative drainage volume, and hospital stay in the study group were (69.52 ± 7.11) min, (102.69 ± 8.54) mL, (167.33 ± 12.35) mL, and (10.93 ± 2.37) days, respectively, which were shorter than those in the traditional group [(78.45 ± 5.98) min, (108.36 ± 10.02) mL, (175.96 ± 14.93) mL, and (12.04 ± 1.95) days] (P<0.05). At 3 months postoperatively, the HKA angle was 178.02° ± 1.05° in the study group and 177.45° ± 1.34° in the traditional group, both were significantly improved compared with those before operation, with the study group showing better improvement (P<0.05). The HSS knee and WOMAC scores in both groups at 3 and 6 months postoperatively were improved compared with preoperative scores (P<0.05). At 3 and 6 months postoperatively, the study group showed higher HSS knee score and lower WOMAC score than the traditional group (P<0.05). The incidence of complications in the study group (3.92%) was lower than that in the traditional group (17.65%) (P<0.05). Conclusion TKA guided by CT three-dimensional reconstruction improved the excellent/good rate, optimized HKA angle and knee function, shortened operation time and hospital stay, reduced intraoperative blood loss and postoperative drainage volume, and decreased the incidence of complications.

Objective To explore the application value of computed tomography (CT) three-dimensional reconstruction in total knee arthroplasty (TKA). Methods A total of 102 patients who underwent TKA in our hospital between April 2022 and April 2025 were enrolled and divided using a random number table into a traditional group (51 cases, routine preoperative evaluation) and a study group (51 cases, CT three-dimensional reconstruction-assisted surgery). The therapeutic efficacy at 3 months postoperatively and perioperative indicators were compared between the two groups. The hip-knee-ankle (HKA) angle was measured preoperatively and at 3 months postoperatively. The Hospital for Special Surgery (HSS) knee score and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score were recorded preoperatively and at 3 and 6 months postoperatively. Complications were documented. Results The excellent/good rate in the study group (92.16%) was higher than that in the traditional group (74.51%) (P<0.05). The operation time, intraoperative blood loss, postoperative drainage volume, and hospital stay in the study group were (69.52 ± 7.11) min, (102.69 ± 8.54) mL, (167.33 ± 12.35) mL, and (10.93 ± 2.37) days, respectively, which were shorter than those in the traditional group [(78.45 ± 5.98) min, (108.36 ± 10.02) mL, (175.96 ± 14.93) mL, and (12.04 ± 1.95) days] (P<0.05). At 3 months postoperatively, the HKA angle was 178.02° ± 1.05° in the study group and 177.45° ± 1.34° in the traditional group, both were significantly improved compared with those before operation, with the study group showing better improvement (P<0.05). The HSS knee and WOMAC scores in both groups at 3 and 6 months postoperatively were improved compared with preoperative scores (P<0.05). At 3 and 6 months postoperatively, the study group showed higher HSS knee score and lower WOMAC score than the traditional group (P<0.05). The incidence of complications in the study group (3.92%) was lower than that in the traditional group (17.65%) (P<0.05). Conclusion TKA guided by CT three-dimensional reconstruction improved the excellent/good rate, optimized HKA angle and knee function, shortened operation time and hospital stay, reduced intraoperative blood loss and postoperative drainage volume, and decreased the incidence of complications.

Objective To explore the application value of computed tomography (CT) three-dimensional reconstruction in total knee arthroplasty (TKA). Methods A total of 102 patients who underwent TKA in our hospital between April 2022 and April 2025 were enrolled and divided using a random number table into a traditional group (51 cases, routine preoperative evaluation) and a study group (51 cases, CT three-dimensional reconstruction-assisted surgery). The therapeutic efficacy at 3 months postoperatively and perioperative indicators were compared between the two groups. The hip-knee-ankle (HKA) angle was measured preoperatively and at 3 months postoperatively. The Hospital for Special Surgery (HSS) knee score and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score were recorded preoperatively and at 3 and 6 months postoperatively. Complications were documented. Results The excellent/good rate in the study group (92.16%) was higher than that in the traditional group (74.51%) (P<0.05). The operation time, intraoperative blood loss, postoperative drainage volume, and hospital stay in the study group were (69.52 ± 7.11) min, (102.69 ± 8.54) mL, (167.33 ± 12.35) mL, and (10.93 ± 2.37) days, respectively, which were shorter than those in the traditional group [(78.45 ± 5.98) min, (108.36 ± 10.02) mL, (175.96 ± 14.93) mL, and (12.04 ± 1.95) days] (P<0.05). At 3 months postoperatively, the HKA angle was 178.02° ± 1.05° in the study group and 177.45° ± 1.34° in the traditional group, both were significantly improved compared with those before operation, with the study group showing better improvement (P<0.05). The HSS knee and WOMAC scores in both groups at 3 and 6 months postoperatively were improved compared with preoperative scores (P<0.05). At 3 and 6 months postoperatively, the study group showed higher HSS knee score and lower WOMAC score than the traditional group (P<0.05). The incidence of complications in the study group (3.92%) was lower than that in the traditional group (17.65%) (P<0.05). Conclusion TKA guided by CT three-dimensional reconstruction improved the excellent/good rate, optimized HKA angle and knee function, shortened operation time and hospital stay, reduced intraoperative blood loss and postoperative drainage volume, and decreased the incidence of complications.

Objective To investigate the effects of scoparone(Sco)on proliferation and invasion of colon cancer cell line HCT116,and its effect on the expression of epidermal growth factor receptor(EGFR).Methods 1)HCT116 cells were divided into control group,50Sco group,100Sco group and 200Sco group.The cells in con-trol group were incubated with culture medium for 48 hrs.The 50Sco group,100Sco group and 200Sco group were incubated with 50,100 and 200 μmol/L scoparone for 48 hrs respectively.2)HCT116 cells were divided into con-trol group,NC-200Sco group,NC-LV+200Sco group and EGFR-LV+200Sco group.The control group was incuba-ted with normal culture medium for 48 hrs.NC-200Sco group was incubated with 200 μmol/L scoparone for 48 hrs.NC-LV and EGFR-LV were infected into HCT116 cells in NC-LV+200Sco group and EGFR-LV+200Sco group,then incubated with 200 μmol/L scoparone for 48 hrs.Cell proliferation was detected by MTT assay and EdU stai-ning,cell apoptosis was detected by flow cytometry and cell invasion was detected by Transwell assay.EGFR mRNA was detected by RT-qPCR,the level of EGFR,Bcl-2,Bax,matrix metalloproteinase(MMP)-2 and MMP-9 protein was detected by Western blot.Results Compared to the control group,the cell viability,proportion of EdU positive cells and counting number of invasive cells in 50Sco group,100Sco group and 200Sco group all decreased(P＜0.05).Cell apoptosis rate and Bax protein expression increased(P＜0.05),the protein expression of Bcl-2,MMP-2 and MMP-9 decreased(P＜0.05).mRNA and protein expression of EGFR were de-creased(P＜0.05).Compared with NC-200 Sco group and NC-LV+200Sco group,the expression level of mRNA and protein of EGFR in EGFR-LV+200Sco group was increased(P＜0.05).Cell viability,proportion of EdU posi-tive cells and counting number of invasive cells all increased(P＜0.05).The cell apoptosis rate and Bax protein expression level were decreased(P＜0.05).The protein expression of Bcl-2,MMP-2 and MMP-9 was increased(P＜0.05).Conclusions Scoparone has anti-colon cancer cell activity and inhibits proliferation as well as invasion of colon cancer cells through inhibition of EGFR.

Objective To establish models for real-time dynamic monitoring of intracellular cytoplasmic adenosine triphosphate(ATP)and mitochondrial ATP levels in cells in order to study the changes in metabolic processes.Methods The lentiviral plasmids of the cytoplasmic chemogenetic green fluorescent protein(GFP)ATP probe(Chemo-G)and those of the mitochondrial-localized chemogenetic GFP ATP probe(mito-Chemo-G)were constructed before being transfected into HEK293T together with the helper plasmids,respectively.Chemo-G and mito-Chemo-G lentiviruses were obtained.HeLa cells were infected with the lentivirus.Puromycin resistance selection and flow cytometry cell sorting were employed to identify and isolate the infected HeLa cells.The growth and GFP expressions of HeLa cells were observed.A live-cell imaging system was used for continuous imaging of the cells,with stimuli added at specific time points to alter intracellular ATP levels to observe changes in the fluorescence intensity of the ATP probe.Results Lentiviral plasmids containing Chemo-G and mito-Chemo-G sequences were constructed.Two cell lines which could stably express Chemo-G and mito-Chemo-G were established that exhibited strong growth and accurate intracellular fluorescence localization.Live-cell imaging revealed that after the addition of 2-deoxy-D-glucose(2-DG)into HeLa-Chemo-G,the fluorescence resonance energy transfer(FRET)/GFP ratio showed a decrease that was partially reversed by the addition of glucose.The FRET/GFP ratio increased after histamine stimulation,but rapidly decreased after the addition of oligomycin.Conclusion The Chemo-G and mito-Chemo-G lentiviral vectors and stably transfected cell lines HeLa-Chemo-G and HeLa-mito-Chemo-G are constructed,which provides reliable experimental models for studying cellular metabolism and changes in intracellular ATP levels.

Objective To establish a stable in vitro model of CD8+T cell exhaustion.Methods CD8+T cells were isolated and purified from the spleens of ovalbumin-specific CD8+T cell receptor(OT-I)transgenic mice and subjected to chronic antigen stimulation to induce exhaustion in vitro.Flow cytometry was employed to evaluate the expressions of exhaustion markers,secretion of effector cytokines,and transcription factor profiles in CD8+T cells.Exhausted and effector(non-exhausted)CD8+T cells were co-cultured with tumor cells before tumor cell viability was measured to assess the cytotoxic potential of CD8+T cells.Additionally,N-acetyl-L-cysteine(N-AC)was used as a positive control during exhaustion induction to validate the model.Results Chronic stimulation resulted in a significant upregulation of inhibitory receptors,including programmed cell death protein 1(PD-1),T cell immunoglobulin and mucin domain-containing protein 3(TIM-3),T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motifdomain(TIGIT),and lymphocyte activation gene 3(LAG-3).Concurrently,the secretion of key effector cytokines such as tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ)was markedly reduced.Exhausted CD8+T cells exhibited diminished cytotoxicity against tumor cells compared to effector CD8+T cells.Notably,treatment with N-AC effectively restored the function of exhausted CD8+T cells and enhanced their anti-tumor activity.Conclusion This study has established an effective in vitro model for CD8+T cell exhaustion.The use of N-AC demonstrates its potential to restore functionality in exhausted CD8+T cells,underscoring the reliability and utility of this model for investigating the anti-tumor potential of exhausted T cells.