Intestinal barrier damage is a prominent feature of non-alcoholic fatty liver disease （NAFLD） and serves as a critical factor driving the progression from simple fatty liver to non-alcoholic steatohepatitis （NASH）， fibrosis， and cirrhosis. The "turbid pathogenic factors entering the blood" theory integrates classical traditional Chinese medicine （TCM） principles with contemporary disease evolution trends and research findings. It posits that endogenous turbid pathogenic factors within the body infiltrate the blood vessels， leading to impure and viscous blood quality， thereby triggering various diseases. Based on this theory， this article elucidated the pathogenic mechanism of NAFLD-associated intestinal barrier damage. It argued that in NAFLD， the liver loses its dredging function， and the spleen becomes obstructed and dysfunctional. Moreover， essential nutrients fail to be properly transformed， resulting in the internal generation of turbid pathogenic factors. This subsequently initiates a series of pathological changes， namely， "infiltration of phlegm-turbidity into the blood， eroding the intestinal mucosa"， "infiltration of glucose-turbidity into the blood， macerating and eroding the intestinal mucosa"， "infiltration of heat-turbidity into the blood， scorching and eroding the intestinal mucosa"， and "infiltration of stasis-turbidity into the blood， stagnating and eroding the intestinal mucosa"， ultimately causing intestinal barrier damage. Furthermore， guided by the "turbid pathogenic factors entering the blood" theory， this article explored TCM intervention strategies： employing medicinals targeting the liver meridian to address the root cause and reduce the generation and deposition of turbid pathogenic factors in the liver， administering blood-system medicinals to clear the blood and purge turbidity， thereby intercepting the progression of the disease mechanism， and applying tonifying medicinals to bolster healthy Qi and defend against turbid invasion， allowing the damaged intestinal mucosa to gradually heal. This article presented novel theoretical and medicinal perspectives for analyzing NAFLD-associated intestinal barrier damage based on the "turbid pathogenic factors entering the blood" theory， aiming to provide new entry points and broader horizons for related research and clinical practice.

ObjectiveTo investigate the protective effect of Huayu Jiedu prescription against PC12 cell damage induced by a combination of neutrophil extracellular traps （NETs） and oxygen-glucose deprivation （OGD）. MethodsFirst， dimethyl sulfoxide （DMSO） was used to induce the differentiation of the human promyelocytic leukemia cell line （HL-60） into neutrophil-like cells （dHL-60）， and cell differentiation was assessed through Giemsa staining and flow cytometry. Next， phorbol myristate （PMA） was used to induce the formation of NETs in dHL-60 cells. The formation of NETs was assessed via SYTOX Green staining， and NETs were extracted by low-speed centrifugation. Finally， the NETs prepared through the conditioned medium were combined with OGD to induce a high-differentiation PC12 neuronal cell damage model through the co-culture method. This model was treated with the Huayu Jiedu prescription-containing serum. Cells were randomly allocated into a normal control group， a model （NETs + OGD） group， a traditional Chinese medicine （NETs + OGD + 5% Huayu Jiedu prescription-containing serum） group， and a NETosis inhibitor （100 mg·L^-1 DNase I + NETs + OGD） group. The cell counting kit-8 （CCK-8） assay was used to evaluate cell viability and screen the optimal intervention concentrations of NETs and Huayu Jiedu Formula-containing serum， as well as the optimal intervention time for OGD. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the levels of myeloperoxidase-DNA complexes （MPO-DNA）， citrullinated histone H3 （CitH3）， and neutrophil elastase （NE）. Western blot was employed to quantify the protein levels of CitH3 and cleaved Caspase-3. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） staining and flow cytometry were employed to assess neuronal apoptosis. ResultsCompared with those in the normal control group， cells in the model group successfully differentiated into dHL60 cells after DMSO induction. Wright-Giemsa staining revealed that the cells in the model group had disappeared nucleoli， distinct reniform and lobulated changes of nuclei， highly condensed chromatin， and the cytoplasm containing pale purple fine granules. Flow cytometry showed that the mean fluorescence intensity （MFI） of CD11b in the model group was enhanced （P<0.01）. Compared with those in the normal control group， dHL60 cells in the model group successfully generated NETs after PMA induction. SYTOX Green staining revealed loss of cell membrane integrity， with chromatin released from the cytoplasm to form abundant extracellular， cloud-like fibrillar structures， and a marked increase in fluorescence intensity （P<0.01）. Through the CCK-8 assay for assessing the viability of highly differentiated PC12 cells， the optimal concentration for NETs was determined as 1 000 ng·L^-1， the optimal concentration of Huayu Jiedu formula-containing serum as 5%， and the optimal duration for OGD as 2 h. Compared with the normal control group， the model group showed elevated levels of MPO-DNA， CitH3， and NE （P<0.01） and upregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. Furthermore， TUNEL staining revealed an increased proportion of apoptotic cells （P<0.01） and flow cytometry showed an increased apoptosis rate （P<0.01） in the model group. Compared with the model group， the Huayu Jiedu formula-containing serum group and the NETosis inhibitor group exhibited declined levels of MPO-DNA， CitH3， and NE （P<0.05，P<0.01） and downregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. In addition， TUNEL staining showed a decrease in the proportion of apoptotic cells （P<0.01）， and flow cytometry revealed a decrease in the apoptosis rate （P<0.01）. ConclusionHuayu Jiedu prescription may exert a protective effect against ischemic neuronal damage by inhibiting NET formation or alleviating NET-induced neurotoxicity

Metabolic dysfunction-associated fatty liver disease （MAFLD） and type 2 diabetes mellitus （T2DM） have a high comorbidity rate， emerging as a major global public health issue. The concept that the middle energizer acting as a balance refers to the role of the middle energizer in maintaining the balance of Qi and blood， dryness and dampness， as well as water and fire （Yin and Yang） in the human body. The core feature of MAFLD with T2DM is the metabolic imbalance， which is closely related to the dysfunction of the middle energizer's balancing role. This article explores the mechanisms of metabolic imbalance in MAFLD with T2DM from the perspective of the middle energizer acting as a balance， proposing that middle energizer dysfunction is the root cause. Qi movement， dryness-dampness， and ministerial fire are the two ends of the balance. MAFLD with T2DM is accompanied by various seemingly contradictory pathological states and imbalances， such as "Qi stagnation and failure of free movement vs. Qi dispersion without retention"， "damp-turbidity obstruction vs. internal dryness and fluid consumption"， and "ministerial fire decline vs. hyperactivity and scorching". On this basis， the traditional Chinese medicine （TCM） intervention strategies and their microscopic mechanisms are discussed. For the root cause of middle energizer imbalance， wind-dispelling herbs and Qi-replenishing and middle energizer-invigorating herbs are used to restore the balancing role of the middle energizer （delaying glucose and lipid absorption， restoring intestinal homeostasis， and ameliorating insulin resistance in skeletal muscle and adipocytes）. For the secondary imbalances， pungent-dispersing and liver-soothing herbs combined with sour-astringent herbs are used to balance Qi movement （alleviating mitochondrial dysfunction in hepatocytes and impaired glucose transport in tissue cells）. Bitter-warm and dampness-draining herbs combined with sweet-cold and moistening herbs are used to balance dryness and dampness （reducing hepatic steatosis， cellular hyperosmotic dehydration， and intracellular metabolic disorders）. Yang-lifting and warm-invigorating herbs combined with clearing-descending and Yin-nourishing herbs are used to balance ministerial fire （ameliorating hepatocyte apoptosis and liver fibrosis processes and alleviating systemic chronic inflammation and related damage）. According to the theory of the middle energizer acting as a balance， this article explores the mechanisms of metabolic imbalance in MAFLD with T2DM and TCM intervention strategies， providing new insights and a framework for related scientific research and clinical practice.

Utilizing CiteSpace and VOSviewer， a bibliometric analysis was conducted on 1，496 articles from the China National Knowledge Infrastructure， Chinese Science and Technology periodical database， Wanfang data， and the Web of Science database spanning 2006 to 2025， systematically revealing the evolutionary patterns of knowledge regarding doctor‐patient shared decision‐making （SDM） in the field of chronic disease management. The research indicated that the number of publications at home and abroad displayed an overall upward trend， yet their development paths differed significantly. International research has formed a “core-periphery” cooperation network， with research hotspots focusing on the improvement of decision-making quality， optimization of decision-making aids， application in primary care settings， and ethical issues in decision-making. Domestic research， driven by policies， developed rapidly and paid more attention to coverage improvement and cultural adaptation， but notable gaps remained in cross-institutional cooperation and tool localization. Currently， China’s practice still encountered multiple challenges， including restricted patients’ participation willingness due to “family-oriented” cultural values， inadequate localization of decision-making aids， and imperfect decision-making support systems in primary healthcare institutions. On this basis， this paper put forward localized suggestions from three dimensions， including cultural adaptation， technological innovation， and institutional coordination. Through multi-dimensional strategies such as advocating for constructing a family-empowered decision-making model， developing decision-making aids integrated with the dialectical logic of traditional Chinese medicine， and promoting the linkage between SDM implementation and the reform of medical insurance payment methods， it was expected to connect international experience with China’s practice. This aimed to provide references for building a chronic disease decision-making model with Chinese characteristics and contribute to the “patient-centered” medical transformation.

ObjectiveTo investigate the protective effect of Huayu Jiedu prescription against PC12 cell damage induced by a combination of neutrophil extracellular traps （NETs） and oxygen-glucose deprivation （OGD）. MethodsFirst， dimethyl sulfoxide （DMSO） was used to induce the differentiation of the human promyelocytic leukemia cell line （HL-60） into neutrophil-like cells （dHL-60）， and cell differentiation was assessed through Giemsa staining and flow cytometry. Next， phorbol myristate （PMA） was used to induce the formation of NETs in dHL-60 cells. The formation of NETs was assessed via SYTOX Green staining， and NETs were extracted by low-speed centrifugation. Finally， the NETs prepared through the conditioned medium were combined with OGD to induce a high-differentiation PC12 neuronal cell damage model through the co-culture method. This model was treated with the Huayu Jiedu prescription-containing serum. Cells were randomly allocated into a normal control group， a model （NETs + OGD） group， a traditional Chinese medicine （NETs + OGD + 5% Huayu Jiedu prescription-containing serum） group， and a NETosis inhibitor （100 mg·L^-1 DNase I + NETs + OGD） group. The cell counting kit-8 （CCK-8） assay was used to evaluate cell viability and screen the optimal intervention concentrations of NETs and Huayu Jiedu Formula-containing serum， as well as the optimal intervention time for OGD. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the levels of myeloperoxidase-DNA complexes （MPO-DNA）， citrullinated histone H3 （CitH3）， and neutrophil elastase （NE）. Western blot was employed to quantify the protein levels of CitH3 and cleaved Caspase-3. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） staining and flow cytometry were employed to assess neuronal apoptosis. ResultsCompared with those in the normal control group， cells in the model group successfully differentiated into dHL60 cells after DMSO induction. Wright-Giemsa staining revealed that the cells in the model group had disappeared nucleoli， distinct reniform and lobulated changes of nuclei， highly condensed chromatin， and the cytoplasm containing pale purple fine granules. Flow cytometry showed that the mean fluorescence intensity （MFI） of CD11b in the model group was enhanced （P<0.01）. Compared with those in the normal control group， dHL60 cells in the model group successfully generated NETs after PMA induction. SYTOX Green staining revealed loss of cell membrane integrity， with chromatin released from the cytoplasm to form abundant extracellular， cloud-like fibrillar structures， and a marked increase in fluorescence intensity （P<0.01）. Through the CCK-8 assay for assessing the viability of highly differentiated PC12 cells， the optimal concentration for NETs was determined as 1 000 ng·L^-1， the optimal concentration of Huayu Jiedu formula-containing serum as 5%， and the optimal duration for OGD as 2 h. Compared with the normal control group， the model group showed elevated levels of MPO-DNA， CitH3， and NE （P<0.01） and upregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. Furthermore， TUNEL staining revealed an increased proportion of apoptotic cells （P<0.01） and flow cytometry showed an increased apoptosis rate （P<0.01） in the model group. Compared with the model group， the Huayu Jiedu formula-containing serum group and the NETosis inhibitor group exhibited declined levels of MPO-DNA， CitH3， and NE （P<0.05，P<0.01） and downregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. In addition， TUNEL staining showed a decrease in the proportion of apoptotic cells （P<0.01）， and flow cytometry revealed a decrease in the apoptosis rate （P<0.01）. ConclusionHuayu Jiedu prescription may exert a protective effect against ischemic neuronal damage by inhibiting NET formation or alleviating NET-induced neurotoxicity

Metabolic dysfunction-associated fatty liver disease （MAFLD） and type 2 diabetes mellitus （T2DM） have a high comorbidity rate， emerging as a major global public health issue. The concept that the middle energizer acting as a balance refers to the role of the middle energizer in maintaining the balance of Qi and blood， dryness and dampness， as well as water and fire （Yin and Yang） in the human body. The core feature of MAFLD with T2DM is the metabolic imbalance， which is closely related to the dysfunction of the middle energizer's balancing role. This article explores the mechanisms of metabolic imbalance in MAFLD with T2DM from the perspective of the middle energizer acting as a balance， proposing that middle energizer dysfunction is the root cause. Qi movement， dryness-dampness， and ministerial fire are the two ends of the balance. MAFLD with T2DM is accompanied by various seemingly contradictory pathological states and imbalances， such as "Qi stagnation and failure of free movement vs. Qi dispersion without retention"， "damp-turbidity obstruction vs. internal dryness and fluid consumption"， and "ministerial fire decline vs. hyperactivity and scorching". On this basis， the traditional Chinese medicine （TCM） intervention strategies and their microscopic mechanisms are discussed. For the root cause of middle energizer imbalance， wind-dispelling herbs and Qi-replenishing and middle energizer-invigorating herbs are used to restore the balancing role of the middle energizer （delaying glucose and lipid absorption， restoring intestinal homeostasis， and ameliorating insulin resistance in skeletal muscle and adipocytes）. For the secondary imbalances， pungent-dispersing and liver-soothing herbs combined with sour-astringent herbs are used to balance Qi movement （alleviating mitochondrial dysfunction in hepatocytes and impaired glucose transport in tissue cells）. Bitter-warm and dampness-draining herbs combined with sweet-cold and moistening herbs are used to balance dryness and dampness （reducing hepatic steatosis， cellular hyperosmotic dehydration， and intracellular metabolic disorders）. Yang-lifting and warm-invigorating herbs combined with clearing-descending and Yin-nourishing herbs are used to balance ministerial fire （ameliorating hepatocyte apoptosis and liver fibrosis processes and alleviating systemic chronic inflammation and related damage）. According to the theory of the middle energizer acting as a balance， this article explores the mechanisms of metabolic imbalance in MAFLD with T2DM and TCM intervention strategies， providing new insights and a framework for related scientific research and clinical practice.

Objective To explore the medication rules and therapeutic principles of Zhang Jingyue's miscarriage prevention therapies based on data mining technique and literature analysis.Methods The prescription data recorded in the chapter about pregnancy and fetal development from the volume of Women's Diseases in Jing Yue Quan Shu(Jingyue's Complete Works)were collected to establish a database.Frequency analysis,association analysis,and cluster analysis of the herbal medicines from the prescriptions for preventing miscarriage were performed using SPSS Modeler 18.0 and SPSS Statistics 26.0 to investigate Zhang's medication rules.Additionally,discussions on miscarriage prevention in the chapter about pregnancy and fetal development were systematically reviewed to summarize his therapeutic principles.Results A total of 76 miscarriage-prevention prescriptions involving 102 herbal medicinals were analyzed.Among them,26 high-frequency herbal medicinals(used≥5 times)were identified,and primarily belonged to the tonifying deficiency category(11 medicinals).Association analysis revealed 10 association rules with high support,such as"Atractylodis Macrocephalae Rhizoma→Ginseng Radix et Rhizoma"and"Glycyrrhizae Radix et Rhizoma Praeparata cum Melle→Ginseng Radix et Rhizoma,Atractylodis Macrocephalae Rhizoma".The network graph of drug association presented high-correlation herbs including Glycyrrhizae Radix et Rhizoma Praeparata cum Melle,Atractylodis Macrocephalae Rhizoma,Poria,Ginseng Radix et Rhizoma,and Angelicae Sinensis Radix.Cluster analysis yielded four core prescription groups:(1)Ziziphi Spinosae Semen,Polygalae Radix,Dioscoreae Rhizoma,and Corni Fructus;(2)Paeoniae Radix Alba,Chuanxiong Rhizoma,Rehmanniae Radix Praeparata,Atractylodis Macrocephalae Rhizoma,Ginseng Radix et Rhizoma,Glycyrrhizae Radix et Rhizoma Praeparata cum Melle,Angelicae Sinensis Radix,Poria,and Citri Reticulatae Pericarpium;(3)Rehmanniae Radix,Glycyrrhizae Radix et Rhizoma,and Scutellariae Radix;(4)Pinelliae Rhizoma,Magnoliae Officinalis Cortex,Zingiberis Rhizoma Recens,and Jujubae Fructus.Zhang's therapeutic principles for preventing miscarriage were as follows:(1)treating primary diseases together by preventing miscarriage;(2)monthly-specific cultivation of the fetus to preemptively address deficiencies;(3)emphasis on maternal health to consolidate fetal vitality;(4)strict adherence to medication and dietary contraindications and lifestyle management during pregnancy.Conclusion Zhang Jingyue's miscarriage-prevention principles emphasize etiological diagnosis with dual focus on treating primary diseases and preventing miscarriage.His therapies prioritize replenishing qi and blood,skillfully regulating spleen-stomach function and qi movement by employing special and mild herbs and prescribing simplified and flexible formulas.These medication rules and therapeutic principles may provide valuable references for modern miscarriage-prevention strategies.

ObjectiveTo explore the regulation of hypothalamic-pituitary-gonadal （HPG） axis by modified Erxian decoction in rats with perimenopausal syndrome （PMS） and to further analyze the expression of proteins related to the silent information regulator 1 （SIRT1）/hypothalamic kisspeptin （Kisspeptin）/gonadotropin-releasing hormone （GnRH） signaling pathway in the arcuate nucleus region （ARC） of the hypothalamus， so as to reveal the potential target of action and molecular biological mechanism of modified Erxian decoction for the treatment of perimenopausal syndrome. MethodsAn animal model was established via the incomplete castration method， with successful modeling confirmed by the exfoliated cervical cell smear method. The 48 rats were divided into six groups based on the randomization principle after successful modeling， including a sham operation group， a model group， an estradiol valerate group （0.09 mg∙kg^-1∙d^-1）， high-， medium-， and low-dose modified Erxian decoction groups （7.614， 3.807，1.903 5 g∙kg^-1∙d^-1）， with 8 rats in each group. The estradiol valerate group and the high-， medium- and low-dose modified Erxian decoction groups were continuously administered by gavage for 28 days， and the indicators were detected 24 hours after the last administration. Body weights and uterine indices were measured. The pathological changes of the uterus were observed by hematoxylin-eosin （HE） staining. Enzyme-linked immunosorbent assay （ELISA） was performed to measure the levels of estradiol （E₂）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and gonadotropin-releasing hormone （GnRH）. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to determine the expression levels of SIRT1， Kisspeptin， kisspeptin receptor （GPR54）， and GnRH in the ARC region of the hypothalamus and gonadotropin-releasing hormone receptor （GnRH-R） in pituitary. ResultsCompared with the sham operation group， rats in the model group had a significantly increased body weight （P0.01）， reduced wet weight and index of uterus （P0.01）， endometrial thinning or atrophy， glandular atrophy， and a decreasing number of glands. Additionally， serum levels of E₂ and the expression of SIRT1 in the ARC region of the hypothalamus significantly decreased （P0.01）. Serum levels of FSH， LH， and GnRH， the expression of Kisspeptin， GPR54， and GnRH in the ARC region of the hypothalamus， and GnRH-R in pituitary significantly increased （P0.01）. Compared with the model group， the estradiol valerate group and the high-， medium-dose modified Erxian decoction groups had significantly reduced body weight， serum levels of FSH， LH， and GnRH， and expression of Kisspeptin， GPR54， and GnRH in the ARC region of the hypothalamus and GnRH-R in pituitary （P0.05， P0.01） and significantly increased wet weight and index of uterus， serum level of E₂， and expression of SIRT1 in the ARC region of the hypothalamus （P0.05， P0.01）. In addition， they showed thickened endometrium， increased number of endometrial glands， and improved glandular atrophy. ConclusionModified Erxian decoction regulates the function of the HPG axis through multi-targets， and its mechanism of action may be related to the up-regulation of the expression of SIRT1 in the ARC region of the hypothalamus， the inhibition of the over-activation of the Kisspeptin/GnRH signaling pathway， the regulation of the expression of GnRH-R in the pituitary， the restoration of secretion balance of gonadotropins， and the elevation of the estrogen level. This study provides an experimental basis for the interpretation of the scientific connotation of modified Erxian decoction in the treatment of perimenopausal syndrome and a theoretical reference for the development of a novel therapeutic strategy based on the SIRT1/Kisspeptin/GnRH pathway.

Objective:To investigate the effects of intravenous lidocaine infusion (IVLI) on the quality of intraoperative neurophysiologic monitoring (IONM) in patients with thyroid tumor.Methods:A prospective randomized controlled study was conducted. A total of 60 patients with thyroid tumor undergoing thyroidectomy in the First Clinical Medical College of Shanxi Medical University between September 2022 and May 2023 were selected. According to the random number table method, all patients were divided into the lidocaine group and the control group, 30 cases in each group. The patients in the lidocaine group were continually given IVLI during the operation and the patients in the control group were continually given the equal 0.9% NaCl solution infusion during the operation. All patients in the 2 groups were induced by the same way of total intravenous anesthesia, and no muscle relaxants were added during anesthesia except the induction dose, and enhanced nerve monitoring tracheal catheter was inserted in the 2 groups. According to the standardized procedure of IONM, the electrode impedance values were measured at the time of eliciting the V1 and V2 signals from the vagus nerve, respectively, and the difference value was defined as the drop in the aggregate impedance level (DAIL). DAIL, perioperative hemodynamic parameters and postoperative recovery quality were compared between the 2 groups.Results:There were no statistically significant differences in the baseline data, operation time, intraoperative dosage of propofol and remifentanil between the 2 groups (all P > 0.05). Compared with the control group, the lidocaine group had a higher proportion of patients with DAIL<50% [80.0% (24/30) vs. 40.0% (12/30), χ2 = 10.00, P = 0.002], a lower hemodynamic fluctuation during extubation [mean arterial pressure: (95±6) mmHg (1 mmHg = 0.133 kPa) vs. (104±7) mmHg, t = 31.00, P < 0.001; heart rate: (73±5) times/min vs. (92±6) times/min, t = 172.58, P < 0.001], a lower visual analog score 24 h after surgery [(2.0±0.7) scores vs. (3.7±0.8) scores, t = -8.86, P < 0.001], a higher score of quality of recovery-15 scale [(127±11) points vs. (118±13) points, t = 2.92, P = 0.005]. Conclusions:IVIL can improve the quality of IONM in patients with thyroid tumor during surgery, reduce perioperative hemodynamic fluctuation and improve postoperative recovery quality of patients.

To prepare a recombinant adeno-associated virus(rAAV9-ZsGreen1-EsASP)capable of expressing the aspartic protease protein of Eimeria stiedai(E.stiedai),and explore its in vitro ac-tivity.The EsASP gene was amplified by PCR,and recombinant adeno-associated viral vector pAAV-IRES-ZsGreen1 was combined with the EsASP gene using homologous recombination to construct the pAAV-ZsGreen1-EsASP expression plasmid;pAAV-ZsGreen1-EsASP expression plasmid,pHelper,and pAAV-RC9 plasmids were cotransfected into HEK-293T cells by liposomal transfection to package and produce recombinant adeno-associated virus rAAV9-ZsGreen1-EsASP capable of expressing EsASP protein.rAAV9-ZsGreen1-EsASP was purified using chloroform treatment-PEG/NaCl precipitation-chloroform extraction method,the purity of the virus was iden-tified by silver staining,the virus morphology was observed by TEM,and virus titer was detected by qRT-PCR;the purified recombinant virus was further infected into HEK-293T cells,and EsASP expression was detected by observing green fluorescent protein ZsGreen1 and Western blot method.The results indicated that double enzyme digestion and DNA sequencing confirmed that the EsASP gene had been successfully constructed into the pAAV-IRES-ZsGreen1 expression plas-mid.The expression of green fluorescent protein in HEK-293T cells suggested that co-transfection was successful.Western blot results of cell protein preparation showed that EsASP protein was successfully expressed;The purified recombinant viral capsid proteins show three distinct bands(VP1-3).The purified rAAV9-ZsGreen1-EsASP was uniform in size,around 20 nm,and had a titer higher than 1.0 × 1012 vg/mL;Green fluorescent protein expression was observed after infection of HEK-293T cells with the recombinant virus,and EsASP expression was detected by Western blot.The results suggest that rAAV9-ZsGreen1-EsASP with in vitro infectious activity was suc-cessfully obtained,providing a material basis for the development of a novel vaccine against Eimer-ia stiedai.