1.Current Status and Prospective of Research on Disease-Syndrome Integrated Animal Models of Spleen and Stomach Diseases in Traditional Chinese Medicine
Jiaqi ZHANG ; Lihui FANG ; Yongtian WEN ; Shan LIU ; Zhuo SHI ; Xintong WANG ; Xinyi DAI ; Meiling SHE ; Lanshuo HU ; Yangxi FU ; Zheng WANG ; Fengyun WANG ; Xudong TANG
Journal of Traditional Chinese Medicine 2026;67(5):510-516
Animal model research on spleen and stomach diseases in traditional Chinese medicine (TCM) is of great significance for elucidating the nature of diseases and syndromes and for revealing the mechanisms of action of Chinese herbal medicinals. At present, studies on classical TCM syndrome models of spleen and stomach diseases mainly focus on spleen deficiency syndrome, liver constraint syndrome, and damp-heat syndrome. Model construction is mostly based on the etiological and pathophysiological characteristics of syndrome, and model evaluation primarily involves macroscopic manifestations and physicochemical indicators. This paper summarizes the current research status of animal models integrating disease and syndrome for seven common spleen and stomach diseases, including chronic gastritis and gastric precancerous lesions, gastroesophageal reflux disease, functional dyspepsia, inflammatory bowel disease, irritable bowel syndrome, functional constipation, and functional diarrhea. The modeling methods and characteristics of disease-syndrome combined animal models for each disease are analyzed. It is proposed that future research on disease-syndrome integration in spleen and stomach diseases should move toward syste-matic, precise, and integrative development, and that interdisciplinary and cross-disciplinary research approaches should be adopted to enhance the predictive value and application efficiency of disease-syndrome combined animal models.
2.Current Status,Challenges,and Strategies of Basic Research on the Brain-Gut Interaction Theory for Spleen and Stomach Diseases in Traditional Chinese Medicine
Ting CHEN ; Jinxia ZHU ; Xiaohua HOU ; Xiaoli ZHANG ; Lifei ZHENG ; Lei ZHANG ; Xinxin WANG ; Xuan LI ; Xudong TANG
Journal of Traditional Chinese Medicine 2026;67(5):517-522
The brain-gut interaction theory is a multidimensional integrative concept based on the brain-gut axis, involving neural, endocrine, and immune regulatory networks as well as the gut microbiota. Zang-fu organs (脏腑) theory in traditional Chinese medicine (TCM) shows a high degree of consistency with the brain-gut interaction theory, and the core functions such as the spleen and stomach governing the ascending of the clear and descending of the turbid, the liver governing the free flow of qi, and the heart governing mental and emotional activities are closely associated with the multi-level regulatory mechanisms of the brain-gut axis. TCM therapy can modulate brain-gut interactions through multiple pathways in the treatment of spleen and stomach diseases, including the regulation of gastrointestinal hormone secretion, neurotransmitter levels, the hypothalamic-pituitary-adrenal (HPA) axis, immune homeostasis and inflammatory responses, as well as the gut microecology. However, current basic research on the brain-gut interaction theory in TCM for spleen and stomach diseases still faces several challenges, such as difficulties in integrating TCM spleen-stomach theory with modern pathophysiology, lack of innovation in research concepts, and limitations in research methodologies. It is therefore proposed that multidisciplinary collaboration, multi-omics technologies, and targeted research approaches should be adopted to provide more comprehensive methods for basic research on TCM spleen and stomach diseases, thereby promoting the in-depth development of brain-gut interaction theory.
3.Neoadjuvant Sintilimab Combined with Gemcitabine and Cisplatin for Muscle-Invasive Bladder Cancer Patients Followed by Selective Bladder Sparing Surgery: A Phase 2 Trial
Zhou TONG ; Guanghou FU ; Feng ZHOU ; Xiaoyan LIU ; Xing XUE ; Hangyu ZHANG ; Yimin WANG ; Xudong ZHU ; Yang GAO ; Lulu LIU ; Xuanwen BAO ; Yi ZHENG ; Weijia FANG ; Peng ZHAO ; Baiye JIN
Cancer Research and Treatment 2026;58(2):581-590
Purpose:
This study aimed to evaluate the safety and efficacy of gemcitabine and cisplatin (GP) regimen in combination with immune checkpoint inhibitor sintilimab as neoadjuvant therapy for muscle-invasive bladder cancer (MIBC) patients and the feasibility of the following selective bladder sparing surgery.
Materials and Methods:
Patients with histopathologically confirmed urothelial carcinoma without distant metastases (T2-4a, N ≤ 1, M0, American Joint Committee of Cancer 8th) and with adequate organ function will be enrolled. The therapeutic regimen was sintilimab 200 mg once on day 8, gemcitabine 1,000 mg/m2 and cisplatin 35 mg/m2 once on days 1 and 8, every 21 days for four cycles. The primary endpoint was pathologic complete response (pCR, pT0N0) rate. The secondary end points were ypT < 2 rate, R0 resection rate, event-free survival, and safety.
Results:
From May 4, 2020, to May 20, 2023, 55 patients were enrolled. Forty-six patients were evaluated for efficacy. Among the 42 patients who underwent surgery, 16 patients (38.0%) achieved pCR. Thirty-three patients (78.6%) achieved pT < 2. With a median follow-up of 15.7 months, the 1-year event-free survival was 91.3%. Notwithstanding the poor pathological baseline characteristic of a high T3-T4a proportion (39.1%), a promising bladder preservation (including 22 patients transurethral resection of bladder tumor, 5 patients partial cystectomy, and 4 surveillances) rate was achieved (67.4%). The most common grade ≥ 3 treatment-related adverse events was neutropenia (n=15, 27.3%), which was related to chemotherapy. There were no grade 3 immune-related adverse events.
Conclusion
Neoadjuvant GP plus sintilimab is a promising regimen for MIBC patients, with relatively high pT < 2 rate and triggering the emerging roles for the multi-disciplinary team decision-making for bladder sparing surgery.
4.Elevated SLC3A2 Expression Promotes the Progression of Gliomas and Enhances Ferroptosis Resistance through the AKT/NRF2/GPX4 Axis
Yuqian ZHENG ; Shaolong ZHOU ; Yiran TAO ; Zimin SHI ; Xiang LI ; Xudong FU ; Jian MA ; Weihua HU ; Wulong LIANG ; Xinjun WANG
Cancer Research and Treatment 2026;58(1):71-94
Purpose:
The aim of this study is to determine the impact of solute carrier family 3 member 2 (SLC3A2) on the malignant phenotype of gliomas and its role in regulating ferroptosis sensitivity.
Materials and Methods:
The malignant phenotype of glioma was assessed by cell proliferation assay, colony formation assay, EdU assay, wound healing, and Transwell experiments. We further validated the impact of reduced SLC3A2 expression on the sensitivity to ferroptosis in glioma cells through Cell Counting Kit-8 assays, flow cytometry, western blotting, and transmission electron microscopy. Western blot was used to explore how SLC3A2 affects glioma sensitivity to ferroptosis through the AKT/NF-E2-related factor 2 (NRF2)/glutathione peroxidase 4 (GPX4) axis. By establishing a subcutaneous xenograft tumor model in BALB/c-nude mice, we investigated the growth of tumors following the knockout of SLC3A2 in glioma cells.
Results:
Downregulation of SLC3A2 suppressed the malignant phenotype of glioma by blocking the cell cycle and epithelial-mesenchymal transition processes. On the other hand, loss of SLC3A2 not only downregulated SLC7A11 but also prevented the activation of the AKT/NRF2/GPX4 axis. These lead to increased accumulation of reactive oxygen species and lipid peroxides, ultimately enhancing the susceptibility of glioma to ferroptosis.
Conclusion
Our findings suggest that SLC3A2 is an oncogene in gliomas, promoting their occurrence and development. It plays a critical role in ferroptosis resistance through the AKT/NRF2/GPX4 axis.
5.Clinical Observation on 45 Cases of Chronic Obstructive Pulmonary Disease in the Stable Phase with Qi Deficiency,Blood Stasis and Phlegm Obstruction Syndrome with Auxiliary Treated with Jinwei Guben Decoction (金卫固本汤) Combined with Bailing Capsule (百令胶囊)
Deyu KONG ; Xudong ZHENG ; Huimin ZHOU ; Ruitao WANG ; Benzhang ZHAO ; Jianjun WU
Journal of Traditional Chinese Medicine 2025;66(4):367-375
ObjectiveTo observe the clinical efficacy of modified Jinwei Guben Decoction (金卫固本汤, MJGD) combined with Bailing Capsule (百令胶囊, BC) in the treatment of chronic obstructive pulmonary disease (COPD) patients in the stable stage with qi deficiency, blood stasis and phlegm obstruction syndrome, in addition to conventional western medicine treatment. MethodsA total of 102 patients with stable COPD and qi deficiency, blood stasis, and phlegm obstruction syndrome were included in the study. According to the patients'preferences, they were divided into treatment group (49 cases) and control group (53 cases). The control group received conventional western medicine treatment, while the treatment group was given MJGD (1 dose daily) combined with BC (2.0 g each time, three times a day) additionally. The treatment period was 3 months, and the patients were followed up for 1 year after the treatment. The acute exacerbation frequency (mild, moderate, severe) before treatment, during treatment, at 6-month follow-up, and at 1-year follow-up was compared between groups. Additionally, the lung function indicators such as FEV1, FEV1%pred, FVC, and FEV1/FVC ratio, traditional Chinese medicine (TCM) syndrome scores, modified British Medical Research Council (mMRC) dyspnea scale, and the COPD Assessment Test (CAT) scores before and after treatment were compared. A logistic regression model was constructed to analyze the impact of MJGD combined with BC on clinical efficacy. ResultsFour patients dropped out from the treatment group and eight from the control group, leaving 45 patients of each group for statistical analysis. The number of mild and moderate acute exacerbations in the treatment group was lower than that in the control group during the treatment period, at 6-month follow-up and within 1 year of follow-up (P<0.05) .The number of severe acute exacerbations was only lower in the treatment group than in the control group at 6-month follow-up (P<0.05). Compared with that before treatment, the number of acute exacerbations of all degrees in the treatment group was significantly reduced within 1 year of follow-up (P<0.05),while only the number of mild acute exacerbations in the control group was significantly reduced within 1 year of follow-up (P<0.05). The treatment group showed significant improvement in FEV1 and FEV1%pred and FEV1/FEV, while the control group showed a significant decline in FEV1 and FVC (P<0.05). After treatment, both groups showed significant reductions in TCM syndrome scores, including coughing, sputum, wheezing, chest tightness, shortness of breath, and fatigue, as well as mMRC and CAT scores (P<0.05), with the treatment group having significantly lower scores than the control group (P<0.05). The overall clinical effective rate of in the treatment group was 93.33% (42/45), significantly higher than that of the control group, 75.56% (34/45, P<0.05). Multivariate logistic regression analysis showed that the use of MJGD combined with BC (OR = 4.68, 95%CI: 1.15 - 19.09, P = 0.03) was positively correlated with clinical efficacy. ConclusionsIn addition to conventional western medicine treatment, the combination of MJGD and BC can reduce the frequency of acute exacerbations, delay the decline of lung function, improve clinical symptoms, and significantly enhance the clinical efficacy in patients with stable COPD and qi deficiency, blood stasis, and phlegm obstruction syndrome.
6.Clinical Efficacy of Jinwei Pingchuan Decoction in Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease with Phlegm-heat Obstruction in Lung Syndrome
Xudong ZHENG ; Deyu KONG ; Benzhang ZHAO ; Huimin ZHOU ; Ruitao WANG ; Jianjun WU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):125-133
ObjectiveTo study the effect of Jinwei Pingchuan decoction combined with conventional Western medicine on the number of acute exacerbations, lung function, and clinical symptoms in patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) with phlegm-heat obstruction in lung syndrome. MethodsA non-randomized controlled trial was conducted to include 60 patients with acute exacerbation of COPD with phlegm-heat obstruction in lung syndrome. Patients were divided into a treatment group and a control group based on whether they received Jinwei Pingchuan decoction, with 30 patients in each group. The treatment group received Jinwei Pingchuan decoction combined with conventional Western medicine therapy, while the control group received conventional Western medicine therapy alone. Both groups received treatment for 7 days. The number of acute exacerbations and lung function indices were followed up and recorded before treatment and three months after treatment. The following outcomes were observed before and after treatment: the number of acute exacerbations, lung function indices (forced expiratory volume in one second [FEV1], percentage of predicted value [FEV1%pred], forced vital capacity [FVC], and FEV1/FVC ratio), the degree of acute exacerbation, TCM syndrome score, COPD assessment test (CAT) score, modified British Medical Research Council Dyspnea Questionnaire (mMRC) score, C-reactive protein (CRP), and white blood cell (WBC) count. ResultsAfter 3 months of follow-up, the treatment group showed a significant reduction in the number of acute exacerbations compared with the pre-treatment values (P<0.05). After treatment, the treatment group had fewer acute exacerbations than the control group (P<0.05). The degree of acute exacerbation in the treatment group improved significantly compared with the pre-treatment values (P<0.05). After treatment, the degree of acute exacerbation in the treatment group was improved compared to the control group (P<0.05). Regarding lung function, FEV1, FEV1%pred, FVC, and FEV1/FVC ratio increased significantly in the treatment group compared with the pre-treatment values (P<0.05), and similar improvements were observed in the control group (P<0.05). After treatment, FEV1 and FVC were higher in the treatment group than the control group (P<0.05). Regarding TCM syndrome scores, the scores for individual symptoms such as wheezing, cough, expectoration, chest tightness, shortness of breath, and fatigue, as well as the total score, decreased significantly in the treatment group compared with the pre-treatment values (P<0.05). In the control group, the scores for cough, expectoration, chest tightness, fatigue, and palpitation, as well as the total score, also decreased (P<0.05). After treatment, the treatment group showed significantly lower scores for wheezing, cough, chest tightness, shortness of breath, and the total score than the control group (P<0.05). Regarding the CAT score, the scores for cough, expectoration, chest tightness, climbing stairs, going out, activity, and energy, as well as the total score, decreased significantly in the treatment group compared with the pre-treatment values (P<0.05). In the control group, the scores for cough, expectoration, chest tightness, sleep, energy, and the total score decreased (P<0.05). After treatment, the treatment group showed significantly lower scores for cough, expectoration, chest tightness, activity, and going out than the control group (P<0.05). Regarding the mMRC score, CRP level, and WBC count, all these parameters decreased significantly in the treatment group compared with the pre-treatment values (P<0.05), and similar reductions were observed in the control group (P<0.05). ConclusionJinwei Pingchuan decoction can reduce the number of acute exacerbations and the degree of acute exacerbation in patients with acute exacerbation of COPD with phlegm-heat obstruction in lung syndrome. It also improves lung function and symptoms such as cough and chest tightness, thereby enhancing the quality of life of patients.
7.Clinical Efficacy of Jinwei Pingchuan Decoction in Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease with Phlegm-heat Obstruction in Lung Syndrome
Xudong ZHENG ; Deyu KONG ; Benzhang ZHAO ; Huimin ZHOU ; Ruitao WANG ; Jianjun WU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):125-133
ObjectiveTo study the effect of Jinwei Pingchuan decoction combined with conventional Western medicine on the number of acute exacerbations, lung function, and clinical symptoms in patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) with phlegm-heat obstruction in lung syndrome. MethodsA non-randomized controlled trial was conducted to include 60 patients with acute exacerbation of COPD with phlegm-heat obstruction in lung syndrome. Patients were divided into a treatment group and a control group based on whether they received Jinwei Pingchuan decoction, with 30 patients in each group. The treatment group received Jinwei Pingchuan decoction combined with conventional Western medicine therapy, while the control group received conventional Western medicine therapy alone. Both groups received treatment for 7 days. The number of acute exacerbations and lung function indices were followed up and recorded before treatment and three months after treatment. The following outcomes were observed before and after treatment: the number of acute exacerbations, lung function indices (forced expiratory volume in one second [FEV1], percentage of predicted value [FEV1%pred], forced vital capacity [FVC], and FEV1/FVC ratio), the degree of acute exacerbation, TCM syndrome score, COPD assessment test (CAT) score, modified British Medical Research Council Dyspnea Questionnaire (mMRC) score, C-reactive protein (CRP), and white blood cell (WBC) count. ResultsAfter 3 months of follow-up, the treatment group showed a significant reduction in the number of acute exacerbations compared with the pre-treatment values (P<0.05). After treatment, the treatment group had fewer acute exacerbations than the control group (P<0.05). The degree of acute exacerbation in the treatment group improved significantly compared with the pre-treatment values (P<0.05). After treatment, the degree of acute exacerbation in the treatment group was improved compared to the control group (P<0.05). Regarding lung function, FEV1, FEV1%pred, FVC, and FEV1/FVC ratio increased significantly in the treatment group compared with the pre-treatment values (P<0.05), and similar improvements were observed in the control group (P<0.05). After treatment, FEV1 and FVC were higher in the treatment group than the control group (P<0.05). Regarding TCM syndrome scores, the scores for individual symptoms such as wheezing, cough, expectoration, chest tightness, shortness of breath, and fatigue, as well as the total score, decreased significantly in the treatment group compared with the pre-treatment values (P<0.05). In the control group, the scores for cough, expectoration, chest tightness, fatigue, and palpitation, as well as the total score, also decreased (P<0.05). After treatment, the treatment group showed significantly lower scores for wheezing, cough, chest tightness, shortness of breath, and the total score than the control group (P<0.05). Regarding the CAT score, the scores for cough, expectoration, chest tightness, climbing stairs, going out, activity, and energy, as well as the total score, decreased significantly in the treatment group compared with the pre-treatment values (P<0.05). In the control group, the scores for cough, expectoration, chest tightness, sleep, energy, and the total score decreased (P<0.05). After treatment, the treatment group showed significantly lower scores for cough, expectoration, chest tightness, activity, and going out than the control group (P<0.05). Regarding the mMRC score, CRP level, and WBC count, all these parameters decreased significantly in the treatment group compared with the pre-treatment values (P<0.05), and similar reductions were observed in the control group (P<0.05). ConclusionJinwei Pingchuan decoction can reduce the number of acute exacerbations and the degree of acute exacerbation in patients with acute exacerbation of COPD with phlegm-heat obstruction in lung syndrome. It also improves lung function and symptoms such as cough and chest tightness, thereby enhancing the quality of life of patients.
8.Population Characteristics of Arsenic-containing TCM Compounds in the Treatment of Platelets in Myelodysplastic Syndrome
Jian LIU ; Wenru WANG ; Peizhen JIANG ; Kaizhi LU ; Qinlong ZHENG ; Haixia DI ; Lijuan YAO ; Bing WU ; Jiangwei WAN ; Qifeng LIU ; Ruibai LI ; Xudong TANG
Chinese Journal of Information on Traditional Chinese Medicine 2025;32(9):154-160
Objective To compare the differences of baseline characteristics of patients with myelodysplastic syndrome(MDS)who achieved platelet(PLT)response after arsenic-containing TCM compounds combined with Western medicine treatment.Methods Totally 72 MDS patients were selected from 12 outpatient departments and wards,such as Xiyuan Hospital,China Academy of Chinese Medical Sciences,Dongfang Hospital,Beijing University of Chinese Medicine from October 2021 to October 2024.Among them,45 patients received arsenic-containing TCM compounds combined with Western medicine treatment,27 patients received Western medicine treatment.The blood routine[white blood cell(WBC)count,hemoglobin,PLT,neutrophil count],TCM syndrome scores,safety indicators,and adverse events were observed before and after three courses of treatment.The efficacy of all patients was evaluated,and the baseline characteristics of patients who achieved PLT response in the arsenic-containing TCM compounds group and the Western medicine treatment group were compared.Results Comparing the differences of baseline characteristics of the two groups,it was found that the patients who achieved PLT response in the arsenic-containing TCM compounds group were compared with those in the Western medicine treatment group:Age<60 years old(P=0.038),longer disease duration(P=0.012),lower WBC(P=0.017),lower reticulocyte percentage(P=0.037),lower blood urea nitrogen(P=0.046),lower high-density lipoprotein cholesterol(P=0.014),and lower N-terminal pro-B-type natriuretic peptide(P=0.034),abnormal electrocardiogram(P=0.013),high blasts(P=0.009),grade 0 reticular fiber staining(P<0.01),normal chromosome karyotype(P<0.01),gene mutation(P<0.01)and high TCM syndrome scores(P=0.013)were found.Conclusion Arsenic-containing TCM compounds consisting of Qinghuang Powder and Bushen Jianpi Decoction combined with Western medicine is used to treat MDS.Patients with age<60 years old,long disease duration,low WBC count,low reticulocyte percentage,low blood urea nitrogen,low high-density lipoprotein cholesterol,low N-terminal pro-B-type natriuretic peptide,abnormal electrocardiogram,high blasts,grade 0 reticular fiber staining,normal chromosome karyotype,gene mutation and high TCM syndrome score are more likely to obtain PLT response.
9.Expert consensus on non-surgical treatment for acute lateral ankle sprain (version 2025)
Hui CHE ; Wenge DING ; Shiming FENG ; Xueping GU ; Qinwei GUO ; Jianchao GUI ; Yinghui HUA ; Yuefeng HAO ; Qinglin HAN ; Bo HU ; Xiaojun LIANG ; Guoping LI ; Yunxia LI ; Qi LI ; Yanlin LI ; Xin MA ; Jun MA ; Xudong MIAO ; Jianzhong QIN ; Xiaodong QIN ; Xu SUN ; Kefu SUN ; Weidong SONG ; Dai SHI ; Zhongmin SHI ; Youlun TAO ; Xu WANG ; Youhua WANG ; Liheng WANG ; Anli WANG ; Aiguo WANG ; Weidong WU ; Yajun XU ; Weidong XU ; Renjie XU ; Yongsheng XU ; Tengbo YU ; Lianqi YAN ; Xiaodong YUAN ; Yuan ZHU ; Mingzhu ZHANG ; Hongtao ZHANG ; Xintao ZHANG ; Xiaofei ZHENG
Chinese Journal of Trauma 2025;41(6):517-529
Acute lateral ankle sprain (ALAS) is one of the most common sport injuries, with high incidence, recurrence and disability rates. Currently, exercise rehabilitation-based non-surgical treatment is the primary management approach for ALAS. However, there remain improper practices such as excessive immobilization or uncontrolled activity, which contribute to recurrent sprains and chronic ankle instability, significantly impairing patients′ athletic function and quality of life. To standardize the non-surgical management of ALAS, improve the cure rates, and reduce the recurrence and disability rates, Chinese Sports Rehabilitation Medicine Training Project of Chinese Medical Association, Foot and Ankle Basics and Orthopedics Group, Orthopedic Branch of Chinese Medical Doctor Association, and Sports Medicine Branch of Jiangsu Medical Association organized relevant experts to formulate Expert consensus on non-surgical treatment for acute lateral ankle sprain ( version 2025), following the principles of scientific vigor, practicality, and innovation. Thirteen recommendations were proposed for standardized treatment protocols across different healing phases, aiming to provide references for standard management of ALAS and improve the therapeutic outcomes.
10.Effects of macrophage migration inhibitory factor on survival,proliferation,and differentiation of human embryonic stem cells
Ting HUANG ; Xiaohan ZHENG ; Yuanji ZHONG ; Yanzhao WEI ; Xufang WEI ; Xudong CAO ; Xiaoli FENG ; Zhenqiang ZHAO
Chinese Journal of Tissue Engineering Research 2025;29(7):1380-1387
BACKGROUND:Macrophage migration inhibitory factor(MIF)is a pleiotropic cytokine,which is secreted in different types of stem cells and can regulate the proliferation,differentiation and migration of various types of stem cells.Our previous research has confirmed that human embryonic stem cells secrete MIF and that its concentration in the culture medium is relatively stable.However,whether MIF is involved in the survival,proliferation and differentiation of human embryonic stem cells remains unclear. OBJECTIVE:To investigate the effects of MIF on survival,proliferation,and differentiation of human embryonic stem cells. METHODS:(1)Human embryonic stem cells H9 were cultured.The growth curve of cells was detected and plotted by CCK-8 assay.Enzyme-linked immunosorbent assay was used to determine the level of MIF in the medium.(2)To determine the effects of exogenous MIF on the survival and proliferation of human embryonic stem cells,different groups were established:the control group,which was cultured in stem cell medium without any modifications;the exogenous MIF group,which was treated with different concentrations(30,100,300 ng/mL)of MIF in the stem cell medium;the MIF inhibitor ISO-1 group,which was treated with different concentrations(2,7,21 μmol/L)of ISO-1 in the stem cell medium;and the MIF+ISO-1 group,which was treated with different concentrations of ISO-1 along with 100 ng/mL of MIF.Cell viability was assessed using the CCK-8 assay.(3)To further elucidate the effect of MIF gene on survival and proliferation of human embryonic stem cell,the MIF knockout H9 cell line was constructed by CRISPR-Cas 9 technology to observe the lineage establishment.(4)To determine the effect of high concentrations of MIF on human embryonic stem cell differentiation,100 ng/mL MIF and 100 ng/mL of CXCR4 neutralizing antibody were separately added to the normal stem cell culture medium.The expression levels of self-renewal factors(KLF4,c-MYC,NANOG,OCT4,and SOX2)and differentiation transcription factors(FOXA2,OTX2)were measured using real-time quantitative polymerase chain reaction,immunofluorescence staining,and western blot analysis. RESULTS AND CONCLUSION:(1)The logarithmic growth phase of H9 cells was between 3-6 days.Under normal growth conditions,human embryonic stem cells secreted MIF at a concentration of approximately 20 ng/mL,independent of cell quantity.(2)Compared to the control group,the addition of different concentrations of MIF had no effect on the proliferation of human embryonic stem cells(P>0.05).ISO-1 significantly inhibited the proliferation of human embryonic stem cells,with a stronger inhibition observed at higher concentrations of ISO-1(P<0.05).The addition of MIF in the presence of ISO-1 reduced the inhibitory effect of ISO-1(P<0.05).(3)Real-time quantitative polymerase chain reaction showed that knocking out 50%of the MIF gene resulted in a significant decrease in the growth vitality of human embryonic stem cells and failure to establish cell lines.(4)Adding 100 ng/mL exogenous MIF to the culture medium resulted in a decrease in the mRNA,protein,and fluorescence expression levels of the self-renewal transcription factor KLF4,while the mRNA,protein,and fluorescence expression levels of the differentiation factor FOXA2 increased.(5)When 100 ng/mL CXCR4 neutralizing antibody was added to the culture medium,the mRNA and protein expression levels of KLF4 increased,while the mRNA and protein expression levels of FOXA2 decreased,contrary to the expression trend observed in the MIF group.In conclusion,the endogenous secretion of MIF by human embryonic stem cells is essential for their survival.The addition of MIF to the culture medium does not promote the proliferation of human embryonic stem cells.However,it can lead to a decrease in the expression of the self-renewal factor KLF4 and an increase in the expression of the transcription factor FOXA2.This provides a clue for further investigation into the effects and mechanisms of MIF on the differentiation of human embryonic stem cells.The MIF-CXCR4 axis plays a regulatory role in this process.

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