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.

ObjectiveTo develop the Evidence Grading Scale for Ancient classical prescriptions in Traditional Chinese medicine， assess its reliability and validity， and apply it in practice to provide multi-source evidence for clinical practice guidelines development. MethodsLiterature retrieval was conducted to extract and screen existing evaluation dimensions， then the initial items were summarized using thematic analysis. Experts in the clinical medicine， medical history and literature participated in the Delphi questionnaire survey to evaluate and refine the items. An expert consensus meeting was conducted to finalize the included items， refine the method for items evaluation and evidence grading. The evidence quality rating scale for ancient classical traditional Chinese medicine （TCM） prescriptions was then established and tested for reliability and validity. ResultsThrough literature review， extraction， screening and summarization， a total of 3 dimensions and 12 initial items were formed. Questionnaires were sent to 69 experts to evaluate the initial items， with a questionnaire response rate of 100% and an expert authority coefficient of 0.92. All 12 items were retained for they had importance scores above 4. The Evidence Grading Scale on Ancient classical prescriptions in Traditional Chinese medicine includes 3 dimensions with 12 items. The 3 dimensions includes ancient evidence， inheritance status， and modern application. Each dimension contains 4 items， and each item has a full score of 5 points. The evidence was rated as high-level， moderate-level， and low-level according to the final scores. The content validity index （CVI） of the 12 items was ＞0.9， the average CVI of the scale was 0.98， and the intraclass correlation coefficient （ICC） was 0.90. ConclusionThe Evidence Grading Scale on Ancient classical prescriptions in Traditional Chinese medicine has good reliability and validity， which is practical for use in the development of TCM clinical guidelines and can better support clinical decision-making.

The advent of chimeric antigen receptor (CAR)-T cell immunotherapies has led to breakthroughs in the treatment of hematological malignancies. However, their success in treating solid tumors has been limited. CAR-natural killer (NK) cells have several advantages over CAR-T cells because NK cells can be made from pre-existing cell lines or allogeneic NK cells with a mismatched major histocompatibility complex (MHC), which means they are more likely to become an "off-the-shelf" product. Moreover, they can kill cancer cells via CAR-dependent/independent pathways and have limited toxicity. Macrophages are the most malleable immune cells in the body. These cells can efficiently infiltrate into tumors and are present in large numbers in tumor microenvironments (TMEs). Importantly, CAR-macrophages (CAR-Ms) have recently yielded exciting preclinical results in several solid tumors. Nevertheless, CAR-T, CAR-NK, and CAR-M all have their own advantages and limitations. In this review, we systematically discuss the current status, progress, and the major hurdles of CAR-T cells, CAR-NK cells, and CAR-M as they relate to five aspects: CAR structure, therapeutic mechanisms, the latest research progress, current challenges and solutions, and comparison according to the existing research in order to provide a reasonable option for treating solid tumors in the future.

Objective:To explore the neuroprotective effect and molecular mechanism of sulforaphane (SFN) on acute carbon monoxide poisoning (ACOP) in rats.Methods:A total of 135 healthy adult male Sprague-Dawley (SD) rats were randomly divided into normal control group, ACOP model group, and SFN intervention group, with 45 rats in each group. The ACOP animal model was reproduced using carbon monoxide (CO) inhalation in a hyperbaric oxygen chamber, while the normal control group was allowed to breathe fresh air freely. The rats in the SFN intervention group received intraperitoneal injection of SFN at a dose of 20 mg/kg once daily starting 2 hours after CO poisoning and continuing until euthanasia. The normal control group and the ACOP model group received equivalent volume of saline injection. Three rats from each group were sacrificed 1 day after intervention to observe the changes in the ultrastructure of neuronal mitochondria in brain tissues under transmission electron microscopy. Six rats from each group were evaluated for cognitive function using neurobehavioral test 7 days after intervention. Brain tissues of 6 rats in each group were collected 1, 3, and 7 days after intervention, and the expressions of phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK), mitofusin 2 (MFN2), and dynamin-related protein 1 (DRP1) were detected using immunohistochemistry staining and Western blotting. Linear regression analysis was performed to assess the correlations between the expression levels of above proteins.Results:In the normal control group, the rats did not exhibit any abnormalities in cognitive function or the ultrastructure of neuronal mitochondria in brain tissues. ACOP induced cognitive impairment and ultrastructural injury to neuronal mitochondria in rats. However, SFN significantly improved cognitive function in poisoned rats and mitigated the extent of neuronal mitochondrial damage. Over poisoning time, the expression levels of p-AMPK and MFN2 in the brain tissues of ACOP rats were gradually decreased, while the expression level of DRP1 was gradually increased. Compared with the normal control group, the ACOP model group showed significant differences in the expressions of p-AMPK, MFN2, and DRP1. After SFN intervention, the expression levels of above proteins were significantly reversed. Compared with the ACOP model group, the SFN intervention group exhibited a marked increase in the expressions of p-AMPK and MFN2 [p-AMPK positive expression ( A value): 0.226±0.003 vs. 0.177±0.033, p-AMPK protein (p-AMPK/GAPDH): 1.41±0.05 vs. 0.89±0.05, MFN2 positive expression ( A value): 0.241±0.004 vs. 0.165±0.007, MFN2 protein (MFN2/GAPDH): 1.33±0.04 vs. 0.79±0.03, all P < 0.05], along with a significant decrease in DRP1 expression [DRP1 positive expression ( A value): 0.103±0.002 vs. 0.214±0.011, DRP1 protein (DRP1/GAPDH): 1.00±0.03 vs. 1.50±0.03, both P < 0.05]. Linear regression analysis revealed a strong negative linear correlation between DRP1 protein expression and MFN2, p-AMPK protein expressions ( R2 values were 0.977 and 0.971, both P < 0.01), and a positive linear correlation between p-AMPK protein expression and MFN2 protein expression ( R2 = 0.985, P < 0.01). Conclusion:SFN can help maintain neuronal mitochondrial homeostasis by activating the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, thereby alleviating neuronal injury caused by ACOP.

Thirty refractory relapsed acute myeloid leukemia (R/R AML) patients who received salvage allo-HSCT with MeCBA conditioning regimen from January 2018 to June 2022 at Henan Cancer Hospital were included, and their clinical data were reviewed. There were 16 males and 14 females among the 30 patients with a median age of 37 (16-53) years. There were 3 sibling allograft donor transplants, 1 unrelated donor transplant, and 26 haplotype transplants. The median course of pre-transplant chemotherapy was 4 (3-22). The time of neutrophil engraftment was 14 (9-22) days and 18 (10-40) days for platelet. The 30-day cumulative incidence of neutrophil engraftment was 100% and the 100-day cumulative incidence of platelet engraftment was 96.7% (95% CI 85.4% -97.5% ). 22 (73.3% ) patients experienced grade 1-2 gastrointestinal reactions, and there was no grade 3-4 organ toxicity. With a median follow-up of 37.1 months, the overall survival (OS) rate, event-free survival (EFS) rate, cumulative recurrence rate (CIR), and non-recurrence mortality (NRM) rate at 3 years after transplantation were 70.0% (95% CI 50.3% -83.1% ), 65.3% (95% CI 44.8% -79.8% ), 21.2% (95% CI 9.2% -44.4% ) and 16.7% (95% CI 7.3% -35.5% ), respectively.

Objective:To establish an ultrasound radiomics model by integrating clinical, pathological, and conventional ultrasound features with radiomics characteristics, and to explore its clinical value in predicting endocrine resistance in hormone receptor(HR)-positive breast cancer.Methods:A retrospective analysis was performed on 478 patients with HR-positive breast cancer from January 2017 to December 2021 in the Second Affiliated Hospital of Harbin Medical University, of which 430 were resistant and 48 were sensitive. The clinical, pathological and immunohistochemical data and ultrasound images were collected.Firstly, the propensity score was used to process and match the data. Secondly, Logistic regression was used to screen clinical, pathological, and conventional ultrasound features associated with endocrine resistance. Then, PyRadiomics was used to extract the radiomic features of grayscale ultrasound images, and a series of methods such as Lasso regression were used to screen the radiomic features related to endocrine resistance. Seven machine learning methods such as random forest were used to build a radiomics model. Finally, clinical, pathological and ultrasound features were added to establish a clinical pathological model, a clinical pathological ultrasound model, a clinical pathological radiomics model and a combined model of the four features, and the model effectiveness was evaluated.Results:①Propensity score matching: 96 patients were matched, including 48 patients in the drug-resistant group and 48 patients in the sensitive group. ②Screening clinical pathological conventional ultrasound features related to endocrine resistance: lymph node metastasis, tumor diameter, posterior echo attenuation, and growth orientation were independent predictors of endocrine resistance (all P<0.05). ③Screening radiomics features related to endocrine resistance: 18 features such as Dependence Entropy. ④Establishing radiomics model: the machine learning model of random forest method (AUC=0.80) performed best. ⑤Radiomics model integrating clinical, pathological and conventional ultrasound features: the AUC of the clinical pathological model was 0.70, the AUC of the clinical pathological ultrasound model was 0.78, the AUC of the clinical pathological radiomics model was 0.82, and the AUC of the combined model was 0.86. Conclusions:The radiomics model established by the random forest method performs best in predicting endocrine resistance in HR-positive breast cancer. The model that integrates multiple features performs best in assessing endocrine resistance.which is expected to provide an objective basis for clinicians to predict endocrine resistance in HR-positive breast cancer.

Objective To investigate the effect of locking plate combined with cortical screw internal fixation on ankle function and quality of life in patients with ankle fracture with tibiofibular separation.Methods A total of 120 patients with ankle fracture and distal tibiofibular separation treated in our hospital from May 2020 to December 2021 were selected and divided into control group and observation group according to random number table method,with 60 patients/group.The control group was treated with cortical screw fixation alone,and the observation group was treated with locking plate combined with cortical screw internal fixation.Before surgery and 6 months after surgery,the recovery function of the two groups was compared.X-ray,operation duration,healing time,intraoperative blood loss,postoperative complications were compared,and the living ability of the two groups of patients was evaluated.Results Before treatment,there was no difference in joint function between the two groups(P＞0.05).After treatment,the longest walking of the control group(15.89±0.85),foot alignment(15.06±0.71),pain response(29.03±4.48)and ground walking(15.65±0.59).The longest walking distance(16.19±0.87),foot alignment(15.29±0.76),pain response(31.24±4.55)and ground walking(15.96±0.68)in the observation group,which were higher than those in control group(P＜0.05).Compared with the control group,the intraoperative blood loss and healing time in the observation group were lower(P＜0.05).BI index of the two groups before treatment had no difference(P＞0.05);After treatment,BI index of observation group was higher than that of control group(P＜0.05).There was no difference in the total complication rate between the two groups(P＞0.05).Conclusion Locking plate combined with cortical screw internal fixation has a good therapeutic effect on improving ankle function,reducing intraoperative blood loss,promoting healing and improving behavioral ability in the treatment of ankle fracture combined with hypotibiofibular syndesmosis injury.

Objective To predict the core targets and action pathways of Hedysari Radix based on UPLC-MS/MS and network pharmacology methods,and to verify the results of network pharmacology by molecular docking and molecular dynamics techniques.This article aims to investigate immune regulation mechanism of effective components absorbed into blood from Hedysari Radix.Methods Qualitative quantification of effective components absorbed into blood from Hedysari Radix were operated by using UPLC-MS/MS technique.The corresponding targets of effective components absorbed into blood from Hedysari Radix were screened by TCMSP and HERB databases.Targets of immune-related disease were obtained through DisGeNET,OMIM,TTD,and MalaCards databases.The network of"components absorbed into blood from Hedysari Radix-immune-related diseases"was then constructed.GO and KEGG enrichment analysis and mapped the PPI network were performed.Molecular docking and molecular dynamics techniques were applied for validation.Results A total of 8 prototype components absorbed into blood,synergistically acting on 101 targets,were identified by UPLC-MS/MS.They mediated 538 biological processes including immune response,positive regulation of gene expression,receptor binding,and cytokine activity.Meanuhile,116 signaling pathways,such as HIF-1,Toll-like receptor,JAK-STAT,T cell receptor,PI3K-Akt,and FoxO etc.were involved.The core targets were MAPK14,PTGS2,MMP9,PPARG,CCND1,etc..The results of molecular docking showed that formononetin and calycosin had strong docking binding activity with MAPK14.And molecular dynamics simulations further demonstrated that the binding between MAPK14 and formononetin or calycosin had good structural stability and binding affinity.Conclusion The results of serum pharmacochemistry,network pharmacology and molecular dynamics were verified to reveal the material basis and mechanism of Hedysari Radix in regulating immunity.The aim of this study is to provide scientific basis for its immunomodulatory mechanism.

OBJECTIVE To study the chemical composition and the function of solid excipients in relieving the "nourishing and spleen-impairing" effect of Zhangbang nine steaming nine sun-dying Rehmanniae Radix(RR) by using UPLC-Q-TOF-MS technique. METHODS UPLC-Q-TOF-MS was used to collect the data of Zhangbang nine steaming nine sun-dying RR, and UNIFI platform and self-built database were used for the rapid qualitative analysis and identification of the chemical components of Zhangbang nine steaming nine sun-dying RR. The effect of solid excipients on the function of RR "nourishing and spleen-impairing" was investigated by establishing a mouse model of low gastric motility and measuring its gastric residual rate and small intestine propulsion rate. RESULTS The UPLC-Q-TOF-MS and UNFI platforms identified 76 chemical components in nine steaming nine sun-dying RR, including 9 iridoid glycosides, 11 phenylethyl glycosides, 31 flavonoids, 6 saccharides and 19 other compounds; the results of gastric emptying and small intestinal propulsion experiments on mice showed that Zhangbang nine steaming nine sun-dying RR had gastric emptying and intestinal propulsion effects. CONCLUSION This method can be used for the rapid identification and analysis of nine steaming nine sun-dying RR, which has solid excipients Citri Reticulatae Pericarpium and Amomi Fructus to alleviate the "nourishing and spleen-impairing" function of RR. This study can provide a reference for quality control and elucidation of the material basis of the medicinal effects of specialty beverage of Zhangbang nine steaming nine sun-dying RR.

Objective To investigate the correlation between MEX3A and differentiation characteristics of gastric cancer and intestinal metaplasia,and its combination with caudal-related homeobox transcription factor 2(CDX2)and mucin 2(MUC2)and mucin 5AC(MUC5AC)to determine the role of carcinogenic intestinal metaplasia.Methods From January 2010 to December 2014,a total of 410 cases of gastric cancer and paracarcinoma paraffin-embedded tissue samples were selected from the Central Hospital Affiliated to Shenyang Medical College and the Second Hospital Affiliated to Shenyang Medical College.According to pathological diagnosis,they were divided into control group(mild superficial gastritis,79 cases),intestinal metaplasia group(149 cases)and gastric cancer group(182 cases).The expressions of MEX3A,CDX2,MUC2 and MUC5AC were detected by immunohistochemistry.Results MEX3A was highly expressed in gastric cancer group and intestinal metaplasia group,especially diffuse gastric cancer,poorly differentiated gastric cancer and type Ⅲ intestinal metaplasia(P<0.05).CDX2 and MUC2 were highly expressed in gastric cancer group and intestinal metaplasia group,especially intestinal type gastric cancer,highly and moderately differentiated gastric cancer,type Ⅰ and type Ⅱ intestinal metaplasia(P<0.05).The expression of MUC5AC was high in control group and low in gastric cancer group and intestinal metaplasia group,especially in intestinal type gastric cancer,type Ⅰ and type Ⅲ intestinal metaplasia(P<0.05).Gastric cancer and intestinal metaplasia differentiation were negatively correlated with MEX3A and MUC5AC expression,but positively correlated with CDX2 and MUC2 expression(P<0.05).MEX3A was negatively correlated with the expression of CDX2 and MUC2,and positively correlated with the expression of MUC5AC in gastric cancer(P<0.05).MEX3A was negatively correlated with the expression of CDX2 and MUC2 in intestinal metaplasia(P<0.05),while CDX2 was positively correlated with the expression of MUC2(P<0.05).Conclusion MEX3A is negatively correlated with gastric cancer and intestinal metaplasia differentiation.Gastric cancer is characterized by high MEX3A expression and low CDX2 and MUC2 expression.