Chemotherapy-induced complications, particularly lethal cardiovascular diseases, pose significant challenges for cancer survivors. The intertwined adverse effects, brought by cancer and its complication, further complicate anticancer therapy and lead to diminished clinical outcomes. Simple supplementation of cardioprotective agents falls short in addressing these challenges. Developing bi-functional co-therapy agents provided another potential solution to consolidate the chemotherapy and reduce cardiac events simultaneously. Drug repurposing was naturally endowed with co-therapeutic potential of two indications, implying a unique chance in the development of bi-functional agents. Herein, we further proposed a novel "trilogy of drug repurposing" strategy that comprises function-based, target-focused, and scaffold-driven repurposing approaches, aiming to systematically elucidate the advantages of repurposed drugs in rationally developing bi-functional agent. Through function-based repurposing, a cardioprotective agent, carvedilol (CAR), was identified as a potential neddylation inhibitor to suppress lung cancer growth. Employing target-focused SAR studies and scaffold-driven drug design, we synthesized 44 CAR derivatives to achieve a balance between anticancer and cardioprotection. Remarkably, optimal derivative 43 displayed promising bi-functional effects, especially in various self-established heart failure mice models with and without tumor-bearing. Collectively, the present study validated the practicability of the "trilogy of drug repurposing" strategy in the development of bi-functional co-therapy agents.

BACKGROUND:Osteonecrosis due to drugs is a serious adverse reaction occurring after the application of such drugs.Increasing evidence suggests that the gut microbiota composition is associated with osteonecrosis due to drugs.However,the causal relationship of the gut microbiota to osteonecrosis due to drugs is still unclear. OBJECTIVE:To evaluate the potential causal relationship between the gut microbiota and the risk of osteonecrosis due to drugs using the Mendelian randomization method. METHODS:A two-sample Mendelian randomization study was performed using the summary statistics of gut microbiota from the largest available genome-wide association study meta-analysis(n=13 266)conducted by the MiBioGen consortium as well as the summary statistics of osteonecrosis due to drugs obtained from the FinnGen consortium R9 release data(264 cases and 377 013 controls).Inverse variance weighted,MR-Egger,weighted median,weighted model and simple model were used to examine the causal association between gut microbiota and osteonecrosis due to drugs.Sensitivity analysis was used to test whether the results of the Mendelian randomization analysis were reliable.Reverse Mendelian randomization analysis was performed on all the bacteria as an outcome for effect analysis and sensitivity analysis. RESULTS AND CONCLUSION:Inverse variance weighted estimates suggested that Lentisphaerae(phylum),Lentisphaeria(class),Melainabacteria(class),Gastranaerophilales(order),Rhodospirillales(order),Victivallales(order)and Bifidobacterium(genus)had protective causal effects on osteonecrosis due to drugs.Methanobacteria(class),Bacillales(order),Methanobacteriaceae(family),Lachnospiraceae(family),Methanobacteriales(order),Holdemania(genus),Holdemania(UCG010 group)(genus),Odoribacter(genus)and Tyzzerella3(genus)had negative causal effects on osteonecrosis due to drugs.According to the results of reverse Mendelian randomization analysis,Clostridiaceae1(family),Peptostreptococcaceae(family),Streptococcaceae(family),Clostridiumsensustricto1(genus)and Streptococcus(genus)showed negative causal effects on osteonecrosis due to drugs.However,Eisenbergiella(genus)showed protective causal effects on osteonecrosis due to drugs.None of the bidirectional sensitivity analysis revealed heterogeneity or horizontal pleiotropy.When gut microbiota were used as exposure and osteonecrosis due to drugs as the outcome,Mendelian randomization analysis found that seven bacterial traits were positively correlated to osteonecrosis due to drugs,nine bacterial traits were negatively related to osteonecrosis due to drugs.When osteonecrosis due to drugs were used as exposure and gut microbiota as the outcome,reverse Mendelian randomization analysis found a negative correlated relationship with five bacterial traits and a positive causal relationship with one bacterial trait.By changing the diversity and composition of gut microbiota,it is expected to improve the incidence and prognosis of osteonecrosis due to drugs,providing new ideas for the study of orthopedic diseases.

BACKGROUND:Osteonecrosis is a common refractory disease in clinical practice,and observational studies have suggested that micronutrients may have a prognostic role in osteonecrosis.However,the specific causal association between micronutrients and osteonecrosis is not known. OBJECTIVE:To explore the causal association between micronutrients and osteonecrosis by Mendelian randomization using summary data from a large population-based genome-wide association study(GWAS)for clinical diagnosis and treatment. METHODS:The required exposure and outcome data(calcium,magnesium,iron,vitamin E,carotenoids,retinol&osteonecrosis)were extracted from the IEU OpenGWAS database,GWAS catalog database,and FinnGen database.Data were analyzed by bidirectional Mendelian randomization with inverse-variance weighted as the primary study method,and weighted median method,simple mode method,weighted mode method,and MR-Egger regression to complement the results.The reliability of the data was then verified through sensitivity analyses. RESULTS AND CONCLUSION:(1)The results found a positive correlation between serum iron concentration and osteonecrosis,while no correlation was found for other micronutrients.There was no reverse causality in all the data.(2)The results of sensitivity analysis showed a robust causality.(3)By Mendelian randomization method,this study provided evidence of causality between serum iron concentration and osteonecrosis,and understanding the causality of micronutrient elements on osteonecrosis can help in the clinical diagnosis and treatment of osteonecrosis,which is of great clinical significance.

Objective:To analyze food intolerance status in children in Qingdao by detecting the serum levels of food-specific IgG (sIgG).Methods:In this cross-sectional study, a total of 4 249 children aged 0 to 14 years (all were permanent residents of Qingdao City) admitted to Women and Children′s Hospital Affiliated to Qingdao University from May 2017 to December 2020 for suspected food intolerance were selected as the study objects with the whole sampling method. According to the age, the objects were divided into 4 groups: 0-<1 year group (440 cases), 1-<3 years group (1 761 cases), 3-<6 years group (1 193 cases), and ≥6 years group (855 cases). Positive condition of serum sIgG antibodies of 14 kinds of food in the children were detected by enzyme-linked immunosorbent assay. Chi-square test was used to compare the positive rate of the antibodies among different foods, gender and age groups.Results:The total positive rate of food sIgG antibody in 4 249 children was 95.32% (4 050/4 249), the highest positive rate was found in eggs (81.50%) and the lowest positive rate was found in pork (1.15%). The positive rates of sIgG antibody in milk (54.98% vs 49.69%, χ2=11.627), crab (5.59% vs 3.71%, χ2=8.049) and shrimp (4.62% vs 2.75%, χ2=9.784) in boys were significantly higher than those in girls, and the positive rates of sIgG antibody in tomato (49.19% vs 45.54%, χ2=5.510), cod (8.53% vs 5.96%, χ2=10.512) and beef (2.58% vs 1.70%, χ2=3.959) in girls were significantly higher than those in boys (all P<0.05). The total positive rate of sIgG antibody in 14 foods was the lowest in 0-<1 year group (89.09%), and it was the highest in 3-<6 years group (96.98%) ( χ2=63.950, P<0.001). The highest positive rate in 0-<1 year group was found in tomato (56.36%), and it was eggs (85.29%, 88.94%, 85.50%) in all the other 3 groups. The positive rates of corn and beef decreased with age ( χ2=44.098, 20.106, P<0.001), while those of cod and mushroom increased with age ( χ2=32.315, 40.338, P<0.001). The positive rate of wheat (57.13%, χ2=42.273), tomato (57.01%, χ2=209.862), soybean (24.99%, χ2=92.580), crab (6.81%, χ2=33.201), shrimp (6.25%, χ2=47.863) were all the highest in 1-<3 years group among the 4 groups (all P<0.001), and the positive rate of chicken was the highest in 3-<6 years group (7.88%, χ2=29.875; P<0.001). Conclusions:Children in Qingdao have a high level of food intolerance, and the highest positive rate is for eggs. Milk, crab and shrimp should be focused on for boys, while tomatoes, cod and beef shoud be paid more attention to for girls. Children of different ages have different kinds of food intolerance, and their diets should be adjusted reasonably according to the characteristics.

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Swine ; Animals ; Induced Pluripotent Stem Cells/metabolism* ; Receptors, Cell Surface/genetics* ; Antigens, CD/metabolism* ; Porcine respiratory and reproductive syndrome virus/genetics*

OBJECTIVE To investigate the mechanism of Mayuan tongbian zhitong decoction on improving slow-transmission constipation（STC）in rats by regulating AMP activated protein kinase （AMPK）/endothelial nitric oxide synthase （eNOS）signaling pathway. METHODS The rats were randomly divided into blank group ，model group ，Mayuan tongbian zhitong decoction low-dose，medium-dose and high-dose groups （6，12，18 mg/kg），with 10 rats in each group. Except for blank group ，other groups were given Compound diphenoxylate suspension to induce STC model. After modeling ，blank group and model group were given normal saline intragastrically ，and Mayuan tongbian zhitong decoction groups were given relevant medicine intragastrically ， once a day ，for consecutive 2 weeks. The number of feces and water content of feces in each group were observed before and after treatment；the carbon powder propulsion rate of rats in each group was calculated ；the pathological structure of colon in each group was observed ；the levels of nitric oxide （NO）and nitric oxide synthase （NOS）in colon tissues of rats in each group were detected；the expressions of AMPK ，eNOS，mammalian target of rapamycin （mTOR），tuberous sclerosis complex 1（Tsc-1）， Tsc-2 and eukaryotic promoter 4E binding protein （4ebp）were also detected. The active ingredients of Cannabis sativa ，Citrus aurantium and Rehmannia glutinosa were screened from Mayuan tongbian zhitong decoction. The active ingredients with high Degree value were docked with AMPK and eNOS ，to verify the interaction. RESULTS Compared with before treatment ，the number and water content of feces were increased significantly in Mayuan tongbian zhitong decoction groups （P＜0.05）. Compared with blank group ，carbon powder propulsion rate of model group was decreased significantly （P＜0.05）； colonic structure was disordered ，and a large number of inflammatory cells were seen in submucosa ；the levels of NO and NOS in colon tissue as well as the protein expressions of AMPK ，eNOS，mTOR，Tsc-1，Tsc-2 and 4ebp were increased significantly （P＜0.05）. Compared with model group ，above indexes of Mayuan tongbian zhitong decoction groups （except for NOS in low-dose group ）were reserved significantly （P＜0.05）. In the molecular docking experiment ，the active components with the highest Degree values in C. sativa ，C. aurantium and R. glutinosa were（Z）-3-（4-hydroxy-3-methoxy-phenyl）-N-[2-（4-hydroxyphenyl）ethyl] acrylamide，nobiletin and stigmasterol. The binding energies of AMPK with these three components were －5.15，－4.61 and －4.83 kJ/mol，the binding energies of eNOS with these three components were －6.11，－5.40 and －5.91 kJ/mol. The conformations of these three compounds with AMPK and eNOS were stable and their binding activities were high. CONCLUSIONS Mayuan tongbian zhitong decoction can improve the constipation symptoms and intestinal function in STC model rats ，and its specific mechanism may be related to the inhibition of AMPK/eNOS signaling pathway.

Conjunctival melanoma (CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B (CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16, CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a K _d value of 0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.

Objective:To investigate the relationship between the mechanism of ulinastatin reducing perioperative myocardial injury and ferroptosis in peripheral blood mononuclear cells (PBMCs) in pediatric patients undergoing heart surgery under cardiopulmonary bypass (CPB).Methods:A total of 60 pediatric patients of either sex, aged 4-8 yr, of American Association of Anesthesiologists physical status Ⅱ or Ⅲ, undergoing elective repair of ventricular septal defect under CPB, were divided into 2 groups by a random number table method: control group (C group) and ulinastatin group (UTI group), with 30 cases in each group.Combined intravenous-inhalational anesthesia was used.In UTI group, ulinastatin 20 000 U/kg was diluted to 100 ml in normal saline, 50 ml was infused through the central vein over 15 min starting from 20 min before skin incision, and the remaining 50 ml was instilled through the CPB pipeline over 15 min starting from 10 min of CPB.The equal volume of normal saline was given instead in C group.Blood samples from the internal jugular vein were collected after anesthesia induction and before skin incision (T 1), at 30 min after start of CPB (T 2), immediately after termination of CPB (T 3) and at 24 h after termination of CPB (T 4) for determination of the levels of amino-terminal B-type pro-brain natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI) and creatine kinase isoenzymes (CK-MB) in plasma by enzyme-linked immunosorbent assay.PBMCs were extracted by modified Ficoll density gradient centrifugation method for determination of the concentrations of Fe 2+ and malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in PBMCs (by colorimetric method) and expression of long-chain acyl-CoA synthase 4 (ACSL4) and glutathione peroxidase 4 (GPX4) in PBMCs (by Western blot). Results:Compared with the baseline at T 1, the levels of NT-proBNP, cTnI and CK-MB in plasma were significantly increased, the concentrations of Fe 2+ and MDA in PBMCs were increased, the expression of ACSL4 in PBMCs was up-regulated, and the activity of SOD was decreased, and the expression of GPX4 was down-regulated at T 2-4 in two groups ( P<0.05). Compared with C group, the plasma levels of NT-proBNP, cTnI and CK-MB were significantly decreased, the concentrations of Fe 2+ and MDA in PBMCs were decreased, the expression of ACSL4 in PBMCs was down-regulated, the activity of SOD was increased, and the expression of GPX4 was up-regulated at T 2-4 in UTI group ( P<0.05). Conclusion:The mechanism by which ulinastatin reduces perioperative myocardial injury may be related to inhibition of ferroptosis in PBMCs in the pediatric patients undergoing open heart surgery under CPB.

OBJECTIVE: To investigate the regulatory effect of metformin on regulatory T cells (Treg) in acidic environment. METHODS: CD4 CD25 Treg cells were obtained by magnetic bead sorting. Treg and conventional T cells (Tcon) cells were cultured for 24-72 h in pH 7.4 or pH 6.7 medium, and the cell proliferation, apoptosis and Foxp3 expression were detected by flow cytometry. Real-time PCR was used to detect the expression levels of the genes related with glucose metabolism. Thirty-two C57BL/6 male mouse models bearing subcutaneous prostate cancer xenograft derived from RM-1 cells were randomized into 4 equal groups for treatment with PBS, metformin, tumor vaccine, or both metformin and the vaccine. The treatment started on the 4th day following tumor cell injection, and metformin (100 mg/kg) or PBS was administered by intraperitoneal injection on a daily basis; the vaccine was intramuscularly injected every 4 days. The tumor size was continuously monitored, and the mice were euthanized on day 25 after tumor implantation to obtain tumor and blood samples. Flow cytometry was used to detect the changes in CD4, CD8, CD4Foxp3 cell subsets in the tumor tissue and peripheral blood. RESULTS: Treg cells showed significantly enhanced proliferation ( < 0.05) while the proliferation of Tcon cells was suppressed in acidic medium ( < 0.001). Treg cells cultured in acidic medium showed significantly increased expressions of OXPHOS-related genes pgc1a ( < 0.001) and cox5b ( < 0.01), which did not vary significantly in Tcon cells in acidic medium. Treg cells exhibited significantly decreased apoptosis in acidic medium ( < 0.01) with increased Foxp3 cells ( < 0.001) and intracellular alkaline levels ( < 0.01). Metformin obviously reversed the acid tolerance of Treg cells without producing significant effect on Tcon cells. In the animal experiment, both metformin ( < 0.05) and vaccine ( < 0.01) alone reduced the tumor volume, but their combined treatment more potently reduced the tumor volume ( < 0.001). Metformin alone did not obviously affect CD4 cells or CD8 cells but significantly decreased the percentage of CD4Foxp3 ( < 0.05); the vaccine alone significantly increased CD4 cells and CD8 cells ( < 0.001) and also the percentage of CD4Foxp3 cells ( < 0.05). The combined treatment, while reducing the percentage of CD4Foxp3cells to a level lower than that in the vaccine group ( < 0.01), produced the strongest effect to increase CD4 cells and CD8 cells ( < 0.01). CONCLUSIONS Metformin can inhibit the proliferation and function of regulatory T cells in an acidic environment and enhance the effect of tumor vaccine by reducing the proportion of Treg cells to achieve the anti-tumor effect.
Animals ; Cell Proliferation ; Forkhead Transcription Factors ; Male ; Metformin ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory

Objective To investigate the prevalence of nonalcoholic fatty liver disease and the risk factors in Jingyuan county of Ningxia Autonomous Region.Methods The population proportionate sampling method was applied to enroll a representative sample of 10 639 adults in Jingyuan county and the study was conducted using questionnaires and physical examinations.A total of 10 553 people were included in the analysis after excluding those with missing data.High-resolution ultrasound was used to examine the liver and fasting blood was collected in the morning for measurement of blood glucose,blood lipid,and uric acid.The participants were divided into two groups of those with and without nonalcoholic fatty liver disease;the difference in blood biochemical indexes between fatty liver and non-fatty liver groups was compared,and the logistic regression model was used to explore the risk factors affecting the prevalence of fatty liver.Results The prevalence of nonalcoholic fatty liver disease was 7.60％.The prevalence of thyroid nodules was higher in men than in women (8.60％ vs.6.82％,x2=1 1.772,P=0.001).The prevalence rate of fatty liver increased with age (x2=57.336,P＜0.001),the prevalence rates among ≥18 years-＜29 years,≥30 years-＜39 years,≥40years-＜49 years,≥50 years-＜59 years,≥60 years-＜69 years,and above 70 years were 2.92％,6.50％,8.81％,9.59％,8.08％,and 4.77％ respectively.The detection rate of overweight,obesity,abdominal obesity,impaired fasting glucose,impaired glucose tolerance,diabetes,hypertension,hyperuricemia,and dyslipidemia were higher in nonalcoholic fatty liver disease group than in the normal group (P＜0.05).Logistic regression analysis showed that nonalcoholic fatty liver disease group had a higher risk for overweight,obesity,abdominal obesity,impaired fasting glucose,impaired glucose tolerance,diabetes,hypertension,hyperurcemia,and dyslipidemia (OR=5.41,12.45,2.99,1.85,2.05,3.30,1.41,2.23,and 1.98).Conclusion The incidence of nonalcoholic fatty liver in Jingyuan county of Ningxia Autonomous Region was higher.The groups of overweight,obesity,abdominal obesity,impaired fasting glucose,impaired glucose tolerance,diabetes,hypertension,hyperuricemia,and dyslipidemia were high risk factors for nonalcoholic fatty liver disease.