Ganoderma lucidum (Leyss.Fr.) Karst is one of the well-known medicinal macrofungi all over the world, and mounting researches have focused on the polysaccharides derived from the spores of G. lucidum. In the present study, BALB/c mice (n = 8–10) were administered with crude polysaccharides of G. lucidum spores (CPGS) and the refined polysaccharides of G. lucidum spores (RPGS) for 30 days to investigate their effect on the adaptive immune system. Results showed that CPGS and RPGS displayed diverse effects on the lymphocyte activity in the spleen. The splenocyte proliferation activity upon mitogen was suppressed by CPGS and RPGS, while the NK cell's tumor-killing ability was promoted by CPGS. Both CPGS and RPGS could increase the proportion of naïve T cells in thymus, but only RPGS significantly uplifted the percentage of T cells, as well as the T cell subsets, in peripheral blood, and promoted the activation by upregulating the expression of costimulatory factor CD28. Moreover, 16S sequencing results showed that the effects of CPGS and RPGS were closely related to the regulation of gut microbiota. β-diversity of the microbiome was evidently changed by CPGS and RPGS. The phytoestrogen/polysaccharide-metabolizing bacteria , and an unclassified Desulfovibrionaceae, were remarkably enriched by CPGS or RPGS, and functions involving carbohydrate metabolism, membrane transport, and lipid metabolism were regulated. Moreover, the enrichments of Adlercreutzia, Prevotella, and Desulfovibrionaceae were positively related to the immune regulation by CPGS and RPGS, while that of Parabacteroides displayed a negative correlation. These findings suggested a promising effect of the polysaccharide from sporoderm-broken spore of G. lucidum in immune regulation to promote health control. Ganoderma lucidum (Leyss.Fr.) Karst belongs to the family of Basidiomycete, Polyporaceae of Ganoderma. G. lucidum is popular worldwide for its health promotion activity. Owing to the multiple regulation on the immune system, G. lucidum has been widely applied as an alternative adjunct to conventional treatment against various kinds of cancers [G. lucidum spore have been recognized as promising medicinal part of G. lucidum, and it has drawn increasing attention for its versatile biological activities owing to the technology development in sporoderm-broken spores [G. lucidum spores include polysaccharides, triterpenoids, peptides, amino acids, fatty acids, and trace elements, which are responsible for the anti-inflammation and antitumor activities [ cancers , 2, suchn spores , 4. The tivities , 6.Gut microbiota is a unique and diverse ecosystem known as the “forgotten organ” of the host . Gut micG. lucidum, as well as those from spore of G. lucidum, exert their activities via the regulation of gut microbiota. Chang et al. [G. lucidum exerted a prebiotic effect to reduce obesity in mice by modulating the gut microbiota. Guo [G. lucidum have the potential to ameliorate lipid metabolic disorders through targeting the specific gut microbiota. Li et al. [G. lucidum polysaccharides displayed an intestinal microbiota-dependent effect on mice with chronic pancreatitis. Su [G. lucidum displayed a synergy with paclitaxel by remodeling the structure of the gut microbiota. In the present study, immunomodulatory activity of the crude polysaccharides of G. lucidum spores (CPGS) and that of the refined polysaccharides of G. lucidum spores (RPGS) were compared in normal mice, as well as the possible involvement of gut microbiota in the immunity regulation of polysaccharide of G. lucidum spores.Emerging evidence has revealed that bioactive components from g et al.  showed tota. Guo  and colli et al.  manifestitis. Su  and collG. lucidum were provided by Guangdong Yuewei Edible Fungi Technology Co. Ltd. Preparation for the crude polysaccharides of G. lucidum spores (CPGS) and that for the refined polysaccharides of G. lucidum spores (RPGS) were as described by Su et al. [The sporoderm-broken spores of u et al. . In brieFor RPGS, the CPGS was dissolved in water and dialyzed with a 3500 Da dialysis tube (MWCO). The content in the 3500 Da dialysis tube was then dialyzed in a 100 kDa dialysis tube (MWCO). The dialysate was pooled, concentrated, and lyophilized to obtain RPGS. Yield of RPGS was 0.4%.Sugar content was determined by the phenol–sulfuric acid method using glucose as standard, and protein was quantified by the BCA protein kit . Weight average molecular weight (Mw) was analyzed by size-exclusive high-performance liquid chromatography (HPLC). Results showed that CPGS contains 64% polysaccharides and 23% protein, and the Mw is 5∼270 kDa. RPGS contains 53% polysaccharides, but the protein was hardly detected, and the Mw is 5 kDa.n = 8–10) were provided by Guangdong Medical Laboratory Animal Center . All animals were housed at 20 ± 2°C with a humidity of 50 ± 5%, using dust-free poplar chips for bedding, and lighting (12 h light–12 h dark cycle). They were fed with standard diet for rodents, and water was provided ad libitum during the whole experiment. The experiment was performed with the approval of the Guangdong Institute of Microbiology Laboratory Animal Ethics Committee according to the guidelines.Male BALB/c mice , CPGS group, and RPGS group. Mice of the normal group received equal volume of distilled water, while mice of the other groups were orally administrated with the corresponding drug once daily for 30 days. According to some previous studies , 21, 22,On the 31st day, peripheral blood was collected from the orbital vein plexus with an EDTA-Li anticoagulant tube, and the feces were collected. Then, the animals were sacrificed by cervical dislocation. Spleen and thymus were harvested, weighed, and then stored according to different purposes immediately. The organ coefficient was calculated as follows: organ coefficient (%) = organ weight/body weight × 100%. Schematic diagram of the experimental design is shown in g for 5 min. The obtained cells were resuspended with 1 mL of homemade ACK  for 5 min to lyse erythrocytes, and then the splenocytes were washed with precooled PBS for 3 times. Splenocytes were resuspended in RPMI-1640 medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin , cultured in incubators at 37°C under an atmosphere of 5% CO2 for assays.The spleen was dissected under sterile conditions, cut into pieces, and ground gently in precooled PBS. The homogenates were filtered through a 200-mesh cell strainer and centrifuged at 250×5 cells/well (100 μL/well) in 96-well culture plates (ninefold wells in each group). The mitogen response assay was performed by coculture of splenocytes with concanavalin A  or lipopolysaccharide  and incubated for 72 h at 37°C. At the same time, blank wells were set, which only contains 200 μL of 1% penicillin/streptomycin RPMI-1640 basic medium. The cultures were treated with 20 μL/well of Cell Titer 96®AQueous MTS Reagent  for 4 h. Absorbances were measured at 490 nm using a Fluorescence Microplate Reader . A proliferation index was calculated by the following formula: Relative activity (%) = (polysaccharide group − blank well)/ × 100%.The splenocytes were seeded at a concentration of 6 × 106 cells/well, 100 μL/well) and YAC-1 cells  were seeded into 96-well culture plates and coincubated for 4 h at 37°C. Wells with YAC-1 cells alone were set as spontaneous release group, and wells with YAC-1 cells incubated with 1% NP40 were set as maximum release group. The plates were centrifuged , and 0.1 mL of the supernatant was aspirated into a new 96-well culture plate. The supernatant was incubated with 0.1 mL of lactate dehydrogenase (LDH) reaction buffer  for another 10 min incubation. Then, 30 μL HCL (1 mol/L) was added to each well, the LDH release activity was quantitated by measuring absorbance at 490 nm. NK cell activity was calculated by the following formula: LDH release activity (%) = /(maximum release − spontaneous release) × 100%.Spleen effector cells .For the analysis of PBMCs, 80 PBMCs were stained with FITC anti-mouse CD3, PerCP anti-mouse CD4, PE anti-mouse CD8, PE-Cyanine7 anti-mouse CD28, APC anti-mouse CD34, FITC anti-mouse CD3, APC anti-mouse CD19, and PE anti-mouse CD3; while lymphocytes in thymus were stained with FITC anti-mouse CD44 and APC anti-mouse CD62 L for flow cytometry. All the above antibodies were purchased from eBioscience, Thermo Fisher Scientific .Sequencing service was provided by Personal Biotechnology Co., Ltd. , and it was performed as described by Su et al.  with milhttp://picrust.github.io/picrust/) [α-diversity between groups. Principle component analysis (PCA), uniFrac distance-based principal coordinates analysis (PCoA), and analysis of similarities (ANOSIM) were applied to compare β-diversity. In addition to taxonomy comparison, analysis of linear discriminant analysis effect size (LEfSe) was employed to figure out the specific bacteria responsible for each group. The predicted genes and their functions were aligned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and differences among groups were compared with STAMP4 [Bioinformatics analysis for the 16S amplicon sequencing data was performed on the Quantitative Insights into Microbial Ecology (QIIME) Version 1.8.0 MOTHUR (version 1.31.2) and PICRUSt (icrust/)  by R sofh STAMP4 .ttest, and the relationship between microbes and immunity was analyzed by Pearson's correlation; otherwise, the data was compared by Mann–Whitney test, and the relationship was analyzed by Spearman's correlation. Statistical differences were considered significant at p < 0.05.Statistical analysis was performed with SPSS 22 . Datasets from each experiment were subjected to a normal distribution test firstly. If in compliance with the normal distribution, the data was analyzed by Student's p < 0.05), while spleen coefficiencies were significantly higher in CPGS group and RPGS group (p < 0.05).Effects of LNT, CPGS, and RPGS on immune organs were compared by organ coefficiency. Results from Figures p < 0.05), but the proliferation by LPS was not affected by LNT. CPGS showed an evident remarkable promotion on the NK cell's tumor-killing ability (p < 0.01), while RPGS did not affect it.However, CPGS and RPGS had diverse effect on the splenocyte activity, including the splenocyte proliferation upon mitogen and the NK cell's tumor-killing ability. Results showed that upon the stimulation of mitogen (ConA or LPS), splenocyte proliferation was evidently suppressed by CPGS and RPGS treatment  in peripheral blood of LNT and RPGS group were obviously higher than those of the normal group (p < 0.05), while the percentage of B cell (CD19+) was not affected by any of them. Data from 3+CD4+) and cytotoxic T cells  of LNT and RPGS group were obviously higher than those of normal group (p < 0.05). Moreover, expressions of the costimulatory factor CD28 in T cell and T-cell subsets (Th cells and Tc cells) of the RPGS group were significantly higher than those of the normal group (p < 0.01). These results indicate that RPGS would be able to promote the activation of T cells.As shown in 3+CD62LCD44+low) (p < 0.05), while lowered that of effector T cell (CD3+CD44+CD62Llow) (p < 0.05). RPGS treatment displayed a more strong increase on naive T cell (p < 0.01), but did not affect effector T cell.Figure 4 showed that CPGS treatment evidently increased the percentage of naive T cell , uniFrac distance-based principal coordinates analysis (PCoA), and analysis of similarities (ANOSIM). By PCA and PCoA, it was found that both microbiome structures of CPGS and RPGS were remarkably different from those of the normal group (p < 0.01) and the weighted R values were 0.3∼0.4 (p < 0.01).al group . The comal group , in whicVerrucomicrobia and Proteobacteria, but RPGS treatment increased that of Actinobacteria (p < 0.05). At the genus level, a total of 78 genera were identified from all samples (SHEET S2 Genus). LEfSe analysis was employed to figure out the enriched genus for each group , indicating that together with the structural modulation, CPGS and RPGS could regulate the function of the gut microbiota to improve immunity.By comparing the sequencing data with those collected in KEGG pathway database by PICRUSt , it was G. lucidum was explored from the perspective of the adaptive immune response.The immune system is a network system that is capable of monitoring, defense, and self-regulation. Generally, innate immunity and adaptive immunity comprise the entire immune system. Innate immunity is an evolutionary defense strategy  against 3+CD62LCD44+low), also known as unsensitized T cells, are enriched in thymus and are able to respond to novel pathogens that the immune system has not yet encountered. On the other hand, effector T cells (CD3+CD44+CD62Llow) are antigen-experienced naïve T cells that would provide an efficient cytotoxic activity. In the spleen, naïve T cells become activated and differentiate into memory/effector T cells once they are presented with the antigen-peptide-MHC-complexes by the antigen-loaded dendritic cells (DCs), and the immune response would be initiated upon the activation of appropriate costimulatory signals [The thymus is an important central immune organ where T cells develop, and the spleen is a key peripheral immune organ for lymphocyte storage and release, as well as for the development of immune memory. Naïve T cells , including Th cells and Tc cells . Th cellFirmicutes : Bacteroidetes ratio and causing endotoxemia mainly by raising the levels of LPS [Firmicutes but more Bacteroidetes, Proteobacteria, and Actinobacteria [α-diversity analysis indicated that treatment of CPGS and RPGS had no impact on community richness diversity or uniformity in comparison with that of normal group, while β-diversity analysis displayed evident structures difference among the overall communities of CPGS, RPGS, and normal group. By taxonomy analysis, it was found that both microbiotas of CPGS and RPGS groups had more unique microbes. Moreover, enrichments of Adlercreutzia, Prevotella, and the unclassified Desulfovibrionaceae were positively related to the immune regulation by CPGS and RPGS, including the percentage of T cell and the subsets, as well as their activation; while those of Parabacteroides displayed a negative correlation. Adlercreutzia, Parabacteroides, and Prevotella are phytoestrogen/polysaccharide-metabolizing bacteria, and they always have a positive relationship with immunity. For instance, Adlercreutzia, Parabacteroides, and Prevotella are higher in healthy control when compared with multiple sclerosis (MS) patients [Prevotella is associated with favorable responses to antiprogrammed death 1 immunotherapy in Chinese patients with non-small-cell lung cancer (NSCLC) [Adlercreutzia within the gut is revealed to be dependent on the type of polysaccharide. Cui et al. [Gelidium pacificumOkamura polysaccharide decreased Adlercreutzia, while animal-derived Cereus sinensis polysaccharide increased it. Parabacteroides is found to be positively related with the attenuation by natural polysaccharide on myocardial injury induced by high-fat diet [Prevotella spp. is a dominant bacterial genus within the human gut. Prevotella spp. exhibit variability in the utilization of diverse complex carbohydrates [Prevotella spp. would be promoted when the host takes in variable natural polysaccharides, which is a benefit for the amelioration of constipation symptoms [Desulfovibrionaceae was not detected in samples of the normal group, while it was evidently increased in samples of CPGS and RPGS groups. It has been revealed to be a positive outcome of the remission of colonic injury or inflammation [G. lucidum spore endowed the mice with a unique gut microbiota structure, which is involved in its immunomodulation activity. In addition to the effect on community structure, it is noteworthy that the carbohydrate metabolism, membrane transport, and lipid metabolism pathways displayed distinct regulation upon the polysaccharide treatment. Taken together, it is probable that by oral supplement, polysaccharide of G. lucidum spores was able to increase the richness of beneficial microorganisms and regulates the microbiome function pathways, thereby improving the balance of adaptive immunity.Nowadays, the gut microbiota has been recognized as a regulator of both immune response and host metabolism . For inss of LPS . And fecbacteria . In the patients . And a h (NSCLC) . Indeed,i et al.  found thfat diet  and immufat diet . Prevotehydrates . As revesymptoms , lipid msymptoms , and so ammation , 41. TheG. lucidum spores would serve as a natural candidate for the improvement of the adaptive immune system, especially the refined polysaccharide. CPGS and RPGS exhibited diverse regulation on the adaptive immune response. The splenocyte proliferation activity upon mitogen was suppressed by CPGS and RPGS, while NK cell's tumor-killing ability was promoted by CPGS. Both CPGS and RPGS could increase the proportion of naïve T cells in the thymus, but only RPGS significantly uplifted the percentage of T cells, as well as the T cell subsets in peripheral blood, and promoted the activation, indicating that RPGS exhibit more prominent regulation on T cell-involving immunity. Moreover, the effects of CPGS and RPGS are closely related to the regulation on gut microbiota, which not only involves the changes in structure and community membership but also affects several metabolic pathways within the microbiome. And the enrichments of Adlercreutzia, Prevotella, and the unclassified Desulfovibrionaceae were positively related to the immune regulation by CPGS and RPGS. These findings suggested a promising effect of the polysaccharide from sporoderm-broken spores of G. lucidum in immune regulation to promote health control.Collectively, the present study revealed that polysaccharides from


Unresponsive thin endometrium caused by Asherman syndrome (AS) is the major cause of uterine infertility. However, current therapies are ineffective. This study is to evaluate the effect of transplantation with collagen scaffold/umbilical cord mesenchymal stem cells (CS/UC-MSCs) on this refractory disease.Eighteen infertile women with unresponsive thin endometrium, whose frozen–thawed embryo transfers (FETs) were cancelled due to reduced endometrial thickness (ET ≤ 5.5 mm), were enrolled in this before and after self-control prospective study. Hysteroscopic examination was performed to confirm no intrauterine adhesions, then twenty million UC-MSCs loaded onto a CS were transplanted into the uterine cavity in two consecutive menstrual cycles. Then uterine cavity was assessed through hysteroscopy after two transplants. FETs were performed in the following cycle. Pregnancy outcomes were followed up. Endometrial thickness, uterine receptivity and endometrial angiogenesis, proliferation and hormone response were compared before and after treatment.P < 0.001). Three of 15 patients after FET got pregnant, of whom 2 gave birth successfully and 1 had a miscarriage at 25 weeks’ gestation. One of 2 patients without FET had a natural pregnancy and gave birth normally after transplantation. Immunohistochemical analysis showed increased micro-vessel density, upregulated expression of Ki67, estrogen receptor alpha, and progesterone receptor, indicating an improvement in endometrial angiogenesis, proliferation, and response to hormones.Sixteen patients completed the study. No treatment-related serious adverse events occurred. Three months after transplantation, the average ET increased from 4.08 ± 0.26 mm to 5.87 ± 0.77 mm (CS/UC-MSCs is a promising and potential approach for treating women with unresponsive thin endometrium caused by AS.NCT03724617. Registered on 26 October 2018—prospectively registered, https://register.clinicaltrials.gov/ClinicalTrials.gov The online version contains supplementary material available at 10.1186/s13287-021-02499-z. Thin endometrium is often found in women with Asherman syndrome (AS) because the basal layer is destroyed, and the functional layer fails to respond to hormonal stimulation, which is the major cause of uterine infertility , 2. AdeqClinically, numerous strategies have been adopted to promote endometrial regeneration, including extended estrogen administration, low-dose aspirin, pentoxifylline, tocopherol, vaginal sildenafil citrate, and intrauterine perfusions with granulocyte colony-stimulating factor –9. HowevIn women at reproductive age, the endometrium undergoes repeated stripping and bleeding during the menses and can be built up without scarring in subsequent cycles . The regMesenchymal stromal cells (MSCs) are being investigated as a potential alternative for cellular therapy. Although bone marrow mesenchymal stem cells (BMSCs) have been used widely, the aspiration of bone marrow is invasive with relatively low cell yield, as is the use of adipose tissue stem cells (ASCs). And with increasing age, cell yield of bone marrow is significantly decreased . As for How to transplant stem cells is an important issue to be solved in clinical applications. Tissue engineering, which involves the use of living human cells on appropriate scaffolds for the repair and reconstruction of various tissue injuries and defects, has provided a new and reliable strategy for the transplant of stem cells and has been widely recognized by the medical community . Collagen = 3) of normal term fetuses  were collected under sterile conditions, soaked in DMEM/F12 medium , and transported on ice to the cell preparation laboratory within 48 h. UC tissues were washed with saline to remove blood stains. Residual blood, capsule, and blood vessels were removed, and the remaining tissue was cut into about 4-mm3 pieces. Tissue fragments were inoculated in a petri dish, cultured in DMEM/F12 medium  supplemented with 10% fetal bovine serum , and then placed in an incubator at 37 °C with 5% CO2 for 7 days. After 7 days of culture, we changed the culture medium and observed that the cells had crawled out under an inverted microscope  and continued the culture to the 14th day. When the cell density reaches 80–90%, the primary cells were passaged and resuspended by serum-free MSC culture medium  for use.The UC-MSCs were of clinical grade, as recognized by the National Institutes for Food and Drug Control  were analyzed with BD FACSCaliburTM flow cytometer  and analyzed using BD CellQuestTM pro software version 6.0. The primary antibodies were anti-human CD105 , anti-human CD73 , anti-human CD34 , anti-human CD45 , anti-human CD90 , and anti-HLA-DR . For each sample, at least 10,000 events were recorded.Flow cytometry was performed to identify the phenotype of UC-MSCs at the 3rd passage. Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and blocked with 2% bovine serum albumin . The cells were stained with primary antibodies, followed by fluorescein isothiocyanate-conjugated antibodies  or phycoetrythrin-conjugated antibodies  diluted in PBS plus 2% BSA. Stained cells , adipogenic differentiation medium , and chondrogenic differentiation medium  in the induction groups. In the non-induction group, MSC culture medium was continued. Culture medium was changed every 3 days. The resulting calcium deposition, fat vesicles, and sulfated glycosaminoglycans were subjected to Alizarin Red , Oil Red O (Solarbio Life Science), and Alcian Blue (Solarbio Life Science) staining 3 weeks later. For UC-MSCs extracted from one umbilical cord, all the above experiments were repeated 3 times independently.UC-MSCs at the 5th passage were detached by trypsin, resuspended by pre-warmed MSC culture medium, and seeded into 24-well plates at 5 × 10This study was approved by the Ethics Committee of Stem Cell Clinical Research Institution of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (No. 20180801-1) and conducted at the Reproductive Medicine Center of Sir Run Run Shaw Hospital from February 2019 to April 2021. All the patients signed informed consents. This study was registered on ClinicalTrials.gov (NCT03724617).Patients were recruited from the Reproductive Medicine Center of Sir Run Run Shaw Hospital from February 2019 to April 2020. The inclusion criteria were as follows: (1) age 20–40 years; (2) infertile patients who had received assisted reproduction treatment and had frozen embryos in store; (3) patients who underwent at least two rounds of hysteroscopic adhesiolysis (HSA) and the uterine cavity returned to normal; (4) women whose ET failed to expand beyond 5.5 mm with the use of 6–8 mg/day estradiol valerate combined with at least one round of treatment with aspirin, granulocyte colony-stimulating factor (G-CSF), heparin, vaginal sildenafil, or Chinese traditional medicine. Patients were excluded from recruitment if they had any of the following issues: (1) those who could not agree to the follow-up conditions required by the study; (2) contraindications for hysteroscopic surgery and estrogen therapy; (3) congenital uterine malformations, adenomyosis, or uterine fibroids that could impair embryo implantation; (4) chromosomal abnormalities; (5) systemic diseases such as thrombosis, cardiopulmonary diseases, hematopoietic diseases, and malignant tumors; and (6) no desire to be pregnant.α) of 0.05 and type II errors (β) of 0.20 and assuming that the dropout rate would be 20%, the sample size should be at least 17.This study was prospective with each patient serving as her own control. We assume that the mean ET would increase from 5 to 7 mm with a standard deviation (SD) of ± 1.2 mm. Accepting type I errors (7/mL (2 mL) UC-MSCs at the 5th passage was dripped uniformly onto the scaffold. The seeded scaffolds were incubated under humid 5% CO2 in air at 37 °C for 1 h before transplantation.The CS/UC-MSCs were fabricated as follows: 4 cm × 6 cm collagen scaffolds with pores of 20–200 μm in diameter  were rinsed with serum-free MSC culture medium; excess fluid was aspirated; and a suspension of 1 × 10The CS/UC-MSC scaffold were spread onto a 10-F Foley catheter and placed into the uterine cavity. After being placed in the uterine cavity, a balloon filled with 3 mL sterile saline was inserted to assist the scaffold in attaching to the inner wall of uterine cavity. B-ultrasonography confirmed that the scaffold had adhered to the uterine wall. The patient was kept in the hospital for 2 h after this procedure to observe vital signs. The balloon was left in place for 3 days before removal. Antibiotics were used to prevent infection in all patients until 6 days after surgery.The patient underwent CS/UC-MSC transplantation on the 7–12th day of menstruation. The study procedure is outlined in the flow chart shown in Fig. Patient follow-up was performed either in the clinic or by telephone consultation, ending in May 2021. Any surgical complications , and the patient’s body temperature and hemograms before and after the transplantation were recorded. The hemograms, and liver and kidney function test results were recorded 1 week after the operation. All patients were followed up to determine whether there was any tumor formation.The primary outcome was the ET measured on the day of starting progesterone before and 3 months after surgery. Secondary outcomes included endometrial morphology under hysteroscopy, uterine receptivity, pregnancy outcomes, and endometrial histology. Ultrasonography was performed to evaluate uterine receptivity with a 5–9 MHz endovaginal probe using a GE Voluson E10  by the same expert examiner at day 3 of progesterone administration during HRT cycles. The evaluation indicators for uterine receptivity in this study mainly included (1) ET; (2) endometrial volume; (3) endometrial and subendometrial blood flow, which were observed and classified using the Applebaum classification ; (4) utePregnant women were followed up until the end of pregnancy, during which fetal conformation and aneuploidy screening and routine prenatal examinations were performed. Any placental complications were monitored by ultrasonography during pregnancy.i + 1), where i indicates the intensity of staining with a value of 1, 2, or 3  and Pi stands for the percentage of stained cells in the whole image, with intensity ranging from 0 to 100%.All endometrial biopsies were taken during the proliferative phase. Human endometrial formalin-fixed and paraffin wax-embedded biopsies obtained before and after treatment was used for hematoxylin and eosin (H&E) staining and immunohistochemistry as previously described . The numt test was used for continuous variables. Fisher’s exact test was performed for comparing categorical variables. A two-sided P value of < .05 was considered statistically significant.Statistical analysis was performed using GraphPad PRISM software . Paired UC-MSCs were spindle-shaped in morphology. UC-MSCs were positive for CD90, CD105, and CD73, and negative for CD34, CD45, and HLA-DR. UC-MSCs were able to differentiate into osteoblasts, adipocytes, and chondrocytes . The difference was statistically significant  after hormone supplements combined with adjuvant treatments. Given that, the pregnancy rate (24%) and live birth rate (18%) are acceptable.In 2011, Nagori et al. reported the first case of AS treated with adult autologous stem cells via intrauterine infusion for endometrial regeneration that resulted in conception after in vitro fertilization and embryo transfer (IVF–ET) . Eight cAnother important difference was in the frequency of stem cells administered. A single stem cell transplantation was performed in almost all patients in the seven studies. Interestingly, in Tan’s study, if the patient’s endometrium exceeds 7 mm after the first transplantation, the second one would be performed, which indicated that increasing the frequency of stem cell transplantation is beneficial to endometrial regeneration . Taking Last but not least, the way stem cells were administered is different. In the above trials, four transplantation methods including intrauterine perfusion, spiral arterioles injection, basal layer injection, and stem cells loaded on collagen scaffold were used as shown in Table The mechanism involved in the restoration of functional endometrium by stem cells is mainly differentiation, angiogenesis, immunosuppression, and anti-inflammatory via paracrine function , 50. We According to the above, this study has some limitations such as lack of control, a small sample size, and knowledge of underlying mechanism. Further studies should be carried out to verify the treatment effect of CS/UC-MSCs in unresponsive thin endometrium in a larger sample or RCT study and to clarify the underlying mechanism.In summary, our work describes that transplantation of CS/UC-MSCs is a promising and potential approach for treating women with unresponsive thin endometrium caused by AS. UC/MSCs may be beneficial for endometrial proliferation and angiogenesis and enhancing the response of endometrium to hormones.Additional file 1: Supplemental Figure 1. Phenotypic analysis and differentiation experiments were performed to characterize the UC-MSCs. A. Morphology of UC-MSCs at the 5th and 15th passage. B. Flow cytometry analysis showed that the positive rates of CD90, CD105 and CD73 were 99%, 99.3%, and 99%, respectively, while the hematopoietic markers CD34, CD45 and HLA were negative, identifying the cells as mesenchymal stem cells with good homogeneity. C. After 21 days of induction culture, Capacity to differentiate into osteocytes, adipocytes and chondrocytes was evaluated staining with Alizarin Red, Oil-red O and Alcian blue.Additional file 2: Supplemental Figure 2. Osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation of UC-MSCs in the non-induction and induction groups.Additional file 3: Supplemental Figure 3. H&E pictures of 17 patients after before and after treatment.Additional file 4: Supplemental Table 1. Biological safety and biological activity analysis of the clinical-grade cells$ recognized by the National Institutes for Food and Drug Control (NIFDC).Additional file 5: Supplemental Table 2. Leukocyte counts, neutrophil percentage and lymphocyte count, liver function and kidney function 7 days after operation in each patient.Additional file 6: Supplemental Table 3. Summary of transferred embryos of 15 patients.Additional file 7. Supplemental Material of the patient #16.


Allopolyploid breeding is an efficient technique for improving the low seed setting rate of autotetraploids in plant breeding and one of the most promising breeding methods. However, there have been few comprehensive studies of the posttranscriptional mechanism in allopolyploids.Oryza sativa, genome AA) with wild rice , we created hybrid rice lines with different ploidy and genome compositions . The genetic differences of the hybrids and the mechanism of allopolyploid breeding dominance were revealed through morphological and cytological observations and single molecule real-time sequencing techniques. The tissues and organs of allotetraploid hybrid F02 exhibited "gigantism" and the highest levels of fertility. The numbers of non-redundant transcripts, gene loci and new isoforms in the polyploid rice lines were higher and the isoform lengths greater than those of the diploid line. Moreover, alternative splicing (AS) events occurred twice as often in the polyploid rice lines than the diploid line. During these events, intron retention dominated. Furthermore, a large number of new genes and isoforms specific to the lines of different ploidy were discovered.By crossing cultivated rice  and AS events contributed to the complexity and superiority of polyploids in the activity of translation regulators, nucleic acid binding transcription factor activities and the regulation of molecular function. Therefore, these APA and AS events in allopolyploid rice were found to play a role in regulation. Our study provides new germplasm for polyploid rice breeding and reveals complex regulatory mechanisms that may be related to heterosis and fertility.The online version contains supplementary material available at 10.1186/s12870-022-03502-2. Rice is one of the most important food crops in the world and is consumed by more than half of the world’s population . Howeverindica and japonica), between species (cultivated rice and wild rice with A genome) and between genomes (cultivated rice and wild rice with non-A genome). An efficient technology that develops synthetic allopolyploid rice based on wide cross, embryo rescue, and in vitro colchicine treatment was established [Oryza sativa) and O.rufipogon, O.barthii, O.nivara, O.longistaminata, O.punctata, O.officinalis, O.alta, O.australiensis, O.meyeriana [Polyploidization, one of the most important evolutionary events in plants, can increase genetic diversity, introduce new genetic combinations, foster adaptation to environments, and create vigorousness effects –7. A greablished . Using teyeriana –14.Hybrid and polyploidy species often undergo alternative splicing (AS) and alternative polyadenylation (APA) events to increase diversity. AS and APA are two important posttranscriptional regulatory mechanisms that can enhance the diversity of the transcriptome and, ultimately, the proteome , 16. AS O. sativa × O. punctata; allotetraploid hybrid  was produced through chromosome doubling of F01; and allotetraploid hybrid  was obtained by crossing tetraploid O. sativa (AAAA) × F02 (AABB). The differences in fertility and morphology of the hybrids were studied using morphology and cytology. Moreover, the full-length transcriptomes of the hybrids were analyzed comprehensively for the first time. AS and APA events in the different hybrids were globally surveyed using PacBio single-molecule long-read isoform sequencing, and the regulatory complexity of allotetraploid rice was explored through a variety of database annotations. This study lays a foundation for the analysis of the molecular mechanism of allopolyploid breeding dominance and provides a useful reference for rice transcriptome annotation resource and genetic diversity.In this study, distant hybrids with different ploidy and genome compositions were investigated. A diploid hybrid F01  was produced from As shown in Fig. The mature and normal pollen grains in the allotetraploid hybrid F02 were full, round, dark and 41.44 μm in mean diameter. The pollen grains of F01 and F03 could not be stained, and typical abortive pollen was the main type, showing characteristics of shrinkage and shriveling. Therefore, the diameters of the pollen grains were smaller, at 21.08 μm and 26.21 μm, respectively -PCR. Isoforms of each gene were aligned in order to design primers that could amplify all predicted transcripts at the same time  were defined as “unannotated isoforms” using Cuffcompare. Figure . APA analysis is subject to certain limitations using conventional RNA-seq short read sequences, and the effect of APA on the complexity in lines of different ploidy are still unknown in rice. The investigation of 3' ends of transcripts using Iso-seq allowed us accurately to identify differential polyadenylation sites in rice  than the diploids, but there was no significant difference between F02 and F03 . In this study, Fig. LOC_Os08g06110 of F02 and F03 in the pollen mature panicle, stem and flag leaf were higher than that of F01. This is consistent with the characteristic that F02 and F03 have a delayed heading date than F01 under long-day conditions (≥ 12 h). The pollen development could be regulated by gene LOC_Os10g38050, resulting in a small amount of pollen. The relative expression levels of LOC_Os10g38050 of F01 were highest, resulting the small amount of pollen and infertility. On the contrary, the pollen number of F02 and F03 was high due to low expression levels.The rice heading period could be regulated by gene O. sativa) and wild rice (O. punctata) as the basic materials, a series of resources created by hybridization can be produced that retain the excellent characteristics of the parents. Currently, our understanding of the rice transcriptome is based mainly on SGS technologies (including resequencing) [Hybridization and polyploidy are effective ways to increase plant biomass, yield and resistance to biotic and abiotic stresses , 29, 30.uencing) , 32. TheOryza species. The results showed that the allotetraploid hybrid AABB (F02) not only overcomes the sterility of the distant hybrid AB (F01) but also displays strong heterosis. Nearly 20 years of sexual reproduction proves that it can be inherited stably in our laboratory. The breeding practice of AABB shows the feasibility of the pathway ‘breeding super rice by using double advantages of distant hybridization and polyploidization’. This demonstrates a new pathway for creating novel crops in the future. Crop polyploidization may play an important role in next-generation crop improvement aimed at facing food security challenges [Under the guidance of the strategy ‘breeding super rice by using double advantages of distant hybridization and polyploidization’ , a numbeallenges .Zea mays and cotton [Hybridization and polyploidization events have frequently occurred throughout plant evolutionary history, and they have played an important role in the evolution of species and the formation of new species. AS is a unique posttranscriptional modification in eukaryotes, by which a gene produces multiple transcriptional translations, creating complexity in the eukaryotic transcriptome. AS in plants can be regulated by many mechanisms initiated at different developmental stages and by environmental signals. These events play an important role in abiotic stress, biotic stress, hormone regulation, development, flowering time and the biological clock response. Therefore, AS may also be an indispensable part of the mechanism of heterosis. In general, the type of AS in plants is mainly IR , 33, 34.d cotton , 35. Wand cotton . The numIn eukaryotes, polyadenylation affects the localization, stability, translation efficiency and function of RNA and is an important method of posttranscriptional modification and regulation, which in turn regulates gene expression through a variety of regulatory mechanisms , 37, incTetraploids generally show vegetative growth superiority, larger organs, higher biological yield, more secondary metabolites and enhanced resistance than diploids , 40, whiIn this study, we comprehensively investigated the transcriptome of the distant hybrids with different ploidy and genome compositions using PacBio third-generation sequencing. Allotetraploid hybrid (AABB) had a good phenotype in plant growth and high rate of seed setting, and could be used as a breeding resource. Furthermore, genetic differences and dominance were revealed by the full-length transcriptome. Allopolyploidy not only increased the number of genes involved in many pathways but also increased the proportion of isoforms involved in energy and carbohydrate use. Alternative transcripts were existed in post-translational modification, protein turnover, chaperones, signal transduction mechanisms and responses to stimuli. These new findings will provide important information for improving genome reannotation of polyploid rice and strengthen our understanding of the rice transcriptome.O. sativa  subsp. japonica was stored at the Polyploid Genetics Lab of Hubei University, Wuhan, China. Wild rice O. punctata,  was kindly provided by the International Rice Research Institute (IRRI), Manila, Philippines. The diploid interspecific hybrid F01  was obtained by crossing O. sativa × O. punctata. F02, an allotetraploid hybrid , was obtained via chromosome doubling of F01(AB), and F03, an allotetraploid hybrid (AAAB) was obtained by crossing tetraploid O. sativa (AAAA) × F02 (AABB).Asian cultivated rice Chromosome root tip squashes were made according to the method described by Wang et al.  with minn = 5), flag leaf length and width (n = 5), panicle number per plant (n = 5), panicle length (n = 25), grain length and width (n = 25), awn length and color (n = 25), total grain number per panicle (n = 25), filled grain number per panicle (n = 25), seed setting rate , stigma color, grain color and shattering traits were measured.Morphological and agronomic characteristics, such as plant height  =  × 100.Mature pollen grains were stained with 1% ITotal RNA was extracted by grinding tissue in TRIZOL reagent. To determine the RNA quality, samples were assessed using a NanoDrop microspectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies). The total RNA from five tissues including the flag leaf, stems, young panicles, meiotic young panicles and pollen mature panicles was pooled together in equal amounts.The mixed RNA samples were reverse transcribed into cDNA using the SMARTer® PCR cDNA synthesis kit and optimized to prepare high-quality and full-length (FL) cDNAs. Subsequently, size fractionation and selection (1–6 kb) were conducted using the BluePippin Size-Selection System . A Single Molecule, Real-Time (SMRT) bell library was constructed with the PacBio DNA Template Prep Kit 2.0. The library was subsequently sequenced on the PacBio RS II platform with P6C4 polymerase enzyme and chemistry.Raw reads were processed into error-corrected reads of insert (ROIs) using the isoform sequencing (Iso-seq) pipeline with min Full Pass = 0 and min Predicted Accuracy = 0.80. Next, full-length, non-chimeric transcripts were determined by searching for the polyA tail signal and the 5' and 3' cDNA primers in the ROIs. Iterative Clustering for Error Correction (ICE) was used to obtain consensus isoforms, and FL consensus sequences were polished using Quiver. High quality FL transcripts were classified with the criteria of post-correction accuracy above 99%.Oryza sativa L. spp. japonica var. Nipponbare MSU_v7.0ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/all.dir/) using Genomic Mapping and Alignment Program. Mapped reads were further collapsed using the pbtranscript-ToFU package with min-coverage = 85% and min-identity = 90%. The 5' difference was not considered when collapsing redundant transcripts.FL consensus sequences were mapped to the reference genome  software  and PRAPhttp://kobas.cbi.pku.edu.cn/index.php) [Corrected isoforms were searched against NCBI non-redundant (NR), NCBI nucleotide sequence (NT), Swiss-Prot , Cluster of Orthologous Groups (KOG/COG)  and Kyotdex.php) , 54, anddex.php) .https://www.invitrogen.com). Complementary DNA was synthesized from 1 μg total RNA in a 20 μl solution using the MonScript™ RTIII Super Mix with dsDNase (Two-Step) .To verify the AS events detected above, we designed primers for the AS regions of these Table S. Total Rhttp://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi) was applied to design the primers for qPCR. The PCR was used to identify the specificity of the primers. Gimmico A4 Fidelity PCR Mix (1.1 ×)  was used for PCR amplification. The PCR products were visualized in agarose gel stained with SuperRed/GelRed . MonAmp™ SYBR® Green qPCR Mix  was used for qPCR amplification. The qPCR was carried out using cDNA, a gene-specific forward primer and a gene-specific reverse primer. The β-actin was amplified as an endogenous control.Primer3Plus webpage (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi) was applied to design the primers for the gene cloning. I-5™ 2 × High-Fidelity Master Mix  was used for PCR amplification. The PCR was carried out using cDNA, a forward primer and a reverse primer. PCR amplification was monitored on 1.5% agarose gel and the PCR products were visualized in agarose gel stained with SuperRed/GelRed . DNA Gel Extraction Kit  was used for acquiring the PCR products and followed by Sanger sequencing.Primer3Plus webpage , and genes with FPKM > 0.3 were selected for analysis.P < 0.05 was used to denote a statistically significant difference.Values of morphological and agronomic characteristics were expressed as means ± standard deviations. Data were analyzed by one-way ANOVA using the SPSS statistical software (version 25.0). The SMRT sequencing raw reads reported in this study have been deposited in the NCBI Sequence Read Archive (SRA) under Bioproject accession number PRJNA699388.Additional file 1: Fig.S1. Flowchart of the experimental design and analysis for PacBio sequencing and RNA sequencing. Fig.S2 Read length with different ploidy and genome compositions hybrids. Fig.S3 The number of isoforms of functional annotation of with different ploidy hybrids. Fig.S4 RT-PCR validation of AS events for two genes. Gel bands in each figure show DNA makers and PCR results in five tissues/samples. Fig. S5 The p-value for GO enrichment of isoforms.Additional file 2:Table S1. Functional annotation of transcripts.Additional file 3:Table S2. Splice junctions and AS modes with different ploidy levels.Additional file 4:Table S3. Functional annotation of transcripts in the polyadenylation events.Additional file 5:Table S4 Summary of primers used in this study.


Antibiotic overuse is the main modifiable driver of antibiotic resistance. Factors associated with overuse have been inconsistently reported and vary across populations. Given the burgeoning occurrence of infectious diseases around the world, there remains a great need to identify barriers and solutions to the control of infections. We examined whether knowledge about infections and antibiotic resistance is associated with antibiotic use in a northern European population sample.> 16 years. Participants were asked whether they had taken an antibiotic in the past 12 months; and six questions were asked concerning knowledge about infections and antibiotic resistance. Correct answers to the six knowledge questions defined a knowledge score (score range 0–6 correct answers). We used multivariable logistic regression to estimate odds of self-reported antibiotic use during the last 12 months in association with knowledge score , and response to each knowledge question. Covariates included sex, age group, smoking, alcohol drinking, deprivation index, self-rated health, and satisfaction with life. Results were outputted as Odds Ratios (OR) and 95% Confidence Intervals (CI).The Health Survey Northern Ireland 2014/15 was completed by a cross-sectional sample of 4135 participants aged p < 0.001), with a P-value < 0.001 for trend with increasing knowledge score. Female sex, age, high deprivation, and poor general health, were independently associated with higher odds of antibiotic use. Stratified analyses showed sex and age group differences.Antibiotic use in the past 12 months was reported by 39.0% (1614/4135); and 84.2% (3482/4135) scored < 6/6 correct on knowledge statements. Compared to the lowest knowledge score (0/6 correct), the highest knowledge score (6/6 correct) was associated with higher odds of antibiotic use (adjusted OR 2.03, 95% CI , Knowledge, and other modifiable and non-modifiable risk factors, were positively associated with antibiotic use in the past 12 months. While the causal direction of these associations could not be determined, given the high prevalence of lesser knowledge, as well as independent contributions of other factors including socioeconomic characteristics, health literacy campaigns to raise awareness of antibiotic resistance should take a multi-pronged approach.The online version contains supplementary material available at 10.1186/s12889-021-11018-x. Antimicrobial resistance (AMR) is a global crisis with serious consequences for the individual, public health, and the economy . It has Global commitment and multi-sector partnerships are critical to implement effective interventions to reduce unnecessary use of antibiotics and control AMR , 6. GlobThe World Health Organization’s (WHO) global action plan on AMR has five objectives, the first of which is to improve knowledge and understanding of AMR through communication, education and training . NationaOne model targeting behavioural change related to AMR suggests that knowledge, environmental change, social/professional roles, social influences, and public beliefs and priorities, among others, are important factors. Thus, multipronged public health interventions are necessary to address AMR and to foster behaviour change . QualitaGiven this backdrop, we took advantage of a big data resource – the Health Survey Northern Ireland 2014/15 (HSNI14/15) – to examine the association between knowledge regarding infections and AMR and antibiotic use. Since multi-prong interventions are necessary to decrease AMR, HSNI data allowed us to consider sex, age group, sociodemographic and behavioural factors, deprivation, and self-rated general health and satisfaction with life. These factors have been previously associated with knowledge and antibiotic use –27. GiveThe HSNI is a series of independent, cross-sectional surveys conducted annually since 2010 by Northern Ireland Statistics Research on behalf of the Northern Ireland Department of Health, Social Services and Public Safety. The HSNI examines a wide range of public health issues that are important for the people of Northern Ireland. In this analysis, we utilised data from HSNI14/15, conducted between April 1, 2014 and March 31, 2015. HSNI14/15 consisted of a random sample of 5850 street addresses that were representative of the general population in Northern Ireland . A vacanEligible households were sent a letter of invitation via post. The letter stated that the survey was being conducted on behalf of the Department of Health, Social Services and Public Safety, Central Survey Unit, which is the leading social survey research organisation in Northern Ireland. The letter also stated that an official interviewer would visit the household to explain more about the study and that the survey could be rescheduled if necessary. Non-response occurred when an eligible address was not reached by an interviewer or when respondents refused to participate. To minimise non-response, an interviewer attempted up to four calls to each eligible household unless the respondents had previously refused to participate.HSNI14/15 surveyed knowledge of infections, antibiotics and antibiotic resistance, and use of antibiotics over the past 12 months. Additionally, the survey collected sociodemographic, behavioural , and general health factors information .The study protocol of HSNI14/15 was approved by Northern Ireland Department of Health. Informed consent was provided when the interviewer visited the household. Participants were able to refuse to participate at any stage of the survey . No inceSurvey data from HSNI14/15 were collected using Computer-Assisted Personal Interviewing (CAPI). CAPI is a face-to-face interviewing method. HSNI14/15 interviewers visited the respondents with a computer, read the questions to the respondents, and entered the responses immediately on the computer during the course of the interview. If a participant did not want to engage in a CAPI interview, they were offered a self-administered, closed booklet version of the HSNI14/15 that contained the questions and response options. Responses were manually entered later .Our primary exposure and outcome variables were located in the ‘Medication’ section of the HSNI14/15. Within this section, there was a subsection dedicated to antibiotics. This section was comprised of seven questions.The answer to the first question in the ‘Medications’ section, subsection ‘Antibiotics’, was the primary outcome used in our analysis. The first question was ‘In the past 12 months, have you taken an antibiotic?’ Response options included: ‘Yes’, ‘No’ or ‘Don’t know’ .The following six questions in the section on ‘Medications’, subsection ‘Antibiotics’, were used as the primary exposure. These six questions assessed knowledge about infections, antibiotics and antibiotic resistance Table . Each quTo examine how knowledge about infections, antibiotics and antibiotic resistance was associated with antibiotic use in the past 12 months, the knowledge variable was considered as a correct answer to each individual statement , as well as a composite knowledge score. The knowledge score was the sum of answers to all six statements. The score range was 0–6, where a score of 0 corresponded to incorrect answers on all 6 statements and a score of 6 was a correct answer to all statements.We selected potential covariates and confounders based on published associations with knowledge, behaviour, and awareness and antibiotic use , as wellDemographic characteristics: included sex  and age group  [Health behaviours: included current cigarette smoking and alcohol drinking [drinking . CurrentSociodemographic factors: included education attained and income [d income . A deprid income  was usedGeneral health status: Self-rated general health and life satisfaction were components of the HSNI [the HSNI . Self-raParticipant characteristics and knowledge about infections and antibiotic resistance were described using frequencies and percentages. The Pearson’s chi-squared test was used to test for associations between categorical variables.To examine the association between the exposure, knowledge about infections and antibiotic resistance and the outcome, self-reported antibiotic use over the past 12 months, weighted logistic regression analyses were used with ‘no reported use’ as the referent. Sampling weights were used to estimate robust standard errors in both unadjusted and adjusted models. The HSNI14/15 sample was weighted by sex and age using the Northern Ireland 2014 mid-year population estimates. Odds ratios (OR) and 95% confidence intervals (CI) were estimated.The primary exposures were: 1) a correct answer to each of six knowledge statements, and 2) a composite knowledge score derived as the sum of correct answers to the six knowledge statements about infections and antibiotic resistance. Knowledge scores range from 0 to 6 with a score of 0 representing 0 correct answers to all 6 statements, and a score of 6 representing 6 correct answers to all 6 statements. First, we ran six models estimating whether the correct answer to each individual statement was associated with antibiotic use in the past 12 months. Second, using the sum of correct answers to the six statements regarding knowledge about infections and antibiotic resistance, we estimated whether knowledge score (maximum score 6/6 correct answers) was associated with antibiotic use in the past 12 months when compared to a lower knowledge score (score < 6/6 correct answers).p < 0.10 in univariate logistic analyses were included in multivariable models. Multivariable models were later stratified by sex and age group . Results were considered statistically significant at p < 0.05 using two-sided tests. All statistical analyses were performed using Stata 16.0.Univariate logistic regression analyses were followed by multivariable analyses. Sex, age group, smoking and alcohol drinking, deprivation index, self-rated general health, and satisfaction with life, were evaluated a priori as potential covariates in the multivariable models. Potential covariates with a n = 72) had missing data for questions comprising knowledge about infections and antibiotic resistance, self-reported antibiotic use, current cigarette smoking status, drinking status, self-rated general health, satisfaction with life or sample weight, and were excluded. This left 4135 participants included in our analysis. The majority of these HSNI14/15 participants were women (58.9%), older than 25 years (94.0%), current non-smokers (79.2%), and current alcohol drinkers (75.3%). There was a relatively equal distribution of participants across deprivation quintiles.Of 5850 randomly selected street addresses, 981 were ineligible since the address was vacant, leaving an effective sample of 4869 households. Among these households, 28.6% (1394 of 4869 households) refused to participate and 8.1% (392 of 4869 households) were not reached. Thus, 63.3% (3083 of 4869) of households completed the survey, with some households providing data from more than one resident. As a result, HSNI14/15 data provided by the UK Data Service included 4207 individuals aged 16 years and older . Of these 4207 participants, 1.8% (n = 1614). Women were more likely to have used an antibiotic compared to men . Participants who provided correct answers to the six statements regarding knowledge about infections and antibiotic resistance, which comprised our primary exposure, are presented in Table n = 334) of participants responded ‘don’t know’ to all six questions.Among all 4135 participants, use of an antibiotic in the past 12 months was self-reported among 39.0% (p’s < 0.10), except alcohol drinking (p = 0.321). Therefore, alcohol drinking was not included in multivariable-adjusted models.HSNI14/15 participant characteristics by self-reported antibiotic use in the past 12 months are presented in Table p < 0.001) of antibiotic use associated with a correct answer to the statement ‘Antibiotics are used to treat bacteria’ and 19% higher odds  of antibiotic use with a correct answer to the statement ‘Chances of resistant bacteria increase if you don’t finish the course’. A breakdown of knowledge score (the sum of correct answers to six statements about infections and antibiotic resistance (range 0–6)) by number correct answers showed that 8.5% (N = 353) of participants had 0/6 correct answers, 2.8% (N = 115) had 1/6 correct answers, 3.8% (N = 160) had 2/6 correct answers, 21.6% (N = 891) had 3/6 correct answers, 24.6% (N = 1011) had 4/6 correct answers, 23.0% (N = 952) had 5/6 correct answers and 15.8% (N = 653) had 6/6 correct answers.The association between knowledge of infections and antibiotic resistance and antibiotic use was estimated using weighted odds of antibiotic use in the past 12 months predicted by knowledge defined by a correct answer to each individual statement, as well as knowledge score Table . Evaluatp for trend < 0.001), with adjustment for sex, age, current smoking, deprivation quintiles, and self-rated general health and satisfaction with life. However, knowledge was not the only characteristic associated with higher odds of antibiotic use. Women compared to men  and those reporting poor versus good general health  were more likely to report using an antibiotic in the past 12 months. Those aged 45–64 years compared to those age 16–24 years  and those comprising the 5th deprivation quintile (the least deprived) compared to the referent 3rd deprivation quintile  were less likely to report antibiotic use.Estimates of the odds of antibiotic use in the past 12 months associated with our primary exposure, knowledge score and each characteristic, were accomplished using weighted multivariable-adjusted models and are presented in Table Sex and age group stratification illustrated some nuances to these data. Among both women and men, the highest knowledge score (6/6 vs. 0/6 correct answers) was associated with 2 times higher odds of antibiotic use in the past 12 months; and poor self-rated health was linked to higher odds of antibiotic use. However, deprivation index was a characteristic associated with antibiotic use among men only, and when compared to the middle deprivation quintile, men comprising the least deprivation quintiles exhibited 42% lower odds of antibiotic use  HSNI14/15 participants comprised the largest proportion  with lesser knowledge about infections and antibiotic resistance, as has been previously reported  years HS. YoungerIn HSNI14/15, there is some evidence of a gradient of association between deprivation and antibiotic use. Compared with the 3rd deprivation quintile, the odds of antibiotic use in the past 12 months were not different among the most deprived (3rd and 2nd deprivation quintiles), but were lower among the least deprived (4th and 5th deprivation quintiles). NIMDM deprivation index is a composite of income, employment, health deprivation and disability, education, skills and training, proximity to services, living environment, and crime and disorder, with consideration for home address. Many studies have shown that individuals comprising lower socioeconomic classes tend to be less knowledgeable about infections and antibiotic resistance compared with individuals in higher socioeconomic classes . While wPoorer self-rated general health was also associated with higher odds of antibiotic use in HSNI14/15. This may have been observed for several reasons, including: 1) co-occurring physical disease or co-morbidities, some of which may increase the risk of a bacterial infection ; 2) psycOur analysis has several strengths. The HSNI14/15 includes a representative, population-based sample of more than 4000 participants, across a wide age range. Self-reported data were collected using well-validated scales and state-of-the-art CAPI methods without reliance on population-based prescribing statistics; and the calculated deprivation index is comprehensive and informative. In addition, multivariable analyses allow evaluation of the association between knowledge and antibiotic use after controlling for potentially relevant confounders, lending to the robustness of our result. Given the relatively few studies in this field, these data are highly valuable and fill a gap in the literature. Despite these strengths, as with many large-scale population-based surveys, there are limitations. First, the HSNI uses a serial cross-sectional design comprised of independent samples. Despite the agreement of our results with other published studies, we cannot determine causality, i.e., whether knowledge influences behaviour (antibiotic use) or vice versa. Second, HSNI data are self-reported, thus, there is inherent recall bias. Regarding the face-to-face efficiency of CAPI, underreporting is more frequent in face-to-face interviewing of sensitive, stigmatized or unpopular behaviours . This riHSNI14/15 data illustrate that there was a positive cross-sectional association between knowledge about infections and antibiotic resistance and antibiotic use. However, we also observed a number of common modifiable and non-modifiable risk factors associated with both self-reported antibiotic use and basic knowledge of treatment of common infectious diseases and antibiotic resistance. These factors represent potential ‘at risk’ groups and thus, allow the creation of targeted campaigns that are critical for increasing knowledge in general, supporting informed choices, and raising the public’s health literacy. In addition, future research should prospectively examine whether knowledge about infections, antibiotics and antibiotic resistance predicts quantity or frequency of antibiotic use; as well as identify optimal types of information and methods of information dissemination that reduce antibiotic overuse. It is of critical importance that future prospective studies include measures of knowledge and attitudes, as well as dedicated measures of health literacy.Additional file 1.Additional file 2.


The Denver Developmental Screening Test II (DDST-II) was applied in 50 six-month-old infants. Metals were detected in 100% of the mother and newborn samples above the limits of detection. Maternal blood lead concentrations were higher in premature newborns . One-third of the infants (n = 17–35.4%) exhibited at least one fail in the neurodevelopment evaluation (fail group). Maternal blood arsenic concentrations were significantly (p = 0.03) higher in the “fail group” (GM: 11.85 µg/L) compared to infants who did not fail (not fail group) (GM: 8.47 µg/L). Maternal and umbilical cord blood arsenic concentrations were higher in all Denver Test’s domains in the “fail group”, albeit non-statistically significant, showing a tendency for the gross motor domain and maternal blood (p = 0.07). These findings indicate the need to further investigate the toxic effects of prenatal exposure to metals on infant neurodevelopment.The PIPA Project is a prospective birth cohort study based in Rio de Janeiro, Brazil, whose pilot study was carried out between October 2017 and August 2018. Arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg) concentrations were determined in maternal ( Metals are significant environmental pollutants and may originate from natural sources or as a result of human activities . The AgeChronic low-dose exposure to these metals may cause toxic human health effects affecting the nervous, hematological, and immune systems . EnvironNeurobehavioral developmental disorders such as autism spectrum disorder, attention deficit hyperactivity disorder, and subclinical decreases in brain function appear to be on the increase worldwide . AccordiIn Brazil, few studies have investigated metals in maternal and umbilical cord blood ,17,18,19The Rio Birth Cohort Study on Environmental Exposure and Child Development (PIPA Project) is a prospective cohort study conducted at the Federal University of Rio de Janeiro Maternity School, in the city of Rio de Janeiro, southeastern Brazil, aiming at the investigation of the effects of environmental pollutants on maternal and child health. A pilot study (PIPA Pilot study) was carried out between September 2017 and August 2018, with 142 pregnant women enrolled in and 135 children born at the Federal University of Rio de Janeiro Maternity School . This stThe PIPA Pilot Study was carried out for 12 months, from September 2017 to August 2018, at the UFRJ Maternity School (UFRJ Maternity). The UFRJ Maternity is located in the south zone of the city of Rio de Janeiro. It is one of the public hospitals of Rio de Janeiro responsible for prenatal assistance and delivery to all pregnant women living in the city, including those with high-risk pregnancies.A total of 135 children were born at the UFRJ Maternity between October 2017 and February 2018 during the PIPA Pilot Study. Five dropouts occurred, so 130 newborns were eligible for follow-up visits. The study population comprised the children that attended the follow-up visit in the sixth month. Exclusion criteria were children admitted to the intensive care unit during the neonatal period, birth weight <2.5 kg, gestational age <34 weeks, and mother drugs use during pregnancy.The infants were evaluated using the Denver Developmental Screening Test II (DDST-II). Children whose parents said they had not had the opportunity to experience the compliance of the DDST-II task were scored as “no opportunity” and were excluded.The study population was 48 children .Arsenic, cadmium, lead, and mercury in maternal blood samples collected during the third trimester of pregnancy and umbilical cord blood collected during delivery were analyzed employing the inductively coupled plasma mass spectrometry technique (ICP-MS), at the National Institute for Quality Control in Health (INCQS) laboratory, at the Oswaldo Cruz Foundation (FIOCRUZ). The limits of quantification (LQ) for As, Cd, Pb, and Hg were, respectively 0.01 μg/L, 0.006 μg/L, 0.05 μg/L, and 0.02 μg/L, while the limits of detection (LD) were 0.003 μg/L, 0.002 μg/L, 0.015 μg/L, and 0.007 μg/L, respectively. The sample collection and analysis procedures have been previously described by Asmus et al. (2020) .Infant neurodevelopment was assessed using the Denver Developmental Screening Test II (DDST-II). The DDST-II is the 1992 revised version of the Denver Screening Test developed by Frankenburg and Dodds in 1967 . The DDSFor all newborns, a follow-up appointment at sixth months was scheduled, when the DDST-II was applied for neurodevelopment assessment, in the presence of the mother by a specially trained examiner who was unaware of the child’s developmental history and of the metal levels at the time of the test.For the analyses, the children were divided into “fail group” and “not fail group”. Following the guidance of the DDST II manual, “fail” and “refuse” scores were considered as “fail”, and “pass” scores were considered as “not fail” . A descriptive analysis of participants characteristics was performed. Frequency distributions for categorical variables and arithmetic mean and median for the continuous variables were calculated. The geometric means, 25th, 50th, 75th, 90th, and 95th percentiles, and the minimum and maximum values of the pollutant concentration levels were calculated for the maternal and umbilical cord blood samples.Spearman’s correlation or non-parametric comparisons (Mann–Whitney-U-test) or parametric (Student) tests were used to evaluate continuous variables, and Chi-squared or Fisher tests were used for categorical variables.p-value < 0.1. No covariates were associated with exposure and outcome simultaneously, so we did not use a regression model to adjust for confounders. All data were analyzed using the Statistical Package for Social Science v.21 software for Windows.To identify confounders and make adjustments, we evaluated the association between metals and maternal and infant sociodemographic characteristics. We also verified the relationship between maternal sociodemographic characteristics and characteristics of birth with the DDST-II outcome. To select which covariates to adjust, we set a The continuous variables—mother age, gestational age, birth weight, per capita income, and education—were counted in years. Categorical variables were ethnicity, gender, alcohol consumption, tobacco exposure, Apgar index, adequacy for gestational age, and prematurity.Maternal sociodemographic data included maternal age, per capita income, education and men, ethnicity, exposure to tobacco, and alcohol consumption, which were collected through questionnaires administered by trained interviewers.Birth information was obtained from the birth record.Family neurological history for autism and ADHD was revealed by the mother at the follow-up visit, but there was no report of these disorders in parents or siblings.Arsenic, Cd, Pb, and Hg were above the LQ in all maternal blood and umbilical cord blood samples . A signip = 0.04) and cord blood (p = 0.08) compared to white women (Mean mother age was 28.54 years (SD ± 7.07), with a mean per capita income of USD 257.00 (SD ± 167.38) and median of 14 years of schooling; 71.4% of the mothers were non-white, 36.7% reported exposure to tobacco , and 50.0% consumed alcohol during pregnancy. A statistically significant positive correlation between maternal age and cord blood concentrations were observed for Hg  and Pb . Non-white pregnant women presented higher geometric Cd means in maternal blood (te women .n = 31) of all infants were male, 11.1% (n = 5) were small for gestational age (SGA), and 6.0% (n = 3) were premature (IG 34–37 weeks). The geometric Pb mean in maternal blood  and in cord blood  was higher in premature babies. An inverse correlation trend was observed between gestational age and Cd concentrations in maternal blood  and in umbilical cord blood  (Mean birth weight was 3.3 kg (SD ± 0.5 kg) and median gestational age was 39 weeks (range: 36–41 weeks), 62.0% ( = 0.16) .In the sixth month follow-up visit, 48 eligible children were evaluated with regard to the four neurodevelopmental Denver Test’s domains. Most (31–64.6%) did not fail in any domain (“not fail” group), and 17 (35.4%) children failed in one or more domains (“fail” group). No differences were observed concerning maternal sociodemographic characteristics and birth characteristics between the “fail” and “not fail” groups .p = 0.03). The maternal and cord blood arsenic concentrations were higher also in the “fail group” for the personal social (p = 0.74), fine adaptative motor (p = 0.27), language (p = 0.36), and gross motor (p = 0.07) Denver Test’s domains,  had at least one failure in the DDST-II. In another study carried out in Acre (Brazil) with 47 children up to 12 months of age, 72.7% of children between 4 and 6 months of age (n = 3) had less than two failures, which were results considered adequate for the age group [In our study, 34.5% of children  . The higp = 0.03) and the gross motor domain (p = 0.07). Some studies have investigated the effect of prenatal exposure to As on neurological infant development. Liang et al. (2020) [n = 892), observed that newborns with a low neonatal behavioral neurological assessment (NBNA) score exhibited higher concentrations  of umbilical cord As  than those with a high NBNA score . Tolins et al. (2014) [In this study, maternal and umbilical cord blood geometric means for As were higher in the “fail” group for DDST-II in the six months. This result was more significant for As maternal blood (. (2020) , in a pr. (2020) , in a cr. (2014)  concludeIn our study, there was no significant difference in maternal blood cadmium concentrations between the fail and not fail groups. Review studies reported that the effects of prenatal exposure to cadmium and childhood neurodevelopment are still not clear, and there is still no consensus about the toxic effect of this metal on cognitive performance ,41.The maternal and cord blood geometric means for Cd were higher in the non-white ethnic group, which was significant only for maternal blood. Some studies suggest that higher biological Cd levels may be associated to a more socioeconomically vulnerable population, including socioeconomically less favored ethnicities ,43.Numerous studies support the concept that Pb affects cognitive function in children exposed prenatally and/or environmentally to low levels of Pb and that no safe parameter has been identified . No assoIn general, the literature points to a more expressive toxic effect of lead exposure on the child neurodevelopment than related to the other metals exposure. One issue to be observed is that the As geometric mean (11.48 µg/L) is much higher than the Pb geometric mean (4.44 µg/dL) in the Fail group. It could be a reason, but the small size of the group does not permit any conclusion.The maternal blood Pb concentrations were significantly higher in preterm infants . One hypothesis of the mechanism of action of lead in prematurity is through oxidative stress ,46.Cord blood lead was positively correlated with maternal age. Other studies have also found that maternal age was associated with higher levels of cord blood lead ,48. Thisp = 0.002).No associations were observed between failures in the neurodevelopment domains and maternal or umbilical cord Hg concentrations in our study population. Nevertheless, in a systematic review study, Asmus et al. (2016)  reportedMercury in umbilical cord blood was positively correlated with maternal age. This correlation can be explained by the bioaccumulation of this metal in the maternal organism and by its transfer across the placental barrier ,51.The strength of this study is to provide information on prenatal environmental exposure to metals and potentially adverse effects on neurodevelopment at six months of age, covering sociodemographic and lifestyle data.The limitations of this study are the small number of participants and the one-time neurological assessment. Many factors can interfere in the child’s process of neurodevelopment, and a long-term evaluation, with monitoring of the multiple intervenient factors, is the best proceeding.This study shows that higher concentrations of arsenic in maternal blood were found in the group of children who failed in DDST-II. Continuous monitoring of concentrations of neurotoxic substances in women of childbearing age is essential to establish preventive measures to eliminate or minimize the risk of fetal exposure during pregnancy. Long-term development studies may reveal other associations. Future investigations should aim to demonstrate the possible neurodevelopmental effects of prenatal exposure to environmental pollutants on child health and identify potential sources of exposure.


In conclusion, ZA enhanced the antitumor effect of cisplatin on osteosarcoma by ROS-PI3K/AKT signaling, reducing the chemoresistance and osteoclast activation to enhance chemotherapy and inhibit osteolysis. And this present study raised the possibility that combining ZA and cisplatin may represent a novel strategy against osteosarcoma.Osteoclasts can interact with osteosarcoma to promote the growth of osteosarcoma. Cisplatin is common in adjuvant chemotherapy of osteosarcoma. However, due to chemoresistance, the efficacy is profoundly limited. Previous studies have found that zoledronic acid (ZA) has osteoclast activation inhibition and antitumor effect. However, the combined effect of ZA and cisplatin on osteosarcoma remains unclear. In vitro, the effects of ZA and cisplatin alone or in combination on 143B cell activity, proliferation, apoptosis, and ROS-PI3K/AKT signaling were detected. At the same time, the effect of ZA and cisplatin on osteoclast formation, survival, and activity was detected by TRAP staining and bone plate absorption test. These were further verified in mice. The results showed that in vitro, compared with the single treatment and control, the combination of ZA and cisplatin could significantly inhibit the activity and proliferation of 143B cells and induced their apoptosis and further promoted the generation of ROS and inhibited the phosphorylation of PI3K and AKT. ROS scavenger and the agonist of the PI3K/AKT pathway could reverse these results. In addition, cisplatin in synergy with ZA could significantly inhibit osteoclast formation and survival to reduce bone plate absorption. In vivo, compared with the single group, the tumor volume and cell proliferation were significantly reduced, apoptosis and necrosis of tumor cells increased, and TRAP Osteosarcoma, common in children and adolescents, is a primary malignant tumor, most of which occur in the metaphysis of long bones . ClinicaCisplatin is a classical chemotherapy drug used to treat osteosarcoma . In mechβ), insulin-like growth factors (IGFs), and fibroblast growth factors (FGFs) [Osteosarcoma cells derive from osteoblasts. However, it has been found that osteoclasts also play an important role in the occurrence and development of osteosarcoma . Osteocls (FGFs) –11. In ts (FGFs) . In mechs (FGFs) , 14. Mors (FGFs) . Preclins (FGFs) , 16, 17.In this study, we examined the efficacy of combined therapy with cisplatin and ZA. We demonstrated that combination with cisplatin and ZA enhanced the chemotherapy effect of cisplatin on osteosarcoma. It could further induce the apoptosis and proliferation inhibition of osteosarcoma cells caused by cisplatin, and the mechanism of synergistic effect enhancement was mainly related to oxidative stress and PI3K/AKT inactivation, which play a crucial role in cell survival and proliferation. Furthermore, we confirmed the synergistic antitumor effect of combined treatment with ZA and cisplatin in mice, and it inhibited osteoclasts to break the “vicious circle” between osteoclasts and osteosarcoma cells and reduce osteolysis (an active resorption of bone matrix by osteoclasts).Zoledronic acid (CAS No. 118072-93-8), cisplatin (CAS No. 15663-27-1), and 740 Y-P (CAS No. 1236188-16-1) were obtained from MedChemExpress . Isoflurane was obtained from Baxter Healthcare Co. . TRAP staining kit (HR0561) was purchased from Biolab Technology Co., Ltd. . Reactive oxygen species (ROS) test kit (DHE) (HR8821) was obtained from Biolab . Phospho-PI3 kinase (p-PI3K) (4228S) and AKT (4691S) antibodies were obtained from Cell Signaling Technology Co., Ltd. Phospho-AKT (AP0637), MRP-1 (A1703), MDR1 (A19093), and PI3K (A0265) antibodies were obtained from ABclonal Co., Ltd. . Goat polyclonal secondary antibody to rabbit IgG-H&L (Alexa Fluor® 488) (ab150077) was obtained from Abcam. Annexin V-FITC/PI kit (BB-4101-50T) was purchased from BestBio ; Ki-67 (GB13030-2), TMR (red) Tunel Cell Apoptosis Detection Kit (G1502), crystal violet (G1014), and CCK-8 kit (G4103) were purchased from Servicebio Co., Ltd. . TRIzol (CSA No. 5346994) was obtained from Invitrogen Co. . Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) kits were obtained from TaKaRa Biotechnology . The SYBR Green dye was purchased from Applied Biosystems by Thermo Fisher Scientific (ABI) . The other chemicals and agents were analytical grade.2 in DMEM medium , including 10% fetal bovine serum  and 100 μg/ml penicillin and streptomycin.Human osteosarcoma 143B cell lines were purchased from Mingzhou Biological Technology Co., Ltd. . Cells resistant to cisplatin were established by exposing the drug-sensitive 143B cells to stepwise increasing concentrations of cisplatin. All cells were cultured at 37°C in a constant temperature incubator with 5% CO3/l cell suspension and seeded in 96-well plates. Then, the cells were treated with ZA  or cisplatin  alone or in combination. Finally, after CCK-8 reagents were added according to CCK-8 kits protocol and incubated for 0.5-4 hours in the cell culture incubator, the absorbance was measured at 450 nm in a microplate reader. After obtaining the data of cell activity, the synergy score was determined by the online application SynergyFinder 2.0 with four reference models with the “viability readout” [50) value of cisplatin was calculated by the survival curve.143B cells were made into 1‐5 × 10readout” . And theμM ZA, 5 μg/ml cisplatin, 5 mM NAC, or 50 μg/ml 740 Y-P for 48 h, respectively. Then, the cells were washed twice with PBS and cultured in a new medium. Eight days later, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. The assay was repeated three times. The staining results were photographed, and the colonies with more than 50 cells were counted under an ordinary optical microscope.The cells were seeded in six-well plates at the appropriate density (500 cells/well) and allowed to culture for 24 h. Once the cells completely adhered, the cells were divided into six groups: control, cisplatin, ZA, ZA+cisplatin, ZA+cisplatin+NAC, and ZA+cisplatin+740 Y-P, which were treated with 32 μl 143B cells into the right tibial plateau of the nude mice with an insulin needle. When the tumor was visible, the mice were randomly assigned to one of four groups: control, cisplatin (2 mg/kg), and combined group (ZA: 2 mg/kg+cisplatin: 2 mg/kg). The above reagents were injected intraperitoneally 3 times once every 5 days. The weight and tumor size of mice were measured every 3 days. The tumor size was then calculated based on the following equation: tumor volume = (length × width2)/2. Finally, all nude mice were sacrificed with 2% isoflurane, the blood samples were collected, and the tumors and lower limbs were isolated for further analysis.All experimental specific pathogen-free BALB/c nude mice (4-6 weeks old) , certification number: 42000800001149) were purchased from Hubei Provincial Center for Disease Control and Prevention . All animal testing procedures comply with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee. The Animal Experimental Ethics Committee of Wuhan University Medical College approved this study (license number: 18026). All animals were kept under sterile standard environmental conditions, allowing free access to food and water. After one week of adaptive feeding, we injected 40 Before the nude mice were sacrificed, the mice were anesthetized with 4% chloral hydrate and then examined tibia with X-rays under the mouse in vivo imager (including X-ray imaging module)  and observed bone destruction. After the nude mice were sacrificed, the bones were soaked in ethanol. Then, we used a micro-CT scan  to scan the tumor side tibia and femur and perform 3D reconstruction. Finally, we used the software  to assess the trabecular bone microstructure with the parameters, including trabecular bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) of the scanned femurs.After the cells were fixed with 4% formaldehyde for 15 min, the membranes were broken with 0.2% Triton X-100 for 5-10 min. Then, samples were incubated with 3% BSA at room temperature for 60 min. Soon afterward, they were incubated overnight at 4°C with primary antibodies in blocking buffer. After washing with TBST three times, the samples were incubated with 1 : 800 secondary antibody-labeled fluorescent for 60 min. The nucleus was then stained with DAPI for 5 min. After washing with PBS three times, fluorescence images were taken with confocal microscopy . 10 visual fields were collected for further analysis of each image. Differently, after the ROS reagent was directly added to the sample according to the protocol, it was immediately observed under a confocal microscope , and the ImageJ software  was used for quantitative analysis.μM ZA and/or 5 μg/ml cisplatin were added for the first two days. The samples were fixed with 4% paraformaldehyde and stained with the TRAP staining. As for bone resorption assay, after we cultured the BMMs in Osteo Assay Surface 24-well plates  for 24 h, a complete inducing medium replaced the previous medium. Similarly, 32 μM ZA and/or 5 μg/ml cisplatin were added for the first two days. The cells were rinsed with 10% sodium hypochlorite and water three times after 6 days. Finally, we used an ordinary light microscope to take pictures, and the ImageJ software was used for quantitative analysis.Primary bone marrow cells were cultured in the complete medium supplemented with 50 ng/ml M-CSF for 3 days to obtain bone marrow-derived macrophages (BMMs). Then, we cultured the cells for 4 days with 50 ng/ml M-CSF and 50 ng/ml RANKL. To assess osteoclast survival, 32 μl of 10% paraformaldehyde. After washing with ddH2O, add tartrate-containing buffer and 50 μl chromogenic substrate. After incubating at 37°C for 40 min, wash with ddH2O. Finally, we used Nikon NIS Elements BR light microscope  for photograph. The staining intensity was analyzed by measuring the mean optical density (MOD) in 10 fields.After the cells were treated, TRAP staining of osteoclasts was performed according to the TRAP staining kit protocol. Briefly, after removing the culture medium, the cells were immobilized by adding 50 μm for further morphological staining analysis. According to the protocol, H&E staining was operated as follows: after dewaxing in xylene for 10 min, the slices were immersed in 100%, 95%, 85%, and 70% ethanol for 3 min. Next, after dyeing with hematoxylin for 10 min, the color was separated with 70% ethanol. After washing with running water for 15 min, dye with 0.2% eosin for 3 min and then dehydrate by 70%, 85%, 95%, and 100% ethanol successively. Finally, xylene was used for transparency. Ki-67 and Tunel staining was followed by an immunohistochemical staining protocol described in this article [The specimens were sectioned at 5  article . All imaμl system  with the Applied Biosystems PCR System : 95°C for 2 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. The cycle threshold (Ct) values were detected by the 2ΔΔCt− method. And the relative mRNA expression was normalized with GAPDH. The primers of related genes are shown in Table The total RNA of cells and bone tissue was extracted by the method of TRIzol according to the manufacturer's guidelines. As for the quantitative real-time PCR (RT-qPCR), the reactions were acted in a 10 μl 1x Annexin V binding buffer, 5 μl Annexin V-FITC staining was added to the cell suspension, and the mixture was gently mixed and incubated at 2-8°C in the dark for 15 min. Then, PI dye solution was added and incubated at 2-8°C in the dark for 5 min. The prepared cells were immediately detected by CytoFLEX  flow cytometry. And the results were analyzed with the CyteExpert software . According to the instructions, the sum of UR and LR quadrant cells is the total apoptosis rate. The experiment was repeated three times for statistical analysis.After different treatments, the experimental cells were centrifuged and collected after digestion with trypsin without EDTA. Then, we used an Annexin V-FITC Apoptosis Detection Kit  to detect apoptotic cells according to the protocol. In brief, after the cells were resuspended with 400 μl) was mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer  and heated at 95°C for 5 min. Finally, the protein sample obtained above was used for further protein quantification . All results were repeated 3 times. The detailed steps of protein molecule separation and visualization were shown in our previous research [The prepared cells were rinsed with PBS twice, and then, RIPA Lysis Buffer was used to lyse the cells for 30 min on ice to extract total protein, and the protein sample was then obtained when the supernatant was collected and transferred to the 1.5 ml precooled centrifuge tube after being centrifuged for 10 min at 12000g and 4°C. An equal amount of protein  and Prism 7  were used for data statistics and graphing. Quantitative data were expressed as the mean ± SD. SNK-q was used for the comparison in two of multiple samples after one-way ANOVA that was used for the comparison of the control, single, and combined treatments. P < 0.05, P < 0.01). Cell clone and immunofluorescence were further used to measure the cell proliferation. The cell clone showed that clone formation was significantly lower in the combined group than either the cisplatin or ZA group, and all of that were lower than control (P < 0.01). The Ki-67 expression by RT-qPCR and immunofluorescence showed that Ki-67 mRNA and protein expressions in the cisplatin or ZA group were significantly lower than those in the control and higher than those in combination . Furthermore, to investigate the apoptosis of 143B cells treated with ZA/cisplatin/combination, flow cytometry and RT-qPCR were used. The results showed that the expression of apoptosis-related genes (caspase 3/9 and Bax) and the apoptosis rate of 143B in the combined treatment group were significantly higher than those in the control and single treatment groups , while the antiapoptosis gene (Bcl-2) expression was the lowest in the combined treatment. Furthermore, the synergy score of cisplatin and ZA was determined by SynergyFinder 2.0 [Firstly, CCK-8 assay was used to detect the cell viability of 143B cells treated with ZA/cisplatin/combination in different concentrations and times. The results showed that the cell activity decreased in a concentration- or time-dependent manner whether in a separate group or the combined group, while the CCK-8 absorption curve in the combined group was significantly higher than alone or control . The PI3K/AKT signaling is a crucial pathway related to the proliferation and apoptosis of osteosarcoma [P < 0.05, P < 0.01). Furthermore, when we treated 143B cells with ROS scavenger N-acetyl-L-Cysteine (NAC) and 740 Y-P . Therefore, these results suggested that ZA+cisplatin could inhibit PI3K/AKT signaling activation by promoting ROS generation to induce apoptosis and proliferation inhibition of osteosarcoma cells.To further explore the mechanism of ZA and cisplatin enhancing antitumor, immunofluorescence was used to detect ROS generation after single or combined action of ZA and cisplatin. The results showed that the ROS generation after ZA or cisplatin treatment was significantly increased compared with the control, but the ROS of the ZA group was lower than that of the cisplatin group, while the ROS production after combined treatment was higher  in combP < 0.05, P < 0.01). We further tested the effects of ZA and cisplatin on the survival of osteoclasts. Consistent with differentiation, the osteoclast apoptosis in the ZA+cisplatin group was more than that in the control, but it was not significantly different from the single treatment group . Moreover, the bone resorption of the combined treatment group was significantly higher than that of the single treatment group, and both were stronger than the control . The above results indicated that either ZA or cisplatin could inhibit osteoclast formation, survival, and activation, but the combination was more effective.It has been reported that ZA can inhibit osteoclast differentiation and survival . HoweverP < 0.01). Furthermore, tumor volume and nude mouse body weight were measured, and tumor growth was significantly slower in the combined treatment group than in the other treatment and control , but there was no significant change in weight (P > 0.05). Further, we exfoliated the tumor and performed H&E, Ki-67, and Tunel staining. The results showed that the apoptosis and necrosis of tumor cells , indicating that the side effects of combined treatment were not higher than those of cisplatin alone.To further explore the effects in vivo, we first established a nude mouse tibia orthotopic osteosarcoma model, and these model mice were then treated with cisplatin/ZA+cisplatin. The results showed that the tumors in the combined treatment group were significantly smaller visually than those in the single treatment and control , and the Figures  were sig Figures  was decr∗P < 0.05, ∗∗P < 0.01). Finally, we detected the mRNA expression of marker genes of osteoclast differentiation (NFATc1) and activity (TRAP and Ctsk) and performed TRAP staining on the tumor side tibia tissue. The results showed that the marker genes of osteoclast differentiation and activity were lower significantly in ZA+cisplatin than those in the group treated with saline or cisplatin . And TRAP staining-positive cells were also significantly reduced, indicating that osteoclasts in bone tissue were inhibited after ZA+cisplatin treatment  . And the apoptosis rate increased, and the mRNA and protein expression of the multidrug resistance gene  decreased in the cisplatin-resistant osteosarcoma cells treated with cisplatin+ZA . In conclusion, the combination of ZA and cisplatin can inhibit ROS-PI3K/AKT signaling to enhance the antitumor efficacy and drug resistance of cisplatin.Cisplatin is one of the most commonly used and classic chemotherapy drugs in the treatment of osteosarcoma , 38. Howβ, IGFs, and FGFs [Physiologically, osteoclasts remove old bone and osteoblasts form new bone, maintaining bone homeostasis. The formation and activation of osteoclasts require two factors: RANKL and macrophage colony-stimulating factor . It has and FGFs –11, to fand FGFs , but we and FGFs . Therefoand FGFs , 51. HowIn conclusion, this study confirmed that ZA can enhance the antiosteosarcoma effect of cisplatin and ameliorate chemoresistance in vivo and in vitro. The mechanism is related to the increased generation of ROS and inhibition of PI3K/AKT signal pathway activation. Moreover, the combination of ZA and cisplatin could inhibit osteoclast differentiation, survival, and activation, thus breaking the “vicious circle” to inhibit tumor growth and osteolysis destruction. Therefore, ZA may be used as an adjuvant chemotherapy drug beneficial to the treatment of osteosarcoma.