BACKGROUND:Studies have shown that patients with premature ovarian failure have changes in the structure of intestinal flora and that imbalance of intestinal microbiota may be one of the important mechanisms in the development of premature ovarian failure. OBJECTIVE:To investigate the effect of Liuwei Dihuang Wan on oxidative stress and intestinal microbiota in premature ovarian failure mice induced by cyclophosphamide. METHODS:Forty-five female ICR mice were randomized into three groups:blank group(normal mice),model group(premature ovarian failure mice),and Liuwei Dihuang Wan group.A mouse model of premature ovarian failure was prepared by one-time intraperitoneal injection of cyclophosphamide(120 mg/kg)in the latter two groups.After successful modeling,the Liuwei Dihuang Wan group was intragastrically administered for 28 continuous days,and the other two groups were intragastrically administered with the same amount of normal saline for 28 days.Mouse body mass was recorded weekly and ovarian index was calculated.The development of mouse follicles was observed using hematoxylin-eosin staining.ELISA method was used to detect serum levels of anti-Mullerian hormone,estradiol,follicle stimulating hormone,superoxide dismutase,glutathione peroxidase,and malondialdehyde.Meanwhile,the gut microbiome of all mice was detected through 16S rDNA sequencing. RESULTS AND CONCLUSION:The mice in the model group had loose hair,decreased vigor and grip strength,almost no increase in body mass,and decreased ovarian index.Whereas,the mouse body mass and ovarian index were increased after treatment with Liuwei Dihuang Wan(P<0.05).The estrous cycle of mice in the model group was disorganized;Liuwei Dihuang Wan could restore the estrous cycle and reduce the number of atretic follicles in mice with premature ovarian failure.The serum levels of follicle stimulating hormone and malondialdehyde in the model group significantly increased(P<0.01),while the levels of estradiol,anti-Mullerian hormone,superoxide dismutase,and glutathione peroxidase significantly decreased(P<0.01).Liuwei Dihuang Wan could significantly decrease the serum levels of follicle stimulating hormone and malondialdehyde(P<0.01),and increase the levels of estradiol,anti-Mullerian hormone,superoxide dismutase,and glutathione peroxidase.According to the 16S rDNA sequencing results,Liuwei Dihuang Wan could regulate the abundance and diversity of intestinal microbiota,and increase the relative abundance of beneficial bacteria.KEGG pathway analysis showed that the intestinal microbiota and metabolic pathways,biosynthesis of secondary metabolites,microbial metabolism in different environments,and biosynthesis of amino acids were regulated by Liuwei Dihuang Wan.To conclude,the changes in the structure of intestinal microbiome may be one of the potential mechanisms of Liuwei Dihuang Wan in treating premature ovarian failure.Liuwei Dihuang Wan can regulate the structure of intestinal microbiome,increase the number of beneficial bacteria,reduce the number of harmful bacteria,and thus improve the balance of intestinal microbiota.This regulatory effect helps to reduce oxidative stress levels and further inhibit ovarian oxidative stress in mice with premature ovarian failure.

BACKGROUND:Most of the formulas for the clinical treatment of premature ovarian insufficiency have evolved from the basic formula of Liuwei Dihuang Pills,and have achieved good therapeutic efficacy.Currently,most of the experimental studies on Liuwei Dihuang Pills focus on morphological observations and physiological and biochemical detection of in vivo animal models,while fewer studies on molecular mechanisms have been reported. OBJECTIVE:To explore the molecular mechanism of Liuwei Dihuang Pills in the treatment of premature ovarian insufficiency based on the receptor gamma coactivator-1 alpha/mitochondrial transcription factor A/reactive oxygen species pathway. METHODS:Premature ovarian insufficiency model was established in mice by intraperitoneal injection of cyclophosphamide 120 mg/kg combined with busulfan 12 mg/kg,and then Liuwei Dihuang Pill suspension was used to intervene in premature ovarian insufficiency mice.After 12 weeks of intervention,the levels of follicle-stimulating hormone,luteinizing hormone,estradiol,anti-Mullerian hormone,8-hydroxydeoxyguanosine,total antioxidant capacity and reactive oxygen species in serum of mice were detected by ELISA method.The morphological changes in mouse ovaries were observed by hematoxylin-eosin staining.The ultrastructure of mouse follicular granulosa cells and the apoptosis of granulosa cell mitochondria were observed by transmission electron microscopy.The expression levels of receptor gamma coactivator-1 alpha and mitochondrial transcription factor A in mouse ovarian granulosa cells were detected by immunohistochemistry. RESULTS AND CONCLUSION:Compared with the model group,serum levels of follicle-stimulating hormone,luteinizing hormone,reactive oxygen species,and 8-hydroxydeoxyguanosine were decreased in the experimental group(P＜0.05),and the levels of estradiol,anti-Mullerian hormone,and total antioxidant capacity were increased(P＜0.05).Hematoxylin-eosin staining showed that in the model group,there were more atretic follicles and corpus luteum forms,some secondary follicles,and interstitial fibrosis and hyperplasia;in the experimental group,a large number of atretic follicles,few corpus luteum forms,primordial follicles were observed at the edges but there were few secondary follicles and no mature follicles.Transmission electron microscopy showed that the organelles in ovarian granulosa cells of mice in the experimental groups were relatively intact.Immunohistochemical results showed that compared with the model group,the expression level of receptor gamma coactivator-1 alpha in the ovarian tissue of mice increased slightly in the experimental group at the 4th week,and there was no significant change at the 8th and 12th weeks.The expression level of mitochondrial transcription factor A in the ovarian tissues of mice in the experimental group was transiently increased at the 4th week,and then slightly decreased,which were all significantly different from those of the model group.To conclude,Liuwei Dihuang Pills inhibit ovarian granulosa cell apoptosis in mice with premature ovarian insufficiency to a certain extent through the receptor gamma coactivator-1 alpha/mitochondrial transcription factor A/reactive oxygen species signaling pathway,thereby improving the endocrine function of the ovary,enhancing the antioxidant capacity,and attenuating the degree of oxidative stress damage.

Objective:To construct a recombinant bioluminescent bacteriophage (HT7) targeting Escherichia coli, and evaluate its ability to identify Escherichia coli. Methods:Initially, pCRISPR-sg (1-10) and PFN-1000 plasmid strains were constructed by genetic engineering, and the most efficient small guild RNA (sgRNA) were screened by bilayer plate. By the gene editing technique, which comprised homologous recombination and clustered regularly interspaced short palin dromic repeats (CRISPR)-Cas system, the Nanoluc luciferase gene was integrated into the downstream non-coding region of 10A gene of T7 phage, to constructe the bioluminescent phage HT7 successfully. The difference of biological characteristics between HT7 phage and T7 phage was evaluated by plaque assay and liquid amplification assay. In addition, 51 strains of Escherichia coli, 20 strains of Klebsiella pneumoniae, 14 strains of Staphylococcus aureus, 6 strains of Enterococcus faecium, 5 strains of Enterococcus faecalis, 3 strains of Acinetobacter baumannii and 1 strain of Pseudomonas aeruginosa were collected and isolated to evaluate the limit of detection and specificity of HT7 phage. Results:Among the 10 CRISPR-targeted cleavage systems constructed, sgRNA8 exhibited the highest cleavage efficiency, with a cleavage rate of 0.18. After three rounds of recombination screening using the pCas9/pCRISPR/PFN-1000 triple-plasmid system, PCR validation yielded recombinant phage bands at 2 798 bp, indicating the successful construction of the HT7 phage. The recombinant phage showed significant differences in biological characteristics in terms of lysis efficiency ( P<0.001), one-step growth curve ( P=0.001), and infection multiplicity ( P=0.031). Both lysis burst time and log growth node were extended by 10 min, with the optimal infection multiplicity being 0.1. Clinical sample testing identified lysis of 6 strains of Escherichia coli within 4.5 h, while other strains remained unaffected, with detection of pathogenic bacteria below 10 CFU/ml. Conclusions:The developed pCas9/pCRISPR/PFN-1000 triple-plasmid editing system efficiently edits the bacteriophage genome. The constructed HT7 fluorescent bacteriophage enables the detection of Escherichia coli below 10 CFU/ml within 4.5 hours, demonstrating low detection limits and high detection specificity.

Objective:Diabetic retinopathy (DR) is the most common cause of adult blindness in China. Screening of DR is important for early detection, prevention, and treatment. However, there is still controversy in the research on the prevalence and risk factors of DR in China. This study was designed to evaluate the prevalence of DR and related risk factors in patients with type 2 diabetes mellitus in Beijing City.Methods:A cross-sectional survey was conducted in in Dongcheng District and Tongzhou District, Beijing City. Patients with type 2 diabetes aged 18-80 years were selected from four communities, and all subjects underwent questionnaires, physical examinations, laboratory examinations and fundus photography. The logistic regression model was used to analyze the associated factors of DR.Results:A total of 1 531 subjects were included, with the median age of 66 years old and the average age of (65.6±7.4) years old, and the glycosylated hemoglobin level in the subjects was 7.2%±1.3%, and the glycosylated hemoglobin compliance rate was 56.0%(857/1 531). A total of 254 patients with diabetic retinopathy were detected, and the prevalence of DR was 16.6%(254/1 531). Among them, there were 218 cases of non-proliferative diabetic retinopathy and 36 cases of proliferative diabetic retinopathy. Compared with the non-DR group, there were statistically significant differences in fasting blood glucose ( Z=-3.74, P<0.001), glycosylated hemoglobin( Z=-10.664, P<0.001), urinary microalbumin excretion rate( Z=-7.767, P<0.001), low-density lipoprotein cholesterol( Z=-2.589, P=0.01), and duration of diabetes( Z=-10.189, P<0.001) between the DR group and the non-DR group. Multivariate regression analysis showed that the duration of diabetes ( OR=1.08, 95% CI: 1.06-1.10, P<0.001), glycosylated hemoglobin ( OR=1.38, 95% CI: 1.23-1.55, P<0.001), and FPG ( OR=1.11, 95% CI: 1.03-1.19, P=0.008) were associated factors for DR. Conclusion:In this study, the prevalence of DR in 4 communities of type 2 diabetes in Beijing City was 16.6%. Besides, this study further confirmed that the duration of diabetes, fasting blood glucose levels, and glycosylated hemoglobin are associated factors for DR in patients with type 2 diabetes.

Objective To explore the effect of MSCT 3D scanning and reconstruction on developmental dysplasia of the hip(DDH)in adult patients with total hip arthroplasty,The predictive value of acetabular prosthesis selection and osteotomy accuracy in THA.Methods Selection in our hospital between February 2021 and February 2023 were 60 adult DDH patients underwent total hip replacement,which USES routine preoperative planning as a control group of 30 patients,and using MSCT + reconstruction of 3 d scanning technology as observation group of 30 patients,planning after the completion of the planning of the surgical operation and implant placement,Relevant information was collected before and after the operation of the two groups.Operation time,intraoperative blood loss,intraoperative fluoroscopy time,acetabular prosthesis selection and matching rate,femur split fracture rate,pelvis and acetabular reduction quality,Majeed score and Harris hip function score were compared between the two groups.The osteotomy distance and sharp shoulder distance of the two groups were compared before planning and in actual conditions,and the correlation between MSCT 3D scanning and reconstruction technology and the accuracy of acetabular prosthesis selection was evaluated.Results Results analysis showed that MSCT 3D scanning+reconstruc-tion technology used in preoperative evaluation of patients in the observation group was correlated with the accuracy of acetabular prosthesis selection(P＜0.05),and the absolute errors of osteotomy distance and sharp shoulder distance of patients in the observation group were smaller than the actual situation before planning(P＜0.05).In addition,compared with the control group,the operative time,intraoperative blood loss,intraoperative fluoroscopy time and incidence of femoral split fracture in the observation group were significantly lower,and the matching rate of acetabular prosthesis selection,the Majeed scores and Harris scores were significantly higher in the following 3 months were significantly higher(P＜0.05).The reduction quality of pelvis and acetabulum was not statistically significant between the two groups(P＞0.05).Conclusion Msct-based 3D scanning + reconstruction has a high predictive value for the selection of acetabular prosthesis and the accuracy of osteotomy in adult DDH total hip replacement,and the use of this technology for preoperative planning has a significant optimization effect on the actual treatment effect and patient recovery.

Objective:Diabetic retinopathy (DR) is the most common cause of adult blindness in China. Screening of DR is important for early detection, prevention, and treatment. However, there is still controversy in the research on the prevalence and risk factors of DR in China. This study was designed to evaluate the prevalence of DR and related risk factors in patients with type 2 diabetes mellitus in Beijing City.Methods:A cross-sectional survey was conducted in in Dongcheng District and Tongzhou District, Beijing City. Patients with type 2 diabetes aged 18-80 years were selected from four communities, and all subjects underwent questionnaires, physical examinations, laboratory examinations and fundus photography. The logistic regression model was used to analyze the associated factors of DR.Results:A total of 1 531 subjects were included, with the median age of 66 years old and the average age of (65.6±7.4) years old, and the glycosylated hemoglobin level in the subjects was 7.2%±1.3%, and the glycosylated hemoglobin compliance rate was 56.0%(857/1 531). A total of 254 patients with diabetic retinopathy were detected, and the prevalence of DR was 16.6%(254/1 531). Among them, there were 218 cases of non-proliferative diabetic retinopathy and 36 cases of proliferative diabetic retinopathy. Compared with the non-DR group, there were statistically significant differences in fasting blood glucose ( Z=-3.74, P<0.001), glycosylated hemoglobin( Z=-10.664, P<0.001), urinary microalbumin excretion rate( Z=-7.767, P<0.001), low-density lipoprotein cholesterol( Z=-2.589, P=0.01), and duration of diabetes( Z=-10.189, P<0.001) between the DR group and the non-DR group. Multivariate regression analysis showed that the duration of diabetes ( OR=1.08, 95% CI: 1.06-1.10, P<0.001), glycosylated hemoglobin ( OR=1.38, 95% CI: 1.23-1.55, P<0.001), and FPG ( OR=1.11, 95% CI: 1.03-1.19, P=0.008) were associated factors for DR. Conclusion:In this study, the prevalence of DR in 4 communities of type 2 diabetes in Beijing City was 16.6%. Besides, this study further confirmed that the duration of diabetes, fasting blood glucose levels, and glycosylated hemoglobin are associated factors for DR in patients with type 2 diabetes.

Objective To investigate the effect of MDM2 inhibitor RG-7388 on the proliferation, cell cycle, and apoptosis of diffuse large B-lymphoma (DLBCL) cells. Methods DLBCL cell strains SUDHL2 and HBL1 were treated with 2, 4, and 8 μmol/LRG7388, respectively. Cell proliferation was detected by CCK8 and EdU methods. Apoptosis was measured by Annexin V–FITC/PI double staining and Caspase 3/7-Glo enzyme activity methods. Cell cycle was assessed by flow cytometry. Changes in the expression of cell cycle and apoptosis-related proteins were determined by Western blot. Results The IC₅₀ of RG7388 for inhibiting SUDHL2 and HBL1 cells were 3.36 and 3.76 μmol/L, respectively, and the inhibitory effect of RG7388 was dose dependent. The proportions of G₁ phase and apoptotic cells in the SUDHL2 and HBL1 cells treated with different doses of RG7388 were significantly higher than those in the control group (all P<0.05). The activity of Caspase 3/7 increased gradually with RG7388 concentration, compared with that in the control group. The expression levels of p53, p27, p21, and PARP increased, whereas the expression of Mcl-1 and Bcl-x_L was down-regulated (all P<0.05). Conclusion MDM2 inhibitor RG-7388 inhibits the proliferation of DLBCL cells, triggers cell cycle arrest in the G₁ phase, and induces apoptosis through the p53 pathway.

Objective:To select human chronic myeloid leukemia (CML) cell line K562 as the experimental object, and use lentivirus mediated CRISPR/Cas9 gene editing technology to construct a stable CML cell line K562/TCRP1-KO that knocks out the tongue cancer resistance related protein 1 (TCRP1) gene; and through functional tests such as cell proliferation, apoptosis, and drug sensitivity, compare the phenotypic differences between K562/TCRP1-KO and control cells (K562/cas9-CTL), and preliminarily explore the possible mechanism of TCRP1 gene involvement in the pathogenesis of CML.Methods:The small guide RNA (sgRNA) targeting TCRP1 was designed at a specific location. After annealing, the oligonucleotide fragments were recombined with the linearized Cas9 expression vector, and the lentivirus packaging system was transfected into 293T cells. The purified virus was collected and infected with K562 cells. Positive polyclons were screened for puromycin pressure, and monoclonal K562/TCRP1-KO was further screened by limited dilution method. Stable cell lines were successfully knocked out by sanger sequencing and Western blot detection; Simultaneously, K562 cells transfected with lentiCRISPR vector were constructed as control cell lines (K562/cas9-CTL); Using cell counting method, cell counting kit 8 (CCK8) method, imatinib (IM) gradient dilution method, and flow cytometry cell proliferation, drug sensitivity, and apoptosis analysis were performed on K562/TCRP1-KO and K562/cas9-CTL, respectively.Results:The sgRNA-Cas9 recombinant plasmid vector for TCRP1 knockout was successfully constructed, and after transfection into 293T cells, TCRP1 knockout monoclonal cell lines were successfully screened using limited dilution method. Compared with K562/cas9-CTL cells, the proliferation ability of K562/TCRP1-KO cells was significantly reduced, IM drug sensitivity was significantly enhanced, and the process of cell apoptosis was significantly accelerated (all P<0.05). Conclusions:A CML cell line with TCRP1 knockout was successfully constructed using CRISPR/Cas9. TCRP1 may act as a cancer related gene to affect the proliferation, IM resistance, and apoptosis process of CML cells.

Abstract Allergic diseases can occur in all systems of the body, covering the whole life cycle, from children to adults and to old age, can be lifelong onset and even fatal in severe cases. Children account for the largest proportion of the victims of allergic disease, Children s allergies start from scratch, ranging from mild to severe, from less to more, from single to multiple systems and systemic performance, so the prevention and treatment of allergic diseases in children is of great importance, which can not only prevent high risk allergic conditions from developing into allergic diseases, but also further block the process of allergy. At present, there is no consensus on the management system of allergic children in kindergartens and primary schools. The "Consensus on Allergy Management and Prevention in Kindergartens and Primary Schools", which includes the organizational structure, system construction and management of allergic children, provides evidence informed recommendations for the long term comprehensive management of allergic children in kindergartens and primary schools, and provides a basis for the establishment of the prevention system for allergic children.

【Objective】 To explore the potential molecular biological mechanism of Belamcanda chinensis in the treatment of glioma based on network pharmacology, molecular docking technology and in vitro cell experiments. 【Methods】 ① The active components, targets of Belamcanda chinensis and targets of glioma were obtained by database search. String database was used to analyze protein-protein interaction relationship, R project was used to analyze gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Cytoscape software was used to build "compound-target-disease" network and PPI network, and AutoDock software was used to verify molecular docking. ② Western blotting, qRT-PCT and apoptosis assay were used to verify the enrichment results of network pharmacology targets and protein pathway. 【Results】 ① We screened out 32 types of active components, 484 types of targets and 464 types of glioma targets, and obtained 62 kinds of therapeutic targets after mapping. We obtained 12 kinds of key pharmacodynamic molecules such as Isoiridogermanal, Iridobelamal A and Rhamnazinand and other key pharmacodynamic molecules, as well as AKT1, STAT3, HRAS and other core targets by network topology analysis. Enrichment analysis results demonstrated that they were mainly involved in biological processes such as peptide serine phosphorylation, protein kinase B signal transduction, peptide serine modification, and pathways including PI3K/AKT signal pathway and Rap1 signal pathway. The results of molecular docking verified the good binding activity of the key pharmacodynamic molecules with the core targets. ② The results of Western blotting showed that the protein expressions of VEGF and MMP9 of Belamcanda chinensis extracts in 8 mg/mL and 16 mg/mL groups were significantly lower than those in the blank control group (P<0.01 or P<0.001). Compared with the blank control group, the early apoptosis rate of Belamcanda chinensis extracts at 8 mg/mL and 16 mg/mL were significantly decreased (P<0.001 or P<0.000 1). qRT-PCR results showed that the mRNA expression levels of VEGF and MMP9 in Belamcanda chinensis extracts at 8 mg/mL and 16 mg/mL were significantly decreased (P<0.001 or P<0.0001). 【Conclusion】 The treatment of glioma with Belamcanda chinensis is the result of multi-component, multi-target and multi-channel interactions. The results of cell experiments confirmed that Belamcanda chinensis extracts can affect the expressions of related target proteins of PI3K/AKT signal pathway and VEGF and MMP9, which verified the results of network pharmacology. The results provide a theoretical basis for the clinical application of Belamcanda chinensis and studies on glioma.