BACKGROUND:Previous studies have shown that puerarin can inhibit the differentiation of osteoclasts,and the expression of Notch signaling pathway-related proteins such as Notch1,HES1,and Jagged1 is decreased.However,the specific mechanism of the Notch1 signaling pathway for the inhibition of osteoclast differentiation by puerarin is not clear. OBJECTIVE:To explore the effect of Notch signaling pathway on puerarin inhibiting the differentiation of mouse macrophage Raw264.7 into osteoclasts. METHODS:Raw264.7 cells were divided into seven groups for intervention culture.Blank control group was cultured in high-sugar DMEM medium;the osteoclast induction group was cultured in osteoclast induction medium;the puerarin intervention group was cultured with 50 μmol/L puerarin at the same time of osteoclast induction;Notch1 siRNA control group,Notch1 siRNA group,Notch1 overexpression control group and Notch1 overexpression group were transfected with Notch1 siRNA control sequence,Notch1 siRNA,Notch1 overexpression control plasmid and Notch1 overexpression plasmid,respectively,and then cultured with osteoclast induction medium and puerarin.The number and size of osteoclasts were observed by tartrate-resistant acid phosphatase staining,the skeleton formation of osteoclasts was observed by F-actin staining,and the gene expression level of osteoclast formation markers was detected by RT-PCR. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining results showed that puerarin intervention could inhibit the generation of osteoclasts,Notch1 silencing could further reduce the number of osteoclasts,while the number of osteoclasts in the osteoclast-induced group increased significantly after Notch1 overexpression.The results of F-actin showed that Raw264.7 cells could form a well-defined F-actin ring after osteoclast induction.Puerarin intervention would inhibit the formation of cytoskeleton,and Notch1 silencing could aggravate the inhibitory effect of cytoskeleton formation,while Notch1 overexpression could alleviate this inhibitory effect of puerarin.RT-PCR results showed that puerarin could inhibit the mRNA expression levels of tartrate-resistant acid phosphatase,Cathepsin K and c-Fos,the expression of the above-mentioned three factors decreased significantly after Notch1 gene silencing,and Notch1 overexpression could upregulate the expression of these factors.These finding indicate that puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway.

Objective To explore the mechanism of 3-methyladenine(3-MA)for alleviating early diabetic renal injury.Methods Mouse models of streptozotocin(STZ)-induced diabetes mellitus were randomized into model group and 3-MA treatment group for daily treatments with normal saline and 10 mg/kg 3-MA by gavage for 6 weeks,respectively.Body weight and fasting blood glucose of the mice were recorded every week.After the treatments,the kidneys of the mice were collected for measurement kidney/body weight ratio,examination of glomerular size with PAS staining,and detection of α-SMA and PCNA expressions using Western blotting and immunohistochemistry.SV40 MES 13 cells cultured in normal glucose(5.6 mmol/L)and high glucose(30 mmol/L)were treated with 24.4 mmol/L mannitol and 5 mmol/L 3-MA for 24 h,respectively,and the changes in cell viability and PCNA expression were examined using CCK8 assay and Western blotting.Bioinformatics analysis of the intersecting gene targets of diabetic kidney disease(DKD)and 3-MA was performed,and the results were verified by Western blotting both in vivo and in vitro.Results In the diabetic mice,treatment with 3-MA produced a short-term hypoglycemic effect,reduced the kidney/body weight ratio and glomerular hypertrophy,and decreased the expressions of α-SMA and PCNA in the renal cortex.In the in vitro study,3-MA significantly lowered the viability and reduced PCNA expression in SV40 MES 13 cells exposed to high glucose.The results of bioinformatic analysis identified AKT1 as the key gene in the therapeutic mechanism of 3-MA for DKD.Western blotting confirmed that 3-MA inhibited the phosphorylation of AKT and S6 in both the renal cortex of diabetic mice and high glucose-treated SV40 MES 13 cells.Conclusion 3-MA suppresses mesangial cell proliferation and alleviates early diabetic renal injury in mice possibly by inhibiting AKT signaling.

Objective To investigate the mechanism mediating the regulatory effect of lncRNA MAGI2-AS3 on cisplatin(DDP)resistance in non-small cell lung cancer(NSCLC).Methods MAGI2-AS3 and miR-1269a expression levels were detected by qRT-PCR in DDP-sensitive lung cancer cell lines(A549 and H1299)and their resistant counterparts(A549/DDP and H1299/DDP).In A549 and H1299 cells with MAGI2-AS3 silencing and A549/DDP and H1299/DDP cells overexpressing MAGI2-AS3,the effects of 20 μmol/L DDP on cell viability and apoptosis were examined with CCK-8 assay,colony formation assay,flow cytometry and Western blotting,and the changes in epithelial-mesenchymal transition(EMT)were assessed with wound healing and Transwell assays.The interaction between MAGI2-AS3,miR-1269a and PTEN was predicted using GEPIA,StarBase and miRDB and verified with luciferase reporter gene assay and radioimmunoprecipitation(RIP)assay.A miR-1269a mimic and pcDNA3.1-PTEN plasmid were used to perform the rescue assay.Results MAGI2-AS3 expression was significantly downregulated in lung cancer tissues(P<0.05)in association with a poor prognosis(P<0.05).In the two DDP-resistant lung cancer cell lines,MAGI2-AS3 expression was significantly lowered as compared with the sensitive cells.Silencing MAGI2-AS3 significantly enhanced cell viability and promoted EMT of A549 and H1299 cells irrespective of DDP treatment,and also decreased DDP-induced apoptosis of the cells.In A549/DDP and H1299/DDP cells,MAGI2-AS3 overexpression strongly repressed cell viability and EMT irrespective of DDP treatment and promoted DDP-induced cell apoptosis.Luciferase reporter gene and RIP assays confirmed the binding of MAGI2-AS3 with miR-1269a and the binding of miR-1269a with 3'-UTR domain of PTEN.The rescue assay demonstrated that MAGI2-AS3 acted as a sponge for miR-1269a to promote PTEN expression and downregulate AKT phosphorylation,thus inhibiting EMT and promoting DDP-induced apoptosis of A549/DDP cells.Conclusion MAGI2-AS3 enhances DDP sensitivity of NSCLC by targeted regulation of the miR-1269a/PTEN/AKT signaling axis.

Objective To investigate the expression,synergistic relationship and clinical significance of alcohol de-hydrogenase(ADH1A)and vascular endothelial growth factor-A(VEGFA)in hepatocellular carcinoma(HCC).Methods The expression and correlation of ADH1A and VEGFA in HCC and adjacent normal tissues were ana-lyzed by GEPIA.TCGA and GSEA were used to analyze the pathway of ADH1A in HCC.The clinical and patho-logical data of 84 patients with HCC were collected,and 54 patients with paracancer normal tissue samples were se-lected as controls to analyze the correlation between ADH1A and VEGFA and clinicopathological parameters of HCC.Immunohistochemistry was used to detect the protein expression of ADH1A and VEGFA in cases and con-trols,and the correlation between the expression of ADH1A and VEGFA and the clinical progression and prognosis of patients with HCC was analyzed based on clinical pathological parameters and Kaplan-Meier.Results Bioinfor-matics analysis found that ADH1A was low-expressed in HCC and VEGFA was highly expressed in HCC,and there was a negative correlation between the two(P<0.001);immunohistochemical detection results showed that the expression of ADH1A in HCC tissue was lower than that in normal tissue adjacent to cancer(P<0.01)while the expression rate of VEGFA in HCC tissue was significantly higher than that of normal tissue adjacent to cancer(P<0.01);The recurrence rate of vascular thrombus and HCC patients in HCC group with high expression of ADH1A was lower(P<0.05).The proportion of tumor diameter>5 cm,high TNM stage,microsatellite and G2-G3 dif-ferentiation in HCC tissues in VEGFA high expression group was higher(P<0.05).Kaplan-Meier survival analy-sis showed that patients with high ADH1A expression and low VEGFA expression had a higher five-year survival rate.Conclusion Low expression of ADH1A and high expression of VEGFA in tumor tissues of patients with HCC indicate tumor progression and can be used as one of the prognostic evaluation indicators for patients with HCC.

Spinal muscular atrophy (SMA) is the most common neuromuscular disease in children, which seriously affects children′s health. At present, gene and molecular modification therapy for SMA have become hot spots. However, there are many uncertainties about when people with SMA should start treatment, how well the drugs can treat, and the prognosis. Therefore, reliable biomarkers for monitoring and evaluation are urgently needed. This review will summarize the progress made in SMA biomarker research in recent years.

Objective To explore the mechanism of 3-methyladenine(3-MA)for alleviating early diabetic renal injury.Methods Mouse models of streptozotocin(STZ)-induced diabetes mellitus were randomized into model group and 3-MA treatment group for daily treatments with normal saline and 10 mg/kg 3-MA by gavage for 6 weeks,respectively.Body weight and fasting blood glucose of the mice were recorded every week.After the treatments,the kidneys of the mice were collected for measurement kidney/body weight ratio,examination of glomerular size with PAS staining,and detection of α-SMA and PCNA expressions using Western blotting and immunohistochemistry.SV40 MES 13 cells cultured in normal glucose(5.6 mmol/L)and high glucose(30 mmol/L)were treated with 24.4 mmol/L mannitol and 5 mmol/L 3-MA for 24 h,respectively,and the changes in cell viability and PCNA expression were examined using CCK8 assay and Western blotting.Bioinformatics analysis of the intersecting gene targets of diabetic kidney disease(DKD)and 3-MA was performed,and the results were verified by Western blotting both in vivo and in vitro.Results In the diabetic mice,treatment with 3-MA produced a short-term hypoglycemic effect,reduced the kidney/body weight ratio and glomerular hypertrophy,and decreased the expressions of α-SMA and PCNA in the renal cortex.In the in vitro study,3-MA significantly lowered the viability and reduced PCNA expression in SV40 MES 13 cells exposed to high glucose.The results of bioinformatic analysis identified AKT1 as the key gene in the therapeutic mechanism of 3-MA for DKD.Western blotting confirmed that 3-MA inhibited the phosphorylation of AKT and S6 in both the renal cortex of diabetic mice and high glucose-treated SV40 MES 13 cells.Conclusion 3-MA suppresses mesangial cell proliferation and alleviates early diabetic renal injury in mice possibly by inhibiting AKT signaling.

Objective To investigate the mechanism mediating the regulatory effect of lncRNA MAGI2-AS3 on cisplatin(DDP)resistance in non-small cell lung cancer(NSCLC).Methods MAGI2-AS3 and miR-1269a expression levels were detected by qRT-PCR in DDP-sensitive lung cancer cell lines(A549 and H1299)and their resistant counterparts(A549/DDP and H1299/DDP).In A549 and H1299 cells with MAGI2-AS3 silencing and A549/DDP and H1299/DDP cells overexpressing MAGI2-AS3,the effects of 20 μmol/L DDP on cell viability and apoptosis were examined with CCK-8 assay,colony formation assay,flow cytometry and Western blotting,and the changes in epithelial-mesenchymal transition(EMT)were assessed with wound healing and Transwell assays.The interaction between MAGI2-AS3,miR-1269a and PTEN was predicted using GEPIA,StarBase and miRDB and verified with luciferase reporter gene assay and radioimmunoprecipitation(RIP)assay.A miR-1269a mimic and pcDNA3.1-PTEN plasmid were used to perform the rescue assay.Results MAGI2-AS3 expression was significantly downregulated in lung cancer tissues(P<0.05)in association with a poor prognosis(P<0.05).In the two DDP-resistant lung cancer cell lines,MAGI2-AS3 expression was significantly lowered as compared with the sensitive cells.Silencing MAGI2-AS3 significantly enhanced cell viability and promoted EMT of A549 and H1299 cells irrespective of DDP treatment,and also decreased DDP-induced apoptosis of the cells.In A549/DDP and H1299/DDP cells,MAGI2-AS3 overexpression strongly repressed cell viability and EMT irrespective of DDP treatment and promoted DDP-induced cell apoptosis.Luciferase reporter gene and RIP assays confirmed the binding of MAGI2-AS3 with miR-1269a and the binding of miR-1269a with 3'-UTR domain of PTEN.The rescue assay demonstrated that MAGI2-AS3 acted as a sponge for miR-1269a to promote PTEN expression and downregulate AKT phosphorylation,thus inhibiting EMT and promoting DDP-induced apoptosis of A549/DDP cells.Conclusion MAGI2-AS3 enhances DDP sensitivity of NSCLC by targeted regulation of the miR-1269a/PTEN/AKT signaling axis.

Objective:To understand the current status of occupational exposure to dust or harmful gases and occupational protection in people aged ≥40 years in China, and provide data support for the prevention and control of occupational dust or harmful gas exposure.Methods:The data were obtained from the surveillance for chronic obstructive pulmonary disease (COPD) in adults aged ≥40 years selected by multi-stage stratified cluster sampling from 125 surveillance points in 31 provinces (autonomous regions and municipalities) during 2014-2015 and 2019-2020, and relevant information about occupational dust or harmful gas exposure and protection measures were collected through face-to-face interviews. Occupational dust or harmful gas exposure rate and occupational protection rate were estimated by using weighting complex sampling methods, and then the results were compared.Results:From 2014 to 2015 and from 2019 to 2020, a total of 71 061 and 71 023 individuals aged ≥40 years were surveyed, respectively. The rate of occupational exposure to dust or hazardous gas was 33.8% (95% CI: 29.9%-37.7%) during 2019-2020. The occupational exposure rate was higher in men than in women and in rural residents than in urban residents. With the increase of education level, the rate of occupational exposure to dust or harmful gas showed a downward trend. The protection rate against occupational dust or hazardous gas exposure was 47.9% (95% CI: 43.2%-52.6%) during 2019-2020. Compared with 2014-2015, the rate of occupational exposure to dust or hazardous gas decreased by 10.7 percentage points in different gender, area and occupational groups and the occupational protection rate increased by 21.9 percentage points during 2019-2020. The decrease in occupational exposure rate was higher in western China than in eastern and central China, and the increase in occupational protection rate was higher in western China than in eastern and central China. Conclusions:The rate of occupational exposure to dust or harmful gas decreased and the rate of occupational protection against dust or harmful gas exposure increased in China during 2019-2020. However, about one-third of the population still suffer from the occupational exposure, and less than half of them take protection measures. It is necessary to pay more attention to the key populations, such as workers with lower cultural level and rural migrant workers, in occupational health practice.

Objective:To understand the passive smoking exposure status in adults aged ≥40 years in China.Methods:Local residents aged ≥40 years were enrolled as study subjects from 125 areas of chronic obstructive pulmonary disease (COPD) surveillance during 2014-2015 and 2019-2020 in 31 provinces of China. A total of 74 559 adults aged ≥40 years were selected through multi-stage stratified cluster sampling for a face to face questionnaire survey and the data from 64 142 study subjects were used for the analysis. The passive smoking exposure rate, the proportions of the adults reporting passive smoking exposure at four types of places and the proportion of the adults living with daily smokers were described by using complicated sampling weighting method, the related factors were analyzed and the results were compared with the data of COPD surveillance during 2014-2015.Results:The passive smoking exposure rate in the adults aged ≥40 years was 46.4% (95% CI: 44.1%-48.8%) in China during 2019-2020, and the rate was higher in women (47.2%, 95% CI:44.8%-49.7%) than in men (44.8%, 95% CI:42.0%-47.6%) and lower in the older people. The office workers had the highest passive smoking exposure rate. The proportions of those reporting passive smoking exposure at homes, workplaces, restaurants, and public transports were 24.3% (95% CI:22.2%-26.4%)、23.3% (95% CI:21.1%-25.5%)、6.6% (95% CI:5.3%-7.9%) and 2.2% (95% CI:1.6%-2.7%). The higher education level the adults had, the less passive smoking exposure at home they reported. The proportions of those living with daily smokers before 14 years old and since 14 years old were 56.4% and 59.2%. Compared with the data during 2014-2015, the overall passive smoking exposure rate in the adults aged ≥40 years during 2019-2020 showed an increase, and the difference was not significant ( P=0.356); The passive smoking exposure rate at homes declined, but the exposure rate at workplaces increased, with the biggest increase found in those being engaged in farming, forestry, husbandry, fishery and water conservancy. Multivariate analysis indicated that the factors influencing the passive smoking exposure and the exposure proportions at different places included gender, age, occupation, and education level. Conclusions:The passive smoking exposure rate in China is still high, especially in those being engaged in farming, forestry, husbandry, fishery and water conservancy. It is necessary to strengthen supervision of the enforcement of current smoking bans in public places and promote the legislation of ban smoking in public places. More attention should be paid to smoking ban and protection against passive smoking exposure in women, people with lower education level and people being engaged in in farming, forestry, husbandry, fishery and water conservancy.

Objective To study expression and prognostic value of Shugoshin-1(SGOL1)in lung adenocarcinoma by bioinformatics method.Methods Expression profile data and clinical data of lung adenocarcinoma and normal tissues were downloaded from The Cancer Genome Atlas database,and expression difference and clinical correlation analysis of SGOL1 were performed.R package"pROC"was used to plot receiver operator characteristic(ROC)curves to evaluate accuracy of SGOL1 expression in predicting clinical diagnosis in lung adenocarcinoma patients.Effects of SGOL1 expression on prognosis of lung adenocarcinoma patients were evaluated by R package"survival","survminer"and univariate and multivariate Cox regression analysis.By searching Tumor Immune Single-Cell Hub and TIMER2.0 databases,expression distribution of SGOL1 in lung adenocarcinoma and its relationship with immune cell infiltration were analyzed,functional enrichment analysis of SGOL1 and its co-expression was performed by using LinkedOmics database.Search tool for the retrieval of interaction gene/proteins was used to construct a protein-protein interaction network for SGOL1.Results Compared with normal tissues,expression level of SGOL1 in tumor tissues was significantly upregulated(P<0.001).Compared with paracancer tissues,expression level of SGOL1 in tumor tissues was significantly upregulated(P<0.001).In different clinical and pathological stages of lung adenocarcinoma,compared with stage Ⅰ,expression levels of SGOL1 in stages Ⅱ,Ⅲ and Ⅳ were significantly higher(P<0.05).ROC curve showed that SGOL1 had a good diagnostic efficiency in lung adenocarcinom patients,with area under the curve of 0.959(95%CI:0.942-0.975).Overall survival,disease specific survival,disease-free survival and progression free interval of high expression group of SGOL1 were significantly shorter than those of low expression group of SGOL1(P<0.05).Univariate and multivariate Cox regression analysis showed that clinical stage(HR=1.629,P<0.001)and SGOL1 expression level(HR=1.447,P=0.002)were associated with poor prognosis in lung adenocarcinoma patients.It can be used as an independent risk factor for the prognosis of lung adenocarcinoma patients.Expression level of SGOL1 was negatively correlated with infiltration level of B cells,CD4+T cells and dendritic cells(P<0.05).Expression level of SGOL1 was positively correlated with infiltration level of macrophages,CD8+T cells and neutrophils(P<0.05).Enrichment analysis showed that SGOL1 may play role in mitosis,cell cycle,p53 signaling pathway and amino acid metabolism pathways.Analysis of protein-protein interaction network suggests that SGOL1 was closely related to multiple molecules such as CBX1,PPP2CA,PPP2R5C,CDCA8,ESPL1,PPP2R1A,BUB1,PPP2R5A,SGO2,CDC20,etc.Conclusion SGOL1 is highly expressed in lung adenocarcinoma tissues,and it is associated with poorer prognosis in lung adenocarcinoma patients.SGOL1 can be used as one of prognostic biomarkers for lung adenocarcinoma patients.