Objective:To investigate the effects of oligodeoxynucleotide (ODN) MT01 on the morphology, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs).Methods:The BMSCs of SD rat were isolated and cultured by direct adherence method . The extracted cells were identified by cell morphology of different generations, the expression of surface markers detected by flow cytometry and osteogenic differentiation potential. ODN MT01 group was set up in a gradient of concentrations (0.5, 1.0, 2.0, 4.0 μg/ml) and PBS group as control. Each group of experiments was repeated three times. The morphological changes of cell nucleus and cytoskeleton were fluorescent stained by DAPI and FITC-phalloidin, respectively. The proliferation activities of the BMSCs in different group were analyzed by CCK-8 assay at 1, 4 and 7 d. The degrees of osteogenic differentiation of BMSCs in different group were assessed via alkaline phosphatase (ALP) staining, ALP activity assay and alizarin red S staining respectively on the 7th and 21st days after cultured in osteogenic induction medium. Statistical differences between two groups and among groups were analyzed by t-test and one-way ANOVA, respectively. Differences were regarded as statistically significant when a P value of less than 0.05. Results:Flow cytometry showed that the BMSCs were positive for CD29 (99.8%) and CD44 (96.1%) while negative for CD11b (1.03%) and CD45 (1.74%). ALP staining and alizarin red S staining were positive at different stages of osteogenesis induction confirmed that BMSCs was able to differentiate into the osteoblast. The nucleus and cytoskeleton staining showed that BMSCs were shrunk and the extensibility was reduced when the concentration of ODN MT01 was 4.0 μg/ml. CCK-8 assay showed that the absorbance value of control group was 0.446±0.018, 1.0 μg/ml ODN MT01 was 0.505±0.019, 2.0 μg/ml ODN MT01 was 0.528±0.014 after cultured for 4 days. Compared with the control group, the difference is statistically significant ( t=2.954, 4.083, P=0.033, 0.008). The absorbance value of control group was 0.514±0.027, 1.0 μg/ml ODN MT01 was 0.607±0.007, and 2.0 μg/ml ODN MT01 was 0.636±0.023 after cultured for 7 days. Compared with the control group, the difference was statistically significant ( t=4.664, 6.091, P=0.009, 0.008). The proliferation ability of BMSCs was significantly higher than that of the control group. However, 4.0 μg/ml ODN MT01 (0.427±0.013) had an inhibitory effect on the proliferation ability of BMSCs ( t=4.332, P=0.0149). The blue mass and mineralized nodule improved significantly with the increase of ODN MT01 concentration during the induction of osteogenic differentiation of BMSCs. After cultured for 4 days, the result of ALP activity assay was similar to ALP staining. The activity value of ODN MT01 in the control group was 1.207±0.023, 0.5 μg/ml ODN MT01 was 1.747±0.095, 1.0 μg/ml ODN MT01 was 2.200±0.136, 2.0 μg/ml ODN MT01 was 3.560±0.088, 4.0 μg/ml ODN MT01 was 3.490±0.144. Compared with the control group, the difference was statistically significant ( t=4.313, 7.934, 18.800, 18.240; P=0.005, 0.001, <0.001, <0.001). But there was no difference between 2.0 and 4.0 μg/ml groups ( t=0.562, P=0.590). Conclusions:ODN MT01 with concentration of 2.0 μg/ml could significantly stimulate the proliferation and osteogenic differentiation of BMSCs without affecting the morphology of BMSCs.

Objective:To investigate the effects of oligodeoxynucleotide (ODN) MT01 on the morphology, proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs).Methods:The BMSCs of SD rat were isolated and cultured by direct adherence method . The extracted cells were identified by cell morphology of different generations, the expression of surface markers detected by flow cytometry and osteogenic differentiation potential. ODN MT01 group was set up in a gradient of concentrations (0.5, 1.0, 2.0, 4.0 μg/ml) and PBS group as control. Each group of experiments was repeated three times. The morphological changes of cell nucleus and cytoskeleton were fluorescent stained by DAPI and FITC-phalloidin, respectively. The proliferation activities of the BMSCs in different group were analyzed by CCK-8 assay at 1, 4 and 7 d. The degrees of osteogenic differentiation of BMSCs in different group were assessed via alkaline phosphatase (ALP) staining, ALP activity assay and alizarin red S staining respectively on the 7th and 21st days after cultured in osteogenic induction medium. Statistical differences between two groups and among groups were analyzed by t-test and one-way ANOVA, respectively. Differences were regarded as statistically significant when a P value of less than 0.05. Results:Flow cytometry showed that the BMSCs were positive for CD29 (99.8%) and CD44 (96.1%) while negative for CD11b (1.03%) and CD45 (1.74%). ALP staining and alizarin red S staining were positive at different stages of osteogenesis induction confirmed that BMSCs was able to differentiate into the osteoblast. The nucleus and cytoskeleton staining showed that BMSCs were shrunk and the extensibility was reduced when the concentration of ODN MT01 was 4.0 μg/ml. CCK-8 assay showed that the absorbance value of control group was 0.446±0.018, 1.0 μg/ml ODN MT01 was 0.505±0.019, 2.0 μg/ml ODN MT01 was 0.528±0.014 after cultured for 4 days. Compared with the control group, the difference is statistically significant ( t=2.954, 4.083, P=0.033, 0.008). The absorbance value of control group was 0.514±0.027, 1.0 μg/ml ODN MT01 was 0.607±0.007, and 2.0 μg/ml ODN MT01 was 0.636±0.023 after cultured for 7 days. Compared with the control group, the difference was statistically significant ( t=4.664, 6.091, P=0.009, 0.008). The proliferation ability of BMSCs was significantly higher than that of the control group. However, 4.0 μg/ml ODN MT01 (0.427±0.013) had an inhibitory effect on the proliferation ability of BMSCs ( t=4.332, P=0.0149). The blue mass and mineralized nodule improved significantly with the increase of ODN MT01 concentration during the induction of osteogenic differentiation of BMSCs. After cultured for 4 days, the result of ALP activity assay was similar to ALP staining. The activity value of ODN MT01 in the control group was 1.207±0.023, 0.5 μg/ml ODN MT01 was 1.747±0.095, 1.0 μg/ml ODN MT01 was 2.200±0.136, 2.0 μg/ml ODN MT01 was 3.560±0.088, 4.0 μg/ml ODN MT01 was 3.490±0.144. Compared with the control group, the difference was statistically significant ( t=4.313, 7.934, 18.800, 18.240; P=0.005, 0.001, <0.001, <0.001). But there was no difference between 2.0 and 4.0 μg/ml groups ( t=0.562, P=0.590). Conclusions:ODN MT01 with concentration of 2.0 μg/ml could significantly stimulate the proliferation and osteogenic differentiation of BMSCs without affecting the morphology of BMSCs.

OBJECTIVE：To establish a method for the content determination of potential genotoxic impurity maleic hydrazide in azintamide raw material. METHODS ：HPLC-FLD method was adopted. The determination was performed on Thermo Syncronis C18 column with mobile phase consisted of 0.2 mol/L acetic acid-methanol （gradient elution ）. The column temperature was set at 30 ℃，the excitation wavelength was 315 nm and emission wavelength was 389 nm. The flow rate was 1 mL/min，and the sample size was 20 μL. RESULTS：The blank solvent and azintamide did not interfere with the determination of maleic hydrazide. The linear range of maleic hydrazide was 19.5-300 ng/mL（r＝0.999 9）. The limit of detection was 4.5 ng/mL and the limit of quantification was 19.5 ng/mL. The recovery ranged from 98.79％ to 103.76％（RSDs were lower than 3.00％，n＝9）. RSDs of precision and stability （24 h）tests were no more than 5.63％，and those of durability tests were less than 2.00％（n＝6）. Maleic hydrazide was not detected in 3 batches of azinamide raw material. CONCLUSIONS ：The method is specific ，sensitive and accurate. It can be used for the trace determination of maleic hydrazide in azintamide or other matrix.

Type 2 diabetes (T2D) is often accompanied with an induction of retinaldehyde dehydrogenase 1 (RALDH1 or ALDH1A1) expression and a consequent decrease in hepatic retinaldehyde (Rald) levels. However, the role of hepatic Rald deficiency in T2D progression remains unclear. In this study, we demonstrated that reversing T2D-mediated hepatic Rald deficiency by Rald or citral treatments, or liver-specific Raldh1 silencing substantially lowered fasting glycemia levels, inhibited hepatic glucogenesis, and downregulated phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6-phosphatase (G6PC) expression in diabetic db/db mice. Fasting glycemia and Pck1/G6pc mRNA expression levels were strongly negatively correlated with hepatic Rald levels, indicating the involvement of hepatic Rald depletion in T2D deterioration. A similar result that liver-specific Raldh1 silencing improved glucose metabolism was also observed in high-fat diet-fed mice. In primary human hepatocytes and oleic acid-treated HepG2 cells, Rald or Rald + RALDH1 silencing resulted in decreased glucose production and downregulated PCK1/G6PC mRNA and protein expression. Mechanistically, Rald downregulated direct repeat 1-mediated PCK1 and G6PC expression by antagonizing retinoid X receptor α, as confirmed by luciferase reporter assays and molecular docking. These results highlight the link between hepatic Rald deficiency, glucose dyshomeostasis, and the progression of T2D, whilst also suggesting RALDH1 as a potential therapeutic target for T2D.

BACKGROUND: LY01005 (Goserelin acetate sustained-release microsphere injection) is a modified gonadotropin-releasing hormone (GnRH) agonist injected monthly. This phase III trial study aimed to evaluated the efficacy and safety of LY01005 in Chinese patients with prostate cancer. METHODS: We conducted a randomized controlled, open-label, non-inferiority trial across 49 sites in China. This study included 290 patients with prostate cancer who received either LY01005 or goserelin implants every 28 days for three injections. The primary efficacy endpoints were the percentage of patients with testosterone suppression ≤50 ng/dL at day 29 and the cumulative probability of testosterone ≤50 ng/dL from day 29 to 85. Non-inferiority was prespecified at a margin of -10%. Secondary endpoints included significant castration (≤20 ng/dL), testosterone surge within 72 h following repeated dosing, and changes in luteinizing hormone, follicle-stimulating hormone, and prostate specific antigen levels. RESULTS: On day 29, in the LY01005 and goserelin implant groups, testosterone concentrations fell below medical-castration levels in 99.3% (142/143) and 100% (140/140) of patients, respectively, with a difference of -0.7% (95% confidence interval [CI], -3.9% to 2.0%) between the two groups. The cumulative probabilities of maintaining castration from days 29 to 85 were 99.3% and 97.8%, respectively, with a between-group difference of 1.5% (95% CI, -1.3% to 4.4%). Both results met the criterion for non-inferiority. Secondary endpoints were similar between groups. Both treatments were well-tolerated. LY01005 was associated with fewer injection-site reactions than the goserelin implant (0% vs . 1.4% [2/145]). CONCLUSION: LY01005 is as effective as goserelin implants in reducing testosterone to castration levels, with a similar safety profile. TRIAL REGISTRATION ClinicalTrials.gov, NCT04563936.
Humans ; Male ; Antineoplastic Agents, Hormonal/therapeutic use* ; East Asian People ; Gonadotropin-Releasing Hormone/agonists* ; Goserelin/therapeutic use* ; Prostate-Specific Antigen ; Prostatic Neoplasms/drug therapy* ; Testosterone