The accurate segmentation of breast ultrasound images is an important precondition for the lesion determination. The existing segmentation approaches embrace massive parameters, sluggish inference speed, and huge memory consumption. To tackle this problem, we propose T ²KD Attention U-Net (dual-Teacher Knowledge Distillation Attention U-Net), a lightweight semantic segmentation method combined double-path joint distillation in breast ultrasound images. Primarily, we designed two teacher models to learn the fine-grained features from each class of images according to different feature representation and semantic information of benign and malignant breast lesions. Then we leveraged the joint distillation to train a lightweight student model. Finally, we constructed a novel weight balance loss to focus on the semantic feature of small objection, solving the unbalance problem of tumor and background. Specifically, the extensive experiments conducted on Dataset BUSI and Dataset B demonstrated that the T ²KD Attention U-Net outperformed various knowledge distillation counterparts. Concretely, the accuracy, recall, precision, Dice, and mIoU of proposed method were 95.26%, 86.23%, 85.09%, 83.59%and 77.78% on Dataset BUSI, respectively. And these performance indexes were 97.95%, 92.80%, 88.33%, 88.40% and 82.42% on Dataset B, respectively. Compared with other models, the performance of this model was significantly improved. Meanwhile, compared with the teacher model, the number, size, and complexity of student model were significantly reduced (2.2×10 ⁶ vs. 106.1×10 ⁶, 8.4 MB vs. 414 MB, 16.59 GFLOPs vs. 205.98 GFLOPs, respectively). Indeedy, the proposed model guarantees the performances while greatly decreasing the amount of computation, which provides a new method for the deployment of clinical medical scenarios.
Humans ; Breast Neoplasms/diagnostic imaging* ; Female ; Ultrasonography, Mammary/methods* ; Image Processing, Computer-Assisted/methods* ; Algorithms ; Neural Networks, Computer ; Breast/diagnostic imaging*

Objective To investigate the effect and mechanism of Efferocytosis Relatived LncRNA (EFRL) on homocysteine-induced atherosclerosis in macrophage efferocytosis. Methods RAW264.7 cells were cultured in vitro, and the Control group (0 μmol/L Hcy) and Hcy intervention group (100 μmol/L Hcy) were set up. After GapmeR transfection of macrophages with Hcy intervention, EFRL knockdown negative control group (Hcy combined with LNA-NC) and EFRL knockdown group (Hcy combined with LNA-EFRL) were set up. High-throughput sequencing was applied for different expression of LncRNA MSTRG. 88917.16 (EFRL), UCSC was used to analyze its conservation, CPC and CPAT were used to analyze its ability to encode proteins, and GO and KEGG were used to analyze related biological functions. The localization of LncRNA EFRL in macrophages was analyzed by nucleoplasmic separation and RNA-FISH. Quantitative real-time PCR was used to detect the expression levels of LncRNA EFRL and its target gene SPAST in Hcy-treated macrophages. The apoptosis rate of Jurkat cells induced by UV was detected by flow cytometry. In vitro efferocytosis assay combined with immunofluorescence technique was used to analyze macrophage efferocytosis. ELISA was used to detect the levels of interleukin 1β(IL-1β) and IL-18. Results The new LncRNA MSTRG.88917.16 was identified and named EFRL(Efferocytosis Relatived LncRNA). UCSC, CPC and CPAT analyses showed that LncEFRL is highly conserved and does not have the ability to encode proteins. GO and KEGG analyses suggested that LncEFRL may be involved in macrophage efferocytosis. LncRNA EFRL was localized in the nucleus of macrophages as determined by nucleoplasmic separation and RNA-FISH. In comparison to the Control group, the expression levels of LncRNA EFRL and its target gene SPAST in the Hcy group were increased. In comparison to the Control group (0 min), the apoptosis rate of the experimental group (15, 30 min) Annexin V is more than 85%. Compared with Hcy combined with LNA-NC group, Hcy combined with LNA-EFRL group had enhanced macrophage efferocytosis and reduced levels of inflammatory factors. Compared with Hcy combined with LNA-NC group, the expression level of SPAST in Hcy combined with LNA-EFRL group was decreased. Conclusion Inhibition of EFRL expression can alleviate the process of Hcy inhibiting macrophage efferocytosis, and the mechanism is related to the regulation of the downstream target gene SPAST by EFRL.
RNA, Long Noncoding/physiology* ; Animals ; Homocysteine ; Mice ; Macrophages/drug effects* ; Humans ; RAW 264.7 Cells ; Atherosclerosis/chemically induced* ; Apoptosis/genetics* ; Phagocytosis/genetics* ; Jurkat Cells ; Interleukin-1beta/genetics* ; Efferocytosis

Objective:To investigate the effect of DEAD-box helicase (DDX) 21 on myocardial ischemia-reperfusion (I/R) injury and its potential mechanisms.Methods:In vivo, adult male Bama pigs and C57BL/6J mice were used to establish a myocardial I/R injury model by ligating the left anterior descending coronary artery, with sham-operated groups set as controls. The expression of DDX21 in myocardium after I/R injury was assessed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. Following the establishment of the myocardial I/R injury model in mice, AAV9 vectors with cardiac-specific expression were injected in situ into the peri-infarct region (The I/R+DDX21 group, I/R+negative control (NC) group, I/R+sh-NC group and I/R+sh-DDX21 group were injected with AAV9:cTnT-DDX21, AAV9:cTnT-NC, AAV9:cTnT-sh-NC and AAV9:cTnT-sh-DDX21, respectively). Additionally, the I/R+A-485 group received intraperitoneal injections of the cAMP response element-binding protein (CREB) binding protein inhibitor A-485, while the I/R+PBS group was injected with an equivalent volume of phosphate-buffered saline (PBS) as the control. Echocardiography was performed on postoperative days 1 and 28 to evaluate cardiac function (left ventricular ejection fraction and fractional shortening). At 28 days post-surgery, mice were euthanized and heart tissues were harvested for histological sectioning. Myocardial fibrosis was evaluated using Masson′s trichrome staining. In vitro, primary cardiomyocytes were isolated from neonatal day 1 C57BL/6J mice using enzymatic digestion method. Cardiomyocytes were transfected with plasmids or small interfering RNA (siRNA). The cardiomyocytes transfected with DDX21-siRNA were assigned to the siDDX21 group, those transfected with the DDX21 plasmid were assigned to the DDX21 group, and those transfected with the corresponding empty plasmid or siRNA were assigned to the NC group. Additionally, cardiomyocytes were treated with A-485 (A-485 group) or PBS (PBS group). An oxygen-glucose deprivation/reoxygenation (OGD/R) model was used to simulate cellular injury. Transcriptome sequencing was performed to identify downstream mechanisms of DDX21. Differential gene expression analysis was conducted using software such as DESeq2, and alternative splicing events in the mRNA transcriptome were analyzed using rMATS software. Mitochondrial superoxide, mitochondrial membrane potential, ATP content, and mitochondrial respiratory chain complex enzyme activity in cardiomyocytes were detected using immunofluorescence staining and commercial assay kits. The oxidative phosphorylation level of the cells was assessed by the Seahorse extracellular flux analyzer. Acetylated DDX21 levels were measured using co-immunoprecipitation and Western blot assays.Results:The expression levels of DDX21 in myocardium from the Bama pigs and mice in the I/R injury model were significantly higher than those in the sham group (all P<0.001). Echocardiographic results showed that at 28 days post-surgery, compared to the I/R+NC group, the I/R+DDX21 group exhibited higher left ventricular ejection fraction and fractional shortening, while the I/R+sh-DDX21 group showed lower values; Masson staining results demonstrated that, compared to the I/R+NC group, the myocardial fibrosis area in the I/R+DDX21 group was significantly reduced, whereas it was significantly increased in the I/R+sh-DDX21 group (all P<0.001). Transcriptomic sequencing results suggested that DDX21 may influence myocardial injury by regulating mitochondrial metabolic activity. In vitro, compared to the OGD/R+NC group, the OGD/R+DDX21 group exhibited lower mitochondrial superoxide levels, higher polymer/monomer ratio, maximal oxygen consumption, reserve capacity, and ATP content. In contrast, the OGD/R+siDDX21 group showed the opposite results, with reduced activity of mitochondrial respiratory chain complex V (all P<0.05). Mechanistically, rMATS software and other analyses indicated that knockdown of DDX21 affected the alternative 3′ splicing sites of ATP5J precursor mRNA, inhibiting the splicing of certain exonic sequences. Overexpression of DDX21 upregulated both mRNA and protein levels of ATP5J. Co-immunoprecipitation experiments showed that, compared to the PBS group, acetylated DDX21 levels were reduced in the A-485 group. Further in vivo experiments showed that, compared to the I/R+PBS group, the I/R+A-485 group exhibited higher left ventricular ejection fraction and fractional shortening, and a lower proportion of left ventricular fibrosis (all P<0.001). Conclusions:DDX21 improves cardiomyocyte energy metabolism and alleviates I/R injury by regulating the alternative splicing of ATP5J. A-485 holds potential as a novel small molecule candidate for the treatment of myocardial injury.

Objective:To investigate the biological role and molecular mechanism of checkpoint kinase 1 (CHK1) in delaying cardiac aging in mice.Methods:In vitro, a senescence model of H9C2 cells (a cardiomyocyte line) was induced using H 2O 2. A control group (without H 2O 2 treatment) and three H 2O 2-treated groups (at concentrations of 10, 30, and 50 μmol/L) were set up. The CCK-8 assay was used to evaluate the proliferative activity of cells in each group; Western blot analysis was employed to detect the expression level of CHK1; and quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to determine the messenger RNA (mRNA) expression levels of P16 and interleukin-1β (IL-1β). In vivo, C57BL/6 wild-type mice aged 2 months ( n=15) and 24 months ( n=40), as well as myocardial-specific CHK1-overexpressing (CHK1-TG) mice aged 2 months ( n=15) and 24 months ( n=40), were selected. The mice were divided into four groups based on age and genotype: 2-month-old wild-type (WT-2M), 24-month-old wild-type (WT-24M), 2-month-old CHK1-TG (CHK1-TG-2M), and 24-month-old CHK1-TG (CHK1-TG-24M). Echocardiography was used to evaluate cardiac function of mice in the WT-24M and CHK1-TG-24M groups. Western blot analysis was conducted to measure the protein expression levels of CHK1, total Ras-related protein 1 (Rap1), NADPH oxidase 4 (Nox4), and Rap1-guanosine triphosphate (Rap1-GTP, the active form of Rap1) in the cardiac tissue of mice in each group. qRT-PCR was used to detect the messenger RNA (mRNA) expression levels of CHK1, collagen type Ⅰ (Coll1), matrix metalloproteinase-2 (Mmp2), alpha-smooth muscle actin (α-SMA), P53, P21, P16, thioredoxin 1 (Trx1), thioredoxin reductase (TrxR), glutathione recluctase (GR), Rap1, and Nox4. Immunofluorescence staining was employed to determine the protein expression levels of P53, P21, and P16, as well as the proportion of histone H2AX phosphorylation-positive cells. Dihydroethidium (DHE) staining was used to detect the relative intensity of DHE. Wheat germ agglutinin staining, HE staining, Masson staining and Sirius red staining were applied to measure the cross-sectional area of cardiomyocytes, cardiac morphology, and myocardial fibrosis area. Mice in the WT-24M and CHK1-TG-24M groups were intraperitoneally injected with the Rap1 activity inhibitor GGTI298 (25 μmol/kg). After injection, the oxidative stress damage in the cardiac tissue of the mice was detected, along with the mRNA expression levels of fibrosis-related indicators (Coll1, Mmp2, and α-SMA) and cell cycle inhibitory proteins (P16, P21, and P53). Results:A concentration of 30 μmol/L was determined as the optimal concentration for establishing an H 2O 2-induced senescence model of myocardial cells in vitro. The expression level of CHK1 in H9C2 cells of the 30 μmol/L H 2O 2 group was lower than that in the control group ( P<0.05). Echocardiographic examination showed that the left ventricular ejection fraction ((61.08±1.13)% vs. (52.55±2.02)%) and fractional shortening ((31.80±1.27)% vs. (25.18±1.59)%) of mice in the CHK1-TG-24M group were higher than those in the WT-24M group (both P<0.05). qRT-PCR and Western blot analysis revealed that, compared with the WT-24M group, mice in CHK1-TG-24M group had higher expression levels of CHK1 and its mRNA, lower expression levels of Nox4 and its mRNA, and higher expression level of Rap1-guanosine triphosphate (Rap1-GTP) (all P<0.05). However, there were no statistically significant differences in the total expression level of Rap1 and its mRNA between the two groups (both P>0.05). In addition, the mRNA expression levels of Coll1, Mmp2, and α-SMA in myocardial tissue of mice in the CHK1-TG-24M group were lower than those in the WT-24M group (all P<0.05). Immunofluorescence staining results showed that the expression levels of P53, P21, and P16 proteins, as well as the proportion of phosphorylated histone H2AX-positive cells in myocardial tissue of mice in the WT-24M group were higher than those in the CHK1-TG-24M group (all P<0.05). qRT-PCR further confirmed that the mRNA expression levels of the above-mentioned proteins in cardiac tissue of mice in the WT-24M group were higher than those in the CHK1-TG-24M group (all P<0.05). DHE staining results indicated that the relative intensity of DHE in cardiac tissue of mice in the CHK1-TG-24M group was lower than that in the WT-24M group ( P<0.05). Meanwhile, the left ventricular internal diameter, cross-sectional area of cardiomyocytes, and myocardial fibrosis area of mice in the CHK1-TG-24M group were all smaller than those in the WT-24M group (all P<0.05). Furthermore, the degree of DNA damage in cardiac tissue as well as the mRNA levels of fibrosis-related indicators (Coll1, Mmp2, and α-SMA) and cell cycle inhibitory proteins (P53, P21, P16) in mice of the WT-24M+GGTI298 group were higher than those in the WT-24M group and the CHK1-TG-24M+GGTI298 group (all P<0.05). Conclusion:CHK1 alleviates oxidative stress-induced damage in mouse cardiomyocytes by activating the Rap1/Nox4 signaling pathway, thereby delaying cardiac aging in mice.

Objective:To explore the distribution characteristics of HBsAg levels in treatment-na?ve and treatment-experienced patients with chronic hepatitis B (CHB) in China.Methods:Data were obtained from the China Registry of Hepatitis B (CR-HepB) platform from the establishment of the platform to April 11, 2024. Patients with CHB who were treatment-na?ve and treatment-experienced with nucleos(t)ide analogs (NAs) were included. Relevant clinical data were collected. The distribution of hepatitis B surface antigen (HBsAg) status, as well as the levels in populations of different age groups after different antiviral treatment durations, were retrospectively analyzed. Normally and non-normally distributed measured data were represented by Mean± SD, and M( Q1, Q3). Results:A total of 13 505 treatment-na?ve patients and 6 390 treatment-experienced patients were included in the analysis. The proportions of treatment-na?ve patients with HBsAg<100, <500, and <1 500 IU/mL were 10.51%, 28.47%, and 46.85%, and the corresponding proportions of treatment-experienced patients were 12.88%, 29.84%, and 52.07%. The proportions of treatment-na?ve patients with HBsAg levels≥1 500, ≥3 000, and≥8 000 IU/mL were 53.15%, 38.17%, and 15.62%, and the corresponding proportions of treatment-experienced patients were 47.93%, 31.77%, and 10.39%. HBsAg level showed a trend of gradual decrease with the increase of antiviral treatment time. The proportion of treatment-experienced patients with HBsAg<100 IU/mL increased from 12.73% when the treatment duration was less than three years to 26.92% when the treatment duration was≥10 years, while the proportion of patients with HBsAg levels≥3 000 IU/mL or≥8 000 IU/mL decreased from 34.66% to 23.08% and from 12.19% to 5.77%, respectively. The proportion of patients with HBsAg<100, <500, and<1 500 IU/mL increased with age, while the proportion of patients with HBsAg≥1 500, ≥3 000, and ≥8 000 IU/mL decreased sequentially.Conclusions:The CR-HepB platform provides a basis for clarifying the serum HBsAg levels in treatment-na?ve and treatment-experienced CHB patients in China. The HBsAg status indicates that with a prolonged antiviral treatment duration, there is a gradual decline trend in HBsAg level.

Objective:To explore the distribution characteristics of HBsAg levels in treatment-na?ve and treatment-experienced patients with chronic hepatitis B (CHB) in China.Methods:Data were obtained from the China Registry of Hepatitis B (CR-HepB) platform from the establishment of the platform to April 11, 2024. Patients with CHB who were treatment-na?ve and treatment-experienced with nucleos(t)ide analogs (NAs) were included. Relevant clinical data were collected. The distribution of hepatitis B surface antigen (HBsAg) status, as well as the levels in populations of different age groups after different antiviral treatment durations, were retrospectively analyzed. Normally and non-normally distributed measured data were represented by Mean± SD, and M( Q1, Q3). Results:A total of 13 505 treatment-na?ve patients and 6 390 treatment-experienced patients were included in the analysis. The proportions of treatment-na?ve patients with HBsAg<100, <500, and <1 500 IU/mL were 10.51%, 28.47%, and 46.85%, and the corresponding proportions of treatment-experienced patients were 12.88%, 29.84%, and 52.07%. The proportions of treatment-na?ve patients with HBsAg levels≥1 500, ≥3 000, and≥8 000 IU/mL were 53.15%, 38.17%, and 15.62%, and the corresponding proportions of treatment-experienced patients were 47.93%, 31.77%, and 10.39%. HBsAg level showed a trend of gradual decrease with the increase of antiviral treatment time. The proportion of treatment-experienced patients with HBsAg<100 IU/mL increased from 12.73% when the treatment duration was less than three years to 26.92% when the treatment duration was≥10 years, while the proportion of patients with HBsAg levels≥3 000 IU/mL or≥8 000 IU/mL decreased from 34.66% to 23.08% and from 12.19% to 5.77%, respectively. The proportion of patients with HBsAg<100, <500, and<1 500 IU/mL increased with age, while the proportion of patients with HBsAg≥1 500, ≥3 000, and ≥8 000 IU/mL decreased sequentially.Conclusions:The CR-HepB platform provides a basis for clarifying the serum HBsAg levels in treatment-na?ve and treatment-experienced CHB patients in China. The HBsAg status indicates that with a prolonged antiviral treatment duration, there is a gradual decline trend in HBsAg level.

Objective:To investigate the effect of DEAD-box helicase (DDX) 21 on myocardial ischemia-reperfusion (I/R) injury and its potential mechanisms.Methods:In vivo, adult male Bama pigs and C57BL/6J mice were used to establish a myocardial I/R injury model by ligating the left anterior descending coronary artery, with sham-operated groups set as controls. The expression of DDX21 in myocardium after I/R injury was assessed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. Following the establishment of the myocardial I/R injury model in mice, AAV9 vectors with cardiac-specific expression were injected in situ into the peri-infarct region (The I/R+DDX21 group, I/R+negative control (NC) group, I/R+sh-NC group and I/R+sh-DDX21 group were injected with AAV9:cTnT-DDX21, AAV9:cTnT-NC, AAV9:cTnT-sh-NC and AAV9:cTnT-sh-DDX21, respectively). Additionally, the I/R+A-485 group received intraperitoneal injections of the cAMP response element-binding protein (CREB) binding protein inhibitor A-485, while the I/R+PBS group was injected with an equivalent volume of phosphate-buffered saline (PBS) as the control. Echocardiography was performed on postoperative days 1 and 28 to evaluate cardiac function (left ventricular ejection fraction and fractional shortening). At 28 days post-surgery, mice were euthanized and heart tissues were harvested for histological sectioning. Myocardial fibrosis was evaluated using Masson′s trichrome staining. In vitro, primary cardiomyocytes were isolated from neonatal day 1 C57BL/6J mice using enzymatic digestion method. Cardiomyocytes were transfected with plasmids or small interfering RNA (siRNA). The cardiomyocytes transfected with DDX21-siRNA were assigned to the siDDX21 group, those transfected with the DDX21 plasmid were assigned to the DDX21 group, and those transfected with the corresponding empty plasmid or siRNA were assigned to the NC group. Additionally, cardiomyocytes were treated with A-485 (A-485 group) or PBS (PBS group). An oxygen-glucose deprivation/reoxygenation (OGD/R) model was used to simulate cellular injury. Transcriptome sequencing was performed to identify downstream mechanisms of DDX21. Differential gene expression analysis was conducted using software such as DESeq2, and alternative splicing events in the mRNA transcriptome were analyzed using rMATS software. Mitochondrial superoxide, mitochondrial membrane potential, ATP content, and mitochondrial respiratory chain complex enzyme activity in cardiomyocytes were detected using immunofluorescence staining and commercial assay kits. The oxidative phosphorylation level of the cells was assessed by the Seahorse extracellular flux analyzer. Acetylated DDX21 levels were measured using co-immunoprecipitation and Western blot assays.Results:The expression levels of DDX21 in myocardium from the Bama pigs and mice in the I/R injury model were significantly higher than those in the sham group (all P<0.001). Echocardiographic results showed that at 28 days post-surgery, compared to the I/R+NC group, the I/R+DDX21 group exhibited higher left ventricular ejection fraction and fractional shortening, while the I/R+sh-DDX21 group showed lower values; Masson staining results demonstrated that, compared to the I/R+NC group, the myocardial fibrosis area in the I/R+DDX21 group was significantly reduced, whereas it was significantly increased in the I/R+sh-DDX21 group (all P<0.001). Transcriptomic sequencing results suggested that DDX21 may influence myocardial injury by regulating mitochondrial metabolic activity. In vitro, compared to the OGD/R+NC group, the OGD/R+DDX21 group exhibited lower mitochondrial superoxide levels, higher polymer/monomer ratio, maximal oxygen consumption, reserve capacity, and ATP content. In contrast, the OGD/R+siDDX21 group showed the opposite results, with reduced activity of mitochondrial respiratory chain complex V (all P<0.05). Mechanistically, rMATS software and other analyses indicated that knockdown of DDX21 affected the alternative 3′ splicing sites of ATP5J precursor mRNA, inhibiting the splicing of certain exonic sequences. Overexpression of DDX21 upregulated both mRNA and protein levels of ATP5J. Co-immunoprecipitation experiments showed that, compared to the PBS group, acetylated DDX21 levels were reduced in the A-485 group. Further in vivo experiments showed that, compared to the I/R+PBS group, the I/R+A-485 group exhibited higher left ventricular ejection fraction and fractional shortening, and a lower proportion of left ventricular fibrosis (all P<0.001). Conclusions:DDX21 improves cardiomyocyte energy metabolism and alleviates I/R injury by regulating the alternative splicing of ATP5J. A-485 holds potential as a novel small molecule candidate for the treatment of myocardial injury.

Objective:To investigate the biological role and molecular mechanism of checkpoint kinase 1 (CHK1) in delaying cardiac aging in mice.Methods:In vitro, a senescence model of H9C2 cells (a cardiomyocyte line) was induced using H 2O 2. A control group (without H 2O 2 treatment) and three H 2O 2-treated groups (at concentrations of 10, 30, and 50 μmol/L) were set up. The CCK-8 assay was used to evaluate the proliferative activity of cells in each group; Western blot analysis was employed to detect the expression level of CHK1; and quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to determine the messenger RNA (mRNA) expression levels of P16 and interleukin-1β (IL-1β). In vivo, C57BL/6 wild-type mice aged 2 months ( n=15) and 24 months ( n=40), as well as myocardial-specific CHK1-overexpressing (CHK1-TG) mice aged 2 months ( n=15) and 24 months ( n=40), were selected. The mice were divided into four groups based on age and genotype: 2-month-old wild-type (WT-2M), 24-month-old wild-type (WT-24M), 2-month-old CHK1-TG (CHK1-TG-2M), and 24-month-old CHK1-TG (CHK1-TG-24M). Echocardiography was used to evaluate cardiac function of mice in the WT-24M and CHK1-TG-24M groups. Western blot analysis was conducted to measure the protein expression levels of CHK1, total Ras-related protein 1 (Rap1), NADPH oxidase 4 (Nox4), and Rap1-guanosine triphosphate (Rap1-GTP, the active form of Rap1) in the cardiac tissue of mice in each group. qRT-PCR was used to detect the messenger RNA (mRNA) expression levels of CHK1, collagen type Ⅰ (Coll1), matrix metalloproteinase-2 (Mmp2), alpha-smooth muscle actin (α-SMA), P53, P21, P16, thioredoxin 1 (Trx1), thioredoxin reductase (TrxR), glutathione recluctase (GR), Rap1, and Nox4. Immunofluorescence staining was employed to determine the protein expression levels of P53, P21, and P16, as well as the proportion of histone H2AX phosphorylation-positive cells. Dihydroethidium (DHE) staining was used to detect the relative intensity of DHE. Wheat germ agglutinin staining, HE staining, Masson staining and Sirius red staining were applied to measure the cross-sectional area of cardiomyocytes, cardiac morphology, and myocardial fibrosis area. Mice in the WT-24M and CHK1-TG-24M groups were intraperitoneally injected with the Rap1 activity inhibitor GGTI298 (25 μmol/kg). After injection, the oxidative stress damage in the cardiac tissue of the mice was detected, along with the mRNA expression levels of fibrosis-related indicators (Coll1, Mmp2, and α-SMA) and cell cycle inhibitory proteins (P16, P21, and P53). Results:A concentration of 30 μmol/L was determined as the optimal concentration for establishing an H 2O 2-induced senescence model of myocardial cells in vitro. The expression level of CHK1 in H9C2 cells of the 30 μmol/L H 2O 2 group was lower than that in the control group ( P<0.05). Echocardiographic examination showed that the left ventricular ejection fraction ((61.08±1.13)% vs. (52.55±2.02)%) and fractional shortening ((31.80±1.27)% vs. (25.18±1.59)%) of mice in the CHK1-TG-24M group were higher than those in the WT-24M group (both P<0.05). qRT-PCR and Western blot analysis revealed that, compared with the WT-24M group, mice in CHK1-TG-24M group had higher expression levels of CHK1 and its mRNA, lower expression levels of Nox4 and its mRNA, and higher expression level of Rap1-guanosine triphosphate (Rap1-GTP) (all P<0.05). However, there were no statistically significant differences in the total expression level of Rap1 and its mRNA between the two groups (both P>0.05). In addition, the mRNA expression levels of Coll1, Mmp2, and α-SMA in myocardial tissue of mice in the CHK1-TG-24M group were lower than those in the WT-24M group (all P<0.05). Immunofluorescence staining results showed that the expression levels of P53, P21, and P16 proteins, as well as the proportion of phosphorylated histone H2AX-positive cells in myocardial tissue of mice in the WT-24M group were higher than those in the CHK1-TG-24M group (all P<0.05). qRT-PCR further confirmed that the mRNA expression levels of the above-mentioned proteins in cardiac tissue of mice in the WT-24M group were higher than those in the CHK1-TG-24M group (all P<0.05). DHE staining results indicated that the relative intensity of DHE in cardiac tissue of mice in the CHK1-TG-24M group was lower than that in the WT-24M group ( P<0.05). Meanwhile, the left ventricular internal diameter, cross-sectional area of cardiomyocytes, and myocardial fibrosis area of mice in the CHK1-TG-24M group were all smaller than those in the WT-24M group (all P<0.05). Furthermore, the degree of DNA damage in cardiac tissue as well as the mRNA levels of fibrosis-related indicators (Coll1, Mmp2, and α-SMA) and cell cycle inhibitory proteins (P53, P21, P16) in mice of the WT-24M+GGTI298 group were higher than those in the WT-24M group and the CHK1-TG-24M+GGTI298 group (all P<0.05). Conclusion:CHK1 alleviates oxidative stress-induced damage in mouse cardiomyocytes by activating the Rap1/Nox4 signaling pathway, thereby delaying cardiac aging in mice.

Objective To study the effect of DNA methyltransferase 1(DNMT1)on sm-like protein-4(LSM4)in hepatocyte apoptosis in mice induced with Hcy.Methods 12 ApoE-/-mice were divided into two groups:normal diet(ND,n=6)and high methionine diet(HMD,n=6)groups.Normal hepatocytes of NCTC1469 were divided into a normal group(control,0 μL/L Hcy),Hcy intervention group(Hcy,100 μL/L Hcy),NC siRNA-transfected control group(si-NC group,0 μmol/L Hcy),LSM4 siRNA-transfected group(si-LSM4 group,0 μmol/L Hcy),DNMT1 siRNA-transfected group(si-DNMT1 group,0 μmol/L Hcy),NC siRNA-transfected Hcy intervention group(Hcy+si-NC group,100 μmol/L Hcy),LSM4 siRNA-transfected Hcy intervention group(Hcy+si-LSM4 group,100 μmol/L Hcy),and DNMT1 siRNA-transfected Hcy intervention group(Hcy+si-DNMT1 group,100 μmol/L Hcy).Analysis of the expression of LSM4 in various tissues was conducted using the NCBI database.Quantitative real-time PCR(qRT-PCR)and Western blot were used to detect differences in LSM4 protein expression in mouse tissues(HMD and ND)and hepatocytes(control and Hcy).Western blot was used to detect the expression of Bcl2-associated X(Bax)and B-cell lymphoma-2(Bcl-2).The cell apoptosis rate in the Control,Hcy,Hcy+si-NC,and Hcy+si-LSM4 groups were detected by flow cytometry.MethPrimer online software was used to analyze the CpG islands of LSM4 promoter region.The expression of LSM4 in the Hcy+si-DNMT1 group was detected by qRT-PCR and Western blot.Results The expression of LSM4 in HMD,Hcy group was higher than that in the ND and Control group(P＜0.05).Bax protein expression was significantly higher,but Bcl-2 was significantly lower in Hcy group compared with those of the Control group(P＜0.05).The expression of Bax protein was significantly lower,but the level of Bcl-2 was significantly higher in the Hcy+si-LSM4 group compared with those in the Hcy+si-NC group(P＜0.05).The cell apoptosis rate in the Hcy group was higher than that in the Control group(P＜0.05),while the apoptosis rate in the Hcy+si-LSM4 group was lower than that in the Hcy+si-NC group(P＜0.05).MethPrimer database analysis showed that the promoter region of LSM4 was GC-rich,and there was one CpG island.Compared with the Hcy+si-NC group,the Hcy+si-DNMT1 group's expression of LSM4 protein was increased(P＜0.05).Conclusions DNMT1 regulates LSM4 hypomethylation to increase its expression,thereby promoting Hcy-induced apoptosis of mouse hepatocytes.

Objective To study the effect of DNA methyltransferase 1(DNMT1)on sm-like protein-4(LSM4)in hepatocyte apoptosis in mice induced with Hcy.Methods 12 ApoE-/-mice were divided into two groups:normal diet(ND,n=6)and high methionine diet(HMD,n=6)groups.Normal hepatocytes of NCTC1469 were divided into a normal group(control,0 μL/L Hcy),Hcy intervention group(Hcy,100 μL/L Hcy),NC siRNA-transfected control group(si-NC group,0 μmol/L Hcy),LSM4 siRNA-transfected group(si-LSM4 group,0 μmol/L Hcy),DNMT1 siRNA-transfected group(si-DNMT1 group,0 μmol/L Hcy),NC siRNA-transfected Hcy intervention group(Hcy+si-NC group,100 μmol/L Hcy),LSM4 siRNA-transfected Hcy intervention group(Hcy+si-LSM4 group,100 μmol/L Hcy),and DNMT1 siRNA-transfected Hcy intervention group(Hcy+si-DNMT1 group,100 μmol/L Hcy).Analysis of the expression of LSM4 in various tissues was conducted using the NCBI database.Quantitative real-time PCR(qRT-PCR)and Western blot were used to detect differences in LSM4 protein expression in mouse tissues(HMD and ND)and hepatocytes(control and Hcy).Western blot was used to detect the expression of Bcl2-associated X(Bax)and B-cell lymphoma-2(Bcl-2).The cell apoptosis rate in the Control,Hcy,Hcy+si-NC,and Hcy+si-LSM4 groups were detected by flow cytometry.MethPrimer online software was used to analyze the CpG islands of LSM4 promoter region.The expression of LSM4 in the Hcy+si-DNMT1 group was detected by qRT-PCR and Western blot.Results The expression of LSM4 in HMD,Hcy group was higher than that in the ND and Control group(P＜0.05).Bax protein expression was significantly higher,but Bcl-2 was significantly lower in Hcy group compared with those of the Control group(P＜0.05).The expression of Bax protein was significantly lower,but the level of Bcl-2 was significantly higher in the Hcy+si-LSM4 group compared with those in the Hcy+si-NC group(P＜0.05).The cell apoptosis rate in the Hcy group was higher than that in the Control group(P＜0.05),while the apoptosis rate in the Hcy+si-LSM4 group was lower than that in the Hcy+si-NC group(P＜0.05).MethPrimer database analysis showed that the promoter region of LSM4 was GC-rich,and there was one CpG island.Compared with the Hcy+si-NC group,the Hcy+si-DNMT1 group's expression of LSM4 protein was increased(P＜0.05).Conclusions DNMT1 regulates LSM4 hypomethylation to increase its expression,thereby promoting Hcy-induced apoptosis of mouse hepatocytes.