Effects of apelin-13 on ferroptosis of the C2C12 skeletal muscle cell line in a high-iron environment / 中华老年医学杂志

Objective:To examine the effects of apelin-13 on ferroptosis of the C2C12 skeletal muscle cell line induced by a high-iron environment and explore potential underlying mechanisms.Methods:C2C12 cells were cultured in Dulbecco's Modified Eagle Medium(DMEM)and divided into a control group, a ferric citrate(FAC)group, an apelin-13 group, an FAC+ apelin-13 group, a ferroptosis inducer RSL3 group and an FAC+ apelin-13+ RSL3 group.Cell viability was detected by the 3-(4, 5-dimethyl thiazole-2)-2, 5-diphenyl thiazolyl blue(MTT)assay.The intracellular concentrations of total iron and divalent iron were measured by colorimetry; the levels of glutathione(GSH), malondialdehyde(MDA)and intracellular reactive oxygen species(ROS)in cells were detected by an enzyme-linked immunosorbent assay, visible spectrophotometry and a chemifluorescence method, respectively.The ultrastructure of C2C12 cells was examined by transmission electron microscopy.The protein expression of glutathione peroxidase 4(GPX-4), ferritin heavy chain 1(FTH-1), heme oxygenase 1(HO-1)and nuclear factor E2-related factor 2(Nrf-2), were detected by Western blotting.Results:Compared with the FAC group, the FAC+ Apelin-13 group had significantly elevated cell viability(optical density: 0.52±0.06 vs.0.28±0.04, t=7.837, P=0.007)and higher concentrations of GSH(2.41±0.35 vs.0.91±0.12 μmol/g Pro, t=9.778, P=0.003), but significantly decreased levels of ROS(22.06±5.79 vs.52.71±7.28 a. u./mg Pro, t=8.064, P=0.006), MDA(4.63±0.51 vs.9.11±0.84 mmol/mg Pro, t=8.642, P=0.006), total iron(1.53±0.24 vs.3.17±0.55 μmol/g Pro, t=6.135, P=0.013)and divalent iron(0.75±0.08 vs.1.94±0.36 μmol/g Pro, t=5.068, P=0.027), as well as reduced intracellular iron deposition.In the control group and the apelin-13 group, the morphology of the mitochondria was clear and normal.In contrast, the mitochondria in the FAC group had increased membrane density, membrane shrinkage and rupture, vacuolar degeneration, and obvious mitochondrial damage, which were consistent with the morphological characteristics of ferroptosis.Compared with the FAC group, the FAC+ apelin-13 group showed significant improvement in mitochondrial damage.Moreover, compared with the FAC+ apelin-13 group, the cell viability of the FAC+ apelin-13+ RSL3 group was significantly decreased(optical density: 0.23±0.04 vs.0.48±0.06, t=7.642, P=0.007). Compared with the FAC group, the FAC+ apelin-13 group had significantly up-regulated cellular expression of GPX-4(relative expression: 0.96±0.14 vs.0.31±0.07, t=7.712, P=0.008), FTH-1(0.57±0.08 vs.0.27±0.05, t=6.944, P=0.011), and HO-1(0.49±0.07 vs.0.28±0.05, t=6.472, P=0.012), as well as increased nuclear expression of Nrf-2(relative expression: 0.42±0.04 vs.0.19±0.05, t=7.114, P=0.008)with a higher ratio of nuclear expression over total cellular expression[(58.36±5.24)% vs.(36.58±5.32)%, t=5.858, P=0.015]and a higher level of HO-1 protein expression(relative expression: 0.49±0.07 vs.0.28±0.05, t=6.472, P=0.012). Conclusions:Apelin-13 inhibits ferroptosis induced by a high iron environment in C2C12 cells, and the underlying molecular mechanisms may be related to the Nrf-2/HO-1 signaling pathway.

The effect and mechanism of apelin-13 on diabetes-associated sarcopenia in rats / 中华老年医学杂志

Objective:To observe the effect of Apelin-13 on diabetes-related sarcopenia in rats model and explore the underlying mechanism.Methods:This experiments were divided into 3 groups.In the normal control group, Wistar rats were fed by ordinary feed, intraperitoneally injected daily with the equal amount of normal saline.In the model+ control group, diabetic-related sarcopenia model was established in rats with fat-fed Goto-Kakizaki rats for 12 weeks and intraperitoneally injected daily with the equal amount of normal saline.In the model+ apelin-13 group, apelin-13(0.1 mol/kg)was intraperitoneally injected into diabetic-related sarcopenia model daily for 12 weeks.The water intake, food intake, and body weight were measured.The fasting blood glucose, fasting insulin and blood lipid levels of rats were measured, and the insulin resistance index was calculated.The wet weight of the rats' gastrocnemius muscle was weighed, and the morphological changes of the gastrocnemius muscle were observed.Western blot was used to detect the protein expressions of phosphatidylinositol-3 kinase(PI3K)and phosphorylated Akt(p-Akt)in the gastrocnemius muscle of rats.Results:Compared with the model+ normal saline group, the model+ apelin-13 group showed that the following parameters were significantly improved.(1)The water intake of rats was significantly decreased at 8, and 12 weeks( F=7.17 and 7.91), and food intake was significantly decreased( F=5.84 and 6.12)and body weight were significantly increased at 8 and 12 weeks( F=5.76 and 6.07)(all P<0.05). (2)The levels of fasting blood glucose were significantly decreased at 8 and 12 weeks( F=8.07 and 8.24, all P<0.05). (3)Serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, the levels fasting insulin and the insulin resistance index were significantly decreased at week 12( F=5.17, 7.94, 10.27, 8.32, 6.94 and 11.31, all P<0.05); (4)The wet weight of gastrocnemius muscle was significantly increased(0.63±0.04 g and 1.02±0.05 g, t=4.32, P<0.05). (5)Compared with the model+ normal saline group, the model+ apelin-13 group showed the protein expressions of PI3K and p-Akt in the gastrocnemius muscle of rats were significantly up-regulated( t=7.32, 8.07, both P<0.05). Conclusions:Apelin-13 has an inhibitory effect on diabetes-related sarcopenia, and its mechanism may be related to the up-regulation of PI3K and p-Akt expression in skeletal muscle.

Establishment and evaluation of a novel mouse model of orthotopic colon cancer in the mesenteric triangle of the cecum / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the feasibility of preparation of a mouse model of orthotopic colon cancer by injecting tumor cell suspension into mesenteric triangle of the cecum.</p><p><b>METHODS</b>Twenty SPF 8-week old BALB/c mice (male:female = 1:1) were used in this study. The mouse caecum was exposed by laparostomy, and suspension of mouse colon adenocarcinoma CT26. WT cells was injected into the mesenteric triangle of cecum for preparation of a mouse model of orthotopic colon cancer.</p><p><b>RESULTS</b>Mouse orthotopic colon cancer was developed by injection of tumor cell suspension into mesenteric triangle of the cecum showing a successful rate of 100%, without intestinal obstruction, and the liver, spleen, diaphragm and mesenteric lymph nodes metastasis rates were high in all the 20 experimental mice.</p><p><b>CONCLUSIONS</b>The establishment of mouse models of orthotopic colon cancer by injection of tumor cell suspension into the mesenteric triangle is a simple, rapid, and easy to master procedure, causing less damage to the colon wall, safe and with less trauma to the mice. This method may provide an ideal mouse model of orthotopic colon cancer for the study of pathogenesis as well as liver metastasis mechanisms of colon cancer.</p>

Preliminary study of the inhibitory effect and mechanism of B16F10-ESAT-6-gpi/IL-21 vaccine on the pulmonary metastasis in mouse models of melanoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effect and mechanism of B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine on pulmonary metastasis in mouse model of melanoma.</p><p><b>METHODS</b>Twelve 8-week old female C57BL/6 mice were used in this study. The mice were injected with wild-type B16F10 cells through tail vein after immunization with B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine, and the pulmonary metastasis was observed. The CD4(+) and CD8(+) T cells were isolated by magnetic activated cell sorting, and then used for the detection of CFSE/7-AAD cytotoxicity by flow cytometry. Serum from the mice immunized with tumor-cell vaccine was used to detect IFN-γ expression by ELISA. The expression of TGF-β2, ZEB1, E-cadherin, and N-cadherin of tumor tissues was detected by RT-PCR and immunofluorescence, respectively.</p><p><b>RESULTS</b>The mice vaccinated with B16F10-ESAT-6-gpi/IL-21 had significantly fewer nodules in the lung and lower lung weight [(285.8 ± 19.01) mg vs. (406.3 ± 27.12) mg], with lower levels of TGF-β2, ZEB1 and N-cadherin proteins but higher level of E-cadherin protein within the tumor tissue, as compared with the control mice. Meanwhile, the immunized mice had significantly increased CD8(+) T cell killing activity [(42.62 ± 3.465)% vs. (22.29 ± 1.804)%] and IFN-γ expression level [(55.200 ± 7.173) pg/ml vs. (6.435 ± 1.339) pg/ml] over the control mice.</p><p><b>CONCLUSIONS</b>The B16F10-ESAT-6-gpi/IL-21 vaccine can inhibit the metastasis of melanoma in the lung in vaccinated melanoma-bearing mice. This inhibitory effect is associated with CD8(+) T cell immune response and a higher level of IFN-γ, which may influence on the mesenchymal-epithelial transition of tumor cells.</p>

Clinical analysis of internal fixation with grip-type of Ni-Ti alloy plate in treatment of multiple ribs fractures / 中国基层医药

ObjectiveTo analyze the clinical effect of internal fixation with grip-type of Ni-Ti alloy plate in treatment of multiple ribs fractures.MethodsClinical data of 50 patients with multiple ribs fractures treated by internal fixation t with grip-type of Ni-Ti alloy plate were retrospectively analyzed.ResultsThe chest pain alleviated,the shape of bony thorax recovered, paradoxical respiration vanished, and dyspnea relieved significantly in all cases after operation.Two months after operation, chest X-ray indicated healed fractures, internal fixation without loosening and breaking off, no chest deformity in all patients.Followed up 2 to 14 months,an average of 9 months,and no complication was found.ConclusionThe internal fixation with grip-type of Ni-Ti alloy plate in treatment of multiple ribs fractures was simple ,convenient,less invasive ,good tissue compatibility, stable fixation, less complications, and clinical results were satisfactory.

Study on anti ovarian cancer efficacy elicited by CD34~+ umbilical cord blood hematopoietic stem cells transfected with IL-21 in nude mouse model / 中华微生物学和免疫学杂志

Objective To investigate the CD34~+ umbilical cord blood hematopoietic stem cells (CD34~+ UBSC) transfected with interleukin 21 (IL-21) against the ovarian cancer effect in tumor-bearing nude mice. Methods CD34~+ UBSC were obtained from the UBSC by a magnetically activated cell sorting technique and then transfected with recombinant plasmid plRES2-IL-21-EGFP after the CD34~+ UBSC were proliferated in vitro. The therapeutic effect was evaluated by the size of the tumor and the life span in nude mice treated with the CD34~+ UBSC-IL-21. The expression of IL-21 and its bioactivity in CD34~+ UBSC-IL-21 and in local neoplasitc tissues were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR), immune fluorescence technique, ELISA, Western blot, immunohistochemistry and splenocyte proliferative activity. The NK cell cytotoxicity and the numbers of NK cells, serum level of IFN-γ and TNF-αwere simultaneouly detected by FCM and ELISA, respectively. Results CD34~+ UBSC were cultured and transfected with pIRES2-IL-21-EGFP successfully. CD34~+ UBSC-IL-21 could inhibit the tumor growth and extended nude mice life span compared with other groups (P < 0.01). The expression of IL-21 in the neo-plastic tissue, serum level of IFN-γ and TNF-α , NK cell activity and the numbers of NK cells of mice origin and of human origin in splenocytes were increased significantly in the nude mice treated with CD34~+ UBSC-IL-21 compared with other groups(P <0.01). Conclusion The CD34~+ UBSC transfected with IL-21 have competent against ovarian cancer in tumor-bearing nude mice. The findings may establish a foundation for gene therapy of the ovarian cancer by CD34~+ UBSC-IL-21 in clinic application.

Anti-tumor mechanisms of lymphopenic mice transferred with tumor-specific lymphocytes and immunized with mIL-21 tumor vaccine / 中华微生物学和免疫学杂志

Objective To explore the mechanism of anti-tumor effects of transferring tumor-specif-ic lymphocytes obtained from pre-immunized BALB/c mice with inactive rolL-21 tumor vaccine (mIL-21-Sp2/0)to syngeneic mice, associated with mIL-21 tumor vaccine immunization, in the condition of cyclo-phosphamide (Cy)-induced lymphopenia. Methods Activated lymphocytes of spleen and lymph nodes ob-tained from pre-immunlzed syngeneic mice with irradiated mIL-21-Sp2/0 cells were infused into BALB/cmice treated with Cy 2 days before, subsequently vaccinated with mlL-21 tumor vaccine, after 7 days, chal-lenged with Sp2/0 tumor cells, observed the growth of tumor of mice. T lymphocyte subsets differentiation was measured by flow cytometry (FCM) analysis. The proliferation and cytotoxie activities of activated lym-phocytes were analyzed by FCM, respectively, staining with CFSE and 7-AAD. The number of IFN-γ-secre-ting cells was evaluated by ELISPOT. Results The lymphopenic mice were transferred with activated lym-phocytes and inoculated with raiL-21 tumor vaccine might provide superior anti-tumor immunoprotection, re-tard tumor growth of the mice. The proliferating capabilities and killing rate of transferred tumor Ag-specific lymphocytes enhanced obviously, the number of IFN-γ-secreting cells was significantly higher compared with the control groups. Conclusion Under Cy-induced lymphopenia condition, tumor Ag-specific lymphocytes sensitized by raiL-21 tumor vaccine were transferred to mice and immunized with mlL-21 tumor vaccine at the same time, benefit the proliferation of transferred effective cells and immune cells itself, assist to form and sustain special anti-tumor effects.