Objective To predict the intervention targets of empagliflozin(EMPA),a specific inhibitor of sodium-glucose cotransporter 2(SGLT2),in gastric adenocarcinoma through comprehensive network pharmacology,and to validate the effects and the molecular mechanisms of EMPA through cellular and molecular biology experiments.Methods Bioinformatics analysis of gastric adenocarcinoma was conducted to assess the correlation between gastric adenocarcinoma prognosis and SGLT2 expression.Network pharmacology was utilized to identify shared targets of EMPA and gastric adenocarcinoma.AGS cells,a human gastric adenocarcinoma cells line,were incubated with EMPA at different concentrations for 24 h and,then,cell proliferation was assessed using the CCK8 assay.After AGS cells were incubated with EMPA at the doses of 0,3,and 6 mmol/L,real-time cell analysis(RTCA)and 5-ethynyl-2-deoxyuridine(EdU)incorporation were used to evaluate EMPA's inhibitory effects on the proliferation of the AGS cells.In addition,wound healing and Transwell assays were performed to assess the inhibitory effect of EMPA on the migration and invasion of the APC cells and Western blot analysis was conducted to examine the expression of mammalian target of rapamycin(mTOR)and phosphorylated mTOR(p-mTOR).BALB/c(nu/nu)nude mice were implanted with 5x106 AGS cells in the axilla.The mice were divided into three groups,a control group,a low-dose group,and a high-dose group,each consisting of 7 mice.After one week,the control group received daily intraperitoneal injections of normal saline,while the low-dose group and high-dose group received daily intraperitoneal injections of EMPA at the doses of 3 mg/kg and 5 mg/kg,respectively.The tumor volume was measured one week after the drug intervention started.Results Gastric adenocarcinoma patients with low expression of SGLT2 exhibited longer survival time and higher survival rate than those with high expression of SGLT2 did.A total of 104 EMPA-related potential targets and 2028 targets associated with gastric adenocarcinoma were identified.Among these,45 targets associated with gastric adenocarcinoma overlapped with potential targets of EMPA.Further analysis revealed 10 relevant pathways and 4 core genes.The core genes were cyclin-dependent kinase 4(CDK4),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),mTOR,and cyclin El(CCNE1).CCK-8 assay revealed that EMPA at concentrations ranging from 0.39 to 50 mmol/L effectively inhibited the proliferation of AGS cells.RTCA results indicated a downward shift in the cell growth curve.In comparison to the findings for the control group,EdU assay demonstrated that EMPA at the concentrations of 3 mmol/L and 6 mmol/L significantly inhibited AGS cell proliferation(P＜0.05).Results from wound healing and Transwell assays indicated a decrease in the levels of cell migration and invasion(P＜0.05)and,notably,there was a significant difference between the high and low-dose EMPA groups(P＜0.05).Western blot showed no statistically significant difference in the expression of total mTOR protein between the groups.However,the expression of p-mTOR in the 3 mmol/L and 6 mmol/L EMPA groups decreased compared to that of the control group(P＜0.05),with the 6 mmol/L EMPA group exhibiting a more pronounced reduction(P＜0.05).Nude mice xenograft tumor experiment demonstrated that,compared to that of the control group,the tumor volumes in the EMPA-treatment groups were significantly reduced(P＜0.05),with the high-dose group showing a more pronounced reduction(P＜0.05).Conclusion EMPA inhibits the abnormal proliferation and migration of gastric adenocarcinoma cells,potentially through the modulation of mTOR protein activation.This study provides new potential medication and intervention targets for gastric adenocarcinoma treatment.

Objective To observe the effects of acupuncture on blood lipids of hyperlipidemia mice, and explore the mechanism of acupuncture against endothelial dysfunction. Methods 40 ApoE (-/- ) mice were randomly divided into control group (C), acupuncture at non-acupoint group (D), acupuncture at acupoint group (E), and simvastatin group (F) with 10 cases in each group. After 8 weeks, total cholesterol (TC), the plasma angiotensin II (Ang II), endothelin-1 (ET-1) and nitric oxide (NO) were measured with enzyme-linked immuno sorbent assay (ELISA). The mice myocardial angiotensin II type 1 receptor (AT1R), and endothelin-1 type A receptor (ETAR) protein were detected with Western blotting. Results The blood lipid, the content of plasma Ang II and ET-1, and the level of AT1R and ETAR in the heart tissue were significantly lower, and the content of NO was significantly higher in groups E and F than in group C (P<0.05). Conclusion Acupuncture can inhibit the level of blood lipids in ApoE(-/-) mice, reduce the levels of Ang II and ET-1, increase the level of NO in peripheral blood, and inhibit the expression of AT1R, ETAR in heart tissue.

Objective The filum terminale(FT)plays an important role in the pathophysiology of tethered cord syndrome(TCS).The study on morphology and structure of fetus FF can provide reference standard for diagnosis of TCS.Methods Ten fresh human aborted fetuses had their fila measured and removed.Transversal and longitudi nal sections of the middle,and distal thirds of FF were submitted to light microscopy analysis with four different techniques.Results The bulk of the Frr is composed of 1～5μm thick spring like longitudinal bundles of colla gen separated by 5～30μm layer intervals and 1～5μm intervals in the layer,although a small quantity of eapillar ies and other elements may be present.Collagen bundles can also be found between layers and bundles.Abundant longitudinally oriented elastic fibers ale found inside or between collagen bundles.Conclusion A complex tridi mensional structure composed by ordered arrangement of spring like fibers and small quantity of capillaries should e licit considerable elastic properties to the FI".Tts alternation of structure and element maybe involved in TCS closely.

Objective To investigate the role of methylated DNA mismatch repair gene MLH1 and MSH2 in the acquired multidrug-resistance of human small cell lung cancer cells H446.Methods The reverse transcription polymerase chain reaction(RT-PCR)and Western blot were applied to measure MLH1 and MSH2 mRNA and protein expressions of the multidrug-resistant cells H446/DDP and its parental cells H446.The promoter methylation status of the genes was assessed by methylation-specific PCR(MSP).Results The expressions of MLH1 and MSH2 significantly decreased both in mRNA level and protein level.Promoter methylation of MLH1 was observed in H446/DDP cells but not in H446 cells.Promoter semi-methylation of MSH2 in H446 cells was transformed to methylation in H446/DDP cells.Conclusion The downregulation of DNA mismatch repair gene MLH1 and MSH2 induced by its promoter methylation may play an important role in the acquired multidrug resistance of human small cell lung cancer.

Objective To establish a multidrug-resistant cell subline NCI-H446/CDDP of human small cell lung cancer and characterize its biological properties. Methods The cell subline NCI-H446/CDDP was derived from human small cell lung cancer cell line NCI-H446 by exposing it to high concentration first followed by being cultured with gradually increasing dose of cisplatin, which used to be the first-line chemotherapeutic drug for lung cancer. The cell growth curve and the doubling time were determined by cell counting. The chemosensitivities of NCI-H446/CDDP and NCI-H446 to cisplatin, hydroxycamptothecine, vincristin, 5-fluorouracil and topotecan were tested and IC 50 measured by MTT. Changes in cellular morphology and ultrastructure were observed under inverted-microscope, scanning electron microscope and transmission electron microscope, respectively. Results Multidrug-resistant cell subline (NCI-H446/CDDP) of human small cell lung cancer was established successfully after culturing NCI-H446 in a high concentration of cisplatin first, followed by subjecting it to increasing concentrations of CDDP until they could stably grow in the culture medium containing 0.5?g/mL CDDP. The rate of cell proliferation of NCI-H446/CDDP was similar to that of NCI-H446, but the number of the former cells exhibiting S phase increased (20.24% vs 18.42% P

Background and purpose:Regulation of MMR activity under hypoxia may play an important role in genetic instability of cancer,but the mechanism is still unclear.We investigated the expression of DNA mismatch repair genes MLH1 and MSH2 in human SCLC cell line H446 under hypoxic condition and explore the role of promoter methylation of genes in hypoxia.Methods:RT-PCR and Western blot were applied to detect MLH1 and MSH2 expression in human SCLC cell line H446 at the mRNA and the protein level,respectively,under either hypoxic condition or after 5-Aza-CdR treatment.Meanwhile,methylation-specific PCR(MSP)was used to determine promoter methylation of MLH1 and MSH2.Results:The expression of MLH1 and MSH2 in H446 cells significantly decreased both at the mRNA and the protein level under hypoxic condition.5-Aza-CdR treatment led to the restoration of MLH1 and MSH2 expression,while,both MLH1 and MSH2 were down-regulated again after removing 5-Aza-CdR.Conclusions:The promoter methylation of MLH1 and MSH2 may play an important role in its defective expression in H446 cells under hypoxic condition.And 5-Aza-CdR could restore MLH1 and MSH2 expression.