Aim To explore the effect of pemetrexed on autophagy and the function of autohpagy activation in A549 cells.Methods The anti-proliferative effect of pemetrexed on A549 cells was detected by MTS. The formation of autophagosome was observed by trans-mission electron microscopy (TEM)and the expression of autophagy-related proteins was determined by West-ern blot.The inhibitory effects of the combination of pemetrexed and a selective autophagy inhibitor on A549 cells were measured by MTS.Results Peme-trexed inhibited the proliferation of A549 cells.Auto-phagy was activated in A549 cells by exposure to pemtrexed.Autophagosome was increased in peme-trexed treatment cells compared with control group. Pemtrexed treatment led to the formation of autophago-some and the conversion of LC3-Ⅰto LC3-Ⅱ.The in-crease of Beclin-1 and the decline of autophagy sub-strate of p62 were observed in pemtrexed treated A549 cells.The pharmacological autophagy inhibitor chloro-quine (CQ)sensitized A549 cells to pemetrexed treat-ment.The inhibitory rate was (28.42 ±2.45 )% for pemetrexed only,and (45 .36 ±3.52 )% for peme-trexed combined with CQ.Conclusions Pemetrexed could activate autophagy in A549 cell lines.The dis-ruption of autophagy facilitates the anti-proliferative effect of pemetrexed on A549 cells,which provides a novel strategy in lung cancer treatment.

Objective To investigate the protective effects of imipramine on alveolar epithelial barrier functiou in mice against LPS-induced acute lung injury (ALI),and explore the possible mechanisms.Methods Total of 32 SPF male Balb/c mice were randomly (random number) divided into four groups:control group,Imipramine group,LPS group,LPS + Imipramine group.To establish an animal model of ALI,mice were administered intraperitoneally with LPS in 20 mg/kg.Mice were treated with imipramine in 25 mg/kg 30 min prior to LPS administration.FITC-FD4 was administered in mice via the tail vein with FITC-FD4 10 min before mice sacrificed under anesthesia at 12 hours after LPS administration,then bronchoalveolar lavage fluid (BALF) and lung tissue were obtained.HE staining was used to observe histopathological changes,and pathology scores;lung tissue wet-to-dry weight ratio and BALF/serum FD4 ratio were used to assess pulmonary edema and alveolar epithelial permeability.Real-time PCR,western blot and immunochemistry were employed to detect the mRNA expressions and protein levels of Occludin,Claudin-4 and ZO-1.Data were analyzed with SPSS 16.0 software,one way analysis of variance (ANOVA) was used to compare multiple sets of variables,the intergroup comparisons were analyzed by the least-significant-difference (LSD) tests with P ＜ 0.05 for the statistically significant difference.Results Compared with LPS group,LPS + lmipramine group had a statistically significant decrease in pathological score [(9.22 ± 0.21) vs.(11.23 ± O.55),P ＜ O.05);the wet-to-dry weight ratio in LPS + Imipramine group was less than that in LPS group and the difference was statistically significant (P ＜ 0.05);compared with LPS group,the ratio of BALF/serum FD4 in LPS + Imipramine was less and the difference was statistically significant (P ＜ 0.05);compared with LPS group,the mRNA expressions and protein levels of Occludin,Claudin-4 and ZO-1 in LPS + Imipramine group were significantly increased (mRNA:Occludin:P ＜ 0.05;Claudin-4:P ＜ 0.05;ZO-1:P ＜ 0.05 . western blot:Occludin:P ＜ 0.05;Claudin-4:P ＜ 0.05;ZO-1:P ＜ 0.05).Immunochemistry showed that Occludin and Claudin-4 were present mainly in alveolar epithelial cell membrane,Z0-1 was found mainly in cytoplasm of alveolar epithelial cell.In control group and Imipramine group,tight junction proteins were obviously expressed.Compared with control group,protein levels in LPS group were significantly decreased (Occludin:P ＜ 0.05;Claudin-4:P ＜ 0.05;ZO-1:t =6.59,P ＜ 0.05);compared with LPS group,the tight junction proteins in LPS + Imipramine group were significantly increased (Occludin:P ＜ 0.05;Claudin-4:P ＜ 0.05;ZO-1:P ＜ 0.05).Conclusion The protective effects of imipramine on alveolar epithelial barrier function by up-regulating tight junction proteins expression in murine LPS-induced ALI.

Objective:To evaluate the feasibility of using cortical bone trajectory (CBT) screws in the osteoporotic thoracolumbar fixation by comparing the bone CT values at the bone-screw interface between traditional trajectory (TT) screws and CBT screws in patients with different bone densities.Methods:The high-resolution CT imaging data of thoracolumbar segments following thoracic or lumbar spine fractures from April 2020 to October 2022 were collected at The Second Hospital Affiliated to Wenzhou Medical University for retrospective analysis. They were divided into 3 groups: a normal bone mass group, an osteopenia group and an osteoporosis group. From each group 30 cases were chosen (90 cases in total, 36 males and 54 females). All the data were imported into Mimics 18.0 for three-dimensional bone reconstruction in which placement of TT and CBT screws was simulated on the vertebrae from T10 to L2 (non-fractured vertebrae). Regions of interest (ROI) where each simulated screw intersected the bone were segmented to measure their CT bone values. For each vertebra in each group, the relative difference percentage in average CT value of ROI between TT and CBT screws was calculated. The CT values of ROI were compared in the same group between TT and CBT screws from T10 to L2; the CT values of ROI were compared in the same screws among the 3 groups from T10 to L2; the CT values of ROI were compared between the CBT screws in the osteopenia and osteoporosis groups and the TT screws in the normal bone mass group; the relative difference percentages in average CT value of ROI between CBT and TT screws were compared between the 3 groups from T10 to L2.Results:The average CT value of ROI for CBT screws was significantly higher than that for TT screws from T10 to L2 in every group ( P< 0.001); as for the CT values of ROI for CBT and TT screws from T10 to L2, the osteoporosis group0.05). At T10, T12, and L1, the relative difference percentage in average CT value of ROI between CBT and TT screws was significantly higher in the osteopenia and osteoporosis groups than that in the normal bone mass group ( P<0.05), but there was no such a difference between the osteopenia and the osteoporosis groups ( P>0.05). At T11 and L2, there was no significant difference between the 3 groups in the relative difference percentage in average CT value of ROI between CBT and TT screws ( P>0.05). Conclusions:As bone mass decreases, both CBT and TT screws lead to a significant decrease in the bone density at the bone-screw interface. In patients with osteoporosis, CBT screws can still lead to a higher bone density at the bone-screw interface than TT screws, thus providing a higher strength at the bone-screw interface.

Objective : To explore the effect of C2 ⁃ceramide , one of the sphingomyelin substances , on apoptosis of non⁃small cell lung cancer cells( A549 and PC9) . Methods : Non⁃small cell lung cancer cell lines ( A549 and PC9) were cultured , total proteins were extracted and Western blot was performed to detect the expression of apoptotic protein Caspase⁃3 and cleaved Caspase⁃3 in the two cells. CCK⁃8 colorimetric method was used to screen drug concentration. Hoechst 33258 apoptosis staining was used to detect apoptosis. The apoptosis rate was detected by flow cytometry , and the expression of apoptotic protein Caspase⁃3 was detected by RT⁃PCR. Results : The cell viability was about 70% when ceramide was treated at 50 μmol/L. Compared with the control group , the expression of apoptotic proteins increased in the ceramide group (P < 0. 05) . Flow cytometry and apoptosis staining showed that the rate of apoptosis increased in the ceramide treatment group compared with the control group (P < 0. 05) . mRNA detection at gene level showed increased the expression of apoptotic protein Caspase⁃3 (P < 0. 05) . Conclusion C2 ⁃ceramide can promote the apoptosis of non⁃small cell lung cancer cells , thus providing a new therapeutic tar⁃ get for clinical lung cancer chemotherapy.