ObjectiveTo investigate the effects of forkhead box protein O4 （FOXO4） expression on gastric precancerous lesions （GPL）， and to clarify its mechanism in mediating the therapeutic effect of Celastrus orbiculatus extract （COE） on GPL by regulating aerobic glycolysis. MethodsReferring to the previously established combined modeling protocol in our research group， a rat model of gastric precancerous lesions （GPL） was constructed through the following procedures： rats were given free access to 170 μg/mL N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） solution for ad libitum drinking， fed with a diet supplemented with 0.03% ranitidine hydrochloride， and treated with a cycling regimen of “2-day feeding followed by 1-day fasting”. Specifically， on the afternoon of each fasting day， the rats received intragastric administration of 2% sodium salicylate at a dose of 10 mL/kg body weight. FOXO4-silenced and overexpression model rats were constructed by tail vein injection of plasmids. The rats were randomly divided into control， model， COE， overexpression negative control （OE-NC）， FOXO4 overexpression （OE-FOXO4）， OE-FOXO4+COE， silencing negative control （shNC）， FOXO4 silencing （shFOXO4） and shFOXO4+COE groups. Gastric mucosal histopathological changes were observed in each group. Lactic acid content in gastric mucosal tissues was detected by colorimetry. The expression levels of FOXO4， HK2， PKM2， LDHA and GLUT1 were evaluated by immunohistochemistry （IHC）， and their mRNA levels were determined by RT-PCR.Results Compared with the control group， the COE， OE-FOXO4 and OE-FOXO4+COE groups exhibited significantly improved gastric mucosal lesions， reduced lactic acid levels， weakened expression of aerobic glycolysis-related proteins （PKM2， HK2， LDHA， GLUT1）， and enhanced FOXO4 expression. The OE-FOXO4+COE group showed the lowest lactic acid level and more pronounced changes in related protein expression compared with the COE and OE-FOXO4 groups. In contrast， the shFOXO4 and shFOXO4+COE groups displayed increased lactic acid levels， enhanced expression of aerobic glycolysis-related proteins， and reduced FOXO4 expression compared with the model group.ConclusionFOXO4 expression is involved in the inhibitory effect of COE on GPL， possibly by regulating the aerobic glycolysis process.

OBJECTIVE To prepare the volatile oil cyclodextrin inclusion compound of Wenjing Decoction to improve its stability and characterize its formation;to compare the inclusion behavior of cinnamaldehyde,paeonol and ligustilide in volatile oil of Wenjing Decoction with different cyclodextrins;and to study the effect of different cyclodextrins on the inclusion of multiple components in the volatile oil of Wenjing Decoction.METHODS The inclusion compounds of volatile oil β-CD and its 5 derivatives in Wenjing Decoc-tion were prepared by ultrasonic method,and the inclusion rate of each component was determined by HPLC.TGA and FT-IR were used to characterize the formation of the inclusion complex.The inclusion behavior and influencing factors were studied by phase solu-bility test and molecular docking.RESULTS The inclusion compounds of volatile oil β-CD and its derivatives in Wenjing Decoction were successfully prepared.TGA results showed that the thermal stability of volatile oil in Wenjing Decoction was improved after inclu-sion by cyclodextrin.FT-IR results showed that some components of the volatile oil were bonded to cyclodextrin through non-covalent bonds such as hydrogen bonds.The results of phase solubility test showed that the three main components in the volatile oil of Wenjing Decoction were combined with β-CD in the molar ratio of 1∶n(n≥1),and with β-CD derivatives in the molar ratio of 1∶1.The re-sults of molecular docking showed that the benzene rings of cinnamaldehyde and paeonol,and lactone rings of ligustilide penetrated into the cyclodextrin cavity to form clathrates.The inclusion rates of β-CD and its derivatives for each component in the volatile oil of Wen-jing Decoction were DM-β-CD>HP-β-CD>β-CD>HE-β-CD>SBE-β-CD>CM-β-CD,which were consistent with the af-finity of phase solubility test and molecular docking.The inclusion rates of the three components with β-CD and its derivatives were cinnamaldehyde>paeonol>ligustilide,which was consistent with the concentration of each component in the volatile oil,but the affin-ity order was opposite to that obtained by phase solubility test and molecular docking.CONCLUSION β-CD and its derivatives can successfully incorporate the volatile oil of the decoction.The components of the volatile oil and cyclodextrin are combined to form the inclusion compound by hydrogen bonding and other non-covalent bonding forces,which increases the stability of the volatile oil.The inclusion rate of volatile oil with different cyclodextrins is related to the affinity between volatile components and cyclodextrins and the concentration of volatile components,which provides a way to ensure the consistency between the main component of volatile oil inclu-sion compound in Wenjing Decoction and the reference sample.

OBJECTIVE To prepare the volatile oil cyclodextrin inclusion compound of Wenjing Decoction to improve its stability and characterize its formation;to compare the inclusion behavior of cinnamaldehyde,paeonol and ligustilide in volatile oil of Wenjing Decoction with different cyclodextrins;and to study the effect of different cyclodextrins on the inclusion of multiple components in the volatile oil of Wenjing Decoction.METHODS The inclusion compounds of volatile oil β-CD and its 5 derivatives in Wenjing Decoc-tion were prepared by ultrasonic method,and the inclusion rate of each component was determined by HPLC.TGA and FT-IR were used to characterize the formation of the inclusion complex.The inclusion behavior and influencing factors were studied by phase solu-bility test and molecular docking.RESULTS The inclusion compounds of volatile oil β-CD and its derivatives in Wenjing Decoction were successfully prepared.TGA results showed that the thermal stability of volatile oil in Wenjing Decoction was improved after inclu-sion by cyclodextrin.FT-IR results showed that some components of the volatile oil were bonded to cyclodextrin through non-covalent bonds such as hydrogen bonds.The results of phase solubility test showed that the three main components in the volatile oil of Wenjing Decoction were combined with β-CD in the molar ratio of 1∶n(n≥1),and with β-CD derivatives in the molar ratio of 1∶1.The re-sults of molecular docking showed that the benzene rings of cinnamaldehyde and paeonol,and lactone rings of ligustilide penetrated into the cyclodextrin cavity to form clathrates.The inclusion rates of β-CD and its derivatives for each component in the volatile oil of Wen-jing Decoction were DM-β-CD>HP-β-CD>β-CD>HE-β-CD>SBE-β-CD>CM-β-CD,which were consistent with the af-finity of phase solubility test and molecular docking.The inclusion rates of the three components with β-CD and its derivatives were cinnamaldehyde>paeonol>ligustilide,which was consistent with the concentration of each component in the volatile oil,but the affin-ity order was opposite to that obtained by phase solubility test and molecular docking.CONCLUSION β-CD and its derivatives can successfully incorporate the volatile oil of the decoction.The components of the volatile oil and cyclodextrin are combined to form the inclusion compound by hydrogen bonding and other non-covalent bonding forces,which increases the stability of the volatile oil.The inclusion rate of volatile oil with different cyclodextrins is related to the affinity between volatile components and cyclodextrins and the concentration of volatile components,which provides a way to ensure the consistency between the main component of volatile oil inclu-sion compound in Wenjing Decoction and the reference sample.

Objective : To observe the effect of Celastrus orbiculatusextract(COE) on gastric precancerous lesions(GPL) and to explore its role in the Notch-1 signaling pathway. Methods : GPL rat models were established using a composite model replication method, and the rats were randomly divided into a control group, a model group, and COE low, medium and high dose groups [COE at 12.5, 25, and 50 mg/(kg·d)]. After 4 weeks of intervention, gastric tissue was collected, and immunohistochemistry(IHC) was performed to detect the expression of mucins(MUC2, MUC5AC, and MUC6), Leucine-rich repeat-containing G-protein coupled receptor 5(Lgr5), Proliferating Cell Nuclear Antigen(Ki67), and Notch-1. Polymerase chain reaction(PCR) was used to determine the mRNA levels of the aforementioned mucins. Human gastric epithelial cells(GES-1) were induced with N-Methyl-N′-nitro-N-nitrosoguanidine(MNNG) to establish a GPL cell model. The cells were randomly divided into control, model, and COE low, medium, and high concentration groups(COE at 5, 10, and 20 μg/ml). After 24 hours of corresponding interventions, changes in cell morphology were observed under an inverted microscope. Western blot was used to measure the expression of Notch-1 and Lgr5, and immunofluorescence(IF) was employed to detect Notch-1 expression. Results : Compared to the control group, the expression of MUC2, Lgr5, Notch-1, and Ki67 in the gastric tissue of the model group rats significantly increased(P<0.000 1), while the expression of MUC5AC and MUC6 decreased(P<0.000 1). In comparison to the model group, the expressions of MUC2, Lgr5, Notch-1, and Ki67 were significantly reduced in the COE groups(P<0.01), while the expression of MUC5AC and MUC6 significantly increased(P<0.01). In the GES-1 model group, the cells exhibited irregular morphology, loose intercellular connections, and disorganized arrangement compared to the control group. In contrast, the cells in the COE groups displayed a more regular morphology and a more organized arrangement than those in the model group. Additionally, compared to the control group, the expression of Lgr5 and Notch-1 in the model group were significantly elevated(P<0.000 1), whereas after COE treatment, their expressions were markedly reduced(P<0.001). Conclusion COE can alleviate GPL, and its mechanism may be associated with the downregulation of the Notch-1 signaling pathway, which improves gastric mucosal mucin barrier function and inhibits the abnormal proliferation of gastric mucosal stem cells.

OBJECTIVE To optimize β-cyclodextrin(β-CD)inclusion process of Cinnamomi Ramulus Formula Granules vola-tile constituents by orthogonal test,based on standard relation and information entropy method.METHODS On the basis of single factor experiments,the ratio of β-CD to aromatic aqueous solution,the inclusion temperature,and the inclusion time were selected as the investigating factors;the inclusion rate,drug loading,and standard relation of cinnamic aldehyde in inclusion complex were used as the evaluation index.The information entropy method was used to determine the weight coefficient of each index,then the comprehen-sive score was calculated,the inclusion process conditions were optimized by orthogonal experiment.The inclusion complex was charac-terized by thin layer chromatography,ultraviolet absorption spectroscopy,Fourier infrared spectroscopy,and X-ray diffraction.RE-SULTS The best inclusion process was that the ratio of β-CD to aromatic aqueous solution was 3∶100(g·mL-1),the inclusion temperature was 50℃,and the inclusion time was 1 h.The average inclusion rate of the obtained inclusion compound was 80.84%,the drug loading was 8.63%,and the standard relation was 0.91.The results of thin-layer chromatography,ultraviolet,infrared spec-troscopy and other characterization experiments showed that the volatile components in the aromatic aqueous solution successfully en-tered the β-CD cavity,and the inclusion complex was successfully prepared.CONCLUSION The optimum inclusion process is sta-ble and feasible,which can provide references for the preparation process of Cinnamomi Ramulus Formula Granules.

ObjectiveThis study aims to investigate the mechanism in which Celastrus orbiculatus extract （COE） affects the proliferation and differentiation of gastric organoids and the expression of Lgr5 and thus reverses the precancerous lesions of gastric cancer （PLGC） by regulating the leucine-rich repeat containing G protein-coupled receptor 5 （Lgr5）/Wingless （Wnt）/β-catenin signaling pathway based on a gastric organoid injury model. MethodGastric organoids were established based on stem cells of the mouse gastric gland. Gastric organoid injury models were constructed by treating gastric organoids with N-methyl-N'-nitro-N-nitrosoguanidine （MNNG， 0.02 mg·L^-1）. Gastric organoid injury models were randomly divided into normal group， model group （0.02 mg·L^-1 MNNG）， low， medium， and high dose （5， 10， 20 mg·L^-1） groups of COE， and Wnt inhibitor Dickkopf-related protein 1 （DKK1） （0.5 mg·L^-1） group， and they were treated with respective agents for 24 h. The number and volume of gastric organoids under different drug concentrations were observed under a microscope. The viability of the gastric organoid injury models was detected by Methyl thiazolyl tetrazolium （MTT） assay. The morphology and pathology of gastric organoids were observed using Hematoxylin and Eosin （HE） staining. The expression levels of Lgr5， Mucin2 （MUC2）， Mucin5AC （MUC5AC）， Mucin6 （MUC6）， Wnt， and β-catenin in gastric organoids under different drug concentrations were detected by Western blot （WB）. ResultCompared with the normal group， the number， volume， and activity of gastric organoids in the model group were decreased （P<0.01）， while the expressions of Lgr5， MUC2， Wnt， and β-catenin were significantly increased （P<0.01）. The expressions of MUC5AC and MUC6 were significantly decreased （P<0.01）. Compared with the model group， the number and volume of gastric organoids in the low， medium， and high dose groups of COE were all improved （P<0.01）， and the vitality of gastric organoids was significantly enhanced （P<0.01）. The effect was the most significant at a COE concentration of 20 mg·L^-1 （P<0.01）. The expressions of Lgr5 and MUC2 in the medium and high dose groups of COE were significantly reduced （P<0.01）， while the expression of MUC5AC and MUC6 were significantly increased in the low， medium， and high dose groups of COE （P<0.05， P<0.01）. Compared with the model group， Wnt inhibitors could promote the expression of MUC5AC and MUC6 in gastric organoids （P<0.05， P<0.01） and reduce the expression of MUC2， Wnt， and β-catenin. In addition， the combined use of COE at high concentrations and Wnt inhibitors could further promote this trend （P<0.01）. ConclusionCOE inhibits the Wnt/β-catenin pathway by inhibiting the expression of Lgr5， MUC2， Wnt， and β-catenin and promoting the expression of MUC5AC and MUC6， thus promoting the proliferation and differentiation of gastric organoids and reversing the PLGC process.

Objective To investigate the expression of transcription factor forkhead box protein O(FOXO)genes in gastric cancer tissues and their correlation and clinical significance with Helicobacter pylori(Hp)infection.Methods The expression levels of FOXOs genes(including FOXO1,FOXO3,FOXO4,and FOXO6)were detected by immunohistochemistry in cancer tissues and paracancerous tissues from 41 gastric cancer patients with Hp(-)and 29 gastric cancer patients with Hp(+),as well as in gastric tissues from 30 healthy individuals.The correlation between FOXOs expression and Hp infection,clinical pathological features was analyzed.The relationship between FOXOs expression and survival prognosis of gastric cancer patients was analyzed using the Kaplan-Meier plotter.Results Compared with those in the Hp(-)gastric cancer tissues,the expression levels of FOXO1,FOXO4,and FOXO6 were higher in the Hp(+)gastric cancer tissues(P<0.05).Meanwhile,the expression levels of FOXO1,FOXO3,and FOXO4 in the Hp(+)gastric cancer tissues were lower than that in the paracancerous tissues(P<0.05)and normal tissues(P<0.0001).The expression of FOXOs in gastric cancer tissues was closely correlated with the degree of differentiation,depth of infiltration,lymph node metastasis,and TNM stage of gastric cancer(P<0.05).Meanwhile,FOXO1/3 was associated with the survival prognosis of patients with gastric cancer.Conclusion Hp infection promotes the expression of FOXO1/4/6 in gastric cancer tissues.The high expression of tumor suppressor genes FOXO1/4 may be one of the reasons for better prognosis in Hp(+)gastric cancer patients.FOXOs genes are widely involved in regulating the disease progression of gastric cancer,which has certain value for disease treatment.

Objective To identify clinical and dosimetric parameters from dose-volume histogram(DVH) relating with incidence of severe acute radiation-induced esophagitis(RE)in patients with non-small cell lung can-cer(NSCLC)underwent tomotherapy with concurrent or sequential chemotherapy. Methods Records about clini-cal information and treatment plan parameters from DVH of 62 NSCLC patients treated with tomotherapy were pro-spectively collected to assess the correlation to severe acute RE from January 2012 to December 2016. Results There were 24.2％patients developed grade 3 RE,grade 4 or 5 in 0％patients. Multivariate analysis indicated that concurrent chemotherapy,esophagus median dose and esophagus V25 and V55 were the influencing factors of RE. The incidence of low frequencies RE was correlated with sequential chemotherapy ,esophagus median dose < 49 Gy,esophagus V25 < 64％ ,V55 < 33％ and V60 < 15％. Conclusions For NSCLC patients treated with tomo-therapy and chemotherapy,the occurrence of acute RE was similar to that of other techniques. It is recommended to balance such parameters for optimizing treatment planning.

Objective To compare the setup errors between single-site and two-site image guidance in treating multiple metastases using Tomotherapy.Methods A total of 1 220 sets of megavoltage CT (MVCT) images from 50 multiple metastases patients were collected.The setup errors of two anatomic sites were determined by registration of MVCT images with planning images.Bland-Altman plot analysis was used to assess the coincidence of these two methods.Pearson correlation analysis was performed to evaluate the correlation of the setup errors determined by two sets of data and to analyze the deviation values of setup errors.Results The deviation values of setup errors more than 3 mm between two sites were 34％,46％ and 28％ in lateral (x),longitudinal (y),vertical (z) directions,respectively.The deviation values of setup errors more than 5 mm were 10％,16％ and 8％,respectively.The BlandAltman plot analysis showed that the 95％ agreement limits of agreement were (9.3,-10.6),(10.5,-11.7),(7.3,-6.9) mm in x,y,z directions,respectively,which were all out of 5 mm tolerance.The Pearson coefficient of correlation along all three directions was less than 0.05,and R2 was 0.074,0.475,and 0.178 in x,y,z directions,respectively.Conclusions To determine the setup errors for patients with multiple metastases,single-site image guidance method is not consistent,and the two site image guidance method would be recommended.

OBJECTIVETo discuss the synergistic mechanism of compatible use of two medicinal herbs, Zingiber offiicinale and Aconitum cainichaeli, by determining single decoction of Z. offiicinale and four gingerols (6-gingerol, 8-gingerol, 6-shogaol, 10-gingerol) contained in compound decoction of Z. offiicinale and A. cainichaeli of different compatibility ratio using HPLC.

METHODKromasil-C18 column (4.6 mm x 250 mm, 5 microm) was adopted. The mobile phase was acetonitrile (B) and 0.1% aqueous acetic acid (A) for gradient elution (0-30 min, 40%-90% B; 30-35 min, 90%-40% B). The flow rate was 1.0 mL x min(-1). The detection wavelength was set at 275 nm. The column temperature was 30 degrees C.

RESULTThe four gingerols were in baseline separation, with a good linearity (r > 0.999), an average recovery of 100.9% -103.5% and RSD < 3.0%. Compared with the single decoction of Z. offiicinale, the content of gingerols in the compound decoction of Z. offiicinale and A. cainichaeli was on the rise and in direct proportion with the increase in the volume of A. cainichaeli.

CONCLUSIONThe synergistic mechanism of the compatibility of Z. offiicinale and A. cainichaeli can be proved with the increased release of gingerols from Z. offiicinale.