OBJECTIVE：To investigate immunotoxicity of the main by-product 2-acetyl-4-tetrahydroxybutylimidazole (THI)，in the process of class Ⅲ caramel color production，on female rats. METHODS： 50 healthy adult female SD rats with SPF grade were randomly divided into 5 groups，10 rats in each group. They were given THI orally at doses of 0，0.1，0.5，2.5 and 5 mg/kg，respectively，continuously for 30 days. The followings were collected/evaluated：body weights，organ coefficients of thymus and spleen，histopathological evaluation of thymuses， number of white blood cells and various types of cells in peripheral blood， proliferation ability of spleen T lymphocytes，activities of NK cells，and flow cytometry evaluation of changes in the proportion of major immune cells in peripheral blood，thymus and spleen. RESULTS：THI exposure reduced the area ratio of thymus cortex/medulum and the proliferation ability of rat spleen T lymphocytes (with 2.5 and 5 mg/kg doses). The results of immunocytopathology showed that THI exposure at a dose of 2.5 mg/kg and above reduced the ratio of the total number of white blood cells and T lymphocytes in the peripheral blood and increase the ratio of NK cells in the spleen. The number of lymphocytes and the ratio of B lymphocytes in the peripheral blood were significantly reduced，and the ratio of T cells in the spleen was also reduced by the 5 mg/kg THI exposure. For the T cell subsets，5 mg/kg THI exposure reduced the ratio of Th cells in the peripheral blood. With doses of 0.5 mg/kg and above，THI increased the ratio of Th cells in the thymus and CD4⁺CD8⁺ double positive. The proportions of non-positive T cells were reduced. With doses of 2.5 mg/kg and above，THI increased the proportion of CTL cells in the thymus. CONCLUSION：Oral exposure to THI caused damage to the immune system of female rats. In addition， changes in the number of lymphocytes in peripheral blood and the proportion of thymus T cell subsets were sensitive indicators. Thus， the no observed adverse effect level of THI immunotoxicity (NOAEL) is 0.1 mg/kg.

OBJECTIVE： To investigate expression and implication of P4HA2 in esophageal squamous cell carcinoma (ESCC). METHODS： Expressions of P4HA2 mRNA in ESCC tissues and normal esophageal tissues were analyzed by Gene Expression Omnibus (GEO) database. P4HA2 protein in ESCC and normal tissues were detected using Immunohistochemistry (IHC) and Western blot. Cell proliferation and colony formation assays were performed to analyze effects of P4HA2 on the malignant phenotypes of ESCC cells in vitro. Effects of P4HA2 on tumor growth were detected using the nude mouse xenograft model in vivo. Western blot was used to analyze changes in downstream pathways from knockdown of P4HA2. RESULTS：Expression levels of both the P4HA2 mRNA and protein were up-regulated in ESCC compared with the normal tissues (P<0.01). In vitro and in vivo results showed that P4HA2 silencing significantly inhibited the proliferation and colony formation of ESCC cells，as well as the growth of tumor xenografts (P<0.01). At the molecular level， P4HA2 knockdown down-regulated EGFR expression，and reduced the phosphorylation levels of AKT and S6 (P<0.05). Moreover，P4HA2 knockdown and over-expression of EGFR significantly restored the phosphorylation levels of AKT and S6 as well as cell proliferation and colony formation (P<0.01). CONCLUSION：P4HA2 was highly expressed in ESCC，and it might enhance cell proliferation and promote development of ESCC via activating the EGFR/AKT/S6 pathway.

OBJECTIVE： The present study aimed to investigate protective effects of angelica keiskei chaclones (AC) on acute alcohol liver injury in mice. METHODS：Sixty healthy mice were divided randomly into five groups (12 mice per group)： normal control，alcohol model，and low-(5 mg/kg)，medium-(15 mg/ kg) and high-dose (30 mg/kg) groups. AC was intragastrically administrated with a volume of 10 μL/g once a day for 30 days. Mice in the normal control and alcohol model groups were given equal volumes of saline. Twelve hours after the last administration，all mice except those in the normal control group were given 50% alcohol by gavage at a dose of 12 mL/kg. After 12 hours， all mice were sacrificed. Blood samples were collected from eyeballs and serum was isolated. Indicators of liver injury in glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST)， lipid metabolism of low-density lipoprotein cholesterol (LDL-C)、 high-density lipoprotein cholesterol (HDL-C)、 total cholesterol(TC)and triglyceride (TG)， oxidative stress of superoxide dismutase (SOD)、 malondialdehyde (MDA)and glutathione peroxidase (GSH-Px)， and cell apoptosis were evaluated. Liver histopathology was examined as well. RESULTS：Compared with the normal control group， the liver indices in the alcohol control group were significantly increased with obvious inflammatory cell infiltration (P<0.05). Their serum transaminase activity and contents of serum TC， T and LDL-C were obviously increased and HDL was significantly reduced (P<0.05). Their activities of SOD and GSH-Px were significantly decreased， and the content of MDA was increased obviously (P<0.05). Their apoptosis rates of hepatocytes were obviously increased (P<0.05). Compared with the alcohol model group，the liver indices for the medium-and high-dose AC groups were significantly decreased (P<0.05)， and inflammatory cell infiltrations were obviously reduced. Their serum transaminase activities and contents of serum TC， TG and LDL-C were significantly decreased (P<0.05). Their activities of SOD and GSH-Px were effectively increased， and the contents of MDA were reduced obviously (P<0.05). Their apoptosis rates of hepatocytes were also significantly lower than the alcohol model group (P<0.05). CONCLUSION：AC exhibited protective effects on acute alcoholic liver injury. A mechanism may be related to reduction of lipid peroxidation，modulation of lipid metabolism and inhibition of hepatocyte apoptosis.

OBJECTIVE： To investigate effects of the blister agent， nitrogen mustard (HN2)， on mitochondrial structure and function of hepatocytes，based on in vitro and in vivo models. METHODS：In the in vitro model，HepG2 cells were divided into normal control and HN2 exposure groups. The normal control group was treated with serum-free medium containing DMSO (0.1%) for 36 h，and the HN2 exposure group was treated with nitrogen mustard (8 μmol/L). After HN2 treatment for 36 h，cell viability was detected by CCK-8 method， mitochondrial membrane potential by JC-1 probe method， ATP content in cell homogenate and liver tissue by NADPH rate method， and mitochondrial function by Seahorse cell energy metabolism analyzer. Twenty C57BL/6 mice were divided into normal control group and HN2 exposure group. The HN2 exposure group was given a single intraperitoneal injection of nitrogen mustard hydrochloride (2 mg/kg)，and the normal control group was injected with normal saline. Serum and liver tissues were used to measure activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST)， and pathological examination of liver tissues and electron microscope observations were performed. Contents of ATP and mitochondrial enzyme complexes I，II and III activities in liver tissues were determined by corresponding kits. RESULTS： In the in vitro model， compared with the normal control group， HN2 exposure decreased cell activity by about 16.2% (P<0.05)， the mitochondrial membrane potential by about 33.2% (P<0.05)， and mitochondrial energy metabolism was significantly abnormal. In the in vivo model，compared with the normal control group，HN2 exposure caused significant increased activities of serum ALT and AST. At the same time， there was large number of inflammatory cell infiltrations into liver tissues accompanied by liver parenchymal cell damage. Mitochondrial morphology was significantly abnormal： manifested as increased number，decreased size，loss or fragmentation of mitochondrial cristae，and significantly decreased activity of mitochondrial enzyme complexes I， II and III (P<0.05). CONCLUSION： HN2 exposure induced mitochondrial damage in hepatocytes and this may be one of the main toxic targets of HN2 poisoning. Results from our study provide new ideas for the prevention and treatment of acute liver injury induced by blister agents such as HN2.

OBJECTIVE： To investigate effects of obese female mice on reproductive ability of male offspring and their possible mechanisms. METHODS： A female mouse obesity model was established by high-fat diet induction for 8 weeks，and caged with normal diet male mice. After birth，the offspring were divided into maternal obesity group and normal control group and fed with normal diet until 8 weeks after weaning. From each group of mice， 8 male offspring were randomly selected and executed after anesthesia. From them，testes and epididymis tissues were isolated，sperm quality was evaluated，testicular sex hormone levels were detected by ELISA，testes were analyzed using by immunofluorescence Expression levels of mRNA and protein for FOXO1 and Nrf2 genes were detected by qPCR and Western blot，respectively. RESULTS： Compared with the control group，sperm concentrations，sperm motilities and forward motion sperm ratios were significantly lower (P<0.01) and the sperm malformation rates were significantly higher (P<0.01) in the maternal obese mice. Other test results showed that maternal obesity could cause testicular tissue damage in the offspring， disturbance in testicular endocrine hormone secretion， elevated testicular tissue ROS levels， significantly increased expression levels of the spermatogonial marker FOXO1， and elevated oxidative stress levels and Nrf2 protein expressions (all P<0.01). CONCLUSION： Due to maternal obesity， oxidative stress levels were elevated in testicular tissues of offspring male mice which might have caused impaired their spermatogonial differentiation and reproductive capacity.

OBJECTIVE： To investigate relationships between vascular endothelial growth factor-A (VEGF-A) protein expression and epidermal growth factor receptor (EGFR) gene mutation in patients’ lung adenocarcinomas， as well as their implications as clinicopathological and prognosis indicators. METHODS： Lung adenocarcinoma tissues from 72 patients were evaluated for EGFR gene mutation VEGF-A protein in the middle-late EGFR mutation type (using immunohistochemical method). Patients were followed up to determine disease progression-free survival. The Chi-square test inspection，Kaplan-Meier survival analysis，single factor and multi-factors Cox regression analysis were used to evaluate the collected data. RESULTS：Among the 72 cases of type EGFR mutations，high expression of VEGF-A rate was 70.83% (51/72)，mainly in the cytoplasm of tumor cells. The difference in the high expression rate of VEGF-A protein expression was not statistically significant between patients with EGFR gene exon 19 deletion and other rare mutation types (P>0.05)， but statistically significant among patients with different clinical stages and lymph node metastasis status (P<0.05) There was no significant correlation with age，sex，and smoking history (P>0.05). Spearman rank correlation analysis showed that there was a significant negative correlation between progression-free survival (PFS) and VEGF-A protein expression in patients with EGFR-mutation (r=-0.513，P=0.009). The results of Kaplan-Meier survival analysis showed that the PFS of patients with high expression VEGF-A were significantly shorter than that of patients with low VEGF-A expression. The results of univariate analysis showed that lymph node metastasis， clinical stage and VEGF-A expression level were the factors affecting the prognosis of patients (HRs were 4.778，11.456，5.676，respectively). Multivariate COX regression analysis showed that clinical stage and VEGF expression (HRs were 2.054，4.949，respectively) were independent risk factors affecting survival and prognosis of patients. CONCLUSION： Relationships between VEGF-A in our patients with high expression of EGFR mutation and lymph node metastasis and clinical stages were statistically significant. VEGF-A expression and EGFR mutations and negative correlation PFS may be useful as an index of judging the prognosis of patients with lung cancer.

OBJECTIVE：To observe cleavage patterns of embryos in early stages by time-lapse imaging system，and to evaluate effects of the cleavage patterns on developmental potential of embryos in early stage. METHODS：From January 2019 to July 2021，development of 1 318 embryos cultured with blastocysts which were recorded by the time-lapse imaging system in the reproductive center were retrospectively analyzed. According to the cleavage mode of two prokaryotic (2PN) fertilized eggs，they were divided into normal and abnormal cleavages. Abnormal cleavages included first cleavage anomaly and non-first cleavage anomaly. Pregnancy outcomes such as rates of high-quality embryo， blastocyst formation， high-quality blastocyst， pregnancy， and abortion of early embryos in different cleavage pattern groups were analyzed on day 3. RESULTS： The D3 high-quality embryo rates were 57.36% and 39.47% respectively； blastocyst formation rates were 66.33% and 36.62% respectively； high-quality blastocyst rates were 60.14% and 27.61% respectively in the normal cleavage group and in the abnormal cleavage group. These were significantly higher than those in the abnormal cleavage group (P<0.01). There was no significant difference in clinical pregnancy and miscarriage rates between the normal cleavage and the abnormal cleavage groups(P>0.05). In the abnormal cleavage group，the rates for D3 high-quality embryos and high-quality blastocysts were 34.45% and 24.64% in the first abnormal cleavage group and they were significantly lower than those in the non-first abnormal cleavage group of 58.02% and 37.66% (P<0.05). There was no significant difference in the rates of clinical pregnancy and abortion between the first cleavage anomaly group and the non-first cleavage anomaly group. CONCLUSION：Abnormal cleavage of embryos reduced developmental potential of early embryo. The earlier the abnormal cleavage occurred，the greater the impact on embryonic development.

OBJECTIVE： To explore relationships between the relative telomere length of blood mononuclear cells (PBMC) and urine arsenic concentration in type-2 diabetic patients exposed to low levels of arsenic. METHODS：A total of 574 patients with type 2 diabetes in South China were selected as the study objects. Basic information was collected and biochemical indicators were detected. Urine arsenic concentrations were detected using inductively coupled plasma mass spectrometer (ICP-MS) technology，and relative telomere lengths of PBMC were detected by real-time fluorescence quantitative PCR (qPCR) technology. The study subjects were divided into 3 groups according to their urine arsenic concentrations. Differences of biochemical indicators and PBMC relative telomere lengths among the 3 groups were compared. Single-and multi-factor ordinal logistic regressions were used to analyze influencing factors for the relative telomere lengths. The mediating effects of Hemoglobin A_1c (HbA_1c) between urine arsenic levels and relative telomere lengths were analyzed using SPSS Process 2.0. RESULTS：The median urine arsenic level in the patients was 29.80 μg/L， and the interquartile interval was 39.81 μg/L，and the relative telomere length was 0.82±0.60. The relative telomere length in low， medium and high arsenic groups were 1.04 ±0.92， 0.77±0.29 and 0.63±0.28， respectively. Differences of relative telomere lengths among the three groups were statistically significant (P<0.05). Multivariate ordinal logistic regression analyses showed that urine arsenic was a risk factor for relative telomere length. Compared with the low arsenic group，the OR (95% CI) of the medium arsenic group was 2.149 (1.451， 3.182)， and that of the high arsenic group was 4.077 (2.687， 6.185)， the differences were statistically significant (P<0.01). The direct effect value of urine arsenic on relative telomere length was -0.287 (-0.369，-0.204). HbA_1c was a mediating variable between urine arsenic and relative telomere length，with the mediating effect value being -0.072 (-0.106，-0.045) and the mediating proportion being 19.99%. CONCLUSION：Presence of arsenic in urine is a risk factor for relative telomere length of PBMC in type-2 diabetic patients： the higher the urine arsenic levels，the shorter the relative telomere lengths. The relative telomere length of PBMC in these patients may be a potential biomarker for arsenic exposures.

OBJECTIVE： To compare the efficacy of neoadjuvant chemotherapy followed by interval debulking surgery with primary debulking surgery for advanced epithelial ovarian cancer，and to analyze the influencing factors of the prognosis. METHODS：From January 2010 to December 2019，advanced epithelial ovarian cancer patients (FIGO stage Ⅲ or Ⅳ) who were initially treated at the Department of Cancer Hospital of Shantou University Medical College，106 were selected and divided into two treatment groups：neoadjuvant chemotherapy with interval debulking surgery (NACT-IDS) and primary debulking surgery (PDS). Among the NACT-IDS group，patients were divided into 2 subgroups according to the preoperative chemotherapy cycles： the neoadjuvant chemotherapy ≤2 cycles (NACT-IDS-1) group and the neoadjuvant chemotherapy >2 cycles (NACT-IDS-2) group. Our patients were included into 67 cases in the NACT-IDS group (40 cases in the NACT-IDS-1 group，and 27 cases in the NACT-IDS-2 group) and 39 cases in the PDS group. Information about the general pathological characteristics， intraoperative bleeding， postoperative outcomes， and survival outcomes of the patients were analyzed retrospectively. Survival analysis was used to compare differences in overall survival (OS) and progression-free survival (PFS) between the groups of NACT-IDS and PDS and the two subgroups of NACT-IDS. T-test，chi-square test and Fisher's exact test were used to compare differences in intraoperative bleeding，operative time，and postoperative complications，etc. The Cox proportional hazards model was used to analyze relationships between clinicopathological characteristics and surgery-related characteristics with prognosis. RESULTS：Among the 106 patients，58 (54.7%) died and the median follow-up time was 46 months. The median OS was 44 and 42 months in the NACT-IDS and PDS groups，respectively， and the median PFS was 21 months in both groups. The differences in median OS and median PFS between the NACT-IDS and PDS groups and between the NACT-IDS subgroups were not statistically significant (all P>0.05). Compared with the PDS group， the NACT-IDS group showed significant differences in intraoperative bleeding，operative time，incidence of postoperative complications，and time to start chemotherapy after surgery (all P<0.05). The results of the multivariable COX proportional hazards model showed that residual disease (incomplete resection) versus no residual disease (complete resection) was statistically significant on OS [HR= 2.82， 95% CI(1.20， 6.63)， P=0.017]； the effect of normalization of CA-125 after 4 cycles of postoperative chemotherapy was statistically significant on both OS and PFS compared with those who did not [HR=0.39， 95%CI(0.18，0.88)，P=0.022；HR=0.34，95%CI(0.17，0.71)，P=0.004]. CONCLUSION：NACT-IDS improves the quality of surgery compared with PDS for advanced epithelial ovarian cancer，but does not significantly enhance the overall survival benefit，and suggests early IDS as soon as conditions permit. Residual disease and whether CA-125 recovered to normal after 4 cycles of postchemotherapy were independent prognostic factors affecting OS in advanced epithelial ovarian cancers.

OBJECTIVE： To investigate relationships among neutrophil/lymphocyte ratios (NLR)， inflammation indices(SII)， tumor markers CEA， CA19-9， CA72-4 and the clinical stages of gastric cancer (GC)；and to develop a nomogram prediction model. METHODS：From January 2019 to May 2022， clinical data from 192 gastric cancer patients who were admitted to the General Surgery Department of the Second Affiliated Hospital of the Zhengzhou University were collected. The preoperative NLR，SII，albumin and serum CEA，CA19-9 and CA72-4 levels of early and advanced gastric cancer groups were determined. Differences in the collected data between the two groups of patients were analyzed. Then，the cut-off value for each test index was determined by the receiver working characteristic (ROC) curve. The nomogram of Logistic regression model was drawn to evaluate the diagnostic value of the markers alone and in combinations for preoperative gastric cancer stages. RESULTS：All preoperative NLR，SII，CEA，CA19-9，and CA72-4 levels were higher in the advanced gastric cancer group than in the early gastric cancer group (P<0.05). The ROC curve analysis showed that the area under the curve (AUC) of NLR，SII，CEA，CA19-9，CA72-4 alone were 0.817，0.792， 0.722， 0.724， and 0.761， respectively， and the best cut-off values were 2.09， 366.73， 2.18， 7.44， and 3.63，respectively. The AUC for each index was 0.944，the sensitivity was 89.3%，and the specificity was 89.2% . A nomogram prediction model including NLR，SII，CEA，CA19-9，CA72-4 and clinical stage was constructed. The internal validation consistency index (C index) was 0.915，and the correction curve suggested that the accuracy of the model was good. CONCLUSION： Preoperative NLR， SII， CEA， CA19-9， and CA72-4 were associated with clinical stages of gastric cancers. In addition，the nomogram model which was constructed based on preoperative NLR， SII， CEA， CA19-9， CA72-4 levels and clinical stage had good accuracy and clinical utility.