BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90％,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P＜0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P＜0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P＜0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P＜0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P＜0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P＜0.05),the mitochondrial membrane potential decreased(P＜0.01),and the degree of damage in the high-fluoride group was more obvious(P＜0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.

Objective To explore the effects of chronic stress and stress cessation on hypothalamic appetite regulators in mice,and to explore the stress-dependent mechanism of appetite change.Methods A total of 32 male C57BL/6 mice were randomly divided into control(Ctrl)group(n=16)and chronic unpredictable mild stress(CUMS)group(n=16).The mice in the CUMS group were given CUMS to establish the stress model,and those in the Ctrl group were fed normally.The food intake and weight of mice were recorded.The CUMS model was verified through tail suspension experiments and forced swimming experiments.Eight mice in the Ctrl group and 8 mice in the CUMS group were randomly sacrificed at the 12th week.The Ctrl group was re-grouped into the cessation-control(C-Ctrl)group(n=8),the CUMS group was re-grouped into the cessation-stress(C-CUMS)group(n=8),and the mice were sacrificed at the 15th week.The mRNA and protein levels of appetite-regulating factors,including orexin 1 receptor(OX1R),leptin receptor(LEPR)and agouti-related protein(AgRP)in the hypothalamus,were detected by quantitative polymerase chain reaction and immunochemistry.Results From week 2 to week 11 of stress,the food intake of the mice in the CUMS group was significantly higher than that in the Ctrl group(all P＜0.05),while there was no significant difference in body weight between the 2 groups within 11 weeks(all P＞0.05).Compared with the Ctrl group,the immobility durations of forced swimming and tail suspension of the CUMS group were markedly longer after 11 weeks(both P＜0.01),indicating successful modeling.AgRP and OX1R mRNA expression in the hypothalamus of the CUMS group was significantly increased(both P＜0.01),while LEPR mRNA expression was significantly decreased(P＜0.01);AgRP protein in the hypothalamic arcuate nucleus of the CUMS group was significantly higher than that of the Ctrl group(P＜0.05),and LEPR protein was markedly lower than that of the Ctrl group(P＜0.01).However,after 3 weeks of stress cessation,the C-CUMS group had less food intake and lower body weight than the C-Ctrl group(both P＜0.05).The LEPR mRNA of the C-CUMS group was significantly increased(P＜0.01),while AgRP and OX1R mRNA were not significantly different(both P＞0.05).There was no significant difference in AgRP protein levels between the C-CUMS group and the C-Ctrl group(P＞0.05),while LEPR protein level of the C-CUMS group was significantly higher than that of the C-Ctrl group(P＜0.01).Conclusion CUMS can lead to increased appetite in mice,which may involve the functional regulation of LEPR and AgRP.After the stress cessation,the appetite decreases,which may involve the functional regulation of LEPR.

AIM:To investigate the effect of Jianpi-Huayu decoction(JPHYD)combined with gemcitabine(GEM)on ferroptosis of pancreatic cancer PANC-1 cells and its mechanism.METHODS:PANC-1 cells were cultured in vitro,and CCK8 method was used to detect the cell viability after different concentrations of JPHYD and GEM,and ap-propriate concentrations were selected for follow-up experiments.EDU assay and clonogenesis assay were used to detect cell proliferation.The apoptosis rate was detected by flow cytometry.Intracellular reactive oxygen species(ROS)were de-tected by DCFH-DA fluorescent probe and lipid peroxidation was detected by BODIPY 581/591C11 staining.The contents of glutathione(GSH),ferrous ion(Fe2+)and malondialdehyde(MDA)in the cells were detected by the kit.The mRNA levels and protein expression levels of Nrf2,HO-1,SLC7A11,GPX4,TFR1 and ACSL4 were detected by RT-qPCR and Western blot.RESULTS:Compared with the control group,the cell viability of PANC-1 treated with JPHYD and GEM was significantly decreased in a concentration-dependent manner.And the combined use of the two can significantly im-prove the cytotoxic effect of GEM and have a synergistic effect;Compared with control group,JPHYD group,GEM group and JPHYD+GEM group can significantly reduce EDU positive efficiency,colony formation numbers and promote cell apoptosis,and the combined group has the most obvious effect.After adding JPHYD+GEM into the cells,the cells be-came rounded and the cell viability decreased.The addition of ferrostatin-1(Fer-1),an inhibitor of ferroptosis,had no significant effect on cell morphology and viability,and the co-treatment with JPHYD+GEM and Fer-1 could reverse the ef-fects of JPHYD+GEM on cell morphology and viability.Compared with control group and GEM group,JPHYD+GEM group can significantly increase the levels of intracellular reactive oxygen species(ROS)and lipid peroxidation,increase the levels of Fe2+and MDA,decrease the levels of GSH,further promote lipid peroxidation and induce ferroptosis.JPHYD+GEM also significantly down-regulated the mRNA and protein expressions of Nrf2,HO-1,SLC7A11 and GPX4,and up-regulated the mRNA and protein expressions of TFR1 and ACSL4.The addition of Fer-1 significantly reversed the activation of iron death in the combined treatment group and reversed its efficacy,and the difference was statistically signif-icant.CONCLUSION:Jianpi Huayu decoction and gemcitabine may induce ferroptosis of PANC-1 cells by inhibiting Nrf2/SLC7A11/GPX4 axis in vitro,thus playing a synergistic anticancer role.

AIM:To investigate the effect of Jianpi-Huayu decoction(JPHYD)combined with gemcitabine(GEM)on ferroptosis of pancreatic cancer PANC-1 cells and its mechanism.METHODS:PANC-1 cells were cultured in vitro,and CCK8 method was used to detect the cell viability after different concentrations of JPHYD and GEM,and ap-propriate concentrations were selected for follow-up experiments.EDU assay and clonogenesis assay were used to detect cell proliferation.The apoptosis rate was detected by flow cytometry.Intracellular reactive oxygen species(ROS)were de-tected by DCFH-DA fluorescent probe and lipid peroxidation was detected by BODIPY 581/591C11 staining.The contents of glutathione(GSH),ferrous ion(Fe2+)and malondialdehyde(MDA)in the cells were detected by the kit.The mRNA levels and protein expression levels of Nrf2,HO-1,SLC7A11,GPX4,TFR1 and ACSL4 were detected by RT-qPCR and Western blot.RESULTS:Compared with the control group,the cell viability of PANC-1 treated with JPHYD and GEM was significantly decreased in a concentration-dependent manner.And the combined use of the two can significantly im-prove the cytotoxic effect of GEM and have a synergistic effect;Compared with control group,JPHYD group,GEM group and JPHYD+GEM group can significantly reduce EDU positive efficiency,colony formation numbers and promote cell apoptosis,and the combined group has the most obvious effect.After adding JPHYD+GEM into the cells,the cells be-came rounded and the cell viability decreased.The addition of ferrostatin-1(Fer-1),an inhibitor of ferroptosis,had no significant effect on cell morphology and viability,and the co-treatment with JPHYD+GEM and Fer-1 could reverse the ef-fects of JPHYD+GEM on cell morphology and viability.Compared with control group and GEM group,JPHYD+GEM group can significantly increase the levels of intracellular reactive oxygen species(ROS)and lipid peroxidation,increase the levels of Fe2+and MDA,decrease the levels of GSH,further promote lipid peroxidation and induce ferroptosis.JPHYD+GEM also significantly down-regulated the mRNA and protein expressions of Nrf2,HO-1,SLC7A11 and GPX4,and up-regulated the mRNA and protein expressions of TFR1 and ACSL4.The addition of Fer-1 significantly reversed the activation of iron death in the combined treatment group and reversed its efficacy,and the difference was statistically signif-icant.CONCLUSION:Jianpi Huayu decoction and gemcitabine may induce ferroptosis of PANC-1 cells by inhibiting Nrf2/SLC7A11/GPX4 axis in vitro,thus playing a synergistic anticancer role.

Objective:To analyze the effectiveness and safety of the second course radiotherapy for unresectable colorectal cancer liver metastases.Methods:We retrospectively collected the data of 28 patients with unresectable colorectal cancer liver metastases who received the second course radiotherapy at Peking University Cancer Hospital and Institute from 2017 to 2023, to analyze the feasibility of re-irradiation.Results:For the 28 patients, the median follow-up time after re-irradiation was 20.2 months. The median time interval between the first- and second-course radiotherapy was 11.1 months. The median biologically effective doses of the first- and second-course radiotherapy were 100 Gy and 96 Gy, respectively. Stereotactic body radiotherapy was administered to 25 patients (89.3%) during the first course and 24 patients (85.7%) during the second course of radiotherapy. The mean equivalent dose in 2 Gy fractions to the normal liver was 10.1 Gy in the first-course radiotherapy and 7.9 Gy in the second-course radiotherapy. The complete response rate, partial response rate, and objective response rate after re-irradiation were 54.5%, 18.2%, and 72.7%, respectively. After re-irradiation, the 2-year cumulative local failure rate was 17.0% when calculated based on patients and 15.1% when calculated based on lesions, the 1-year progression-free survival rate was 27.4%, and the 3-year overall survival rate was 46.7%. The second-course radiotherapy was well tolerated, with most patients (75.0%) experiencing grade 1-2 acute adverse reactions and only one case (3.6%) experiencing grade 3 acute adverse events.Conclusions:Second course radiotherapy is an effective and safe treatment approach for selected patients with unresectable colorectal cancer liver metastases.

A patient aged 68 years old presented urinary frequency, urgency, and gross hematuria for 1 month, with initial PSA of 72.72 ng/ml and alkaline phosphatase (ALP)of 114 U/L. Prostate biopsy pathology showed small cell neuroendocrine carcinoma of prostate. The patient was immediately administered 6 cycle of chemotherapy including etoposide and cisplatin combined with medical castration. The CDK4 gene was detected 1.99 times amplification by peripheral blood free DNA (cfDNA)gene analysis. The chemotherapy was followed by parbosini therapy. The number and density of bone metastases continued to decrease significantly by bone scan at 3 and 6 months after treatment, with a continuous decline of ALP and PSA. After 1 year of follow-up, pelvic MRI and bone systemic imaging indicated stable lesions, with PSA of 0.05 ng/ml and ALP of 59 U/L.

Background: Arctic-like (AL) lineages of rabies viruses (RABVs) remains endemic in some Arctic and Asia countries. However, their evolutionary dynamics are largely unappreciated. Objectives: We attempted to estimate the evolutionary history, geographic origin and spread of the Arctic-related RABVs. Methods: Full length or partial sequences of the N and G genes were used to infer the evolutionary aspects of AL RABVs by Bayesian evolutionary analysis. Results: The most recent common ancestor (tMRCA) of the current Arctic and AL RABVs emerged in the 1830s and evolved independently after diversification. Population demographic analysis indicated that the viruses experienced gradual growth followed by a sudden decrease in its population size from the mid-1980s to approximately 2000.Genetic flow patterns among the regions reveal a high geographic correlation in AL RABVs transmission. Discrete phylogeography suggests that the geographic origin of the AL RABVs was in east Russia in approximately the 1830s. The ancestral AL RABV then diversified and immigrated to the countries in Northeast Asia, while the viruses in South Asia were dispersed to the neighboring regions from India. The N and G genes of RABVs in both clades sustained high levels of purifying selection, and the positive selection sites were mainly found on the C-terminus of the G gene. Conclusions The current AL RABVs circulating in South and North Asia evolved and dispersed independently.

Objective:To investigate the effects of microRNA (miR)-513a-3p regulating hexokinase 2 (HK2) on proliferation and glycometabolism in colorectal cancer cell.Methods:From May 2019 to February 2020, the miR-513a-3p simulant, miR-513a-3p inhibitor and miR control were transfected into colorectal cancer SW480 cell respectively. Real-time quantitative polymerase chain reaction was used to detect the expression levels of miR-513a-3p in colorectal cancer SW480 cell, normal colorectal cell and all transfected colorectal cancer SW480 cell. The effect of miR-513a-3p on cell proliferation was detected by CCK-8 assay. Brd/PI incorporation assay was used to detect the effect of miR-513a-3p on glycometabolism. Western blot was used to detect the expression of HK2.Results:The expression level of miR-513a-3p in colorectal cancer SW480 cell was significantly lower than that in normal colorectal cell (0.43 ± 0.06 vs. 1.00 ± 0.02), and there was statistical difference ( t = 7.024, P = 0.003). The expression level of miR-513a-3p in colorectal cancer SW480 cell transfected with miR-513a-3p simulant was significantly higher than that in colorectal cancer SW480 cell transfected with miR control and transfected with miR-513a-3p inhibitor (1.18 ± 0.24 vs. 0.45 ± 0.04 and 0.22 ± 0.03), the expression level of miR-513a-3p in colorectal cancer SW480 cell transfected with miR control was significantly higher than that in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor, and there were statistical differences ( P<0.05). The proliferation ability in colorectal cancer SW480 cell transfected with miR-513a-3p simulant at 24, 48, 72 and 96 h was significantly lower than that in colorectal cancer SW480 cell transfected with miR-513a-3p control group, the proliferation ability in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor at 24, 48, 72 and 96 h was significantly higher than that in colorectal cancer SW480 cell transfected with miR-513a-3p control, and there were statistical differences ( P<0.05). The glucose intake, lactate and thioredoxin-interacting protein (TXNIP) expression levels in colorectal cancer SW480 cell transfected with stimulant were significantly lower than those in colorectal cancer SW480 cell transfected with miR control (1.02 ± 0.04 vs. 1.90 ± 0.06, 0.88 ± 0.03 vs. 1.45 ± 0.04 and 0.16 ± 0.02 vs. 0.86 ± 0.06), the indexes in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor were significantly higher than those in colorectal cancer SW480 cell transfected with miR control (2.35 ± 0.09 vs. 1.90 ± 0.06, 1.67 ± 0.08 vs. 1.45 ± 0.04 and 2.01 ± 0.15 vs. 0.86 ± 0.06), and there were statistical differences ( P<0.05). The expression level of HK2 in colorectal cancer SW480 cell transfected with miR-513a-3p stimulant was significantly lower than that in colorectal cancer SW480 cell transfected with miR control (0.20 ± 0.01 vs. 1.02 ± 0.04), and there was statistical difference ( t = 8.367, P<0.05); the expression level of HK2 in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor was significantly higher than that in colorectal cancer SW480 cell transfected with miR control (1.91 ± 0.07 vs. 1.02 ± 0.04), and there was statistical difference ( t = 4.279, P<0.05). Conclusions:MiR-513a-3p can significantly inhibit the proliferation and glycometabolism of colorectal cancer cell, and its regulatory mechanism is related to the inhibition of the HK2 protein expression in the cell by miR-513a-3p.

Objective To review the failure patterns and clinical outcomes for patients with cervical esophageal carcinoma (CEC) undergoing definitive radiotherapy (RT).Methods Medical records,clinical characteristics and outcomes of patients with CEC treated by definitive RT from August 2008 to May 2017 were retrospectively reviewed and analyzed.Results A total of 97 patients with squamous cell CEC were enrolled in this study with a median age of 59 years old (range 18-78 years old).There were 34 patients with limited cervical esophagus,and 63 patients with diseases beyond cervical region,respectively.There were 69,7,and 6 patients with Bronchi invasion,thyroid lobes involvement and aortic involvement,respectively.There were 11,80 and 6 patients with stage Ⅱ,Ⅲ and Ⅳ (non-regional lymph node metastases),respectively.The median dose to the gross tumor volume (GTV) was 66 Gy,in which 46 patients received above 66 Gy and 51 patients received less than 66 Gy,respectively.The median progression free survival (PFS) and overall survival (OS) were 16.03 and 23.30 months,respectively,with a median follow-up of 14.90 months.The 1,2,3-year PFS and OS were 56.86％,30.35％,26.34％,and 72.54％,47.94％,40.81％,respectively.Sixty-one patients had treatment failure at their last follow-up,in which 40,27,and 18 patients developed local failure,regional failure,and distant metastasis,respectively.Univariate analysis revealed that thyroid lobes involvement resulted in lower PFS (x2 =5.773,P＜0.05) and OS (x2 =13.461,P＜0.05),and bronchi involvement (x2 =4.283,P＜0.05) was associated with lower OS.Multivariate analysis indicated that aortic involvement and thyroid lobes involvement were associated with lower PFS (x2 =6.796,4.548,P＜0.05) and OS (x2 =13.421,10.581,P＜0.05),and GTV dose above 66 Gy was associated with higher OS (x2=5.296,P＜0.05).Conclusions Local-regional recurrence was the main failure pattern for patients with CEC after definitive RT.Aortic,thyroid lobes,and/or bronchi involvement were associated with poor prognosis,and GTV dose ≥66 Gy tended to improve OS.Prospective studies with larger population were needed to further confirm this study.