Objective:To explore the changes of aquaporin-4 (AQP4) expression in rat astrocytes (RA) and rat model of radiation-induced brain injury (RIBI) after X-ray radiation, as well as its impact on cell functions.Methods:Logarithmic-phase RA were divided into four groups: Sham group (no treatment), AQP4 siRNA group (transfected with AQP4 lentivirus), IR group (single 20 Gy X-ray irradiation), and IR+AQP4 siRNA group (transfected with AQP4 lentivirus followed by single 20 Gy X-ray irradiation). The cell proliferation viability after radiation was detected using the CCK-8 assay. The relative expression of AQP4 mRNA in each group was measured by quantitative reverse transcription polymerase chain reaction, and the optimal AQP4 siRNA lentiviral sequence was selected for further studies. Western blot was used to detect the relative expression levels of AQP4, phosphorylated histone H2A family member X (γH2AX), autophagy-related proteins, glial fibrillary acidic protein (GFAP), proteins in the phosphatidylinositol-3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) pathway, and apoptosis-related proteins in cells from each group. Immunofluorescence (co-expression) staining was used to detect the expression of AQP4, γH2AX, GFAP, LC3, phosphatase and tensin homolog-induced kinase 1 (PINK1) in cells from each group, as well as the expression of AQP4 and LC3 in brain tissues from the rat RIBI model. Flow cytometry was used to detect the apoptosis rate of cells in each group. The differences between two groups were compared using the t-test, while the differences among multiple groups were evaluated using analysis of variance (ANOVA) and Tukey's multiple comparison test. Results:The proliferation viability of RA decreased to its lowest level at 24 h after irradiation. At the 24 h post-irradiation time point, the expression levels of γH2AX and AQP4 were significantly increased in the IR group compared with the Sham group. The fluorescence intensity of AQP4 in the AQP4 siRNA group was lower than that in the Sham group. The cells in the IR group showed significant enlargement and irregular morphology, with a significant increase in AQP4 fluorescence intensity, while the IR+AQP4 siRNA group showed mild cell enlargement and lower AQP4 fluorescence intensity compared with the IR group. Compared with the Sham group, the expression of microtubule-associated protein1 light chain 3 (LC3) and PINK1 proteins were decreased in the IR group, while the IR+AQP4 siRNA group showed higher expression levels compared with the IR group. Compared with the Sham group, the IR group showed increased expression of AQP4, GFAP, and sequestosome 1 (P62) proteins, decreased expression of Beclin-1 (BECN1) and LC3 proteins, and a reduced LC3-II/LC3-I ratio, while the IR+AQP4 siRNA group exhibited a restored LC3-II/LC3-I ratio. The apoptosis rate in the AQP4 siRNA group was similar to that in the Sham group, while the IR group showed a significantly increased apoptosis rate, and the IR+AQP4 siRNA group had a lower apoptosis rate than the IR group. The expression of caspase-3 (Caspase3) decreased in the IR group, while the expression of PI3K, AKT, mTOR, extracellular signal-regulated kinase (ERK)1/2, and cleaved Caspase3 protein increased; these changes were partially reversed in the IR+AQP4 siRNA group. In the rat RIBI model, the expression area and intensity of AQP4 were higher in the IR group compared with the Sham group, while LC3 expression showed the opposite pattern.Conclusions:The possible molecular mechanism of RA apoptosis caused by X-ray irradiation is that irradiation promotes the expression of AQP4, causes cell swelling, inhibits autophagy, and prevents cells from clearing damaged organelles in a timely manner, thereby promoting cell apoptosis.

Objective:To explore the changes of aquaporin-4 (AQP4) expression in rat astrocytes (RA) and rat model of radiation-induced brain injury (RIBI) after X-ray radiation, as well as its impact on cell functions.Methods:Logarithmic-phase RA were divided into four groups: Sham group (no treatment), AQP4 siRNA group (transfected with AQP4 lentivirus), IR group (single 20 Gy X-ray irradiation), and IR+AQP4 siRNA group (transfected with AQP4 lentivirus followed by single 20 Gy X-ray irradiation). The cell proliferation viability after radiation was detected using the CCK-8 assay. The relative expression of AQP4 mRNA in each group was measured by quantitative reverse transcription polymerase chain reaction, and the optimal AQP4 siRNA lentiviral sequence was selected for further studies. Western blot was used to detect the relative expression levels of AQP4, phosphorylated histone H2A family member X (γH2AX), autophagy-related proteins, glial fibrillary acidic protein (GFAP), proteins in the phosphatidylinositol-3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) pathway, and apoptosis-related proteins in cells from each group. Immunofluorescence (co-expression) staining was used to detect the expression of AQP4, γH2AX, GFAP, LC3, phosphatase and tensin homolog-induced kinase 1 (PINK1) in cells from each group, as well as the expression of AQP4 and LC3 in brain tissues from the rat RIBI model. Flow cytometry was used to detect the apoptosis rate of cells in each group. The differences between two groups were compared using the t-test, while the differences among multiple groups were evaluated using analysis of variance (ANOVA) and Tukey's multiple comparison test. Results:The proliferation viability of RA decreased to its lowest level at 24 h after irradiation. At the 24 h post-irradiation time point, the expression levels of γH2AX and AQP4 were significantly increased in the IR group compared with the Sham group. The fluorescence intensity of AQP4 in the AQP4 siRNA group was lower than that in the Sham group. The cells in the IR group showed significant enlargement and irregular morphology, with a significant increase in AQP4 fluorescence intensity, while the IR+AQP4 siRNA group showed mild cell enlargement and lower AQP4 fluorescence intensity compared with the IR group. Compared with the Sham group, the expression of microtubule-associated protein1 light chain 3 (LC3) and PINK1 proteins were decreased in the IR group, while the IR+AQP4 siRNA group showed higher expression levels compared with the IR group. Compared with the Sham group, the IR group showed increased expression of AQP4, GFAP, and sequestosome 1 (P62) proteins, decreased expression of Beclin-1 (BECN1) and LC3 proteins, and a reduced LC3-II/LC3-I ratio, while the IR+AQP4 siRNA group exhibited a restored LC3-II/LC3-I ratio. The apoptosis rate in the AQP4 siRNA group was similar to that in the Sham group, while the IR group showed a significantly increased apoptosis rate, and the IR+AQP4 siRNA group had a lower apoptosis rate than the IR group. The expression of caspase-3 (Caspase3) decreased in the IR group, while the expression of PI3K, AKT, mTOR, extracellular signal-regulated kinase (ERK)1/2, and cleaved Caspase3 protein increased; these changes were partially reversed in the IR+AQP4 siRNA group. In the rat RIBI model, the expression area and intensity of AQP4 were higher in the IR group compared with the Sham group, while LC3 expression showed the opposite pattern.Conclusions:The possible molecular mechanism of RA apoptosis caused by X-ray irradiation is that irradiation promotes the expression of AQP4, causes cell swelling, inhibits autophagy, and prevents cells from clearing damaged organelles in a timely manner, thereby promoting cell apoptosis.

Objective:To evaluate the impact of sarcopenia on survival and treatment-related toxicity in postoperative recurrent esophageal squamous cell carcinoma (ESCC) patients treated with chemoradiotherapy.Methods:Clinical data of 147 patients with postoperative locoregional recurrent ESCC receiving chemoradiotherapy in Huai'an First People's Hospital from 2016 to 2017 were retrospectively analyzed. Pectoralis muscle area (PMA) was determined using routine pre-radiotherapy CT simulation scan above the aortic arch level. Sarcopenia was defined as a cut-off value of pectoralis muscle index (PMI) (PMA/height 2) <11.55 cm 2/m 2 for males and <8.69 cm 2/m 2 for females. The incidence of toxicity, 1- and 3-year overall survival (OS) rates were statistically compared between patients with and without sarcopenia. Results:Sarcopenia was detected in 49 of 147 (33.3%) patients. The incidence of grade 3-4 toxicities in sarcopenic patients was significantly higher compared to that in their counterparts without sarcopenia (40.8% vs. 18.4%, P=0.005). In addition, patients with sarcopenia had significantly worse 1-year (61.2% vs. 82.7%) and 3-year OS rates (10.2% vs. 28.6%) than those without sarcopenia (both , P<0.001). Multivariate analysis showed that sarcopenia was an independent prognostic factor for poor OS ( P<0.001). Conclusion:PMI based on CT simulation scan has prognostic value in postoperative locoregional recurrent ESCC patients treated with chemoradiotherapy, which probably serves as a novel diagnostic tool for sarcopenia.

Objective To investigate the dynamic changes of nuclear factor-κB(NF-κB) during brain injury of irradiated rats.Methods According to the random number table method,82 healthy Sprague-Dawley rats were divided into two groups:50 rats in the irradiation group and the other 32 rats in the control group given sham-irradiation.The whole brains of rats in the irradiation group were irradiated with a single dose of 20 Gy electron beam to establish an animal model of radiation-induced brain injury.All brain tissues were respectively taken out 1,3,7,14 and 28 d after irradiation.The quantificational real-time-polymerase chain reaction (QRT-PCR) was used to detect the expressions of the NF-κB mRNA while the expression of NF-κB protein was analyzed by ELISA and Western blot.The positioning expression of NF-κB protein in the hippocampus was measured with immunohistochemical staining.Results In the irradiation group,the expression of NF-κB mRNA significantly increased at 3 and 7 d following irradiation (t =37.79,35.30,P ＜ 0.05) and it approached a peak value on the third day following irradiation.The expression of NF-κB protein in whole brain tissues after irradiation was up-regulated and reached a peak on the first day post-irradiation and it was significantly higher than controls at 1,3,7,14 d after irradiation (t =30.94,14.87,27.17,13.27,P ＜ 0.05) then declined and dropped to the normal level by 28 d.The results of immunohistochemical staining showed that the number of NF-κB positive cells in the hippocampus increased at 1,3 and 7 d following irradiation (t =-8.49,-4.47,-3.46,P ＜ 0.05).Conclusions After irradiation,the expressions of NF-κB mRNA and corresponding protein first increase and then later decrease,which promotes the development of brain injury.