In order to elucidate the mechanisms of p53 overexpression in nasopharyngeal carcinoma (NPC) and detect proteins associated with the function of p53 in high throughout screening, p53 which knockdown human NPC CNE2 cell line (CNE2sip53) were successfully established by using stable RNA interference (RNAi). Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of CNE2sip53 and its control cell line CNE2/pSUPER, and PDQuest software was applied to analyze 2-DE images. Twenty-two differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3? etc.) , and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, the differential expression levels of the partial proteins (HSP27, 14-3-3?, GRP75) were confirmed by Western blot analysis and compared with CNE2 and CNE2 cells transfected with pcDNA3.1-FLAG, CNE2 cells transfected with pcDNA3.1-FLAG-p53 had obvious down-regulations of HSP27 and 14-3-3?, and an up-regulation of GRP75. The 22 differentially expressed proteins could be divided into five groups based on their functions: signal transduction, chaperone, transcription and translation, metabolism and cytoskeleton, which were involved in cell cycle, the transcription regulation, cell adherence,cellular metabolism and so on. The data suggest that these differential proteins may be associated with the function of p53 in NPC, and will be valuable for further to study the mechanisms of p53 overexpression and inactivation in NPC.

Objective Screening of modified Foshou San to improve cerebral ischemia,determine the best prescription composition and the proper dose.Methods Using data mining method,the traditional Chinese medicine for hypertensive intracerebral hemorrhage(HICH)were screened,and cluster analysis was carried out to obtain Jiawei Foshou San combinations;According to the mixed uniform design U14(42×33×22),the auricular artery dilatation experiment and acute cerebral ischemia induced by saturated magnesium chloride in mice were used to observe the auricular artery dilatation index and survival time of mice,and the pharmacological effects of each prescription to improve cerebral ischemia were evaluated.On the basis of visual analysis of pharmacodynamics,stepwise multiple regression analysis was carried out,and the regression equation was used to calculate the best prescription composition and dose of Jiawei Foshou San.The optimized prescription was verified by acute cerebral ischemia experiment in mice.Results A total of 51 prescriptions that may be used for the treatment of HICH were selected by data mining,including 103 Chinese herbs,with a total frequency of 535 times.The results of mixed uniform design U14(42×33×22)test showed that the auricular artery index of mice in each prescription group of Jiawei Foshou San increased to varying degrees.The differences between prescription 4,8,12 and 13 were significant(P<0.05),and the differences between prescription 3,7,9,11 and 14 were extremely significant(P<0.01).The survival time of acute cerebral ischemia in mice was prolonged to varying degrees in each group of Jiawei Foshou San,and the difference between prescription 2-14 groups and the blank group was statistically significant(P<0.01).The prescription of the new Jiawei Foshou San was determined as 60 g of Astragalus membranaceus,60 g of Angelica sinensis,24 g of Ligusticum chuanxiong,15 g of Paeonia lactiflora and 9 g of Dilong from the intuitive analysis.The validation results showed that the high and middle dosage of the new Jiawei Foshou San could significantly prolong the breathing time,increase the number of breaths,and prolong the time of normal pressure hypoxia tolerance in mice with acute cerebral ischemia(P<0.01,P<0.05).Conclusion Using mixed uniform design combined with improving cerebral ischemia effect index can determine the best prescription composition and the best dose of Jiawei Foshou San,and the effect of improving cerebral ischemia is certain.

Objective:Iron accumulation is related to the occurrence of postmenopausal osteoporosis. Meanwhile, autophagy abnormality of bone marrow hematopoietic cells is observed in hip osteoporotic fracture. This study is performed to investigate correlation between iron accumulation induced bone loss and hematopoietic autophagy dysfunction to explore the new risk factor of osteoporosis.Methods:Male iron accumulation mice model was established by intraperitoneally injecting ferric ammonium citrate. Serum ferritin and osteogenic indicator P1NP were tested by ELISA. Bone mineral density was measured by micro-CT. Femur and tibia bone marrows were collected for hematopoietic stem and progenitor cells proportion and cell apoptosis analysis. Autolysosome formation was measured by image flow cytometry. We used conditional mouse model Atg7 flox/flox; Vav-Cre(Atg7 -/-) in which Atg7 had been genetically deleted in the hematopoietic system. Bone marrow hematopoietic stem and progenitor cells were collected for RNA sequence. micro-CT scan was conducted for Atg7 -/- femur. Results:Ferritin level of iron accumulation mice was significantly higher than control group( P<0.05). Iron accumulation inhibited P1NP and induced decreased bone mineral density( P<0.05). Iron accumulation bone marrow displayed enhanced hematopoietic stem and progenitor cells proportion( P<0.05), with more cell apoptosis( P<0.05). Hematopoietic autophagy was deteriorated in iron accumulation bone marrow. Transcriptomic profiling showed up-regulation of iron activity in Atg7 -/- mice, with increased iron homeostasis and iron membrane transporter genes, including Lcn2, Tfr2, Slc40a1(Fpn1), Steap3, and Cpox. micro-CT revealed severe bone loss and decreased bone mineral density in Atg7 -/- mice( P<0.05). Conclusion:Iron accumulation induced bone loss is related to inhibition of hematopoietic cells. Hematopoietic autophagy dysfunction is associated with bone loss.

Objective:To investigate the clinical outcome after surgery and first 131I treatment in patients with moderate-risk papillary thyroid cancer (PTC), and analyze the relevant factors that affect the therapeutic effect. Methods:From January 2018 to April 2019, 135 patients (48 males, 87 females; age (42.7±11.1) years) with moderate-risk PTC in the Second Affiliated Hospital of Chongqing Medical University were retrospectively analyzed. According to the 2015 American Thyroid Association (ATA) guidelines, patients were divided into excellent response (ER) group, inderteriminate response (IDR) group, biochemical incomplete response (BIR) group and structural incomplete response (SIR) group, of which IDR, BIR, SIR were collectively referred to as the non-ER group. χ2 test and Mann-Whitney U test were used to compare the general clinical features between the ER and non-ER groups, and then multivariate logistic regression analysis was performed. The predicted value of pre-ablation stimulated thyroglobulin (ps-Tg) to ER was assessed by ROC curve analysis. Results:The treatment responses of 94 patients were ER, and those of 41 were non-ER. The differences in tumor size (0.80(0.50, 1.10) vs 1.00(0.55, 1.50) cm; U=1 491.50, P=0.036), the number of metastatic lymph nodes (3(2, 5) vs 4(2, 12); U=1 422.00, P=0.015), metastatic lymph node size (0.50(0.30, 0.65) vs 0.50(0.30, 1.45) cm; U=1 396.50, P=0.013), metastatic lymph node involvement rate (50%(30%, 70%) vs 60%(50%, 85%); U=1 441.50, P=0.024), metastatic lymph node location (central/lateral: 76/18 vs 24/17; χ2=7.40, P=0.007) and ps-Tg level (2.1(0.8, 5.3) vs 14.0(3.2, 35.2) μg/L; U=680.00, P<0.001) were statistically significant between the ER and non-ER groups. Multivariate logistic regression analysis showed that ps-Tg (odds ratio ( OR)=1.200, 95% CI: 1.107-1.302, P<0.001) was an independent factor influencing ER. The cut-off value of ps-Tg for predicting ER was 7.38 μg/L, with the sensitivity and specificity of 68.3%(28/41) and 87.2%(82/94) respectively. Conclusion:Moderate-risk PTC patients with smaller tumor size, fewer metastatic lymph nodes, lower metastatic lymph node involvement rate, metastatic lymph nodes in central area, smaller metastatic lymph node size, and ps-Tg<7.38 μg/L have better therapeutic effect after initial 131I treatment.

Background Lipid metabolism imbalance is tightly linked to the development and progression of multiple diseases. The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is important for the regulation of lipid metabolism. However, whether silicosis is associated with lipid metabolic abnormalities has yet to be explored. Objective To observe the changes of lipid deposition, cholesterol, and phosphorylated proteins of PI3K/AKT/mTOR pathway in silicon dioxide (SiO₂)-induced MLE-12 cells and to explore potential mechanism of lipid composition regulated though the pathway. Methods (1) MLE-12 cells were stimulated with 50 mg·L⁻¹ SiO₂ suspension, and divided into fourgroups: a control group and three SiO₂ groups (12, 24, and 48 h of stimulation). (2) Cellproliferation was detected to determine an optimal dose of LY294002, an inhibitor of PI3K protein. LY294002 at 5 μmol·L⁻¹ was used for further study, in which MLE-12 cells cultured for 48 h were divided into four groups: a control group; a 50 mg·L⁻¹ SiO₂ suspension stimulation group; a 50 mg·L⁻¹ SiO₂ suspension and 5 μmol·L⁻¹ LY294002 treatment group; a 5 μmol·L⁻¹ LY294002 treatment group. Total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE; total cholesterol minus free cholesterol), and triglycerides (TG) were measured with enzyme assay kits. Lipid deposition was observed using Oil Red O staining. The expressions of p-PI3K, p-AKT, and p-mTOR proteins were detected by Western blotting. Results (1) The contents of TC, FC, and CE in the 50 mg·L⁻¹ SiO₂-induced MLE-12 cells were increased compared to those of the control group in a time-dependent manner by trend analysis, and the increment at 24 and 48 h were significant. By 48 h, the contents of cholesterol indicators were all elevated: TC from (2.242±0.181) mg·g⁻¹ to (5.148±0.544) mg·g⁻¹, FC from (1.923±0.158) mg·g⁻¹ to (4.168±0.433) mg·g⁻¹, and CE from (0.318±0.067) mg·g⁻¹ to (0.978±0.134) mg·g⁻¹, compared with the control group (P<0.01). The changes of TG were not significant (P>0.05). The SiO₂ suspension induced orange-red particle deposition in the MLE-12 cells, especially at 48 h (P<0.01). The protein expression levels of p-PI3K, p-AKT, and p-mTOR in SiO₂-stimulated MLE-12 cells were higher than those of the control groups with the prolongation of stimulation time, which peaked at 48 h (P<0.01). (2) The contents of TC, FC, and CE in MLE-12 cells of the SiO₂ + LY294002 group were decreased, comparing to those of the SiO₂ stimulation only group (P<0.01), companied with less orange-red lipid deposition, and suppressed protein expression levels of p-PI3K, p-AKT, and p-mTOR (P<0.01). Conclusion SiO₂ could induce increases of cholesterol and lipid deposition through activation of PI3K/AKT/mTOR signaling pathway in MLE-12 cells.

ObjectiveTo investigate the clinical efficacy and possible mechanism of Erzhi Tiangui prescription on repeated implantation failure （RIF） of kidney deficiency syndrome. MethodSeventy patients with RIF of kidney deficiency syndrome who underwent natural cycle frozen-thawed embryo transfer （FET） in the Reproductive and Genetic Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine were enrolled and randomly divided into a treatment group （35 cases） and a control group （35 cases）. Patients in the treatment group took oral Erzhi Tiangui prescription from the third day of each menstrual cycle two months before the FET cycle and continued to take it until the day of transplantation from the third day of the menstrual cycle in the month of transplantation. Those in the control group did not accept traditional Chinese medicine （TCM）. In addition，10 patients who successfully achieved clinical pregnancy after the first natural cycle FET were screened from the reproductive medical record bank of this hospital and assigned to the normal group. Peripheral blood samples of patients in the three groups on the day of embryo transfer were collected from the specimen bank of the Reproductive and Genetic Center. Serum soluble programmed death molecule-1 （sPD-1），soluble programmed death molecule-ligand 1 （sPD-L1），transforming growth factor-β （TGF-β），interleukin-17 （IL-17）， and interleukin-10 （IL-10） levels were measured by enzyme-linked immunosorbent assay （ELISA）. The changes in kidney deficiency syndrome scores， the final biochemical pregnancy rates， clinical pregnancy rates， and embryo implantation rates of the treatment group and the control group before and after treatment were observed. ResultCompared with the normal group，the model group showed increased serum levels of sPD-1 and IL-17（r=0.347，P<0.05），decreased levels of IL-10 and TGF-β （P<0.01），and non-significant change in sPD-L1 level. Serum sPD-1 was positively correlated with IL-17 （P<0.05） and negatively correlated with IL-10（r=-0.521，P<0.01） and TGF-β（r=-0.457，P<0.01） in RIF patients with kidney deficiency syndrome. After TCM treatment，compared with the control group， the treatment group showed improved TCM syndrome score （P<0.05） and increased clinical pregnancy rate and embryo transfer rate（P<0.05），but there was no statistically significant difference in the biochemical pregnancy rate between the two groups. ConclusionAbnormal expression of sPD-1 in patients with RIF of kidney deficiency syndrome breaks the balance of T helper 17 （Th17）/regulatory T cell （Treg），which is not conducive to embryo implantation and pregnancy maintenance. Erzhi Tiangui prescription，a TCM for tonifying the kidney，can significantly improve the symptoms of kidney deficiency in patients with RIF of kidney deficiency syndrome，reduce the concentrations of sPD-1 and IL-17 in the peripheral serum，increase the levels of TGF-β and IL-10，regulate the peripheral Th17/Treg immune balance，and increase the implantation rate and clinical pregnancy rate，which has a high clinical value.

The morbidity and mortality of lung cancer ranks among the top cancers in the world. Non-small cell lung cancer (NSCLC) is the main pathological type of lung cancer, with limited treatment options and poor prognosis. The nuclear factor E2-related factor 2 (NRF2) signaling pathway is highly mutated and activated in NSCLC, and promotes the malignant progression of lung cancer through various mechanisms. NRF2-targeted therapy will provide new treatment strategies for patients with NSCLC. This article will review the basic structure and response pathways of the NRF2 pathway, the mechanism of NRF2 regulating lung cancer cell proliferation, and the research and development progress of NRF2 inhibitors.
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Humans ; Carcinoma, Non-Small-Cell Lung/pathology* ; NF-E2-Related Factor 2/metabolism* ; Lung Neoplasms/pathology* ; Gene Expression Regulation, Neoplastic ; Signal Transduction ; Cell Proliferation

Lung cancer is one of the most common cancers in the world, and its treatment strategy is mainly surgery combined with radiotherapy and chemotherapy. However, long-term chemotherapy will result in drug resistance, which is also one of the difficulties in the treatment of lung cancer. Ferroptosis is an iron-dependent and lipid peroxidation-driven non-apoptotic cell death cascade, occurring when there is an imbalance of redox homeostasis in the cell. Nuclear factor erythroid 2-related factor 2 (Nrf2) is key for cellular antioxidant responses. Numerous studies suggest that Nrf2 assumes an extremely important role in regulation of ferroptosis, for its various functions in iron, lipid, and amino acid metabolism, and so on. In this review, a brief overview of the research progress of ferroptosis over the past decade will be presented. In particular, the mechanism of ferroptosis and the regulation of ferroptosis by Nrf2 will be discussed, as well as the role of the Nrf2 pathway and ferroptosis in tumor drug resistance, which will provide new research directions for the treatment of drug-resistant lung cancer patients.
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Humans ; Ferroptosis ; NF-E2-Related Factor 2/genetics* ; Lung Neoplasms/genetics* ; Drug Resistance, Neoplasm ; Iron

Salmonella enterica serovar Enteritidis (SE) is one of the most important zoonotic pathogens that cause enteritis and systemic infection in animals and human. Understanding invasive capacities of SE isolates is of vital importance to elucidate pathogenesis of Salmonella infection. To improve the throughput capacity and repeatability of classical gentamicin protection assay (GPA), a modified PGA was developed by taking high-throughput advantage of 96-well cell plates and multichannel pipettes. In addition, drop plate technique rather than spread plate method was applied in the modified GPA protocol for bacterial enumeration. The modified GPA protocol was evaluated by phenotyping intracellular replication of a high virulent and a low virulent SE isolates, JL228 and LN248, in a phagocytic cell line RAW264.7. The protocol was then applied in invasive phenotype determination of 16 SE strains to non-phagocytes (HT-29) and the intracellular replication of 43 SE strains to phagocytes (RAW264.7). Significant lower intra-group and inter-group coefficient of variations of the modified GPA was observed, implying good repeatability and reproducibility over traditional protocol. Further, replication phenotypes were also correlated with those from direct observation by confocal microscopy. Collectively, the improved GPA protocol had advantages of high throughput capacity, good repeatability and reliability, it was also noticed that the protocol also represented a fast and labor-saving alternative scheme for the invasive phenotype determination of Salmonella Enteritidis, and providing reliable phenotype profiles for Salmonella-host interplay interpretation.
Animals ; Gentamicins/pharmacology* ; Humans ; Phenotype ; Reproducibility of Results ; Salmonella Infections, Animal ; Salmonella enteritidis

ObjectiveTo investigate the protective mechanism of Qianyang Yuyin granules （QYYY） on aldosterone-induced podocyte injury. MethodA total of 30 C57BL/6J mice were randomly divided into five groups： control group， model group， QYYY low dose （QYYY-L） group， QYYY high dose （QYYY-H） group， and spironolactone （SPL） group， with six mice in each group. Except for the control group， mice were implanted with osmotic minipumps and injected continuously with aldosterone （300 μg·kg^-1·d^-1） to induce renal injury. The drug administration group was given low and high doses （2.6， 5.2 g·kg^-1·d^-1） of QYYY and SPL （18 mg·kg^-1·d^-1） for 28 days. The renal pathological changes of mice were observed by hematoxylin-eosin （HE） staining and Masson staining. The expression levels of Nephrin， Desmin， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cleaved Caspase-3， nuclear receptor subfamily 3 group C member 2 （NR3C2）， extracellular regulated protein kinases （ERK）， and phospho-ERK （p-ERK） in kidney tissue were detected by Western blot. The apoptosis levels of kidney tissue were detected by TdT-mediated dUTP nick and labeling （TUNEL） staining， and the superoxide dismutase （SOD） levels were detected. In vitro， the mice were divided into five groups： Control group， model group （aldosterone concentration of 200 nmol·L^-1）， QYYY-L group， QYYY medium dose （QYYY-M） group， and QYYY-H group （25， 50， and 100 mg·L^-1）. The effect of different concentrations of QYYY on the relative viability of aldosterone-induced podocytes was detected by cell proliferation and viability assay （CCK-8）. The expressions of Nephrin， Desmin， Bax， Bcl-2， cleaved Caspase-3， NR3C2， and p-ERK/ERK were detected by Western blot. AnnexinV-FITC/PI flow cytometry was used to detect the apoptosis levels of podocytes. Reactive oxygen species （ROS） in podocytes were observed by DCFH-DA. ResultCompared with the control group， the model group showed structural pathological changes and fibrotic conditions in the kidney， increased apoptosis levels （P<0.01）， and decreased SOD levels （P<0.01）. Aldosterone concentration at 200 nmol·L^-1 showed a significant decrease in podocyte activity （P<0.05）. Podocytes in the model group showed structural pathological changes， disordered arrangement of intercellular microfilaments， increased apoptosis levels （P<0.01）， and increased intracellular ROS levels （P<0.01）. The protein expressions of Nephrin， Bcl-2， and p-ERK/ERK in kidney tissue and podocytes were decreased （P<0.05， P<0.01）. The protein expressions of Desmin， Bax， cleaved Caspase-3， and NR3C2 were increased （P<0.05， P<0.01）. Compared with the model group， QYYY alleviated the structural damage and fibrosis of the kidney， decreased the apoptosis levels （P<0.05， P<0.01）， and enhanced the SOD content of the kidney （P<0.05， P<0.01）. QYYY improved the activity of podocytes （P<0.05， P<0.01）， restored the foot process structure of podocytes， and decreased apoptosis levels （P<0.01） and ROS levels of podocytes （P<0.01）. The protein expressions of Nephrin， Bcl-2， and p-ERK/ERK in kidney tissue and podocytes were increased （P<0.05， P<0.01）， and the protein expressions of Desmin， Bax， cleaved Caspase-3， and NR3C2 were down-regulated （P<0.05， P<0.01）. ConclusionQYYY improves aldosterone-induced podocyte injury by regulating the NR3C2/ROS/ERK pathway.