Background::Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma.Methods::The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro. A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry. Results::Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro. AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 +/CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion. Conclusions::This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.

Objective: The mechanism of Gualou Guizhi Decoction (GGD) in the treatment of lower limb spasm after stroke was analyzed based on network pharmacology. Methods: Using the TCMSP and SwissTarget Prediction platform to screen the main components and targets of GGD, the obtained target information was imported into Gephi software to build the active components-targets network of GGD. Genecard, DisGeNET, and TTD disease databases were used to screen target genes for stroke and lower limb spasm; Target protein interaction network was constructed by using STRING platform; KEGG pathways, and GO (Gene Ontology) function were analyzed by using ClueGO software and DAVID v 6.8 platform. Results: Flavonoids, organic acids, and saponins of GGD was applied to 49 key target genes, such as IL6, TNF, EGFR, ESR1, TP53, STAT3, and F2, etc; Forteen category biological processes including proliferation of striated muscle cells, bone remodeling, smooth muscle cells, muscle cells and endothelial cells proliferation were involved; Thirty signaling pathways, including TNF, MAPK, PI3K-Akt, ALS, serotonergic synapse, and neurotrophin, etc, were regulated. Conclusion: Various active ingredients of GGD has pharmacological effects such as anti-inflammatory, antioxidant, neurotrophic, and participate in some biological processes, such as muscle cell proliferation, so as to treat lower limb spasm after stroke from central nervous system and peripheral muscle tissue two-ways.

Objective: To investigate the effect and related mechanism of homocysteine (Hcy) on calcium overload in neonatal rat atrial cells (NRICs). Methods: NRICs were assigned to 9 groups after culture for 3 days: (1) control group; (2) Hcy group (0, 50, 100, 200, 500 μmol/L for 48 hours); (3) antioxidant group (NAC, 10 μmol/L for 24 hours); (4) Hcy+NAC group (500 μmol/L Hcy for 48 hours, then treated with 10 μmol/L NAC for 24 hours); (5) calcium/calmodulin dependent protein kinase Ⅱδ (CaMKⅡδ) inhibitor group (KN-93, 3 μmol/L KN-93 for 5 hours); (6) specific sodium current inhibitor group (ELE, 1 μmol/L ELE for 5 hours); (7) Hcy+KN-93 group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 for 5 hours); (8) Hcy+ELE group (500 μmol/L Hcy for 48 hours, then treated with 1 μmol/L ELE for 5 hours; (9) Hcy+KN-93+ELE group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 and 1 μmol/L ELE for 5 hours). Moreover, NRICs were also treated with CaMKⅡδ-siRNA lentivirus, and Nav1.5-siRNA lentivirus, negative lentivirus carrier containing green fluorescent protein (GFP) for 24 hours. The MOI values of the three groups were 10. Infection efficiency of lentivirus was determined by observing the percentage of GFP fluorescence under inverted fluorescence microscope after transfection for 24 hours, and cultured regularly with simultaneous Puro screening, then cells were grouped as Hcy+CaMKⅡδ-siRNA group, Hcy+Nav1.5-siRNA group and Hcy+negative group. The concentration of Ca²⁺ in NRICs ([Ca²⁺]i) of various groups was detected through Fluo-4/AM fluorescence probe, then 2', 7'- two chlorofluorescein diacetate (DCFH-DA) was used as a probe to detect reactive oxygen species (ROS) in NRICs by flow cytometry. The malondialdehyde (MDA) was detected by the activity of superoxide dismutase (SOD) and xanthine oxidase was detected by thiobarbituric acid colorimetry. The protein and mRNA expression level of CaMKⅡδ and Nav1.5 in NRICs were detected by Western blot and quantitative real-time PCR. Results: (1) ROS, MDA and SOD were similar between NAC group and control group, ROS and MDA were significantly increased, while SOD was significantly reduced in Hcy group in a concentration-dependent manner. (2) [Ca²⁺]i: The level of [Ca²⁺]i was (155.57+7.25), (187.43+13.07), (248.98+27.22) and (307.36+15.09) nmol/L in 50, 100, 200 and 500 μmol/L Hcy groups, which was significantly higher than that in the control group ((123.18+7.24) nmol/L, P<0.01). In addition, the level of [Ca²⁺]i in Hcy+NAC group ((222.87+23.71)nmol/L) was significantly lower than that in Hcy 500 μmol/L group ((305.15+39.45) nmol/L, P<0.05), while [Ca²⁺]i level was similar between NAC group and the control group. (3) The protein expression of CaMKⅡδ and Nav1.5 was significantly upregulated in Hcy groups than in the control group. The protein expression level of CaMKⅡδ-Thr287 was significantly lower in NAC group than in Hcy 500 μmol/L group (P<0.01), however, there was no significant difference on the protein expression levels of CaMKⅡδ-Thr287 and Nav1.5 between NAC group and control group (all P>0.05). (4) The protein expression levels of CaMKⅡδ-Thr287 and the concentration of [Ca²⁺]i were significantly lower in Hcy+KN-93 group and Hcy+KN-93+ELE group than in Hcy 500 μmol/L group (P<0.05). [Ca²⁺]i concentration was significantly lower in Hcy+KN-93 group, Hcy+ELE group and KN-93+ELE+Hcy group than in Hcy 500 μmol/L group (P<0.05). (5) The mRNA and protein expression levels of CaMKⅡδ and Nav1.5 in each group infected with lentivirus: the GFP expression was ideal post lentivirus transfection for 24 hours (up to 90%), which was significantly lower in the CaMKⅡδ-siRNA group and Nav1.5-siRNA group than in the negative infection group (all P<0.05), which was similar between negative infection group and control group (P>0.05). Moreover, the mRNA and protein expression levels of CaMKⅡδ and CaMKⅡδ-Thr287 was significantly lower in Hcy+Nav1.5-siRNA group than in Hcy+negative infection group (P<0.05). The protein and mRNA levels of Nav1.5 were similar between Hcy+CaMKⅡδ-siRNA group and Hcy+negative infection group (P>0.05). Conclusions Hcy can induce calcium overload in NRICs by increasing oxidative stress, upregulating the sodium channel protein, and activating the late sodium current and phosphorylating CaMKⅡδ.

The present study carried out a phytochemical investigation of the methanol extract of the branches and leaves of Clausena lansium and afforded nine carbazole alkaloids (compounds 1-9) including two new carbazole alkaloids, claulansiums A and B (compounds 1 and 2). The new compounds were elucidated on the basis of extensive spectroscopic data (MS, NMR, IR, and UV) and the known compounds were identified by comparing spectroscopic data with those reported in literature. All the isolated compounds were tested for their cytotoxic activity against A549 and Hela cancer cell lines. Our results showed that compounds 2-6 exhibited varying degrees of cytotoxicity to cancer cells, with IC values ranging from 8.67 to 98.89 μmol·L.
A549 Cells ; Alkaloids ; chemistry ; isolation & purification ; toxicity ; Antineoplastic Agents ; chemistry ; isolation & purification ; toxicity ; Carbazoles ; chemistry ; isolation & purification ; toxicity ; Cell Line, Tumor ; Cell Survival ; drug effects ; Clausena ; chemistry ; HeLa Cells ; Humans ; Molecular Structure ; Plant Extracts ; chemistry ; toxicity ; Plant Leaves ; chemistry ; Plant Stems ; chemistry ; Plants, Medicinal ; chemistry

Objective To investigate the effect of sperm preparation in male infertility on sperm morphology and DNA fragmentation index(DFI). Methods Four hundred men were divided into the groups of fertile , teratozoospermics and unexplained subfertility. Sperm morphology and DFI were analyzed before and after the sperm preparation. Results Sperm abnormal morphology and DFI in the infertility group were higher than those in the fertile group ,but significantly decreased after sperm preparation. The method of density gradient separation results in obtaining better sperm(P<0.01). Conclusions Compared to the method of swimming up ,the method of density gradient separation could result in obtaining sperm with improved normal morphology and DNA integrity.

Glucocorticoid receptor (GR) is identified as a member of nuclear receptor family. To exert its biological action, the ligand bound GR is translocated from the cytoplasm into the nucleus by regulating transcriptional signals of related genes. In clinical practice, the effects of glucocorticoid are often mediated by GR signaling pathways. An increasing number of studies have indicated that GR signaling pathways play an essential role in the proliferation, invasion and prognosis of bladder cancer. Meanwhile, the new-generation selective GR activator improves its anti-tumor effects, and at the same time reduces the adverse reactions of hormones, which probably raises the prospect for the treatment of bladder cancer.
Animals ; Antineoplastic Agents ; pharmacology ; Cell Nucleus ; genetics ; Humans ; Prognosis ; Protein Transport ; genetics ; Receptors, Glucocorticoid ; agonists ; physiology ; Signal Transduction ; genetics ; Transcriptional Activation ; drug effects ; physiology ; Urinary Bladder Neoplasms ; genetics ; physiopathology

OBJECTIVETo establish an accurate, fast and simple screening method for AZF microdeletions using capillary technology and use it for clinical testing.

METHODSFor each pair of primers, the 5' end of either forward or reverse primer was labeled with a FAM, JOE or TAMRA fluorescence dyes to establish multiplex quantitative fluorescence PCR systems for the establishment of a screening method of Y chromosome AZF microdeletions by capillary technology. The detection of Y chromosome AZF microdeletion was carried out on 725 cases of non-obstructive azoospermia, oligospermia or asthenospermia.

RESULTSA screening method for Y chromosome AZF microdeletions using capillary technology was established. Thirty eight cases of AZF microdeletions were found among 725 cases of non-obstructive azoospermia, oligospermia or asthenospermia, which gave a deletion rate of 5.24%. Y chromosomal microdeletions were found in 8.62% of the azoospermia group, 6.75% of the oligozoospermic group, and 2.23% of the asthenospermia group.

CONCLUSIONAn accurate, fast and simple screening method of Y chromosome AZF microdeletions by capillary technology has been established, which may have an important clinical value.

Adult ; Azoospermia ; genetics ; Capillary Action ; Chromosome Deletion ; Chromosomes, Human, Y ; Humans ; Infertility, Male ; Male ; Multiplex Polymerase Chain Reaction ; Sex Chromosome Aberrations ; Sex Chromosome Disorders of Sex Development ; diagnosis

OBJECTIVETo explore the mechanism of Tuina for treatment of chronic fatigue syndrome.

METHODSA total of 90 patients were randomly divided into a Tuina group, a Taijiquan (take exercise) group and a Fluoxetine group, 30 cases in each group. They were treated with Tuina, Taijiquan and Fluoxetine, respectively. After a month, the therapeutic effects and the changes of malondialdehyde (MDA) content and the activity of serum superoxide dismutases (SOD) and serum glutathione peroxidase (GSH-Px) were ohserved.

RESULTSThe total effective rate of 93.3% (28/30) in the Tuina group was better than 80.0% (24/30) in the Taijiquan group and 73.3% (22/30) in the Fluoxetine group (both P < 0.05). After treatment, MDA contents in the three groups were all decreased (P < 0.01, P < 0.05), and the activity of SOD. GSH-Px in both the Tuina group and the Fluoxetine group were increased (P < 0.01, P < 0.05), and especially in the Tuina group with a significant difference as compared with the other two groups (P < 0.05, P < 0.01).

CONCLUSIONThe therapeutic effect of the Tuina group is superior to that of the Taijiquan group and the Fluoxetine group. Tuina can regulate oxygen free radicals metabolism and clean superfluous oxygen free radicals to alleviate fatigue, which may be one of the mechanisms of Tuina in treating chronic fatigue syndrome.

Adult ; Fatigue Syndrome, Chronic ; blood ; enzymology ; therapy ; Female ; Glutathione Peroxidase ; blood ; Humans ; Male ; Malondialdehyde ; blood ; Massage ; Middle Aged ; Oxygen ; Reactive Oxygen Species ; blood ; Superoxide Dismutase ; blood

Objective To investigate mechanism of chronic bacterial prostatitis (CBP) causes increased oxidative stress and oxidative damage in the patients. Methods Seventy CBP patients (CBPPs) sampled randomly, and seventy healthy adult volunteers(HAVs) sampled randomly, were enrolled in a case-control study, in which the levels of nitric oxide (NO), vitamin C (VC), vitamin E (VE), and (-carotene ((-CAR) in plasma, as well as the level of malondialdehyde (MDA), and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) in erythrocytes were determined by spectrophotometry. Results Compared with the average values of NO and MDA in the HAVs group((378?33)nmol/L和(28.6?4.4)nmol/g?Hb),the average values in the CBPPs group ((426?31)nmol/L and (34.7?4.7)nmol/g?Hb)were significantly increased, while VC, VE, ?-CAR, SOD, CAT, and GPX in the CBPPs group((47.4?11.6)?mol/L,(18.1?4.8)?mol/L,(1.4?0.4)?mol/L, (1 912?221)U/g?Hb, (254?67) K/g?Hb and (25.0?5.0) U/g?Hb) were significantly decreased compared with the average values in the HAVs group((55.1?13.4)?mol/L、(25.7?4.5)?mol/L、(1.7?0.5)?mol/L、(2 081?222)U/g?Hb、(294?77)K/g?Hb和(28.8?5.1)U/g?Hb)).The partial correlation analysis between the course of disease and the said each parameter for 70 CBPPs while controlling for the age suggested that with prolonged course of disease the values of NO and MDA were gradually increased (rNO=0.480 1,rMDA=0.436 4,P

【Objective】To investigate the effect of Anti-virus Capsule(AVC) on quality of life(QOL) of patients with recurrent genital herpes(RGH).【Methods】The universal QOL measurement scale of WHOQOL-BREF was used to evaluate the QOL status of 40 RGH patients before and after treatment.The recurrence rate and skin lesion healing time were also observed before and after treatment.【Results】After treatment,QOL was much improved,mean recurrence rate was decreased and skin lesion healing time was shortened,the difference being significant(P