Objective To observe the eotaxin expression of rat airway smooth muscle cells ( ASMCs) induced by serum from asthmatic rats, and explore the possible mechanism. Methods ASMCs isolated fromrat tracheas were cultured in vivo. Then they were treated with serum from asthmatic rats, or treated with serum and dexamethasone simultaneously. The level of eotaxin protein in supernatant and eotaxin mRNA in ASMCs were measured by ELISA and reverse transcription-polymerase chain reaction. The expression of cAMP in ASMCs was examined by radioimmunoassay. Results After the treatment with sensitized serum, the eotaxin level in supernatant and mRNA expression in ASMCs were significantly higher [ ( 107. 09 ±7. 12) ng/L vs. ( 0. 63 ±0. 56) ng/L, P lt; 0. 05; 1. 39 ±0. 04 vs. 0. 05 ±0. 01, P lt;0. 05] , and the level of cAMP in ASMCs was significantly lower compared with the control group [ ( 17. 58 ±3. 62) ng/L vs. ( 32. 39 ±3. 36) ng/L, P lt; 0. 05] . After intervened by the sensitized serum and dexamethasone simultaneously, the protein and mRNA expressions of eotaxin were lower compared with those intervened by sensitized serumalone [ ( 64. 18 ±4. 04) ng/L and 0. 77 ±0. 19] . The level of eotaxin in supernatant was negatively correlated with cAMP level in ASMCs ( r = - 0. 788, P lt; 0. 01) . Conclusions There is anautocrine function in ASMCs as inflammatory cells after stimulation with sensitized serum. Eotaxin may play an important roll in the pathogenesis of asthma via a cAMP-dependent pathway.
Objective To investigate the effect of S-adenosyl-l-methionine (SAM) on oxidative stress and alveolar septal cell apoptosis in mice with emphysema after smoking cessation. Methods Twenty-two male SPF C57BL/6J mice aged 6 - 8 weeks were randomly divided into 4 groups, ie. a healthy control group, an emphysema group, a smoking cessation group, and a SAM intervention for 8 weeks after smoking cessation group, with 8 mice in each group. The mice model of emphysema was established by intraperitoneal injection of cigarette smoke extract (CSE) combined with cigarette smoke exposure. Smoking cessation started after the emphysema model was successfully constructed and lasted for 8 weeks. After smoking cessation, the mice in SAM intervention groups were intraperitoneally injected with SAM mg·kg–1·d–1 for 8 weeks. The right lung sections of the mice were taken for hematoxylin-eosin staining to observe pathological changes, and the mean linea rintercept (MLI) and mean alveola rnumber (MAN) of lungs were measured. The concentrations of malondialdehyde (MDA), superoxide-dismutase (SOD) and glutathione (GSH) in alveolar lavage fluid of left lung were detected by spectrophotometry. Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) technique was carried out to detect the alveolar septal cells apoptosis. Results MLI, apoptosis index of alveolar septal cell and MDA concentration in bronchoalveolar lavage fluid (BALF) increased significantly in the emphysema group compared with healthy controls, increased significantly in the smoking cessation group compared with the emphysema group, and decreased in the SAM intervention group compared with the smoking cessation group (all P<0.05). GSH concentration and SOD activity in BALF and MAN was significantly lower in the emphysema group compared with the healthy control group, significantly lower in the smoking cessation group compared with the emphysema group, and significantly higher in the SAM intervention group compared with the smoking cessation group (all P<0.05). Conclusions Lung histopathology and apoptosis of alveolar septal cells in emphysema mice progress continuously after smoking cessation. SAM may reduce oxidative stress and improve apoptosis of alveolar septal cells, so as to protect emphysema mice after smoking cessation.
Objective To assess the effectiveness and safety of meglumine adenosine cyclophosphate (MAC) for chronic heart failure. Methods The databases such as Cochrane Central Register of Controlled Trials (Issue 3, 2011), MEDLINE (1950 to March 2011), EMbase (1980 to March 2011), CNKI (1995 to March 2011), and VIP (1989 to March 2011) were searched, and the relevant journals and conference proceedings were also manually retrieved. Then the studies were screened according to predefined inclusion and exclusion criteria, and their quality was evaluated. Meta-analyses were performed by using RevMan 5.0 software. Results Seventeen randomized controlled trials (RCTs) involving 1 281 patients were included. All of the included RCTs were Grade C in methodological quality. The results of meta-analyses showed that MAC plus routine treatment was superior to routine treatment in improving the left ventricular ejection fraction (WMD=5.75, 95%CI 3.61 to 7.89), stroke volume (SV) (WMD=5.55, 95%CI 3.71 to 7.38), E/A (WMD=0.09, 95%CI 0.05 to 0.14) and 6 min walk test (WMD=43.52, 95%CI 21.00 to 66.04). But MAC plus routine treatment was similar to routine treatment in regulating cardiac index (CO) (WMD=0.20, 95%CI –0.31 to 0.71) and heart rate (WMD=0.64, 95%CI –7.49 to 8.77). No significant adverse effects or allergic reactions were reported. Conclusion The current evidence shows that MAC may improve the left ventricular ejection fraction, stroke volume, E/A and 6 min walk test. Due to a high risk of selection bias and detection bias in the included studies, the evidence is insufficient to determine the effectiveness of MCA. Further large-scale trials are required to define the role of MAC in the treatment of chronic heart failure.
ObjectiveTo explore the clinical value of age/pleural fluid adenosine deaminase (age/ADA) ratio and serum lactate dehydrogenase/pleural fluid adenosine deaminase ratio (Cancer Ratio, CR) in the diagnosis of malignant pleural effusions (MPE). MethodsThe study collected 44 patients with MPE and 48 patients with benign pleural effusion (BPE) to compare the differences in age, gender, carcinoembryonic antigen (CEA), age/ADA ratio and CR between the groups. The receiver operating characteristic (ROC) curve of CEA, age/ADA and CR was constructed and the area under the ROC curve (AUC), sensitivity and specificity was calculated to identify the diagnostic performance of the three indicators alone or in combination in MPE. ResultsCEA, age/ADA and CR were significant higher in the MPE group than those in the BPE group (all P<0.05), the AUCs of CEA, age/ADA and CR were 0.768, 0.837 and 0.866, respectively; the sensitivity was 61.36%, 88.64% and 81.82%, the specificity was 85.42%, 75.00%, 83.33%, respectively. The AUCs of CEA combined with age/ADA, CEA combined with CR, age/ADA combined with CR, CEA combined with age/ADA and CR were respectively 0.892, 0.911, 0.837 and 0.907; the sensitivity was 81.82%, 86.36%, 88.64% and 90.91%, the specificity was 79.17%, 79.17%, 75.00% and 77.08%, respectively. ConclusionsAge/ADA and CR demonstrated good diagnostic performance in MPE, moreover, the diagnostic performance can be further improved when combined with the traditional tumor marker CEA, and more research about its diagnostic value is needed in the future.
ObjectiveTo investigate the effects of overexpression of alpha/beta hydrolase domain-containing protein 5 (ABHD5) on the invasion and migration of human colon cancer cell line HCT116 and the pathway of adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR).MethodsThe expression of ABHD5 in colon cancer tissues and its relationship with clinicopathological features was analyzed by UALCAN database. HCT116 cells were cultured in vitro and transfected with ABHD5 recombinant plasmid, then they were divided into control group, negative transfection group and ABHD5 transfection group. Real time quantitative PCR (qRT-PCR) was used to detect the expression of ABHD5 mRNA in HCT116 cells. The proliferation of HCT116 cells was detected by CCK-8 method. Transwell assay was used to detect the invasion and migration of HCT116 cells. The expression of matrix metalloprotein 9 (MMP-9), E-cadherin, Snail, and AMPK/mTOR pathway proteins p-AMPK, AMPK, p-mTOR and mTOR were detected by Western blot.ResultsThe results of the UALCAN showed that compared with normal colon tissues, the expression of ABHD5 mRNA in colon cancer tissues was decreased (P<0.05), and which in the adenocarcinoma and the N1 stage was lower than that of the mucinous adenocarcinoma (P<0.05) and N0 stage (P<0.05), respectively. Compared with the control group and the negative transfection group, the expression of ABHD5 mRNA in the ABHD5 transfection group was increased (P<0.05), the proliferation inhibition rate of HCT116 cells in the ABHD5 transfection group was increased (P<0.05), the numbers of migration and invasion cells in the ABHD5 transfection group were decreased (P<0.05), the expressions of MMP-9, Snail, p-mTOR and mTOR were reduced, and the expressions of E-cadherin, p-AMPK and AMPK were increased (P<0.05).ConclusionsThe overexpression of ABHD5 can inhibit the invasion and migration of colon cancer HCT116 cells, activate AMPK, and inhibit the expression of mTOR. It suggests that ABHD5 may play a role in inhibiting colon cancer by affecting AMPK/mTOR pathway.
Objective To investigate the effects of sodium valproate (VPA) in inhibiting Erastin-induced ferroptosis in bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. Methods BMSCs were isolated from bone marrow of 8-week-old Spragur Dawley rats and identified [cell surface antigens CD90, CD44, and CD45 were analyzed by flow cytometry, and osteogenic and adipogenic differentiation abilities were assessed by alizarin red S (ARS) and oil red O staining, respectively]. Cells of passage 3 were used for the Erastin-induced ferroptosis model, with different concentrations of VPA for intervention. The optimal drug concentration was determined using the cell counting kit 8 assay. The experiment was divided into 4 groups: group A, cells were cultured in osteogenic induction medium for 24 hours; group B, cells were cultured in osteogenic induction medium containing optimal concentration Erastin for 24 hours; group C, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA for 24 hours; group D, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA, and 8 μmol/L EX527 for 24 hours. The mitochondrial state of the cells was evaluated, including the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS). Osteogenic capacity was assessed by alkaline phosphatase (ALP) activity and ARS staining. Western blot analysis was performed to detect the expressions of osteogenic-related proteins [Runt-related transcription factor 2 (RUNX2) and osteopontin (OPN)], ferroptosis-related proteins [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11)], and pathway-related proteins [adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1)]. Results The cultured cells were identified as BMSCs. VPA inhibited Erastin-induced ferroptosis and the decline of osteogenic ability in BMSCs, acting through the activation of the AMPK/SIRT1 pathway. VPA significantly reduced the levels of ROS and MDA in Erastin-treated BMSCs and significantly increased GSH levels. Additionally, the expression levels of ferroptosis-related proteins (GPX4, FTH1, and SLC7A11) significantly decreased. VPA also upregulated the expressions of osteogenic-related proteins (RUNX2 and OPN), enhanced mineralization and osteogenic differentiation, and increased the expressions of pathway-related proteins (AMPK and SIRT1). These effects could be reversed by the SIRT1 inhibitor EX527. ConclusionVPA inhibits ferroptosis in BMSCs through the AMPK/SIRT1 axis and promotes osteogenesis.
Objective To study the effects of morroniside (MOR) on the proliferation and osteogenic differentiation of mouse MC3T3-E1 cells. MethodsThe 4th generation MC3T3-E1 cells were randomly divided into 6 groups: control group (group A), MOR low dose group (10 μmol/L, group B), MOR medium-low dose group (20 μmol/L, group C), MOR medium dose group (40 μmol/L, group D), MOR medium-high dose group (80 μmol/L, group E), and MOR high dose group (100 μmol/L, group F). The proliferation activity of each group was detected by cell counting kit 8 (CCK-8) assay; the bone differentiation and mineralized nodule formation of each group were detected by alizarin red staining; real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect cyclin-dependent kinase inhibitor 1A (P21), recombinant Cyclin D1 (CCND1), proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP), collagen type Ⅰ (COL-1), bone morphogenetic protein 2 (BMP-2), and adenosine A2A receptor (A2AR) mRNA expressions; Western blot was used to detecte the expressions of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and adenosine A2AR protein. ResultsThe CCK-8 assay showed that the absorbance (A) values of groups B to F were significantly higher than that of group A at 24 hours of culture, with group C significantly higher than the rest of the groups (P<0.05). The MOR concentration (20 μmol/L) of group C was selected for the subsequent CCK-8 assay; the results showed that the A values of group C were significantly higher than those of group A at 24, 48, and 72 hours of culture (P<0.05). Alizarin red staining showed that orange-red mineralized nodules were visible in all groups and the number of mineralized nodules was significantly higher in groups B and C than in group A (P<0.05). RT-qPCR showed that the relative expressions of P21, CCND1, and PCNA mRNAs were significantly higher in group C than in group A (P<0.05). The expressions of ALP, BMP-2, COL-1, and adenosine A2AR mRNAs in groups B to E were significantly higher than those in group A, with the expressions of ALP, BMP-2, COL-1 mRNAs in group C significantly higher than the rest of the groups (P<0.05). Compared with group A, the expressions of OPN and RUNX2 proteins in groups B and C were significantly increased, while those in group D and E were significantly inhibited (P<0.05). There was no significant difference between groups B and C and between groups D and E (P>0.05). The relative expression of adenosine A2AR protein in groups B to E was significantly higher than that in group A, with group C significantly higher than the rest of the groups (P<0.05). Conclusion MOR can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells; the mechanism of MOR may be achieved by interacting with adenosine A2AR.
Objective To investigate the role of adenosine A2A receptor plays in retinal pathological neovascularization in mice. Methods A total of 202 mice were divided into room-air group (n=66) and oxygen induced retinopathy (OIR) group (n=136). Among room-air group, there were 18 A2A knock-out (KO) mice (KO subgroup) and 24 C57BL/6 mice as wide type (wide type subgroup). OIR group were divided into OIR control subgroup (n=48), A2A-OIR subgroup (n=24) and Caffeine-OIR subgroup (n=64). The retinal neovascularization of OIR group was induced by oxygen. The pathological neovascularization was determined by retinal sections. Fluorescent quantitative polymerase chain reaction (PCR) was used to measure the mRNA expression of A2A and vascular endothelial growth factor (VEGF). 0.1, 0.3, 1.0 g/L Caffeine was dissolve in drinking water of lactating females in Caffeine-OIR subgroup, non-perfusion areas of retina in mice at the age of 0 - 17, 0 - 7, 7- 17, 7-12, and 12- 17 days were analyzed in different dosage and when the dosage as 1.0 g/L. Results Compared with OIR control subgroup, the retinal non-perfusion areas and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in A2A- OIR subgroup were reduced significantly (t=7.694, 7.747;P＜0.001). Compared with wide type subgroup, the level of A2A and VEGF mRNA in OIR control subgroup increased significantly (t=4.036, 2.230;P＜0.05). Compared with OIR control subgroup, the level of VEGF mRNA in A2A- OIR subgroup decreased significantly (t=3.122,P＜0.01). Compared with OIR control subgroup, the retinal non-perfusion areas in mice at the dosage of 0.1 and 1.0 g/L (t=2.397, 4.533) and at the age of 0 -17, 0 -7 days when the dosage as 1.0 g/L (t=4.070, 2.399) were reduced significantly (P＜0.05). Conclusions The expression of adenosine A2A receptor increases in oxygen-induced retinal pathological neovascularization. Adenosine A2A receptor may regulate the expression of VEGF. A2A receptor inactivation can inhibit oxygen-induced retinal pathological neovascularization.
Objective To investigate the protective effects of ischemic postconditioning (IPo) on ischemiareperfusion (I/R) myocardium and the relationship with mitochondrial adenosine triphosphate (ATP) sensitive K+ channels (mitoKATP) and provide evidences to the development of druginduced postconditioning. Methods Langendorff models were established in 40 Wistar rats which were divided into 5 groups by random number table with 8 rats in each group. Normal control group(NC group): the rat hearts were continuously reperfused by KrebsHenseleit bicarbonate buffer (K-HB) for 100 min without any other treatment; I/R group: the rat hearts underwent a 40-min global ischemia followed by a 60-min reperfusion; IPo group: after a 40-min global ischemia, the process of 10-second reperfusion followed by a 10-second ischemia was repeated 6 times, then there was a continuous 58min reperfusion; 5-hydroxydecanoic acid(5-HD) group: after a 40min global ischemia, hearts with 5HD(100 μmol/L) K-HB were reperfused for 15min and then perfused without 5HD for 45min；IPo+5-HD group: after a 40-min global ischemia, the process that the isolated hearts with 5-HD(100 μmol/L) KHB were reperfused for 10second followed by a 10second ischemia was repeated 6 times, then the hearts with 5-HD(100 μmol/L) KHB were continuously [CM(159mm]perfused for 13-min followed by reperfusion without 5-HD(100 μmol/L) K-HB for 45-min. The cardiac function,coronary flow(CF), cardiac troponin I(cTnI) content in coronary effluent, the area of acute myocardial infarction (AMI) and myocardial ultrastructure were observed. Results Left ventricular developed pressure(74.3±3.3 mm Hg vs. 57.1±3.3 mm Hg,t=1300, P=0.000),+dp/dtmax(1 706.6±135.6 mm Hg/s vs. 1 313.3±96.2 mm Hg/s,t=6.28,P=0.000),－dp/dtmax(1 132.8±112.1 mm Hg/s vs. 575.7±67.7 mm Hg/s,t=13.48, P=0.000) and CF(6.49±0.30 ml/min vs. 3.70±0.24 ml/min,t=28.6,P=0.000) in IPo group were higher than those in I/R group. Left ventricular enddiastolic pressure(10.9±1.7mm Hg vs. 26.2±1.5 mm Hg,t=－19.21, P=0000)and cTnI content in coronary effluent (0.62±0.01 ng/ml vs. 0.71±0.01 ng/ml, t=-12.00,P=0.000) were lower than those in I/R group; the area of AMI decreased 20.8％ compared with that in I/R group (Plt;0.05). The myocardial protective effect in IPo+5HD group was similar with that in IPo group, but lower than that in IPo group. The electron microscope showed that IPo and IPo+5HD could reduce myocardial fiber damage and mitochondrial damage caused by I/R. Conclusion IPo can protect I/R myocardium, which is achieved mainly by activating mitoK-ATP channels.
Objective To compare the difference in the expressions of forkhead box protein 3 (FoxP3) and adenosine 2a receptor (A2aR) in gastric cancer tissues and its adjacent tissues, and to investigate the relationship between the elevated expression of FoxP3/A2aR and clinicopathological features in gastric cancer. Methods Gastric cancer tissues and their adjacent tissues from 52 patients with gastric cancer were collected, who underwent surgery in the Affiliated Hospital of Xuzhou Medical University from July 2015 to November 2016, immunohistochemical staining was used to detect the expressions of FoxP3 and A2aR. Results ① The high-expression rate of FoxP3 in gastric cancer tissues was 69.2% (36/52), which was higher than that of adjacent tissues (11.5%, 6/52), P<0.001. The high-expression rate of A2aR in gastric cancer tissues was 69.2% (36/52), which was higher than that of adjacent tissues (25.0%, 13/52),P<0.001. ② The expression of FoxP3 was positively correlated with the expression of A2aR in gastric cancer tissues (r=0.76, P<0.05). ③ In gastric cancer tissues, high-expressions of FoxP3 and A2aR were not related to gender, age, diameter of tumor, tumor location, degree of differentiation, gross type, and histological type (P>0.05), but both associated with TNM stage, T stage, number of lymph node metastasis, and distant metastasis (P<0.05), the high-expression rates of FoxP3 and A2aR in patients with stage Ⅲ+Ⅳ were higher than those of patients with stage Ⅰ+Ⅱ, the high-expression rates of FoxP3 and A2aR in patients with stage T3+T4 were higher than those of patients with stage T1+T2, the high-expression rates of FoxP3 and A2aR in patients with distant metastasis were higher than those of patients without distant metastasis, and the high-expression rates of FoxP3 and A2aR increased gradually with the increase in the number of lymph node metastasis. Conclusion There are high expressions of FoxP3 and A2aR in gastric cancer tissues, and both of them may play important role in promoting the occurrence and development of gastric cancer.