Research progress on association between macrophages and ischemia-reperfusion injury / 器官移植

Ischemia-reperfusion injury (IRI) is an extremely complicated pathophysiological process, which may occur during the process of myocardial infarction, stroke, organ transplantation and temporary interruption of blood flow during surgery, etc. As key molecules of immune system, macrophages play a vital role in the pathogenesis of IRI. M1 macrophages are pro-inflammatory cells and participate in the elimination of pathogens. M2 macrophages exert anti-inflammatory effect and participate in tissue repair and remodeling and extracellular matrix remodeling. The balance between macrophage phenotypes is of significance for the outcome and treatment of IRI. This article reviewed the role of macrophages in IRI, including the balance between M1/M2 macrophage phenotype, the mechanism of infiltration and recruitment into different ischemic tissues. In addition, the potential therapeutic strategies of targeting macrophages during IRI were also discussed, aiming to provide reference for alleviating IRI and promoting tissue repair.

Buzhong Yiqitang Regulates Apoptosis via Nrf2/ROS Pathway to Alleviate Cisplatin Resistance in A549/DDP Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Buzhong Yiqitang-containing serum in alleviating the cisplatin resistance in human non-small cell lung cancer （A549/DDP） cells via regulating the nuclear factor E2-related factor 2 （Nrf2）/reactive oxygen species （ROS） signaling pathway. MethodThe serum containing Buzhong Yiqitang was prepared and A549/DDP cells were cultured and randomly grouped： blank （10% blank serum）， cisplatin （10% blank serum+20 mg·L-1 cisplatin）， Buzhong Yiqitang （10% Buzhong Yiqitang-containing serum+20 mg·L-1 cisplatin）， ML385 （10% blank serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， Buzhong Yiqitang+ML385 （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， tertiary butylhydroquinone （TBHQ） （10% blank serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）， and Buzhong Yiqitang+TBHQ （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）. The median inhibitory concentration （IC50） of cisplatin in each group was determined by the cell counting kit-8 （CCK-8） method and the resistance index （RI） was calculated. The apoptosis rate was detected by flow cytometry. The ROS content of each group was determined with the DCFH-DA fluorescence probe. Western blot was employed to determine the protein levels of Nrf2， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）， cytochrome C （Cyt C）， and B-cell lymphoma-2 （Bcl-2）. ResultCompared with those in the cisplatin group， the IC50 and RI of A549/DDP cells to cisplatin in Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups decreased （P˂0.05）. Compared with the blank group， the cisplatin， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups showed increased apoptosis rate of A549/DDP cells （P˂0.05）. Compared with the blank group， cisplatin promoted the expression of Nrf2 （P˂0.05）. Compared with the cisplatin group， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 inhibited the expression of Nrf2 （P<0.05）， elevated the ROS level （P˂0.05）， up-regulated the protein levels of cleaved Caspase-3 and Cyt C， and down-regulated the protein level of Bcl-2 （P<0.05）， which were the most significant in the Buzhong Yiqitang+ML385 group. Compared with the cisplatin group， the TBHQ group showed increased IC50 and RI of cisplatin （P<0.05）， decreased apoptosis rate of A549/DDP cells （P<0.05）， up-regulated protein levels of Nrf2 and Bcl-2 （P<0.05）， lowered level of ROS （P˂0.05）， and down-regulated protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. Compared with the TBHQ group， Buzhong Yiqitang+TBHQ decreased the IC50 and RI of cisplatin in A549/DDP cells （P<0.05）， increased the apoptosis rate （P<0.05）， down-regulated the protein levels of Nrf2 and Bcl-2 （P<0.05）， increased ROS （P˂0.05）， and up-regulated the protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. ConclusionBuzhong Yiqitang induced apoptosis by inhibiting Nrf2/ROS pathway to alleviate cisplatin resistance in A549/DDP cells.

Hypoxia-induced ROS aggravate tumor progression through HIF-1α-SERPINE1 signaling in glioblastoma / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Hypoxia, as an important hallmark of the tumor microenvironment, is a major cause of oxidative stress and plays a central role in various malignant tumors, including glioblastoma. Elevated reactive oxygen species (ROS) in a hypoxic microenvironment promote glioblastoma progression; however, the underlying mechanism has not been clarified. Herein, we found that hypoxia promoted ROS production, and the proliferation, migration, and invasion of glioblastoma cells, while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI). Hypoxia-induced ROS activated hypoxia-inducible factor-1α (HIF-1α) signaling, which enhanced cell migration and invasion by epithelial-mesenchymal transition (EMT). Furthermore, the induction of serine protease inhibitor family E member 1 (SERPINE1) was ROS-dependent under hypoxia, and HIF-1α mediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region, thereby facilitating glioblastoma migration and invasion. Taken together, our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, and that targeting ROS may be a promising therapeutic strategy for glioblastoma.

PRE-084 ameliorated learning and memory impairment in T1DM via regulating neuronal MAM / 西安交通大学学报(医学版)

【Objective】 Diabetic mice could show learning and memory dysfunction, and we aimed to investigate the effect of Sigma-1 receptor agonist, PRE-084, on neurons and cognitive impairment in mice with type 1 diabetes (T1DM). 【Methods】 Twenty mice with T1DM induced by streptozocin, aged 8-10 weeks, and 20 control mice (CON) were randomly divided into four groups (CON+Vehicle, CON+PRE-084, T1DM+Vehicle and T1DM+PRE-084). Mouse primary neurons were cultured in high glucose medium with PRE-084 and control solvent, respectively. The body weight, food and water intake, and fasting blood glucose level of mice in each group were detected and recorded. The learning and memory abilities of mice were detected by new object recognition experiment. The mitochondria-associated endoplasmic reticulum membrane (MAM) structure of neurons in hippocampal CA1 area of mice was detected by transmission electron microscope. And the expression levels of ATP and reactive oxygen species (ROS) in hippocampus of mice were detected by biochemical kit. Cell viability and ROS level of primary neurons were detected by CCK8 and cellular ROS kit. 【Results】 PRE-084 reduced the increase of body weight, food and water intake, and blood glucose caused by diabetes. PRE-084 significantly ameliorated the learning and memory impairment of the mice with T1DM, improved the changes of MAM structure in neurons of hippocampal CA1 area of diabetic mice, increased the level of ATP in hippocampus of diabetic mice, and decreased the increase of ROS expression in diabetic hippocampus and neurons under high glucose conditions. 【Conclusion】 Sigma-1 receptor agonist, PRE-084, could improve learning and memory impairment in the mice with T1DM, which might be related to the structural changes of MAM, the increase of ATP production, and the decrease of ROS production in hippocampal neurons.

Effect of a disintegrin and metalloprotease 17 deletion on production of reactive oxygen species and mitochondrial function in nasopharyngeal carcinoma cells / 中国生物制品学杂志

@#Objective To investigate the effects of a disintegrin and metalloproteinase 17(ADAM17) deletion on the production of reactive oxygen species(ROS) and mitochondrial function in nasopharyngeal carcinoma(NPC) cells.Methods Three groups of ADAM1 7 interfering plasmid ADAM17 shRNA and empty plasmid ADAM17-shRNA-NC were transfected into NPC cell line(CNE1) and detected for the interference efficiency by RT-PCR and Western blot to select shRNA with the best interference effect for the follow-up experiments.The cell proliferation was detected by CCK-8 assay,while the cell growth by clone formation test,the apoptosis and changes in mitochondrial membrane potential(MMP) by flow cytometry,the level of mitochondrial oxidative damage product ROS by fluorescence microscope,the contents of oxidative stress markers MDA and SOD by malondialdehyde(MDA) kit and superoxide dismutase(SOD) kit and the expression of mitochondrial damage markers Bax/Bcl-2,cleaved-caspase 9/caspase 9,cleaved-caspase 3/caspase 3 and c-Myc by Western blot.Results ADAM17-shRNA2 group showed the best interference effect.Compared with shRNA-NC group,the proliferation rate of cell in ADAM17-shRNA 2 group decreased significantly(t=8.964,P=0.036);the number of colonies were significantly reduced(t=10.351,P=0.014);the number of apoptosis increased significantly(t=11.25,P=0.008);the fluorescence intensity representing ROS level in cells increased obviously;the mitochondrial membrane potential decreased significantly(t=9.233,P=0.013);the SOD content decreased(t=7.233,P=0.034) and MDA content increased(t=7.415,P=0.038) significantly;the levels of Bax/Bcl-2,cleaved-caspase 9/caspase 9 and cleaved-caspase 3/caspase 3 significantly increased(t=8.985,9.021 and 7.789,P=0.023,0.011 and 0.031,respectively),while the expression of c-Myc proteins significantly decreased(t=10.352,P=0.004).Conclusion Interfering with ADAM1 7 induced SOD decrease and MDA increase by promoting oxidation,thereby alleviating oxidative damage of cell membrane,which also promoted the expression level of ROS in mitochondrion,reduced MMP,inhibited cell proliferation in vitro,and promoted apoptosis.

AAZ2 induces mitochondrial-dependent apoptosis by targeting PDK1 in gastric cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Drastic surges in intracellular reactive oxygen species (ROS) induce cell apoptosis, while most chemotherapy drugs lead to the accumulation of ROS. Here, we constructed an organic compound, arsenical N-‍(4-(1,3,2-dithiarsinan-2-yl)phenyl)acrylamide (AAZ2), which could prompt the ROS to trigger mitochondrial-dependent apoptosis in gastric cancer (GC). Mechanistically, by targeting pyruvate dehydrogenase kinase 1 (PDK1), AAZ2 caused metabolism alteration and the imbalance of redox homeostasis, followed by the inhibition of phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and leading to the activation of B-cell lymphoma 2 (Bcl2)/Bcl2-associated X (Bax)/caspase-9 (Cas9)/Cas3 cascades. Importantly, our in vivo data demonstrated that AAZ2 could inhibit the growth of GC xenograft. Overall, our data suggested that AAZ2 could contribute to metabolic abnormalities, leading to mitochondrial-dependent apoptosis by targeting PDK1 in GC.

A Comparative Investigation of the Activity of Superoxide Dismutase in Patients with Leukoplakia and Healthy Controls

Context and Aim: Oral leukoplakia has recently been re?defined as “a predominantly white lesion of the oral mucosa that cannot be characterised as any other definable lesion”. Superoxide dismutase (SOD) is one of the enzymatic anti?oxidants which form the first line of defence in the cell. There are very limited data available on the analysis of SOD in leukoplakia patients. Therefore, the present study was planned to analyse anti?oxidant SOD levels in leukoplakia patients. Methods and Material: The study group consisted of 29 subjects of oral leukoplakia, and the control group consisted of 25 healthy individuals. All the subjects were evaluated for SOD enzyme in plasma. Statistical Analysis Used: The results for each determinant were calculated using one?way ANOVA test, Student ‘t’ test, Pearson Chi square test, and ‘t’ test for equality of means wherever applicable. Results: The study revealed that among the different clinical entities of oral leukoplakia, the enzymatic anti?oxidant SOD is shown to have decreased in all forms of oral leukoplakia, but without any statistical significance. A statistically highly significant decrease (at P < 0.0001) of SOD in oral leukoplakia patients compared to normal healthy patients was observed in the present study. Conclusions: The study revealed a difference in the free?radical activity and oxidative stress in blood of leukoplakia patients compared to healthy patients, which is reflected by the variation in the levels of blood SOD, thus

Research progress on the role of oxidative stress in ischemia-reperfusion injury of marginal donor liver transplantation / 器官移植

Common marginal donor liver mainly consists of fatty donor liver, elderly donor liver, small volume donor liver and liver graft from donation after cardiac death (DCD), etc. The application of marginal donor liver may resolve the severe shortage of donor liver to certain extent. Nevertheless, marginal donor liver yields a higher risk of ischemia-reperfusion injury (IRI) and causes more severe IRI than normal donor liver, which is a main cause for the failure of transplantation. In addition, oxidative stress is a major risk factor causing IRI of marginal donor liver. Therefore, how to mitigate oxidative stress and alleviate IRI of marginal donor liver has become a hot spot in clinical practice. Reactive oxygen species (ROS)-mediated oxidative stress occurs throughout the whole process of IRI. In this article, the role of oxidative stress in IRI of marginal donor liver transplantation and the ROS-targeted prevention and treatment were reviewed, aiming to provide reference for clinical practice.

Protective Mechanism of Youguiwan through AOPPs Regulating RAGE/ROS/NF-κB Axis and Wnt/β-catenin Signal in Rats with Adriamycin-induced Nephrotic Syndrome / 中国实验方剂学杂志

ObjectiveTo explore the effect of Youguiwan on the rats with adriamycin-induced nephrotic syndrome （NS） and its mechanism. MethodSD rats were randomly divided into a normal group， a model group， three Youguiwan low， medium， and high-dose groups， and a prednisone group. Rats in the model group were intravenously injected with adriamycin in the tail vein to induce the NS model. Rats in the Youguiwan low， medium， and high-dose groups were given 2.8， 5.6， 11.2 g·kg-1·d-1 of crude drugs， respectively， and rats in the prednisone group were given 6.3 mg·kg-1·d-1 of prednisone acetate. Each administration group was given continuous medicine for 6 weeks， and the normal group and model group were given an equal volume of normal saline. Bicinchoninic acid （BCA） assay was used to detect 24 h urine protein （24 h UP）. Automatic biochemical analyzer was used to detect serum urea nitrogen （BUN）， creatinine （SCr）， albumin （ALB）， total cholesterol （TC）， and triglyceride （TG） levels. Hematoxylin-eosin （HE） staining was used to observe renal tissue morphology， and kit was used to detect serum advanced oxidized protein products （AOPPs） and reactive oxygen species （ROS）. Western blot was used to detect the receptor of advanced glycation endproducts （RAGE） of renal tissue， nuclear factor-κB （NF-κB） phosphorylation levels， Wnt， and β-catenin protein expression. ResultAs compared with the normal group， 24 h UP， serum BUN， SCr， TC， TG， AOPPs， and ROS levels in the model group increased significantly （P<0.01）， whereas ALB decreased （P<0.01）. There were typical pathological injuries in the renal tissue， and the expressions of RAGE， phosphorylation（p）-NF-κB， Wnt1， and β-catenin protein were significantly increased （P<0.01）. As compared with the model group， the 24 h UP， serum BUN， SCr， TC， TG， AOPPs， and ROS levels of rats in the Youguiwan low， medium， and high-dose groups significantly reduced （P<0.01）， and ALB significantly increased （P<0.01）. The renal tissue damage was reduced， and the expressions of RAGE， p-NF-κB， Wnt1， and β-catenin protein were significantly decreased （P<0.01） in a dose-dependent manner. ConclusionYouguiwan improves the kidney injury of rats with adriamycin-induced NS. The mechanism may be related to the reduction of AOPPs level， inhibition of RAGE/ROS/NF-κB axis， and activation of Wnt/β-catenin signal.

Roles of neutrophil reactive oxygen species (ROS) generation in organ function impairment in sepsis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Sepsis is a condition of severe organ failure caused by the maladaptive response of the host to an infection. It is a severe complication affecting critically ill patients, which can progress to severe sepsis, septic shock, and ultimately death. As a vital part of the human innate immune system, neutrophils are essential in resisting pathogen invasion, infection, and immune surveillance. Neutrophil-produced reactive oxygen species (ROS) play a pivotal role in organ dysfunction related to sepsis. In recent years, ROS have received a lot of attention as a major cause of sepsis, which can progress to severe sepsis and septic shock. This paper reviews the existing knowledge on the production mechanism of neutrophil ROS in human organ function impairment because of sepsis.

Molecular hydrogen is a promising therapeutic agent for pulmonary disease / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Molecular hydrogen exerts biological effects on nearly all organs. It has anti-oxidative, anti-inflammatory, and anti-aging effects and contributes to the regulation of autophagy and cell death. As the primary organ for gas exchange, the lungs are constantly exposed to various harmful environmental irritants. Short- or long-term exposure to these harmful substances often results in lung injury, causing respiratory and lung diseases. Acute and chronic respiratory diseases have high rates of morbidity and mortality and have become a major public health concern worldwide. For example, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. An increasing number of studies have revealed that hydrogen may protect the lungs from diverse diseases, including acute lung injury, chronic obstructive pulmonary disease, asthma, lung cancer, pulmonary arterial hypertension, and pulmonary fibrosis. In this review, we highlight the multiple functions of hydrogen and the mechanisms underlying its protective effects in various lung diseases, with a focus on its roles in disease pathogenesis and clinical significance.

Polyphenols from Securidaca inappendiculata alleviated acute lung injury in rats by inhibiting oxidative stress sensitive pathways / 中草药·英文版

Objective: Securidaca inappendiculata is a medicinal plant frequently used in the treatment of inflammatory diseases in south China. In this study, we aimed to explore its bioactive constituent which contributes to the anti-inflammatory activity. Methods: Polyphenol-enriched and polyphenol-deprived fractions (PRF and PDF, respectively) were separated from the ethanolic extract by HPD300 macroporous resin-based method, and their anti-inflammatory activities were investigated on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model in rats. The possible mechanism of action in alleviating acute inflammation was studied using RAW264.7 cells. Results: Both Folin-Ciocalteu and

Mechanism of human umbilical cord mesenchymal stem cells alleviating ischemia-reperfusion injury of hepatocytes through mitochondrial transfer / 器官移植

Objective To explore the mechanism of human umbilical cord mesenchymal stem cell (HUC-MSC) alleviating ischemia-reperfusion injury (IRI) of liver cells through mitochondrial transfer. Methods Normal human liver cell line L02 was divided into the blank control group, oxygen-glucose deprivation (OGD) group, experimental control group, and L02 and HUC-MSC co-culture group (L02+HUC-MSC group). L02+HUC-MSC group was further divided into 10:1 co-culture subgroup (group A), 4:1 co-culture subgroup (group B), 2:1 co-culture subgroup (group C), 1:1co-culture subgroup (group D) and 1:2 co-culture subgroup (group E) according to different co-culture ratio of L02 and HUC-MSC. The apoptosis rate and relative reactive oxygen species (ROS) level of L02 cells were detected by flow cytometry. The MitoTracker positive rate of L02 cells was detected by flow cytometry. The mitochondrial transfer from HUC-MSC to L02 cells was observed by laser confocal microscope. Results The apoptosis rate and relative ROS level of L02 cells in the OGD group were significantly higher than those in the blank control group (both P < 0.05). Compared with the OGD group, the apoptosis rates of L02 cells in group B, C, D and E were significantly decreased (all P < 0.05), and the relative ROS level of L02 cells in group E was significantly declined (P < 0.05). The MitoTracker positive rate of L02 cells did not significantly differ between group A and experimental control group (P>0.05), whereas the MitoTracker positive rates of L02 cells in group B, C, D and E were significantly higher than that in the experimental control group in a concentration-dependent manner (all P < 0.05). Under the laser confocal microscope, mitochondrial transfer fromHUC-MSC to L02 cells could be observed through tunneling nanotube (TNT). Conclusions HUC-MSC may alleviate cell apoptosis and reduce ROS level of liver cells after IRI via direct mitochondrial transfer between cells.

Effect of Dihydroartemisinin on Oxidative Damage and Energy Metabolism of HepG2 Cells and Its Synergistic Effect with Sorafenib / 中国实验方剂学杂志

Objective:To explore the inhibitory effect of dihydroartemisinin （DHA） on the proliferation of HepG2 cells， elucidate the mechanism from the perspectives of oxidative damage and energy metabolism， and discuss the possibility of combined use of DHA with sorafenib （Sora）. Method:Cell counting kit-8 （CCK-8） assay was used to obtain the 50% inhibitory concentration （IC₅₀） of DHA and Sora on HepG2 and SW480 cells and Chou-Talalay method was used to obtain the combination index （CI） of DHA and Sora. HepG2 cells were classified into the control group， DHA group （10 µmol·L^-1）， Sora group （5 µmol·L^-1）， and DHA + Sora group （DHA 10 µmol·L^-1， Sora 5 µmol·L^-1） and then incubated with corresponding drugs for 8-12 h. Seahorse XF glycolytic rate assay kit and cell mito stress test kit were employed to respectively detect the glycolysis function of cells and oxidative phosphorylation function of mitochondria. DCFH-DA and lipid peroxidation MDA assay kit were separately used to analyze the intracellular levels of reactive oxygen species （ROS） and malondialdehyde （MDA）. Western blot was applied to determine the intracellular levels of heme oxygenase-1 （HO-1） and glutamate-cysteine ligase catalytic subunit （GCLC）. Result:Compared with the control group， DHA alone inhibited the ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis （<italic>P</italic><0.01）， increased the levels of intracellular ROS and MDA （<italic>P<</italic>0.05）， and decreased the levels of HO-1 and GCLC （<italic>P<</italic>0.05） in HepG2 cells. DHA and Sora had synergistic inhibitory effect on proliferation of HepG2 and SW480 cells， with CI < 0.90. The DHA + Sora group showed stronger suppression of ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis （<italic>P</italic><0.01）， higher levels of intracellular ROS and MDA （<italic>P<</italic>0.01）， and lower levels of intracellular antioxidation-related proteins HO-1 and GCLC in HepG2 cells （<italic>P<</italic>0.01） than the DHA group. Conclusion:DHA may increase the level of MDA by reducing HO-1 and GCLC and increasing ROS in HepG2 cells， which results in mitochondria oxidative damage， restricts cell glycolysis and mitochondrial oxidative phosphorylation， and thus finally inhibits the proliferation of HepG2 cells. DHA and Sora have synergistic inhibitory effect on the proliferation of HepG2 and SW480 cells， and the mechanism may be related to the synergistic oxidative damage that affects the mitochondrial electron transport chain and suppresses cell energy metabolism.

Effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma Extract on HUVECs Senescence Mediated by Endothelial Microparticles / 中国实验方剂学杂志

Objective:To investigate the effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract on endothelial microparticles （EMPs）-induced vascular endothelial cell senescence， and explore the possible mechanism. Method:Human umbilical vein endothelial cells （HUVECs） were used as the research objects， and the aged model was established with 10-12 passages of replicative senescence cells. The experimental cells were divided into young group （2-4 passage cells）， aged group （10-12 passage cells）， only EMPs intervention group （extract EMPs produced by aged cells to intervene young cells） and low dose， middle dose and high dose drug intervention groups （200， 300， 400 mg·L^-1）. Senescence related <italic>β</italic>-galactosidase （SA-<italic>β</italic>-gal） staining and cell cycle propidium iodide （PI） staining were used to determine cell senescence. Cell counting kit-8 （CCK-8） assay was used to screen the drug concentration. EMPs were extracted by two-step centrifugation， EMPs labeled with phycoerythrin （PE） anti-human CD31 antibody or fluorescein isothiocyanate （FITC） annexin V were detected by flow cytometry， intracellular reactive oxygen species （ROS） were detected by 2'，7'- dichlorofluorescein diacetate （DCFDA） staining. Result:After treatment with the drug， SA-<italic>β</italic>-gal activity of the aged cells significantly decreased （<italic>P</italic><0.01）， the S phase arrest was restored （<italic>P</italic><0.01）， and the number of CD31⁺ EMPs and annexin V⁺ EMPs secreted by aged cells decreased （<italic>P</italic><0.05）. Compared with the young group， only EMPs intervention group could induce increased SA-<italic>β</italic>-gal activity and S phase arrest in young cells （<italic>P</italic><0.05，<italic>P</italic><0.01）. However， after intervention of EMPs and the drug， EMPs-mediated increase of SA-<italic>β</italic>-gal activity was significantly inhibited and S phase arrest was restored （<italic>P</italic><0.05）. The increase of intracellular ROS induced by EMPs was also significantly inhibited by the drug （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract can delay the senescence of vascular endothelial cells by influencing EMPs， and the mechanism may be related to the inhibition of increased intracellular ROS induced by EMPs.

Study of impact of Wolbachia surface protein on reactive oxygen species production in HepG2 cell line exposed to ethanol

Objective: To study the impact of Wolbachia surface protein (WSP) on reactive oxygen species (ROS) level inethanol (EtOH)-exposed HepG2 cells.Materials and Methods: Increase in ROS level was induced in HepG2 cells by subjecting HepG2 cells toEtOH exposure. Impact of WSP on ROS level was examined by staining of intracellular ROS in cells usingthe specific ROS-detecting dye 2ʹ, 7ʹ-dichlorodihydrofluorescein diacetate (H2DCFDA), followed by flowcytometric analysis.Results and Conclusion: Flow cytometry analysis using H2DCFDA-based staining study of ROS level inHepG2 cells revealed that EtOH caused oxidative stress in HepG2 cells by inducing production of high levelsof ROS. However, EtOH-induced increased ROS production in cells decreased with treatment of WSP.From the current study, we can culminate that WSP provides cytoprotective action against EtOH-inducedincreased ROS production and oxidative stress in HepG2 cells by decreasing ROS production. This will beof significance for the treatment of EtOH-related liver ailments. Thus, this article emphasizes that WSP withprotecting ability could be used as a powerful therapeutic drug to treat EtOH-related liver ailments.

Passion fruit (Passiflora edulis) leaf extract modulates the oxidative metabolism of rat peritoneal neutrophils in a model of inflammation

This study was conducted to evaluate the effect of extracts of Passiflora edulis Sims leaves on the oxidative metabolism of rat peritoneal neutrophils using a model of acute inflammation. The extract was obtained by maceration in 70% ethanol, evaporation under reduced pressure and lyophilisation. Total phenolic content (TP) was determined by the Folin-Ciocalteu assay. The P. edulis extract, in different doses, was administered by gavage 1 h prior to inflammation induction by carrageenan (8 mg/kg, i.p.); five hours later, the neutrophils were obtained by intraperitoneal lavage. The tests performed in neutrophils were cytochrome C and chemiluminescence assay as well as myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) activities. The administration of the extract reduced the number of neutrophils recruited to the site of inflammation; however, the extract did not alter the activity of NADPH oxidase as well as SOD activity in these cells. The MPO and CAT activities in peritoneal neutrophils of rat treated with extract was lower than in the control group, and the GPx activity was increased. Based on the experimental model utilised, the anti-inflammatory potential of P. edulis leaf extract could be related to the presence of phenolic compounds in the extract.

Selenoprotein thioredoxin reductase mediated menadione reduction: catalytic properties & inhibition effects / 生物工程学报

Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec⁴⁹⁸ is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys⁴⁹⁸ is mainly responsible for menadione catalysis in comparison to Cys⁴⁹⁷, while the N-terminal Cys⁶⁴ is slightly stronger than Cys⁵⁹ regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.

Effect of Qingzao Jiufei Tang on Enzymatic Activity and Regulatory Factor of Key Enzyme G6PD in Pentose Phosphate Energy Metabolism Pathway in Lung Cancer / 中国实验方剂学杂志

Objective::To discuss the effect of Qingzao Jiufei Tang on enzymatic activity and regulatory factor of glucose 6-phosphatedehydrogenase(G6PD) in pentose phosphate energy metabolism pathway in lung cancer. Method::Fifty male C57BL6J mice were randomly divided into five groups. Animal models were induced through axillary injection with Lewis cells. The Qingzao Jiufei Tang group was given drugs (11, 5.5, 2.8 g·kg-1·d-1) two weeks before modeling, the cyclophosphamide(CTX) group was intraperitoneally injected with CTX (50 mg·kg-1), and the model group was intraperitoneally injected with the same volume of saline after molding. At 14 d after modeling, the mice were sacrificed, and the tumor tissues were collected. The enzymatic activity of G6PD, content of reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay(ELISA) method. Expressions of gp91phox and p22phox mRNA were detected by real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) method. Result::Compared with the model group, the enzymatic activity of G6PD in high-dose group, medium-dose group and low-dose group were reduced obviously (P<0.05, P<0.01). Content of ROS, mRNA expressions of gp91phox and p22phox in high-dose group, medium-dose group and low-dose group were reduced obviously (P<0.01). Conclusion::Qingzao Jiufei Tang may inhibit the expression of G6PD by inhibiting the expression of gp91 phox, p22phox oxidase, and then reduce content of ROS, so as to reduce the energy metabolism and hyperplasia of lung cancer cells.

Inhibitory Effect of Magnolol Analogue CT2-3 on Colon Cancer Cells and Its Mechanism / 中国实验方剂学杂志

Objective:To study the anti-colon cancer effect and mechanism of magnolol analogue CT2-3， in order to lay a foundation for the application of CT2-3 in anti-colon cancer area. Method:Colon cancer cells SW480 and LoVo were cultured in vitro. Different concentrations （10， 20， 40， 80 μmol·L-1） of CT2-3 and magnolol were used to stimulate colon cancer cells for 24， 48 h. The effect of CT2-3 and magnolol on the cell viability of colon cancer cells was detected by cell counting kit （CCK-8）. Colony formation assay was used to detect the colony formation capacity of CT2-3 on colon cancer cells. Flow cytometry and Western blot were used to determine the effect of CT2-3 on the apoptosis of colon cancer cells and the expression of DNA damage marker phosphorylated histone H2AX （γH2AX）. Reactive oxygen species （ROS） generation was measured by ROS assay kit. Real time quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of CT2-3 on expressions of mitochondrial apoptosis-related genes B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X （Bax） in colon cancer cells. Result:The half maximal inhibitory concentration （IC50） of magnolol in two kinds of colon cancer cells after treatment for 24， 48 h were both higher than 80 μmol·L-1. While the IC50 of CT2-3 in SW480 cells after treatment for 24， 48 h were （54.59±1.73） μmol·L-1 and （29.82±1.13） μmol·L-1， respectively. The IC50 of CT2-3 in LoVo cells after treatment for 24，48 h were （66.68±2.11） μmol·L-1 and （46.70±1.81） μmol·L-1， respectively. Compared with the blank group， the colony formation capacity of colon cancer cells in CT2-3 groups （20， 40 μmol·L-1） was significantly decreased in a dose-dependent manner （P<0.01）， apoptotic colon cancer cells were significantly increased （P<0.01）， relative expression of DNA damage marker γH2AX was significantly increased （P<0.01）， ROS was significantly increased （P<0.01）. In addition， relative mRNA expression of Bcl-2 was significantly decreased （P<0.01）， while relative mRNA expression of Bax was significantly increased （P<0.01）. Conclusion:CT2-3 can remarkably inhibit colon cancer cells， and the underlying mechanism might be that CT2-3 promotes mitochondria dysfunction and ROS generation by regulating expressions of mitochondrial apoptosis-related genes， so as to further induce DNA damage and finally lead to apoptosis.