Mycoplasma pneumoniae induces IL-1β production through activating NLRP3 inflammasome by ROS in RAW264.7 cells

AIM: To investigate whether Mycoplasma pneumoniae (Mp)-induced interleukin-1β (IL-1β) production in RAW264.7 cells is through the activation of NLRP3 inflammasome via reactive oxygen species (ROS). ME-THODS: RAW264.7 cells were randomly divided into 3 groups. In normal group, RAW264.7 cells were treated without Mp. In model group, RAW264.7 cells were treated with 1∶ 10 multiplicity of infection (MOI) of Mp. In NAC group, RAW264.7 cells were pretreated with N- acetylcysteine (NAC) at a concentration of 5 mmol/L for 30 min before infection with Mp. The RAW264.7cells were infected with Mp (1∶ 10 MOI) for 4, 8, 16 and 24 h in model group and NAC group, respectively. The intracellular ROS level was analyzed by flow cytometry. The mRNA expressions of NLRP3, ASC and caspase-1 were detected by real-time PCR. The protein levels of NLRP3, ASC and caspase-1 p20 were determined by Western blot. The levels of pro-inflammatory cytokine IL-1β in the supernatant were measured by ELISA. RESULTS: Compared with normal group, the production of ROS were significantly increased at 4, 8, 16 and 24 h after infection, the mRNA expression of NLRP3, ASC and caspase-1 were increased at 8, 16 and 24 h after infection, the protein levels of NLRP3, ASC and caspase-1 p20 were increased at 16 and 24 h after infection, and the releases of IL-1β were increased at 24 h after infection in model group (P<0.01). Compared with the model group, the level of ROS in NAC group decreased, so as the expression of NLRP3, ASC and caspase-1 at mRNA and protein levels and the releases of IL-1β in the supernatant at the corresponding time points. CONCLUSION: Mp may stimulate the ROS production to activate NLRP3 inflammasome in RAW264.7 cells.     

Intervention effects of apyrase on silica-induced pulmonary fibrosis in mice

AIM: To investigate the effect of apyrase on the experimental silicosis. METHODS: C57BL/6 male mice were randomly divided into control group, silica treatment group, silica+apyrase group and silica+NS group. A mouse model of lung fibrosis was induced by crystalline silica particles (50 mg/kg, via oropharyngeal instillation), and were sacrificed at 3 h, 7 d, 14 d and 28 d. Apyrase was delivered by oropharyngeal aspiration at the same time and 4 h after silica challenge. The lung indexes were calculated and the concentration of ATP was detected by bioluminescent assay. The mRNA expression levels of collagen type Ⅰ(Col Ⅰ), collagen type Ⅲ (Col Ⅲ) and transforming growth factor β1 (TGF-β1) were examined by real-time PCR. The protein levels of TGF-β1 in bronchoalveolar lavage fluid were measured by ELISA. RESULTS: The elevated lung index and collagen levels showed that silicosis model was established successfully. Compared with silica group, apyrase treatment significantly alleviated silica-induced inflammation, reduced inflammation score on day 7, and decreased the lung index, collagen volume fraction and the mRNA expression of Col Ⅰand Col Ⅲ on day 28. Treatment with apyrase effectively down-regulated the mRNA levels of TGF-β1 in the lung tissues and TGF-β1 protein levels in bronchoalveolar lavage fluid on day 7.CONCLUSION: Apyrase attenuates the pulmonary inflammation and fibrosis of silicosis, which may be related with down-regulation of ATP and TGF-β1 in the lung tissues.     

甲基紫精滴注法快速衡量香蕉抗氧化能力的初步试验

 滴注在香蕉(Musa paradisiaca var.sapientum)叶片表面上的甲基紫精(Methyl Viologen, 0.25 mmol·L^-1 )液滴，经过50 μmol·m^-2 ·s^-1 光照10 h，可在滴注位点周围形成明显的褐色损伤斑点。通过测定香蕉叶片主要抗氧化酶SOD和CAT活性，发现褐斑直径与SOD和CAT活性呈明显的负相关，说明甲基紫精褐斑直径与香蕉抗氧化能力之间具有密切的关系。外源活性氧处理引起的叶片膜脂过氧化损伤与甲基紫精褐斑直径呈正相关，表明利用甲基紫精滴注法快速衡量香蕉抗氧化能力的可行性。     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Dihydroartemisinin enhances antitumor effect of 5-fluorouracil against gastric cancer by down-regulating SIRT1 expression

AIM: To investigate the effect of dihydroartemisinin (DHA) adjuvant treatment on enhancing the antitumor effect of 5-fluorouracil (5-FU) against gastric cancer. METHODS: The gastric cancer BGC-823 cells were divided into control group, DHA group, 5-FU group, 5-FU+DHA group and 5-FU+DHA+SIRT1 plasmid group. The viability of BGC-823 cells treated with DHA and 5-FU was measured by MTT assay. The expression of SIRT1 and NADPH oxidase, activation of caspase-9 and caspase-3, and phosphorylation of ASK1 and JNK in the BGC-823 cells treated with DHA and 5-FU were determined by Western blot. The production of ROS and the apoptosis of the BGC-823 cells treated with DHA and 5-FU were analyzed by flow cytometry. RESULTS: Dihydroartemisinin significantly inhibited the expression of SIRT1 and increased NADPH oxidase protein level (P<0.05). DHA increased the sensitivity of BGC-823 cells to 5-FU, thus decreasing the IC₅₀ of 5-FU to the gastric cancer cells. However, transfection with SIRT1 plasmid decreased the cytotoxicity of DHA and 5-FU co-treatment to the BGC-823 cells. DHA promoted the production of ROS and phosphorylation of ASK1 and JNK induced by 5-FU in the BGC-823 cells (P<0.05). However, ROS scavenger N-acetylcysteine (NAC) or JNK specific inhibitor SP600125 inhibited the cell death and activation of caspase-9 and caspase-3 induced by DHA and 5-FU co-treatment (P<0.05). In addition, NAC significantly inhibited the phosphorylation of JNK in the BGC-823 cells co-treated with DHA and 5-FU. However, treatment with SP600125 did not influence the ROS production in the BGC-823 cells, indicating that JNK was the downstream target of ROS pathway. CONCLUSION: Combination of DHA with 5-FU induces caspase-dependent apoptosis in gastric cancer cells through the SIRT1/NADPH oxidase/ROS/JNK signaling pathway.     

Inhibition of HMGB1 expression promotes apoptosis of hemangioma endothelial cells

AIM: To investigate the effect of inhibiting high-mobility group box protein 1 (HMGB1) expression on the viability and apoptosis of hemangioma endothelial cells (HemECs). METHODS: Human HemECs were isolated and cultured, and HMGB1 small interfering RNA (HMGB1-siRNA) was transfected into the cells. The cell viability was detected by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) content were analyzed by flow cytometry. The protein levels of HMGB1, NF-κB p65, p-IκBα, cyclin D1 and survivin were determined by Western blot. RESULTS: The protein expression of HMGB1 in the HemECs transfected with HMGB1-siRNA was significantly lower than that in blank control group (P<0.05). Compared with NC group, the cell viability was decreased significantly in the HemECs transfected with HMGB1-siRNA, the apoptotic rate was significantly increased, the content of ROS increased significantly, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were significantly decreased (P<0.05). After exposure to NF-κB signaling pathway inhibitor PDTC, the cell viability was inhibited, the apoptosis was increased, ROS content, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were down-regulated significantly, as compared with si-HMGB1 group (P<0.05). CONCLUSION: Inhibition of HMGB1 reduces the viability of HemECs and induces apoptosis by increasing the content of ROS and down-regulating the activity of NF-κB signaling pathway.     

Effects of silymarin on homocysteine-induced apoptosis in human umbilical vein endothelial cells

AIM:To investigate the effect of silymarin on homocysteine-induced cell viability and apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS:Cell viability was analyzed by using MTT and LDH assay. Apoptotic cells were detected by using DNA fragmentation and flow cytometric analysis. The level of intracellular reactive oxygen species (ROS) and the potential of mitochondrial membrane were determined by flow cytometric assay. The activity of caspase-3, -6 and -9 were measured with microplate spectrofluorometer. Protein levels were examined by Western blotting. RESULTS:Treatment of cultured HUVECs with HCY for 48 h induced a significant decrease in cell viability, and the percentage of apoptosis increased to 76.8%. The level of intracellular ROS and activity of caspase-3, -6 and -9 enhanced, and the red/green ratios of mitochondrial membrane decreased. However, simultaneous treatment with silymarin exhibited cytoprotective effects, reduced formation of the DNA ladder, prevented the levels of Bax and Bcl-2 proteins and the accumulation of ROS as well as caspase-3, -6 and -9 activation, reconverted the potential of mitochondrial membrane, and the percentage of apoptosis/necrosis was significantly decreased to 12.7% in a dose-dependent manner. CONCLUSION:These results demonstrate that silymarin has the protective capacity to antagonize HCY-induced apoptosis in HUVECs. The antiapoptotic action of silymarin may be partially dependent on an anti-oxidative stress effects, inhibition of caspases activity, and maintenance of mitochondria function.     

白菜耐光氧化能力鉴定及其与活性氧代谢关系的研究

 采用人工光氧化技术对30个常见白菜栽培品种进行光氧化处理, 以总叶绿素存留率作为评价指标, 利用系统聚类分析将供试品种划分为5个级别(1～5级) 。其中耐光氧化能力强的品种有青优4号、暑绿、抗热605、绿扬青、矮抗6号、华王; 不耐光氧化的品种有四倍体矮脚黄、绿星。在此基础上, 以耐光氧化品种抗热605与不耐光氧化品种绿星为材料, 研究了光氧化过程中活性氧代谢的特征。结果表明:在光氧化条件下, 白菜叶片内的抗氧化酶SOD、POD、CAT、APX活性受光氧化诱导升高, 但光氧化敏感性品种绿星的酶活性很快下降, 耐光氧化品种抗热605的酶活持续保持在较高的水平。因此, 绿星的O₂·生成速率、H₂O₂ 含量及MDA的积累量均高于抗热605, 最终导致品种绿星在光氧化下总叶绿素存留率低。表明抗热605植株内较高的抗氧化酶活性对提高耐光氧化能力起了重要作用。     

Role of vascular NAD(P)H oxidase in vascular remodeling

NAD(P)H oxidase was initially found in phagocytes and it participates in the generation of reactive oxygen species(ROS). Recent researches have showed that NAD(P)H oxidase also expresses in other tissues including blood vessels and it plays a critical role in vascular remodeling through ROS which are important signaling molecules in vascular cells.This article reviews the biochemical characterization, activation paradigms, structure, and function of this enzyme.     

Rebamipide repairs injury of small intestinal epithelial barrier induced by aspirin in mice

AIM: To investigate whether rebamipide repairs the small intestinal epithelial barrier in aspirin-induced small intestinal injury (SⅡ) in mice and its mechanism.METHODS: Small intestinal injury was induced by aspirin (200 mg·kg^-1·d^-1 for 5 d) in BALB/c mice. Based on the treatment with aspirin and/or rebamipide (320 mg·kg^-1·d^-1), the mice were divided into 4 groups (n=18 in each group). The living mice in each group (n=6) were sacrificed via cervical dislocation method at day 0, day 5, and day 10. The structure and function of intestinal barrier and the levels of the signaling pathway factors were measured by transmission electron microscopy, immunohistochemistry, qPCR, and Western blot.RESULTS: Tight junctions between intestinal epithelial cells improved significantly after reba-mipide treatment. The expression of ZO-1 and occludin in the injured small intestine showed a gradually increasing trend after rebamipide administration (P<0.05). There was a decreased trend of D-lactate level in rebamipide-treated SⅡ mice (P<0.05). The expression of cyolooxygenase-2 (COX-2), β-catenin, and c-Myc, and prostaglandin E₂ concentration in small intestinal tissues were significantly increased in rebamipide treatment group (P<0.05). However, down-regulated COX-1 expression in the SⅡ mice was sustained at a low level after rebamipide administration.CONCLUSION: Rebami-pide repairs the injury of small intestinal mucosa and improves the structure and function of small intestinal barrier in aspirin-induced SⅡ mice by up-regulating the expression of COX-2.     

Role of juglone in regulating oxidative stress and apoptosis in ovarian cancer SKOV3 cells

AIM: To study the cytotoxicity of juglone on ovarian cancer SKOV3 cells. METHODS: The activity of SKOV3 cells was detected by MTT assay. The cells apoptosis was examined by flow cytometry. The level of reactive oxygen species (ROS) was measured by DCF-DA staining. The protein levels of cytochrome C (Cyt C) and activated caspase-3 were evaluated by Western blot. RESULTS: MTT assay showed that juglone significantly inhibited the growth of SKOV3 cells in dose- and time-dependent manners (P<0.05). The early apoptotic rate and late apoptotic rate of SKOV3 cells in 50 μmol/L juglone group at 24 and 48 h were higher than those in control group (P<0.01).Moreover, juglone induced ROS accumulation, and increased the protein levels of Cyt C and activated caspase-3.CONCLUSION: Juglone inhibits the cell growth and induces the apoptosis of SKOV3 cells by ROS accumulation.     

Effects of norepinephrine on the activity of NF-κB in cardiomyocytes

AIM: To study the effects and mechanisms of norepinephrine (NE) on the activity of nuclear factor-kappa B (NF-κB) in cardiomyocytes. METHODS: Using the cultured neonatal rat cardiac myocytes, the levels of reactive oxygen species and the nuclear DNA binding activity of NF-κB were measured in cardiomyocytes before and after stimulated with NE alone, NE+prazosin+propranolol, and NE+vitamin E, respectively. RESULTS: NE increased significantly the levels of reactive oxygen species and NF-κB activity in cardiac myocytes. These effects of NE were attenuated by vitamin E pretreatment, as well as prazosin and propranoloe. CONCLUSION: The effects of NE on cardiomyocytes might be exerted partly through NF-κB activation, associated with NE-induced overproducion of reactive oxygen species via the adrenergic receptor pathway.     

草莓白化相关病毒中国分离物全基因组分析

草莓白化相关病毒(strawberrypallidosis-associatedvirus,SPa V)属于长线形病毒科(Closteroviridae)毛形病毒属(Crinivirus),可引起草莓病害,2017年在中国首次报道。采用高通量测序、RACE和RT-PCR技术获得了SPa V中国分离物(FJ)的基因组全长。该病毒含有两条正单链基因组RNA1和RNA2。RNA1全长8 048 nt,5′和3′非编码区序列分别为264和197 nt,含有3个开放阅读框(ORF),分别编码ORF 1a/1b融合蛋白和p9蛋白。RNA2全长7 977 nt,5′和3′非编码区序列分别为248和186 nt,含有8个开放阅读框(ORF),分别编码HSP70h、CPh、CP、CPm、p7、p6、p9和p28等8个蛋白。RNA1和RNA2与美国M1分离物分别具有98.5%和99.0%的核苷酸一致性;系统发育分析结果表明,SPa V中国分离物(FJ)单独处在一个分支。对SPa V来源的小RNA的分析表明,来源于SPa V的小RAN长度以21和22 nt为主。     

不同光质LED光源对鱼腥草幼苗膜脂过氧化及抗氧化酶的影响

以鱼腥草为材料,以日光灯处理为对照,分析了不同光质LED光源(白、红、黄、绿、蓝LED灯)对鱼腥草叶片活性氧、类黄酮含量及抗氧化酶的影响。结果表明,蓝光下叶片中TBARS(硫代巴比妥酸)含量最高,较对照高30.18%,与希夫试剂染色结果相一致;过氧化氢产生最多,较对照高190.21%,与DAB染色结果一致;超氧阴离子产生速率在蓝光下最高,为对照组的1.54倍,白光下最低;蓝、绿光下脯氨酸含量是对照的2倍;蓝光和黄光处理下,黄酮含量较对照提高了21.4%。蓝、绿光提高了SOD2同工酶的表达,且在蓝光下最高;蓝、绿、红、黄光处理分别诱导了更多的POD基因位点的表达;不同LED光质均提高了CAT抗氧化酶活性,其中蓝、绿光下CAT同工酶的表达量明显高于对照。综上所述,蓝色光处理可以有效提高幼苗的抗氧化能力,可为鱼腥草品质的控制提供参考。     

Effects of spermine on myocardial ischemia/ reperfusion injury in rats

AIM: To observe the effect of exogenous spermine (low concentration) on myocardial ischemia/reperfusion injury in rats.METHODS: 40 Wistar rats were randomly divided into 4 groups: sham- operation group (Sham), ischemic reperfusion group (I/R), spermine group (Sp) and natural saline group (NS). The model of ischemic/reperfusion injury was established by ligating rat coronary artery. In Sp group, spermine (0.5 mmol/L, 2 mL/kg) was injected slowly into rat vein. During the process, we recorded the electrocardiogram and the LV functional parameters, assayed the levels of SOD, LDH, NO and MDA in serum, and examined the ultrastructure of the myocardium. RESULTS: In I/R group, the incidence of arrhythmia was 90%, myocardial ultrastructure was injured seriously, values of LVSP and ±dp/dtmax decreased, levels of LDH, NO and MDA increased while SOD activity decreased (P<0.05 or P<0.01, compared with Sham group). Compared with I/R and NS group, all those indexes in Sp group changed significantly (P<0.05 or P<0.01). CONCLUSION: Exogenous spermine alleviates myocardial ischemia/ reperfusion injury in rats. The mechanism may be related to its antioxidant effect and relieving the injury caused by oxygen free radical.     

Role of autophagy in formation of intrarenal crystals induced by ethylene glycol in rats

AIM: To investigate the role of autophagy in the formation of intrarenal crystals induced by ethy-lene glycol in rats. METHODS: Healthy male SPF SD rats (n=40) were divided into 5 groups (n=8):normal control group, stone model group, rapamycin treatment group (0.75% ethylene glycol + rapamycin), chloroquine treatment group (0.75% ethylene glycol + chloroquine) and N-acetylcysteine (NAC) treatment group (0.75% glycol + NAC). After 4 weeks of intervention, Western blot and immunohistochemistry were applied to detect the expression levels of autophagy protein LC3-Ⅱ in the kidney tissues of each group. The number of autophagic vacuoles in each group was observed under transmission electron microscope. The level of total superoxide dismutase (T-SOD) in urine from each group was detected by T-SOD kit, while the level of glutathione peroxidase (GSH-Px) was measured by GSH-Px kit. The serum levels of creatinine and urea nitrogen in each group were examined by automatic biochemical analyzer. The levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (Kim-1) in 24 h urine of each group were measured by ELISA. Von Kossa staining was used to observe the deposition of renal crystals in each group for evaluating the pathological changes of renal tissues. RESULTS: Compared with model group, the expression level of autophagy protein LC3-Ⅱ and the quantity of autophagic vacuoles in rapamycin treatment group and chloroquine treatment group were increased, while those in NAC treatment group were reduced (P<0.05). Compared with model group, the urine levels of T-SOD and GSH-Px were decreased in rapamycin treatment group, while those in chloroquine treatment group and NAC treatment group were increased (P<0.05). Compared with normal group, the serum levels of creatinine, urea nitrogen, NGAL and Kim-1, and the crystal deposition in the kidneys in model group were increased. The renal damage and crystal deposition were further increased in rapamycin treatment group, while those in chloroquine and NAC treatment groups were significantly reduced. CONCLUSION: Ethylene glycol-activated autophagy promotes the formation of intrarenal crystals in rats. After application of antioxidants and autophagy inhibitors, the level of autophagy in the kidney, the renal tissue damage, the crystal deposition and the formation rate of kidney stones are reduced.     

Role of reactive oxygen species in 23-hydroxybetulinic acid-induced apoptosis in LoVo cells

AIM: To investigate the changes of reactive oxygen species (ROS) in apoptosis of LoVo cells induced by 23-hydroxybetulinic acid.METHODS: LoVo cells were treated with 23-hydroxybetulinic acid. The apoptotic morphological change was observed under the light microscope. Intracellular ROS production and the rate of apoptosis were detected by flow cytometry.RESULTS: LoVo cells improved apoptotic morphological changes treated with 23-hydroxybetulinic acid for 48 h. At concentrations of 25, 50, 100, 200 μmol/L of 23-hydroxybetulinic acid, the apoptotic rates of LoVo cells were (7.17±2.31)%, (15.60±4.02)%, (32.47±5.25)% and (52.71±5.93)%, respectively. The results indicated a certain concentration-dependent relationship. 23-hydroxybetulinic acid caused an increase in the ROS production, and the ROS levels were 2.83±0.80, 5.97±1.72, 12.53±2.57 and 16.73±4.58. Compared with the control group (2.13±0.32), the increase in ROS production in LoVo cells at the concentration of 100, 200 μmol/L of 23-hydroxybetulinic acid treatment was significant (P<0.05). CONCLUSION: 23-hydroxybetulinic acid induces LoVo cell apoptosis. The production of ROS may play a crucial role in the process of the LoVo cell apoptosis.