芍药黑斑病病原菌鉴定及其对杀菌剂敏感性分析

对在江苏省扬州市芍药(Paeonialactiflora)叶片上发现的一种黑斑病,采集其病叶,分离和纯化病原物,根据柯赫氏法则明确其致病性。基于病原物形态特征和多基因(r DNA-ITS、endo PG、OPA2-1、Alt a 1)序列联合分析,鉴定该病原物为链格孢(Alternaria alternata)。该病原菌的最适生长条件是:温度为28℃,p H 7.0,最适碳源为可溶性淀粉,最适氮源为硝酸钾。室内毒力测定发现,在测试的4种杀菌剂中,嘧菌酯和吡唑醚菌酯乳油对病原菌离体生长的抑制作用较强,而戊唑醇和异菌脲对其生长的抑制作用较差。     

枣炭疽病菌鉴定及9种杀菌剂对其菌丝生长的影响

为明确枣炭疽病的病原菌,从病样中分离病原菌并选择获得代表性的2个菌株,研究病原菌的形态学特征、rDNA-ITS序列、致病性及9种杀菌剂对其菌丝生长的影响。结果表明:引起枣炭疽病的病原菌为胶孢炭疽菌(Colletotrichum gloeosporioides),该病原菌主要侵染枣果实,发病初期果皮病斑为淡黄色,逐渐变成黑褐色圆形或近圆形斑,病斑中央凹陷,边缘呈黄色晕圈。吡唑醚菌酯和咪鲜胺对炭疽菌菌株YY-02和YY-05的菌丝生长抑制作用最强,其平均EC_(50)值分别为0.27,0.23μg/ml,2种药剂的毒力显著高于其他药剂;戊唑醇、苯醚甲环唑、醚菌酯、三唑酮4种药剂对胶孢炭疽菌YY-02和YY-05菌丝也具有一定的抑制作用,其平均EC_(50)值分别为2.85、2.97、3.17、5.69μg/mL。建议在枣炭疽病发病的不同时期,轮换使用吡唑醚菌酯、咪鲜胺、戊唑醇、苯醚甲环唑、醚菌酯和三唑酮等杀菌剂,以有效控制该病害。     

梨梢枯病菌生物学特性及对药剂敏感性研究

通过对在中国新发现的梨梢枯病病原菌(Aureobasidium pullulans var. pullulans)在不同温度、pH、碳源、氮源和光照等培养条件和孢子致死温度的测定,明确病原菌的生长规律和死亡条件,并采用生长速率法对该病原菌进行9种杀菌剂的毒力筛选。结果表明:梨梢枯病病原菌菌丝生长的适宜温度为15～25 ℃,最适温度为20 ℃,温度高于35 ℃时,菌丝生长缓慢;菌丝生长最佳培养基为PDA培养基,产孢最佳培养基为OA培养基;甘露醇和酵母粉分别为菌丝生长和产孢的最佳碳源和氮源;菌丝可在pH 4～10条件下生长,pH 5为生长最适pH;光照对菌丝生长有促进作用,并且有利于病原菌产孢,在光暗交替的培养条件下,菌落会形成明显的轮纹。病原菌分生孢子的致死温度为48.9 ℃。该病原菌对75%肟菌·戊唑醇水分散粒剂的敏感性较高,其EC₅₀ <0.01 mg·L^-1;对70%甲基硫菌灵可湿性粉剂、50%多菌灵可湿性粉剂、43%氟菌·肟菌酯悬浮剂和325 g·L^-1苯甲·嘧菌酯悬浮剂的敏感性次之,其0.01 mg·L     

莲藕腐败病病原旋柄腐霉的生物学特性及药剂室内毒力测定

以山东莲藕腐败病新病原旋柄腐霉(Phytopythium helicoides)为研究对象,采用菌丝体生长速率法,研究了P.helicoides的生物学特性和12种杀菌剂对该菌菌丝生长的抑制作用,以期为莲藕腐败病防控提供参考依据。结果表明:旋柄腐霉在15~43℃均能生长,最适温度为35℃;在pH 5~11范围内均能生长,最适pH为7。室内毒力测定结果表明,9种杀菌剂对旋柄腐霉具有抑制作用,毒力测定结果显示60%唑醚·代森联WG抑制效果最好,EC50为85.33mg·L~(-1);68%精甲霜·锰锌WG、80%代森锰锌WP抑制效果次之,EC50分别为98.12、193.46mg·L~(-1);77%硫酸铜钙WP、25%嘧菌酯SC、72.2%霜霉威盐酸盐AS的抑制效果较差,EC50高达到1 878.85、1 119.73、965.86mg·L~(-1)。     

引致桂花叶斑病的葡萄座腔菌生物学特性及药剂毒力测定

以桂花为试材,利用组织分离法分离桂花叶斑病菌,并对其进行形态特征鉴定和rDNA-ITS序列分析,研究了该病原菌的生物学特性及其对9种杀菌剂的敏感性。结果表明:该病原菌为葡萄座腔菌;光照对病原菌的生长无明显影响;菌丝生长的最适温度20~30℃;最适pH6~9;最适碳源为葡萄糖、蔗糖和麦芽糖;最适氮源为硝酸钾和天冬酰胺。9种杀菌剂对桂花叶斑病菌的生长均表现出抑制作用,其中苯醚甲环唑、戊唑醇和氟硅唑的毒力最强,EC50分别为0.6839、0.8754、1.2236μg/mL;百菌清毒力最弱,EC50为57.0778μg/mL。     

哈密瓜镰刀果腐菌的鉴定、生物学特性和室内防治药剂的筛选北大核心CSCD

【目的】鉴定引起哈密瓜(Cucumis melo L.)果蒂处的果肉腐烂病原菌种类,以及对不同宿主的致病性检测,探讨光照、温度、pH值、碳源、氮源及不同药剂处理对该菌生长的影响,并筛选出哈密瓜镰刀果腐病的有效防治方法。【方法】采用组织分离法,从发病的哈密瓜果实中分离病原菌,结合形态学与核糖体内转录间隔区(rDNA-ITS)、转录延伸因子EF-1α和组蛋白Histone3序列的分子鉴定确定病原菌种类。测定不同的培养温度、pH、碳源、氮源、光照及需氧条件对其生长的影响。用NaHCO;、硼酸、施保功、苯甲·嘧菌酯和纳他霉素处理,观察各种药剂的抑菌效果。再用罗丹明123、健那绿B、线粒体红色荧光探针(Mito Tracker;Red CMXRos)及溴化丙啶(PI)染色,了解不同药剂处理对病原菌细胞膜透性及线粒体功能的影响,用安全性较高的纳他霉素对哈密瓜进行室内防效实验,了解纳他霉素对病原菌的防治效果。【结果】病原菌为哈密瓜果腐变红镰刀菌(Fusarium incarnatum,编号ZJHM-01),孢子形态呈镰刀状,长度为25.4~35.5μm,宽度为3.3~5.4μm;病原菌生物学特性测定结果表明,宿主具广谱性,光照能促进生长,最适生长温度为30℃,最适p H为7~9,最适碳源为淀粉、山梨醇和葡萄糖,最适氮源为蛋白胨和牛肉膏。1.0%NaHCO;、1.2%硼酸、0.025%施保功、0.016%苯甲·嘧菌酯和40 mg·L^(-1)纳他霉素均能完全抑制病原菌生长。罗丹明123染色结果发现,1.0%NaHCO;、1.2%硼酸和30 mg·L^(-1)纳他霉素处理使菌丝的细胞膜透性增加;健那绿B和Mito Tracker;Red CMXRos染色发现,药剂处理降低了菌丝的线粒体活性;PI染色结果表明,病原菌细胞膜完整性遭到破坏,线粒体活性和线粒体膜电位受损。纳他霉素室内防效表明,纳他霉素处理的果实发生病害时间明显延后且症状减轻,说明纳他霉素具有较好的防病效果。【结论】果实采收期,生产上用施保功和苯甲·嘧菌酯防治哈密瓜镰刀菌果腐病。果实采收后,用1.0%NaHCO;、1.2%硼酸和40 mg·L^(-1)纳他霉素浸泡果实处理以降低哈密瓜镰刀菌果腐病的发生率。从食品安全性考虑,纳他霉素应该作为哈密瓜采后镰刀菌果腐病防治的首选药剂。研究结果为哈密瓜镰刀菌果腐病防治提供了新的方法。     

杧果畸形病病菌(Fusarium mangiferae)生物学特性及杀菌剂对其室内毒力测定

为进一步了解杧果畸形病病原菌Fusarium mangiferae的生物学特性,筛选出防治效果较好的药剂应用于生产,系统研究了该菌在不同培养基、温度、光照和碳氮源条件下的生长特性,并测定了9种杀菌剂对该菌的抑制作用。结果表明,病菌在OMA培养基上生长速度最快,在液体PDA中生长量最大,最佳产孢培养基为PSA;菌丝生长和孢子萌发的最佳温度均为25～28℃;光照时间和酸碱(pH 4～10)对病菌菌丝生长速度、孢子产生及萌发的影响不明显。病原菌生长的最佳碳源和氮源分别为蔗糖和酵母提取物。室内9种杀菌剂病原的毒力测定结果表明:25%咪鲜胺乳油对病菌菌丝生长的抑制效果最好,EC50和EC95分别为1.200 4 mg·L-1和2.623 9 mg·L-1;25%嘧菌酯悬浮剂能够强烈抑制病菌分生孢子的萌发,EC50和EC95分别为0.531 2 mg·L-1和2.974 1 mg·L-1。25%咪鲜胺乳油和25%嘧菌酯悬浮剂对杧果畸形病病菌具有较好的抑制作用,可进一步进行田间药效试验。     

百合枯萎病菌Fusarium commune的鉴定及其对杀菌剂敏感性研究

采集龙牙百合(Lilium brownii var. viridulum)枯萎病典型症状的鳞茎进行病原物分离、纯化和致病性测定,结合病原菌形态学观察及其r DNA-ITS、EF-1α、mt SSU和IGS基因的序列分析,确定病原菌为共享镰孢菌Fusarium commune。测定了光照、温度、p H、碳源和氮源等处理对该病原菌生长和产孢的影响,并采用菌丝生长速率法测定其对5种杀菌剂的敏感性。结果表明,最适菌丝生长的条件包括温度为25~30℃、p H 8.0、碳源为乳糖、氮源为蛋白胨;最适产孢条件包括24 h黑暗、温度为30℃、p H 10.0、碳源为乳糖、氮源为蛋白胨;另外,24 h光照、24 h黑暗和12 h光照/12 h黑暗条件均适宜该菌的菌丝生长。在5种参试杀菌剂中,肟菌·戊唑醇抑制作用最强,EC50值为0.294 mg·L-1,噁霉灵的效果最差,EC50为39.359 mg·L-1。     

细辛精油对六种苹果病害的离体抑菌活性

细辛是一种重要的中草药,活性物质主要是挥发油.研究采用生长速率法和孢子萌发法测定了细辛精油对引起苹果病害的6种病原菌(灰斑病菌、褐斑病菌、斑点落叶病病菌、炭疽病菌、苹果轮纹病菌和干腐病菌)的菌丝生长和4种病原菌(灰斑病菌、褐斑病菌、斑点落叶病病菌、炭疽病菌)的孢子萌发的影响.结果表明,细辛精油对两者均有一定的抑制作用:对褐斑病菌菌丝生长的抑制效果最好,EC90仅为397.37mg/L;对炭疽病菌孢子萌发的抑制效果最好,EC90仅为276.77mg/L.对于同一种病原菌来说,细辛精油对其孢子萌发的抑制效果好于对其菌丝生长的抑制效果.     

葡萄炭疽病防治药剂筛选试验

采用菌丝生长速率法和孢子萌发法在室内条件下测定了8种杀菌剂对葡萄炭疽病病原菌的抑制作用。试验结果表明,咪鲜胺、吡唑醚菌酯、丙环唑、甲基硫菌灵和戊唑醇对葡萄炭疽病病原菌的菌丝生长有明显的抑制作用,EC50分别为0.025 8、0.087 0、0.492 7、0.623 4、1.420 1 mg/L;吡唑醚菌酯、福美双、甲基硫菌灵对葡萄炭疽病病原菌的孢子萌发有很好的抑制作用,EC50分别为1.19×10-6、2.34×10-4、5.94×10-4mg/L。室内测定结果表明,嘧菌酯和福美双1?10混配时有较强的增效作用。田间试验结果表明,55%嘧菌酯.福美双可湿性粉剂1 500倍液涂抹枝条后对葡萄炭疽病的防效达75.78%;25%吡唑醚菌酯乳油3 000倍液喷洒果穗后对葡萄炭疽病的防效达87.62%。     

Relationship between rat hepatic fibrosis and regulation of hepatic stellate cell autophagy by microRNA-181a

AIM To observe the changes of liver structure, the levels of transforming growth factor-β1 （TGF-β1）, microRNA-181a, LC3-II/-I, beclin-1 and collagen deposition in hepatic fibrosis （HF） rats induced by carbon tetrachloride （CCl₄）, and the effect of microRNA-181a on autophagy of rat hepatic stellate cells （HSCs） induced by TGF-β1, and to explore the possible mechanism of microRNA-181a in regulating HSC activation and HF. METHODS Wistar rats （n=40） were randomly divided into 5 groups （with 8 in each）： control group （subcutaneous injection of olive oil, 3 mL/kg, twice a week）, and CCl₄-induced HF groups of 2, 4, 6 and 8 weeks （subcutaneous injection of 40% CCl₄, 3 mL/kg, twice a week for 2, 4, 6 and 8 weeks, respectively）. Masson staining was used to evaluate the changes of HF in rats. The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA. The level of microRNA-181a in rat liver tissues was detected by RT-qPCR. The protein levels of LC3-II/-I, beclin-1, α-smooth muscle actin （α-SMA）, collagen type I （Col I） and collagen type Ⅲ （Col Ⅲ） in rat liver tissues were measured by Western blot. HSC-T6 cells were transfected with microRNA-181a inhibitor, or pretreated with the autophagy inhibitor 3-methyladenine （3-MA）, before treatment with TGF-β1 to stimulate autophagy. The expression of microRNA-181a, LC3-II/-I, beclin-1, α-SMA, Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot. RESULTS The levels of TGF-β1, microRNA-181a, LC3-II/-I ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF, and microRNA-181a expression showed a positive correlation with autophagy-associated proteins （P<0.01）. MicroRNA-181a level was significantly increased, which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181a inhibitor, along with suppression of autophagy and cell activation （P<0.01）, which were similar to the effects of 3-MA treatment. CONCLUSION CCl₄ promotes rat HF, the microRNA-181a expression of liver tissue, and autophagy in a time-dependent manner. Reducing the expression of microRNA-181a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1. The regulation of HSC autophagy by microRNA-181a may be involved in rat HF.     

NOD8 inhibits autophagy in human pancreatic cancer cells and effect of apoptosis on autophagy regulatd by NOD8

AIM: To explore whether NOD8 inhibits autophagy in human pancreatic cancer cells and its underlying mechanisms, and to investigate the effect of apoptosis on the autophagy regulated by NOD8. METHODS: The empty plasmid pEGFP-C2 and recombinant plasmid pEGFP-NOD8 were transfected into the Panc-1 cells using JetPRIME reagent.The untransfected cells served as control group. The protein levels of NOD8, autophagy-related proteins beclin-1 and LC3-II, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins Akt, p-Akt, mTOR and p-mTOR were determined by Western blot 48 h after transfection. Meanwhile, the number of LC3 spots was quantified by immunofluorescence staining. Furthermore, after a broad caspase inhibitor Z-VAD-FMK was applied to NOD8-over-expressing cells, the protein expression levels of beclin-1 and LC3-II were detected by Western blot and the number of LC3 spots was observed by immunofluorescence staining. RESULTS: The protein level of NOD8 in pEGFP-NOD8 group was significantly higher than that in control group and pEGFP-C2 group (P<0.01). The protein expression of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8 group were significantly decreased as compared with control group and pEGFP-C2 group. Moreover, the protein levels of p-AKT and p-mTOR in pEGFP-NOD8 group were higher than those in control group and pEGFP-C2 group, while no significant difference of mTOR and AKT protein expression was found among these 3 groups. Furthermore, the protein levels of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8+Z-VAD-FMK group were significantly increased compared with pEGFP-NOD8 group. CONCLUSION: NOD8 inhibits autophagy in the Panc-1 cells and its mechanism may be related to the activation of PI3K/Akt/mTOR pathways. Apoptosis enhances the inhibitory effect of NOD8 on autophagy.     

Hydrogen inhibits C/EBP homologous protein-mediated macrophage apoptosis by activating autophagy

AIM: To investigate the protective effect of hydrogen (H₂) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H₂-saturated medium was added to murine RAW264.7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine (3-MA) and 3 μmol/L rapamycin (Rap) for 1 h, and then treated with ox-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI staining, respectively. The activity of lactate dehydrogenase (LDH) in medium was detected. The protein levels of beclin-1 (a molecular marker of autophagy) and C/EBP homologous protein (CHOP, a key signaling component of endoplasmic reticulum stress-associaed apoptosis pathway) were determined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope. RESULTS: Hydrogen attenuated the reduction of cell viability, LDH leakage, apoptosis and CHOP upregulation induced by ox-LDL. Hydrogen promoted ox-LDL-induced autophagy in macrophages as assessed by beclin-1 upregulation, and LC3 granulation, and this promotion effect of hydrogen was inhibited by 3-MA (an autophagy inhibitor) and further enhanced by Rap (an autophagy inducer). Moreover, the inhibitory effect of hydrogen on ox-LDL-induced macrophage apoptosis, reduction of cell viability and CHOP upregulation were also blocked by 3-MA and enhanced by Rap. Similar results were obtained in human THP-1-derived macrophages, as assessed by the inhibition of ox-LDL-induced apoptosis and CHOP upregulation, and the promotion of beclin-1 expression by hydrogen. CONCLUSION: Hydrogen may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression, and the upstream mechanism may partially involved in the activation of autophagy.     

Effect of cytarabine on curcumin-induced autophagy in human acute myeloid leukemia cells

AIM:To observe whether autophagy occurs in curcumin-induced human acute myeloid leukemia KG1a cells in the presence of chemotherapeutic drug cytarabine and the possible mechanism. METHODS:KG1a cells were cultured in vitro. The ultrastructural changes of the cells were observed under transmission electron microscope. Autophagy was detected by acridine orange staining. The cell viability was measured by MTT assay. The cell cycle distribution was analyzed by flow cytometry. The expression of autophagy-related molecules beclin-1 and LC3 at mRNA and protein le-vels was determined by RT-qPCR and Western blot. RESULTS:Curcumin dose-dependently inhibited the viability of KG1a cells (P<0.05). The growth inhibition rate in combination group was significantly higher than that in single reagent group and control group (P<0.01). Electron microscopical observation showed that curcumin induced the occurrence of autophagosomes, and cytarabine increased curcumin-induced autophagosomes. Acridine orange staining showed that the combined treatment with cytarabine increased the autophagy induced by curcumin, and the number of autophagic acid vesicles and cells containing autophagic acid vesicles were increased. Curcumin blocked the cell cycle in the G₀/G₁ phase. The mRNA expression levels of beclin-1 and LC3 in combination group were significantly higher than those in single reagent group and control group(P<0.01). The results of Western blot showed that the protein expression of beclin-1 was significantly up-regulated in combination group (P<0.05), and the ratio of LC3-Ⅱ/LC3-I was higher than that in control group (P<0.01). CONCLUSION:Curcumin inhibits the viability of KG1a cells and induces autophagy. Cytarabine promotes autophagy, which is superior to curcumin alone. It may be related to the up-regulation of beclin-1 and LC3-Ⅱ by the two reagents.     

Effect of rapamycin on proliferation,invasion, adhesion,apoptosis and autophagy of human lung adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum

AIM: To study the effect of rapamycin (Rap) on the proliferation, invasion, adhesion, apoptosis and autophagy of human adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum (DDP). METHODS: Human adenocarcinoma A549 and resistant A549/DDP cell lines were cultured. The inhibitory effects of Rap alone or combined with DDP on A549 and resistant A549/DDP cells were detected by MTT assay. The in vitroinvasion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by Transwell methods. The in vitroadhesion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by adhesion experiments. The apoptosis of A549 and resistant A549/DDP cells induced by Rap alone or combined with DDP was analyzed by flow cytometry. The cell autophagy marker proteins beclin-1 and LC3 in A549 and resistant A549/DDP cells treated with Rap alone or combined with DDP were detected by Western blotting.RESULTS: Compared with Rap or DDP alone group, the combination of Rap and DDP significantly inhibited the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells in vitro, and promoted the cell apoptosis and autophagy marker proteins beclin-1 and LC3 expression (all P＜0.05).CONCLUSION: Rap enhances the effect of DDP through promoting the cell autophagy, thereby inhibiting the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells and inducing the cell apoptosis with a synergistic effect.     

Effects of PDCD5 on hypoxia/reoxygenation-induced autophagy and apoptosis of cardiomyocytes

AIM: To investigate the influence of programmed cell death 5 (PDCD5) on apoptosis and autophagy in the cardiomyocytes exposed to hypoxia/reoxygenation (H/R) and its potential mechanism.METHODS: H9c2 cells were exposed to H/R. PDCD5 was downregulated by RNA interference. The cell viability was measured by MTT assay. TUNEL assay was used to detect cell apoptosis. The mRNA and protein levels were determined by RT-qPCR and Western blot.RESULTS: The expression of PDCD5 was upregulated in the cardiomyocytes after H/R injury. Furthermore, H/R injury obviously reduced the cell viability and enhanced the apoptosis and autophagy of the cardiomyocytes. However, knockdown of PDCD5 increased the cell viability, and attenuated H/R-induced apoptosis, accompany with reduction of Bax and augment of Bcl-2 expression. Additionally, silencing PDCD5 markedly inhibited H/R-induced autophagy by regulating the expression of LC3-II/LC3-I and beclin-1. Moreover, downregulation of PDCD5 suppressed NF-κB signaling by redu-cing the protein level of p-P65.CONCLUSION: Silencing PDCD5 suppresses H/R-induced H9c2 cells apoptosis and autophagy by blocking NF-κB signaling pathway. The result indicates a new strategy for the prevention and treatment of myocardial I/R injury.     

Adiponectin inhibits decrease in autophagy of rat H9c2 cardiomyocytes induced by β1-adrenergic receptor autoantibodies

AIMTo investigate whether adiponectin inhibits the decrease in autophagy of rat H9c2 cardiomy?ocytes induced by β₁-adrenergic receptor (β₁-AR) autoantibodies (β₁-AA), and to explore its mechanism. METH?ODS: SD rats were actively immunized with β₁-AR extracellular second loop (β₁-AR-ECII) antigen peptide. Affinity chromatography was used to purify β₁-AA in serum of the SD rats. The viability of H9c2 cells was measured by CCK-8 assay. The mRNA levels of LC3B and beclin-1 in the H9c2 cells were detected by real-time PCR. The protein levels of LC3-II, P62, AMP-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) were determined by Western blot. RESULTSPretreatment with adiponectin at 10 μg/L for 1 h reversed the decreased viability of H9c2 cells induced by β₁-AA. Compared with control group, β₁-AA decreased the mRNA expression of LC3B and beclin-1, decreased the protein level of LC3-II, and increased the expression of P62 protein in the H9c2 cells, suggesting that β₁-AA decreased the autophagic flux in cardiomyocytes. Adiponectin obviously reversed β₁-AA-induced decline in autophagic flux, and up-regulated the phosphorylation level of AMPK decreased by β₁-AA. Treatment with AMPK inhibitor Compound C for 30 min, we observed that the mRNA expression of LC3B and beclin-1 and the protein level of LC3-II in the H9c2 cells decreased by β₁-AA were not effectively reversed by adiponectin, but the increase in P62 protein expression was still effectively reversed, indicating that adiponectin increased autophagosome production dependent on the AMPK pathway, but increased autophagosome clearance independent on the AMPK pathway. CONCLUSION Adiponectin inhibits the decreased autophagy of H9c2 cardiomyocytes induced by β₁-AA.     

Effects of miR-337 targeting p53 expression on autophagy and migration ability of colon cancer cells

AIM: To investigate the effect of microRNA-337 (miR-337) on the autophagy and migration ability of colon cancer cells, and to explore its possible mechanism involving targeting p53 expression. METHODS: The me-thod of immunohistochemistry was used to detect the protein expression of beclin-1, LC3B and p53 in colon cancer tissues. The correlations between the protein expression of beclin-1/LC3B and clinicopathological features, and the correlations between the protein expression of p53 and beclin-1/LC3B were analyzed. After knock-down of p53 expression by small interfering RNA, the formation of autophagiosomes was observed under electron microscope in colon cancer cell line HCT116, and the protein expression of beclin-1 and LC3B was determined by Western blot. The miRNAs targeting p53 were predicted and screened by bioinformatics, and their expression in HCT116 cells was verified by RT-qPCR. Luciferase reporter assay was used to detect the regulatory effect of miR-337 on p53 gene. The protein expression of p53, beclin-1 and LC3B was determined by Western blot, and the migration ability of HCT116 cells after miR-337 over-expression was detected by Transwell assay. RESULTS: The protein expression of beclin-1 and LC3B in the colon cancer tissues was decreased, which was significantly related to the occurrence, development, invasion and metastasis of colon cancer. The expression of p53 was increased in the colon cancer tissues, which was negatively correlated with the protein expression of beclin-1 and LC3B. Knock-down of p53 gene expression increased the protein expression of beclin-1 and LC3B (P<0.05). Over-expression of miR-337 down-regulated the expression of p53, up-regulated the protein expression of beclin-1 and LC3B, and decreased the migration ability of HCT116 cells (P<0.05). CONCLUSION: miR-337 promotes autophagy and inhibits migration ability of colon cancer cells, and the mechanism may be related to targeted inhibition of p53 expression.     

Angiotensin II type 1 receptor autoantibodies induces INS-1 islet β-cell apoptosis by upregulation of autophagy

AIM: To explore whether the angiotensin Ⅱ type 1 receptor autoantibodies (AT1-AA) induces islet β-cell apoptosis and whether autophagy is involved in the process. METHODS: The INS-1 cells treated with AT1-AA at 10^-6 mol/L for 24 h, and then the apoptosis was analyzed by flow cytometry, Western blot and Hoechst 33258 staining. In addition, the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot. The effects of AT1-AA on the apoptosis, autophagy and viability of INS-1 cells with or without 3-methyladenine (3-MA; a common autophagy inhibitor) or telmisartan (an angiotensin Ⅱ type 1 receptor blocker) pretreatment, were detected by flow cytometry, Western blot and CCK-8 assay. RESULTS: Treatment with AT1-AA at 10^-6 mol/L for 24 h significantly reduced the cell viability (P<0.05). Compared with the negative IgG control group, the apoptotic cells increased after incubation with AT1-AA for 12 h, 24 h and 36 h, respectively (P<0.05). Moreover, the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time, and the elevation of apoptosis and autophagy were blocked by telmisartan. After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone. CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet β cells by upregulating autophagy via the angiotensin Ⅱ type 1 receptor pathway.     

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy. METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3 groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group. The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 mL/kg, suspended in corn oil, ig). After 24 h of operation, the rats from each group were sacrificed and their brains were collected. TTC staining and HE staining were used to define the infarct areas and brain structure. The autophagy-related proteins beclin-1, P62, LC3-Ⅱ/-I and Atg5 in the cortex in each group were compared by Western blot. In vitro, PC12 cells were divided into 3 groups:control group, CoCl₂ group and CoCl₂+EA pretreatment group. CoCl₂ at 800 μmol/L was added to the PC12 cells to induce an anoxic environment. The PC12 cells were pretreated with EA at 8 μmol/L and the cell viability was measured by CCK-8 assay. The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining. MDC staining and TMRE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively. The autophagy-related proteins in PC12 cells were also investigated. RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining. HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage. EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group. Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-Ⅱ/-I in the cortex were increased (P<0.01), and P62 protein expression was decreased (P<0.01) in HIE group. Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-Ⅱ/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01). In vitro, compared with CoCl₂ group, the PC12 cells in CoCl₂+EA pretreatment group showed a lower ROS level. Moreover, the cells in CoCl₂+EA pretreatment group exhibited higher mitochondrial membrane potential than that in CoCl₂ group. MDC staining in CoCl₂ group showed high value of fluorescence and increased number of autophagosomes. EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12 cells. Furthermore, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I in CoCl₂ group were higher (P<0.01), and the protein expression of P62 was lower (P<0.01) than those in control group. In CoCl₂+EA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with CoCl₂ group (P<0.01). CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.