Role of endoplasmic reticulum stress and autophagy mediated by endocannabinoid N-arachidonic acid aminoethanol in ovarian cancer cells

AIM To study the effect of endocannabinoid N-arachidonic acid aminoethanol （AEA） on ovarian cancer and its mechanism. METHODS The serum levels of AEA in healthy control group and ovarian cancer group were analyzed by ELISA, and the diagnostic value of AEA in ovarian cancer patients was evaluated by ROC curve. The effects of AEA on the viability, migration and invasion abilities of the ovarian cancer cells were detected by CCK-8 assay, Transwell cell invasion test and Scratch test. The effect of AEA on ovarian cancer was further verified by the measurement of tumor volume, tumor weight and visual map of tumor tissue. Meanwhile, flow cytometry and Western blot were used to determine the effect of AEA on the apoptosis of ovarian cancer cells, so as to explore the mechanism of AEA promoting the apoptosis of ovarian cancer cells through detecting the endoplasmic reticulum stress and autophagy related proteins by Western blot. RESULTS The serum levels of N-arachidonic aminoethanol in the patients with ovarian cancer were significantly decreased. ROC results suggested that AEA was a sensitive biological marker to distinguish the patients with ovarian cancer from healthy dedividuals. In addition, AEA inhibited the cell viability, migration and invasion abilities of ovarian cancer cells, and inhibited the growth of ovarian cancer tissues. CONCLUSION By promoting endoplasmic reticulum stress and affecting autophagy of ovarian cancer cells, AEA promotes apoptosis of ovarian cancer cells.     

Effect of continuous renal replacement therapy on mRNA expression of autophagy-related molecules and prognosis in patients with acute kidney injury

AIM To explore the effect of continuous renal replacement therapy （CRRT） on mRNA expression of autophagy-related molecules and the prognosis in the patients with acute kidney injury （AKI）. METHODS A total of 174 patients from our hospital who were diagnosed to have AKI and underwent CRRT between February 2015 and March 2018 were involved in this study. The plasma levels of interleukin-1β （IL-1β） and interleukin-6 （IL-6）, the serum creatinine （SCr） level, and the mRNA expression levels of autophagy-related molecules, including microtubule-associated protein 1 light chain 3-II （LC3-II）, autophagy-related protein 5 （Atg5） and beclin-1, in the monocytes from peripheral blood were compared before and after CRRT. According to the survival of AKI patients after 4 weeks of CRRT, the enrolled patients were divided into death group （n=43） and survival group （n=131）, and the mRNA expression levels of the above molecules were compared between the 2 groups. RESULTS After CRRT treatment, the plasma levels of IL-1β and IL-6, the level of SCr, and the mRNA expression levels of LC3-II, Atg5 and beclin-1 in the monocytes were significantly lower than those before CRRT treatment （P<0.05）. The mRNA expression levels of LC3-II, Atg5 and beclin-1 in death group were significantly higher than those in survival group （P<0.05）. The positive correlation between SCr and IL-1β, IL-6, LC3-II or beclin-1 was observed （P<0.05）, and no correlation between SCr and Atg5 was found （P>0.05）. CONCLUSION CRRT decreases the mRNA expression levels of autophagy-related molecules in the patients with AKI and reduces the autophagy activity, which is protective for the patients. These autophagy-related molecules may be applied as a potential markers to predict the prognosis of CRRT.     

Baicalein induces autophagy in breast cancer cells

AIM:To investigate the autophagy of breast cancer cells induced by baicalein and to explore its mechanism.METHODS:The effects of baicalein on the viability of MCF-7 cells and 4T1 cells were investigated by MTT assay,and the dosage of the drug was determined.The expression levels of microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ) and LC3-I in the MCF-7 cells and 4T1 cells treated with baicalein at doses of 25,50 and 100 μmol/L,or combined with autophagy inhibitor 3-methyladenine (3-MA) were determined by Western blot.In order to confirm the role of baicalein in autophagy,the effect of 3-MA on the apoptosis of both MCF-7 cells and 4T1 cells induced by baicalein was analyzed by flow cytometry.The protein levels of p-mTOR,mTOR,p-AKT and AKT were examined by Western blot and the role of AKT-mTOR pathway in the induction of autophagy in breast cancer induced by baicalein was determined by the combination of activators.RESULTS:Baicalein at 50 μmol/L and above doses significantly inhibited the viability of breast cancer cells in a dose-and time-dependent manner.The expression of LC3-Ⅱ/LC3-I in both MCF-7 cells and 4T1 cells was significantly enhanced after the action of baicalein,and the ratio of LC3-Ⅱ/LC3-I was significantly decreased after 3-MA addition.The results of flow cytometry showed that,compared with baicalein group,the combination of baicalein and 3-MA promoted the levels of necrosis and apoptosis.Moreover,the protein levels of p-mTOR and p-AKT were significantly decreased and were rescued by EGF,while their total protein levels were not changed.CONCLUSION:Baicalein induces autophagy through AKT-mTOR pathway both in MCF-7 cells and 4T1 cells.     

Low-intensity ultrasound combined with microbubbles activates auto-phagic death of thyroid cancer cells by promoting ROS production

AIM: To investigate the effect of low-intensity ultrasound combined with microbubble contrast agent on autophagic death of thyroid cancer cells, and to analyze the mechanism of autophagy activation and its effect on cell viability. METHODS: Human thyroid cancer cell line TPC1 was treated with low-intensity ultrasound at 20 kHz frequency and 80 mW intensity combined with microbubbles. The cell death and viability were analyzed by Live/Dead assay and CCK-8 assay 60, 120 and 240 s after the treatment. The protein levels of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ), autophagy-related protein 5 (ATG5) and SQSTM1/P62 were determined by Western blot. The number of intracellular autophagosomes was measured by the methods of monodansylcadaverine (MDC) staining, green fluorescent protein (GFP)-LC3 transfection and transmission electron microscopy. The level of reactive oxygen species (ROS) was mea-sured and the effect of ROS on autophagy activation was evaluated by N-acetyl-L-cysteine (NAC) treatment. The effect of ATG5 siRNA transfection on autophagy was analyzed for determining the role of autophagic death. RESULTS: Low-intensity ultrasound combined with microbubbles significantly promoted TPC1 cell death and inhibited TPC1 cell viability (P<0.05) in a time-dependent manner. Compared with low-intensity ultrasound group and microbubble group, ultrasound combined with microbubbles significantly increased the protein levels of LC3-Ⅱ and ATG5, but inhibited the protein level of P62 (P<0.05). The results of MDC staining, GFP-LC3 transfection and transmission electron microscopy showed that ultrasound combined with microbubbles significantly increased the number of autophagosomes in the TPC1 cells. Compared with low-intensity ultrasound group and microbubble group, ultrasound combined with microbubbles increased the level of ROS, while NAC significantly reduced the protein level of LC3-Ⅱ (P<0.05). Thansfection with ATG5 siRNA inhibited the autophagy, significantly decreased the percentage of cell death and increased cell viability (P<0.05). CONCLUSION: Low-intensity ultrasound combined with microbubbles promotes the autophagic cell death by increasing the level of ROS in thyroid cancer cells, leading to death of thyroid cancer cells.     

Rapamycin reduces SH-SY5Y cell damage induced by oxygen-glucose deprivation

AIM: To observe the effect of rapamycin (Rapa) on human neuroblastoma SH-SY5Y cell injury induced by oxygen-glucose deprivation (OGD), and to explore the role of autophagy in this process. METHODS: The SH-SY5Y cells were randomly divided into 4 groups:normal control group:the cells were cultured without OGD treatment; Rapa group:the cells were pretreated with Rapa for 1 h; OGD group:the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O₂, 94% N₂ and 5% CO₂ for 12 h; Rapa+OGD group:the cultured cells were treated with Rapa for 1 h, and then were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by determining the leakage of lactate dehydrogenase (LDH). The enzyme activity of caspase-3 was detected. TUNEL staining were used to detect the variation of cell apoptosis. The protein levels of apoptosis-related proteins Bax and Bcl-2, autophagy-related protein beclin-1 and autophagy marker protein LC3B were determined by Western blot. RESULTS: Compared with OGD group, the viability of the SH-SY5Y cells was significantly increased, and the activity of caspase-3 was significantly reduced in Rapa+OGD group (P<0.05). The SH-SY5Y cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in Rapa+OGD group (P<0.05). Compared with control group, the protein level of Bcl-2 was significantly decreased (P<0.05) and the protein level of Bax was significantly increased in OGD group. Compared with OGD group, the levels of Bcl-2, beclin-1 and LC3B-Ⅱ were significantly increased and the protein level of Bax was significantly increased in Rapa+OGD group (P<0.05). CONCLUSION: Rapamycin has a protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the promotion of autophagy.     

杏衰退萎黄病病毒的siRNA高通量测序和RT-PCR鉴定

为了明确引起杏衰退萎黄病的病毒种类，利用小干扰RNA（small interfering RNA，siRNA）高通量测序技术，结合生物信息寻找杏衰退萎黄样品中的病毒序列，发现存在亚洲李属病毒1（Asian prunus virus 1，APV1）和亚洲李属病毒3（Asian prunus virus 3，APV3）。为了验证该结果，采用RT-PCR对10个待测杏样品进行APV1、APV2和APV3检测，所有样品中未检测到APV2，检出APV1和APV3的样品数分别为3和4个。对检测到病毒的外壳蛋白（coat protein，CP）进行测序和系统进化分析，分别获得了3条653 bp的APV1和4条1 031 bp（或1 032 bp）的APV3 CP基因片段；3条APV1 CP基因核苷酸序列一致性为99.2% ~ 99.8%，与NCBI中已发表的26个APV1 CP基因序列一致性为44.0% ~ 99.8%；4条APV3 CP基因序列一致性为78.5% ~ 99.5%，与NCBI中已发表的26个APV3 CP基因序列一致性为44.7% ~ 99.5%。     

Effect of resveratrol on autophagy and renal interstitial fibrosis in kidney of diabetic mice

AIMTo observe the effect of resveratrol (Res) on renal autophagy level and renal interstitial fibrosis in the mice with diabetes mellitus (DM), and to discuss the possible mechanism. METHODSThe wild-type C57BL/6 mice were randomly divided into 3 groups, including normal control (NC) group, DM group and Res group (8 in each group). The diabetic mouse model was established by injection of streptozotocin. After 8 weeks of successful replication of the diabetic model, Res was given to the mice in Res group by continuous gavage for 12 weeks, and then the mice in each group were sacrificed to detect the relevant biochemical parameters. The pathological changes of the kidney tissues were observed by HE staining and Masson staining. The levels of the proteins related to autophagy, epithelial-mesenchymal transition (EMT) and fibrosis were determined by Western blot. The mRNA expression of collagen type IV (Col IV), α-smooth muscle actin (α-SMA) and E-cadherin were detected by real-time PCR. RESULTSCompared with NC group, fasting blood glucose (FBG), kidney index (KI), serum creatinine, 24-hour urinary albumin excretion rate and 24-hour urine total protein were remarkably increased in DM group (P<0.05). The results of HE and Masson staining indicated that renal tissue presented fibrosis in DM group. The protein levels of E-cadherin, beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II) were reduced in DM group, while the levels of α-SMA, Col IV and Snail1 were increased (P<0.05). After intervention with Res for 12 weeks, all the relevant biochemical parameters and KI were reduced (P<0.05) except FBG (P>0.05), and renal fibrosis lesions were obviously alleviated. Compared with DM group, the protein levels of E-cadherin, beclin-1 and LC3-II were increased in Res group, but the protein expression levels of α-SMA, Col IV, Snail1 were reduced (P<0.05). Compared with DM group, the mRNA level of E-cadherin was increased in Res group , but the mRNA levels of Col IV and α-SMA were reduced (P<0.05). CONCLUSION Resveratrol significantly inhibits EMT and reduces renal interstitial fibrosis in diabetic mice, and its mechanism may be related to the promotion of renal autophagy.     

Effect of miR-22 secreted by macrophage exosomes on cardiomyocyte autophagy under uremic toxin stimulation

AIM To investigate the effect of microRNA-22 (miR-22) secreted by macrophage exosomes on the autophagy of H9c2 cardiomyocytes under uremic toxin stimulation. METHODS The macrophage-derived exosomes stimulated by indoxyl sulfate (IS) were collected and co-cultured with H9c2 cells. The levels of miR-22 in the macrophages， macrophage-derived exosomes and H9c2 cells were detected by RT-qPCR. The viability of H9c2 cells was measured by CCK-8 assay. The expression of exosome surface marker protein CD63 and autophagy-related proteins LC3 and P62 was determined by Western blot. RESULTS Under IS stimulation, the expression of exosome surface marker protein CD63 in the macrophages was significantly higher than that in control group (P<0.05), and the levels of miR-22 in the macrophages and macrophage-derived exosomes were significantly increased (P<0.01). With the increase in macrophage exosome concentration, the viability of H9c2 cells was decreased gradually (P<0.05), and the stimulation of macrophage exosomes reduced P62 expression and promoted the conversion of LC3-I to LC3-II in a dose-dependent manner (P<0.05). Macrophage-derived exosomes increased the ratio of LC3-II to LC3-I but decreased P62 protein expression in the H9c2 cells transfected with miR-22 mimic compared with the cells transfected with corresponding negative control miRNAs (P<0.05). However, miR-22 inhibitor yielded contrasting results. CONCLUSION IS-stimulated macrophages increase expression of miR-22 in cardiomyocytes through exosomes, and promote autophagy of the cardiomyocytes.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

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。