Identification of novel juvenile-hormone signaling activators via high-throughput screening with a chemical library

Juvenile hormone (JH) is an insect-specific hormone that regulates molting and metamorphosis. Hence, JH signaling inhibitors (JHSIs) and activators (JHSAs) can be used as effective insect growth regulators (IGRs) for pest management. In our previous study, we established a high-throughput screening (HTS) system for exploration of novel JHSIs and JHSAs using a Bombyx mori cell line (BmN_JF&AR cells) and succeeded in identifying novel JHSIs from a chemical library. Here, we searched for novel JHSAs using this system. The four-step HTS yielded 10 compounds as candidate JHSAs; some of these compounds showed novel basic structures, whereas the others were composed of a 4-phenoxyphenoxymethyl skeleton, the basic structure of several existing JH analogs (pyriproxyfen and fenoxycarb). Topical application of seven compounds to B. mori larvae significantly prolonged the larval period, suggesting that the identified JHSAs may be promising IGRs targeting the JH signaling pathway.     

程序性细胞死亡因子10抑制Ⅰ型干扰素表达并促进FMDV复制

旨在探究宿主蛋白程序性细胞死亡因子10（programmed cell death factor 10，PDCD10）通过抑制Ⅰ型干扰素表达进而促进口蹄疫病毒（foot-and-mouth disease virus，FMDV）的复制。首先，本研究验证了过表达和沉默PDCD10对FMDV复制的影响，接着利用双荧光素酶报告系统探究PDCD10对Ⅰ型干扰素信号通路活化的影响，最后，利用实时荧光定量PCR探究PDCD10对Ⅰ型干扰素通路下游刺激基因（IFN-stimulated genes，ISGs）转录的影响。结果表明，过表达PDCD10显著促进FMDV的复制，沉默PDCD10显著抑制FMDV的复制。与对照相比，过表达PDCD10后感染仙台病毒（Sendai virus，SeV）的细胞培养液上清液显著促进FMDV复制，进一步，PDCD10显著抑制SeV诱导的IFN-β启动子以及NF-κB的激活且呈剂量依赖性，并且PDCD10负调控Ⅰ型干扰素通路信号分子转录，最后还发现PDCD10负调控Ⅰ型干扰素下游ISGs转录。本研究结果为深入探究PDCD10在抗病毒天然免疫中的作用积累了资料。     

轮状病毒感染肠上皮细胞激活宿主先天免疫研究进展

轮状病毒(RV)是引起婴幼儿、幼畜禽急性肠胃炎的人畜共患病原,常与其他病原体混合感染,多以呕吐,严重水样腹泻,脱水为临床症状,感染后具有较高病死率,对人类公共卫生以及养殖业造成极大危害。RV病原相关分子模式(PAMP)可被肠上皮细胞(IECs)中一组可遗传的模式识别受体(PRR)识别,通过IECs、先天免疫细胞与RV互作,激活细胞内信号级联,从而迅速诱导炎症和多种抗病毒基因表达。论文就轮状病毒特性、肠道先天免疫等方面进行综述,探讨RV感染宿主IECs后诱导不同抗病毒信号通路,为利用先天免疫途径预防轮状病毒感染提供了一定的参考。     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

Study on the Hepatoprotective Effect of Oxalis coriniculata L. and Related Mechanism by Regulating Oxidative Stress and TLR-2/NF-κB Signaling Pathway

[Objectives]This study aimed to explore the protective effect of Oxalis coriniculata L.on rats with acute liver injury induced by carbon tetrachloride(CCl4)and ...     

Salvianolate attenuates oxidative stress injury of human endothelial EA.hy926 cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups:control group, damage group, and anti-damage groups (salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide (NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor (VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased (P<0.05). The apoptotic rate was significantly decreased (P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased (P<0.05), and the intracellular ROS level was significantly decreased (P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.     

Role of PERK pathway in morphine-induced myocardial protection of H9c2 cells

AIM: To explore whether morphine protects oxidative stress-damaged myocardial cells by inhibiting the PERK pathway to reduce endoplasmic reticulum stress and prevent mitochondrial permeability transition pore (mPTP) opening. METHODS: Rat myocardial H9c2 cells were cultured to establish an oxidative stress model, and then randomly divided into control group, H₂O₂ group, H₂O₂+morphine group, H₂O₂+morphine+PERK pathway inhibitor GSK2656157 group, morphine group and GSK2656157 group. Immunohistochemical method was used to detect the effects of morphine on expression of glucose-regulated protein (GRP) 78 and GRP94 induced by oxidative stress. The protein levels of PERK signaling pathway-related molecules were determined by Western blot. Confocal microscopy was used to observe the effects of morphine on mPTP opening and endoplasmic reticulum induced by oxidative stress. Cellular toxicity was detected by lactate dehydrogenase (LDH) kit and cell viability was measured by MTT assay. RESULTS: Compared with control group, GRP78 and GRP94 proteins in H₂O₂ group were strongly expressed, and the brown-yellow particles were significantly increased, but morphine significantly inhibited this process. Compared with control group, the phosphorylation of PERK was significantly reduced with GSK2656157 treatment at different concentrations, among which 2 μmol/L had the most significant effect (P < 0.05). Oxidative stress significantly increased the protein levels of GRP78, GRP94, p-PERK and CHOP, but significantly decreased p-GSK-3β level. These changes were inhibited by morphine, and the effects of morphine were further enhanced by GSK2656157 (P < 0.05). Compared with control group, oxidative stress significantly reduced the fluorescence intensity of mitochondrial TMRE and ER-Tracker Red. Morphine significantly inhibited this effect even when mitochondrial membrane potential was reduced, mPTP was open, and endoplasmic reticulum was damaged, while GSK2656157 further enhanced the effect of morphine (P < 0.05). Compared with control group, H₂O₂ significantly increased cellular toxicity and decreased the cell viability. Morphine inhibited this effect and GSK2656157 significantly enhanced the effect of morphine (P < 0.05). CONCLUSION: Morphine protects cardiac H9c2 cells under oxidative condition by inhibiting endoplasmic reticulum stress through PERK pathway and preventing the mPTP opening via GSK-3β inactivation.     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway， thus promoting the apoptosis of cardiomyocytes.     

Role of IP3R in LH-EGFR-induced mouse oocyte meiotic resumption

AIM: To explore the effect of inositol 1, 4, 5-trisposphate receptor (IP₃R) in luteinizing hormone-epidermal growth factor receptor (LH-EGFR)-induced oocyte meiotic resumption. METHODS: Models of mouse cumulus-oocyte complexs (COCs) culture and follicle culture in vitro were generated to study the effects of 2-aminoethyl diphenyl borate (2-APB) and heparin (IP₃R specific inhibitors) on LH/EGF-induced oocyte meiotic resumption and EGF-induced cumulus cell expansion. Real-time PCR was used to detect the mRNA expression of cumulus expansion-related factors. The changes of the intracellular calcium level were monitored using Fluo 3-AM, and the cGMP level was measured by ELISA. RESULTS: The inhibitors of IP₃R, 2-APB and heparin, dramatically reversed EGF-induced oocyte maturation (P<0.05) and decreased cGMP levels in COCs (P<0.05). In addition, 2-APB and heparin reversed EGF-induced cumulus expansion, and significantly inhibited EGF-induced cumulus expansion-related factor expression (P<0.05). The activation of IP₃R increased intracellular calcium level, and the study found that 2-APB and heparin dramatically reversed EGF-induced elevation of calcium level in cumulus cells (P<0.05). Follicular culture in vitro showed that 2-APB and heparin significantly reversed the LH-induced oocyte maturation (P<0.05). CONCLUSION: LH-EGFR signaling pathway increases calcium level in cumulus cells through IP₃R, resulting in meiotic resumption.