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

ZHAO Miao  HAN Ya-ru  HE Yi-fei  FU Yu  LIU Yu-lin  XI Jin-kun  HE Yong-gui 《园艺学报》2020,36(1):9-16

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.  相似文献   

Minimally modified low-density lipoprotein up-regulates endothelin receptors in mouse mesenteric artery through p38 MAPK pathway     

XIE Xi  CHEN Chen  LIN Jie  TANG Hong-xia  QIN Xu-ping  LI Jie 《园艺学报》2020,36(5):779-787

AIMTo investigate whether minimally modified low-density lipoprotein (mmLDL) affects the quantity and activity of endothelin （ET） type A (ET_A) and type B (ET_B) receptors in mouse mesenteric artery by activating p38 mitogen-activated protein kinase (MAPK) inflammatory pathway. METHODSThe KM mice were divided into normal saline (NS) group (injection of NS via caudal vein), mmLDL group (injection of mmLDL via caudal vein), LDL group (injection of LDL via caudal vein), mmLDL+SB 203580 group (injection of mmLDL via caudal vein and intraperitoneal injection of p38 MAPK pathway specific inhibitor SB 203580) and mmLDL+DMSO group (injection of mmLDL via caudal vein and intraperitoneal injection of DMSO). Mesenteric artery ring segment vasoconstriction dose-response curves affected by sarafotoxin 6c (S6c) and ET-1 were recorded by the myography system. The mRNA levels of ET_B receptor, ET_A receptor and interleukin-6 (IL-6) were detected by RT-qPCR. The protein levels of ET_B receptor, ET_A receptor, IL-6, p38 MAPK, p-p38 MAPK, NF-κB and p-NF-κB were determined by Western blot. The serum concentration of IL-6 was measured by ELISA. RESULTSThe contractile responses of the blood vessel segments to S6c and ET-1 were significantly increased by mmLDL (P<0.01). The mRNA and protein expression levels of ET_A receptor, ET_B receptor, and IL-6 significantly increased (P<0.01). The protein levels of p-p38 MAPK and p-NF-κB were significantly increased (P<0.01). The serum level of IL-6 was significantly increased (P<0.01). These effects of mmLDL were inhibited by p38 MAPK inhibitor SB 203580. CONCLUSION mmLDL increses the serum concentration of IL-6, up-regulates the expression of IL-6, ET_A receptor and ET_B receptor in mouse mesenteric artery, and enhances the vasoconstriction function medi?ated by ET_A and ET_B receptors, which is related to the activation of p38 MAPK inflammatory pathway and downstream NF-κB pathway.  相似文献   

Down-regulation of miR-153-3p attenuates H₂O₂-induced H9C2 cell injury by promoting Nrf2 expression     

HUANG Zhao-qi  XU Wei  HUANG Jiong-hua  LU Xiong 《园艺学报》2020,36(7):1249-1254

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.  相似文献   

Effects of platelet-rich plasma on chondrocyte apoptosis and NLRP3/IL-1β signaling pathway in rabbits with osteoarthritis     

QU Yi-ming  SHAO Gao-hai  ZHANG Ming-hua  LI Bo  ZHU Feng-chen  HE Chao  CAO Chun-feng  ZHAO Bo 《园艺学报》2020,36(6):1089-1095

AIM To explore the effect of platelet-rich plasma (PRP) on rabbit osteoarthritis and its possible mechanism. METHODS The rabbits with knee osteoarthritis were prepared and then divided into model group, sodium hyaluronate (SH) group and PRP group, and another sham operation group was set up, with 6 rabbits in each group. The gross morphological changes of rabbit cartilage were observed. HE staining was used to evaluate the pathomorphological changes of the cartilage. TUNEL staining was used to detect the apoptosis of chondrocytes. The expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin-1β （IL-1β） signaling pathway-related molecules was observed by immunohistochemical staining, and the protein levels of caspase-3, Bcl-2 and Bax were determined by Western blot. Chondrocytes were isolated and processed according to grouping, and the NLRP3 and IL-1β levels of the cells were measured by ELISA. RESULTS Compared with sham operation group, Pelletier score, Mankin score, chondrocyte apoptotic rate, the positive protein expression rates of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in model group were increased significantly (P<0.05), while the protein expression of Bcl-2 was decreased significantly (P<0.05). Compared with model group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in SH group and PRP group were decreased significantly (P<0.05), while the protein expression of Bcl-2 was increased significantly (P<0.05). In PRP group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax were lower than those in SH group, while the protein expression of Bcl-2 was higher than that in SH group (P<0.05). Compared with control group, the expression of NL?RP3 and IL-1β in MCC950 (NLRP3 ihibitor) group were significantly reduced (P<0.05), the expression of NLRP3 in eucalyptol (IL-1β inhibitor) group was not significantly changed (P>0.05), and the expression of IL-1β was significantly reduced (P<0.05). CONCLUSION Platelet-rich plasma promotes the repair of cartilage in osteoarthritis rabbits, which has better effect than SH. The mechanism may be related to the inhibition of NLRP3/IL-1β pathway and the reduction of chondrocyte apoptosis.  相似文献   

CD36/Src/ERK pathway is involved in process of monocyte differentiation into macrophages     

XIA Jun  YU Ting  ZHAO Lei 《园艺学报》2020,36(6):1020-1026

AIM To investigate the role of fatty acid translocase (FAT/CD36) on differentiation of monocytes to macrophages. METHODS Human monocyte THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 0, 100 and 200 μg /L. Small interfering RNA (siRNA) targeting CD36 (siCD36) was employed to knock down the expression of CD36 in THP-1 cells. The CD36 over-expression (CD36OE) cell line was constructed by transfection with a recombinant lentivirus containing CD36 cDNA. Optical microscopy and crystal violet staining were used to detect the monocyte morphological changes and adhesion ability. The protein expression of CD36 was measured by flow cytometry and Western blot. The mRNA levels of CD36, CD11b and CD80 were detected by real-time PCR. The protein levels of extracellular signal-regulated kinase (ERK） and Src tyrosine kinase were determined by Western blot. RESULTS The cellular adhesiveness of THP-1 cells was elevated in the process of monocytes differentiation, and the expression of CD36 was increased in this process as well (P<0.01). siCD36 was transfected into the THP-1 cells (CD36i group) and the silencing efficiency was approximately 80%. The cell surface area and cellular adhesiveness were significantly decreased in CD36i group compared with scrambled siRNA (NCi) group (P<0.01). The mRNA levels of CD11b and CD80 were decreased in CD36i group compared with NCi group (P<0.01). The cell surface area and cellular adhesiveness were increased in CD36OE group compared with empty vector (vector) group (P<0.05). The mRNA levels of CD11b and CD80 were increased in CD36OE group compared with vector group (P<0.01). The phosphorylation levels of ERK and Src were decreased in CD36i group compared with NCi group (P<0.05). CONCLUSION CD36 promotes the differentiation of human monocyte THP-1 cells to macrophages by increasing the phosphorylation of Src and further activating ERK.  相似文献   

Hyperbaric oxygen therapy activates CREB/BDNF signaling pathway to reduce synaptic damage in hippocampal neurons of Alzheimer disease mice     

YIN Chuan-hong  YAN Yu-hui 《园艺学报》2020,36(6):1063-1070

AIM To investigate the effect of hyperbaric oxygen (HBO) on synaptic damage of hippocampal neurons in APP/PS1 transgenic (TG) mice and its possible mechanism. METHODS The 6-month-old male APP/PS1 TG mice were randomly divided into TG group, HBO group and cAMP response element binding protein (CREB) inhibitor H89 group, with 10 mice in each group. Ten male wild-type (WT) C57BL/6 mice of the same age were used as negative control group (WT group). The mice in HBO and H89 groups were treated with HBO for 6 cycles, while the mice in WT group and TG group were not treated. The learning and memory abilities were observed by Morris water maze. The nesting ability of the mice was detected by nesting test. The Nissl bodies in hippocampal neurons were observed by Nissl staining. The mRNA expression of CREB and brain-derived neurotrophic factor (BDNF) in hippocampus was detected by real-time PCR. The protein levels of synapsin (SYN), postsynaptic density protein 95 (PSD95), growth-associated protein 43 (GAP43), CREB, phosphorylated CREB (p-CREB) and BDNF in the hippocampus were determined by Western blot. RESULTS Compared with WT group, the learning and memory abilities of the mice in TG group were signilficantly reduced (P<0.05). In addition, the nesting score, the number of Nissl bodies in the hippocampal neurons, the mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). Compared with TG group, the learning and memory abilities of the mice in HBO group were improved (P<0.05). Meanwhile, the nesting scores of the mice were significantly increased (P<0.05), the neurons in the hippocampus were arranged neatly, and the number of Nissl bodies, the relative mRNA expression of CREB and BDNF,and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also increased significantly (P<0.05). Compared with HBO group, the mice in H89 group had poor learning and memory abilities, lowered nesting scores and decreased number of Nissl bodies. Futhermore, the relative mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). CONCLUSION HBO improves the learning and memory abilities of APP/PS1 TG mice, and its mechanism may be related to activating the CREB/BDNF signaling pathway to reduce synaptic damage of hippocampal neurons in mice.  相似文献   

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

HAO Xiao-qiong  LI Yan-kang  FU Xu-yang  JIA Fang-yi 《园艺学报》2020,36(3):532-538

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.  相似文献   

Molecular mechanisms of Belinostat inhibiting viability of osteosarcoma cells     

PENG Bin  HE Min  LI Zheng-mao  FU Yong  TAN Wen-fu 《园艺学报》2020,36(3):552-556

AIM: To observe the effect of histone deacetylase inhibitor (HDACi) Belinostat on the viability of osteosarcoma cells and to study the underlying mechanism. METHODS: Osteosarcoma cell lines SAOS-2 and U2OS were incubated with Belinostat at different concentrations in vitro. The viability of the cells was measured by MTT assay. The activity of caspase-3/-7 and the DNA fragmentation were detected by fluorescence probe and ELISA, respectively. Western blot was used to detect the levels of histone acetylation, expression of PTEN, caspase-3, Bcl-xL and Akt, and phosphorylation of glycogen synthetase kinase 3β (GSK-3β) and Akt. Finally, the cells were incubated with Belinostat and doxorubicin at different concentrations, and then the combination index (CI) was calculated by MTT. RESULTS: Belinostat at 0.5, 1, 2.5 and 5 μmol/L inhibited the viability of U2OS cells and SAOS-2 cells in a dose-dependent manner, induced DNA fragmentation, enhanced caspase-3/-7 activity, and promoted the activation of caspase-3. At the same time, in the SAOS-2 cells, the expression of Bcl-xL was reduced, and the acetylation of histones H3 and H4 was increased. The results of Western blot showed that phosphorylation levels of Akt and GSK-3β in U2OS cells and SAOS-2 cells were decreased significantly after treatment with Belinostat (P<0.05). MTT results showed that combination of Belinostat and doxorubicin further reduced the viability of U2OS and SAOS-2 cells (CI<1). CONCLUSION: Belinostat inhibits the viability of osteosarcoma cells treated with doxorubicin, and the mechanism may be related to the inhibition of Akt signaling pathway.  相似文献   

植物病原丝状真菌寄生性与RGS蛋白的关系研究   总被引：1，自引：0，他引：1  

祝友朋  韩长志 《华中农业大学学报》2020,39(6):23-29

以49个全基因组序列已经公布的真菌为研究对象,通过OrthoVenn2同源基因簇比对、BLASTp比对和关键词搜索3种方法对G蛋白信号调控因子（regulators of G-protein signaling,RGS）同源基因进行比对分析,并结合SMART进行保守结构域分析,结果发现,49个真菌中共有229个RGS蛋白,每种真菌中所含有RGS蛋白的数量范围为3~9个,且死体营养型病原菌和半活体营养型病原菌的RGS蛋白数量高于活体营养型病原菌;根据RGS蛋白中保守结构域进行分类,可以划分为DEP-RGS、RGS-TM、PXA-RGS-PX、RGS、RGS-PAS-PAC、TM-RGS等6种,其中,具有RGS-PAS-PAC和TM-RGS这2种特殊保守结构域的RGS蛋白主要集中在半活体营养型病原菌和死体营养型病原菌;进一步对229个RGS蛋白进行遗传关系分析,发现具有同种保守结构域的RGS蛋白亲缘关系较近。上述研究结果表明植物病原丝状真菌的寄生性与RGS蛋白数量和种类之间存在着一定的关联性。  相似文献   

胚胎干细胞诱导分化研究进展     

马勇江  杨学义  窦忠英 《西北农林科技大学学报(自然科学版)》2003,31(3):25-29

哺乳动物胚胎干细胞一般可从胚胎囊胚内细胞团分离得到 ,具有在体外保持不分化的无限增殖能力 ,在合适的培养条件下 ,胚胎干细胞可定向诱导分化形成多种细胞类型。人们对胚胎干细胞进行体外培养与定向分化可以得到大量同源细胞 ,以应用于患疾病的细胞或器官移植治疗等研究。文章概述了胚胎干细胞增殖与分化的信号调节机制、诱导分化方法 ,国内外研究概况及展望 4个方面的内容  相似文献