细胞自噬在植物天然免疫反应中的作用

在介绍植物细胞自噬的基础上,分析了细胞自噬对超敏反应细胞程序化死亡（HR-PCD）的控制及可能作用于启动HR—PCD的促死亡信号分子等方面作用,指出在植物免疫反应过程中,细胞自噬通过降解对病原菌诱导的HR—PCD产生负调控作用,对于将细胞死亡限制在感染部位具有重要的控制作用。     

rhPGRN promotes cell proliferation by regulating autophagy and endoplasmic reticulum stress through MAPK/PI3K pathway

AIM To extract and purify recombinant human progranulin (rhPGRN) and to examine its effect on the proliferation, autophagy and endoplasmic reticulum stress (ERS) in human chondrocyte C28I2 and mouse macrophage RAW264.7. METHODS The histidine-tagged protein was specifically affinity-purified using Ni-NTA Sefinose resin, and the concentration and purity of the target protein were verified by Coomassie blue staining, BCA method and Western blot. The effects of rhPGRN on the proliferation, autophagy and ERS in C28I2 and RAW264.7 cells were detected by cell counting, Western blot and RT-qPCR. RESULTS The highly purified biologically active human recombinant protein rhPGRN was successfully extracted from the cell line with stable PGRN transfection. rhPGRN promoted the proliferation of the C28I2 cells and RAW264.7 cells, up-regulated the mRNA expression of cell cycle-related molecules (PCNA, cyclin B1 and cyclin D1) and the protein expression of Ki67, and increased the phosphorylation levels of proliferation-related signaling molecules ERK and Akt. Treatment with ERK pathway inhibitor U0126 inhibited rhPGRN-promoted proliferation, autophagy and ERS in the cells. The rhPGRN-induced autophagy of the cells was also inhibited by PI3K/Akt pathway inhibitor 3-methyladenine. The rhPGRN-promoted protein expression of Ki67 was down-regulated by autophagy inhibitor bafilomycin A1 and ERS inhibitor 4-phenylbutyric acid. CONCLUSION These results not only established a method for stable extraction of biologically active high-concentration high-purity recombinant protein rhPGRN, but also confirmed that the biological effect of rhPGRN on promoting cell proliferation was achieved through regulating autophagy and ERS via MAPK and PI3K/Akt pathway.     

Nuciferine promotes autophagy and reduces macrophage foaming by inhibiting PI3K/Akt/mTOR signaling pathway

AIM To investigate the effect of nuciferine （NUF) on the formation of foam cells and its possible molecular mechanism. METHODS Human monocyte-macrophage cell line THP-1 was induced by oxidized low-density lipoprotein (Ox-LDL) to establish foam cell model, and simultaneously treated with NUF at 5, 10 or 20 μmol/L. Oil red O staining was used and total cholesterol content was measured to observe the effect of NUF on foam cell formation. Autophagy flow was detected by immunofluorescence， and autophagosomes were detected by transmission electron microscopy. The protein levels of microtubule-associated protein 1 light chain 3 (LC3), P62, phosphorylated protein kinase B (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) were determined by Western blot. 3-Methyladenine (3-MA), an autophagy inhibitor, was used to inhibit autophagy and to observe whether NUF inhibited foam cell formation by regulating autophagy. RESULTS Compared with control group, the intracellular lipid deposition and total cholesterol content in Ox-LDL group were increased. Compared with Ox-LDL group, the intracellular lipid deposition and total cholesterol content in NUF group were decreased, while autophagy flow and number of autophagosomes were increased. The inhibitory effect of NUF on cell foaming was weakened after 3-MA treatment. Moreover, NUF decreased the protein levels of p-mTOR and p-Akt. CONCLUSION Nuciferine may promote autophagy by inhibiting phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway, thus reducing intracellular lipid deposition and formation of foam cells.     

Tanshinone IIA inhibits doxorubicin resistance in gastric cancer cells

AIM: To investigate the inhibitory effect and the specific mechanism of tanshinone IIA on doxorubicin (DOX)-resistant gastric cancer cells. METHODS: The sensitivity of gastric cancer cells lines to DOX was determined by MTT assay. DOX-resistant gastric cancer cell lines were established by step selection with increasing concentrations of DOX. The cell cycle arrest, apoptosis and autophagy related-markers were analyzed by flow cytometry and Western blot. The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multi-drug resistance-associated protein 1 (MRP-1) was determined by RT-qPCR and Western blot. RESULTS: DOX-sensitive cell lines SNU-719 and SNU-601 as well as the cell lines relatively resistant to DOX including SNU-638, SNU-668, SNU-216 and SNU-620 were identified according to the IC₅₀ values of DOX for different cell lines. Two DOX-resistant cell lines SNU-719R and SNU-601R were also established. Tanshinone IIA inhibited the expression of MRP-1 in DOX-resistant cell lines. Compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in cancer cells decreased the G₂/M phase cell number, increased the protein expression of p21, decreased the protein expressions of cyclin B1 and cyclin-dependent kinase 1 (CDK1) in the SNU-719 R cells and SNU-620 cells. In addition, compared with DOX treatment alone group, combined treatment of DOX and tanshinone IIA in the cancer cells increased the protein expressions of p53, Bax and LC3B-II, decreased the protein expression of Bcl-2 and p62 (P<0.05). CONCLUSION: Tanshinone IIA is an effective drug in the inhibition of DOX resistance in gastric cancer.     

Delaying aging of AMPK by inhibiting NLRP3 inflammasome

AMP-activated protein kinase (AMPK) is a central regulator of many metabolic pathways involved in the pathophysiological processes of aging and related diseases, and has become an important integrator influencing inflammation (including inflammasome). The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome has recently become a highly concerned marker of stress and metabolic risk. It is associated with neurodegenerative and cardiovascular diseases and type 2 diabetes mellitus. However, the signal pathways regulating NLRP3 inflammasome have not yet been clarified. Studies have shown that many AMPK-dependent signaling pathways, such as autophagy (including mitophagy) and endoplasmic reticulum stress (ERS), regulate the expression of NLRP3 inflammasome during aging. Inhibiting the expression of NLRP3 seems to be a common factor in many complex-mediated aging responses. Therefore, eliminating the common triggers of autophagy/ERS and NLRP3 inflammasome, and suppressing the signal cascade of inflammatory cytokines such as IL-1β and TNF-α, will be an important research direction to explore new anti-aging strategies.     

Shikonin induces apoptosis and autophagy of human cervical cancer HeLa cells by PI3K/Akt/mTOR signaling pathway

AIM:To observe the effects of shikonin on the apoptosis and autophagy of human cervical cancer HeLa cells, and to explore the possible role of PI3K/Akt/mTOR signaling pathway in these processes. METHODS:The HeLa cells were treated with shikonin, and the cell viability was detected by CCK-8 assay. The apoptosis was detected by Annexin V/PI double staining. The autophagosome was observed by transfection with GFP-LC3 into the HeLa cells. After the treatment with shikonin combined with autophagy inhibitor 3-methyladenine (3-MA) or apoptosis inhibitor Z-DEVD-FMK, the protein levels of autophagy-and apoptosis-related molecules microtuble-associated protein 1 light chain 3 (LC3) and cleaved caspase-3 in the HeLa cells were determined by Western blot. The protein levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) were also determined by Western blot. RESULTS:Shikonin significantly inhibited the viability of HeLa cells (P<0.05). Compared with control group, shikonin significantly induced apoptosis of HeLa cells (P<0.05). The results of GFP-LC3 plasmid transfection analysis showed that green dot-like congregate autophagosomes appeared in the cytoplasm of the HeLa cells after shikonin treatment, while the autophagosomes were rarely observed in control group. Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly decreased and cleaved caspase-3 was significantly increased in shikonin+3-MA group (P<0.05). Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly increased and cleaved caspase-3 was significantly decreased in shikonin+Z-DEVD-FMK group (P<0.05). Compared with control group, shikonin significantly decreased the protein levels of p-PI3K, p-Akt and p-mTOR (P<0.05). CONCLUSION:The apoptosis and autophagy of the HeLa cells are induced by shikonin, these two processes are complementary. The mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway.     

Tanshinone ⅡA attenuates doxorubicin-induced injury in H9c2 cardiomyocytes via AMPK-mediated autophagy

AIM: To investigate the effect of tanshinone ⅡA on doxorubicin-induced rat H9c2 cardiomyocyte injury. METHODS: The H9c2 cardiomyocytes were treated with tanshinone ⅡA and/or doxorubicin with or without AMPK inhibitor dorsomorphin. The cell viability was measured by CCK-8 assay, the LDH release was examined for evaluating the cell injury, autophagy was analyzed by immunofluorescence staining, and AMPK activation was determined by Western blot. RESULTS: Compared with control group, the viability of H9c2 cells was decreased, the release of LDH was increased, the autophagy degree was increased, and AMPK activation was inhibited after treatment with doxorubicin (P<0.05). Compared with doxorubicin group, the treatment with tanshinone ⅡA restored the cell viability, reduced the release of LDH, further increased autophagy degree, and activated AMPK in the H9c2 cells (P<0.05). AMPK inhibitor dorsomorphin attenuated the abilities of tanshinone ⅡA to restore the cell viability, reduce the release of LDH, and increase autophagy degree in the H9c2 cells (P<0.05). CONCLUSION: Tanshinone ⅡA attenuates doxorubicin-induced injury in H9c2 cardiomyocytes, and its mechanism might be related to AMPK-mediated autophagy, which provides experimental and theoretical basis for the clinical application of tanshinone ⅡA in the prevention and treatment of doxorubicin-induced cardiomyocyte injury.     

Injury pathways of hypoxia/reoxygenation in AC16 cardiomyocyte

AIM:To investigate the effects of hypoxia/reoxygenation (H/R) for different reoxygenation times on cardiomyocyte injury. METHODS:Human cardiomyocyte AC16 was cultured in glucose-free and serum-free DMEM with 1% O₂ for 24 h, 10% fetal bovine serum and low glucose DMEM combined with 21% O₂ were used to establish reoxygenation for 2 h, 6 h and 12 h, respectively. The cell viability was measured by CCK-8 assay. The protein levels of different cell injury pathway related molecules, such as LC3-Ⅱ/-I (autophagy), caspase-1 and gasdermin D (pyroptosis) and caspase-3 and Bax/Bcl2 (apoptosis), were determined by Western blot. RESULTS:Compared with blank control group, the cell viability in each H/R group was continuously decreased with the extension of reoxygenation time (P<0.05). The expression of LC3-Ⅱ/-I was up-regulated in hypoxia group and H/R group compared with blank control group (P<0.05). In addition, the protein levels of cleaved caspase-1 and cleaved gasdermin D were increased in H/R groups for 6 h and 12 h, respectively (P<0.05). Cleaved caspase-3 and Bax/Bcl2 were increased after reoxygenation for 12 h (P<0.05). CONCLUSION:Autophagy in hypoxia-induced AC16 cells is up-regulated, and then decreased by reoxygenation. The cell pyroptosis is activated earlier than the apoptosis during reoxygenation.     

类枯草杆菌蛋白酶的生物学功能及其在农牧业疾病控制中的应用

综述国内外研究类枯草杆菌蛋白酶基因在生物生长过程中参与表型的稳定性、致病性和自噬等重要生理功能及其在农牧业疾病控制中的应用前景。     

Induction of programmed death and cytoskeletal damage on Trichoplusia ni BTI-Tn-5B1-4 cells by azadirachtin

Plenty of insect cells are sensitive to azadirachtin, an ideal biopesticide. However, the mode of action of azadirachtin on insect cells still remains unexplained. In this study, Trichoplusia ni BTI-Tn-5B1-4 cell line was used as the model system to evaluate induction of programmed death and cytoskeletal damage by azadirachtin. The result of colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) assay indicated that the effect of azadirachtin on cells growth was time-dependent with different concentrations (0.75, 1.5 and 3 μg/mL). As autophagic vacuoles were observed obviously by transmission electron microscopy, azadirachtin could induce autophagy in cells. Furthermore, lysosomal function was affected after treatment with azadirachtin in time-dependent manner. Aberrant morphological figures appeared in treated cells, such as contraction and turning round, especially the appearance of apoptotic bodies and membrane blebbing, which was the evidence of azadirachtin-induced apoptosis, with apoptosis rate of 13.98% at 1.5 μg/mL for 120 h. As the stress fibers changed after treatment, the cytoskeletal function could be influenced. The effect on cell cycle was also analyzed by flow cytometry, with the result that azadirachtin enhanced cell cycle arrest at the G2/M phase gradually.