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1.
AIM: To investigate the effect and mechanism of sodium selenite (Na2SeO3) on the proliferation of endometrial cancer cells. METHODS: Endometrial cancer Ishikawa cells and HEC-1A cells were treated with Na2SeO3. The effect of Na2SeO3 on cell proliferation was determined by MTT assay. The effects of Na2SeO3 on cell cycle distribution and apoptosis were tested by flow cytometric analysis. The expression of cyclin A was detected by Western blotting. RESULTS: Na2SeO3 inhibited the proliferation of Ishikawa cells and HEC-1A cells. For Ishikawa cells, IC50 was 3.26 μmol/L, and for HEC-1A cells, IC50 was 4.77 μmol/L. After treated with Na2SeO3, the cells in G0/G1 phase were reduced and the cells in S phase and G2/M phase were increased. Na2SeO3 also increased the percentage of apoptosis cells. The result of Western blotting showed that the expression of cyclin A was increased. CONCLUSION: Na2SeO3 inhibits the proliferation of endometrial cancer Ishikawa cells and HEC-1A cells via up-regulating the expression of cyclin A, arresting cell cycle and inducing apoptosis.  相似文献   

2.
AIM: To explore the effect of glucocorticoid on autophagy and senescence in the chondrocytes. METHODS: The collagen II in the normal chondrocytes isolated from the SD rats was checked. After stimulation with glucocorticoid, LysoTracker Red staining, MDC staining and Western blot were used to detect the level of autophagy in the chondrocytes. The mTOR pathway related molecules were investigated by Western blot. Cell senescence was analyzed by SA-β-gal staining. RESULTS: A dose-dependent increase in the number of autophagic vacuoles was observed in the dexamethasone-treated chondrocytes, which was demonstrated by the LysoTracker Red and MDC staining. The expression of LC3-II and beclin-1 was increased by dexamethasone, especially in the cells treated with dexamethasone for 4 d. However, P62 expression was decreased. SA-β-gal staining showed that the percentage of cell senescence was increased by dexamethasone. Surprisingly, the cell senescence induced by dexamethasone was exacerbated by the autophagic inhibitor 3-MA. CONCLUSION: Autophagy induced by dexamethasone protects chondrocyte from senescence. The mTOR pathway may be involved in the autophagy activation.  相似文献   

3.
AIM：To study the autophagy of prostate cancer PC-3 cells induced by CD147 in vitro. ME-THODS：The method of amino acid starvation to induce autophagy was used. The expression of CD147was detected by Western blotting. To study the functional effects of CD147 on autophagy in prostate cancer PC-3 cells, the down-regulation of CD147expression was induced by the technique of RNAi. The conversion of autophagic marker protein LC3-I to LC3-II was determined by Western blotting. The cell death after starvation-induced autophagy was analyzed by trypan blue exclusion assay. RESULTS：The CD147 expression gradually increased in starvation-induced autophagy. The down-regulation of CD147 significantly increased the expression of autophagy-related protein LC3-II compared with control group. Meanwhile, the cell death rates increased from (19.3±3.1)% and (22.3±3.5)% in control groups to (38.4±3.1)% in silencing the expression of CD147in the PC-3 cells (P<0.05). CONCLUSION：CD147 inhibits starvation-induced autophgy and autophagy death in the prostate cancer PC-3 cells.  相似文献   

4.
AIM: To explore the therapeutic effect of adriamycin combined with rapamycin on endometrial cancer cells. METHODS: Two endometrial carcinoma cell lines with different PTEN gene states were chosen: HEC-1A (wild type) and Ishikawa (mutant type). Before adriamycin administration, the cells were pretreated with low concentration of rapamycin for 24 h. The cell viability and 50% inhibitory concentration (IC50) of adriamycin at 24 h were determined by MTT assay. Multiple drug effect/combination index (CI) was used to evaluate the interaction between adriamycin and rapamycin. Apoptotic rate was measured by flow cytometry. The effects of the drugs on phosphorylation of PI3K/Akt and apoptosis protein caspase-3 were detected by Western blotting. RESULTS: Both adriamycin and rapamycin showed obvious growth inhibitory effects on the 2 endometrial cancer cell lines in a time- and dose-dependent manner. After pretreated with rapamycin, IC50 of adriamycin decreased sharply. In Ishikawa cells, it decreased from (21.3±3.8) μmol/L to(11.9±1.2) μmol/L,P<0.05. In HEC-1A cells, it decreased from (14.3±2.8) μmol/L to (8.2±0.9) μmol/L,P<0.05. Combination index value of the 2 drugs was more than 1.15 in the 2 endometrial cancer cell lines, indicating synergistic effects. The combination therapy of adriamycin with rapamycin increased apoptotic rates in the 2 cell lines, and induced the down-regulation of phosphorylated Akt and over-expression of caspase-3 as compared with single drug treatment (P<0.05). CONCLUSION: Adriamycin combined with rapamycin significantly enhances the chemosensitivity of endometrial cancer cells and reduces drug resistance, which will become a new trend for treating endometrial cancer.  相似文献   

5.
AIM: To investigate the effect of advanced glycation end products (AGEs) on autophagy in human umbilical endothelial cells (HUVECs) and to identify the role of autophagy in advanced glycation end product-induced cell apoptosis. METHODS: HUVECs were cultured and treated with AGEs or bovine serum albumin. The protein expression was detected by Western blotting. Autophagosomes were observed under electron microscope. The cell apoptotic rate was determined by flow cytometry. The cell viability was quantified by MTT assay. RESULTS: After treated with AGEs, the level of autophagy-associated protein LC3-Ⅱ in HUVECs was up-regulated, and the number of autophagosomes was increased. Compared with control group, the apoptotic rate of HUVECs increased and the viability of HUVECs was decreased in AGEs treatment group. Furthermore, pretreating the cells with an autophagy inhibitor 3-methyladenine aggravated these effects. The levels of phospho-protein kinase B(Akt) and phospho-mammalian target of rapamycin(mTOR) in HUVECs were also decreased by treatment with AGEs. Pretreatment with Akt activator insulin-like growth factor 1 (IGF-1) increased Akt phosphorylation and suppressed the AGE-induced LC3-Ⅱ expression. CONCLUSION: AGEs induce autophagy in HUVECs through PI3K/Akt/mTOR signal pathway. Autophagy plays a protective role in AGE-induced apoptosis in HUVECs.  相似文献   

6.
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.  相似文献   

7.
AIM:To study the effects of maternal expressed gene 3 (MEG3) and adenosine on the autophagy and proliferation of human hepatocellular carcinoma HepG2 cells, and to explore the possible mechanisms of autophagy and the effect on the proliferation of HepG2 cells induced by MEG3 and adenosine. METHODS:HepG2 cells were cultured according to the conventional cultural method, and divided into control group, MEG3 group (the cells were transfected with MEG3 lentivirus), adenosine group (the cells were treated with 1 mmol/L adenosine) and MEG3+adenosine group (the cells were treated with 1 mmol/L adenosine and MEG3 lentivirus). The protein expression of LC3-Ⅱ, LC3-Ⅰ and mammalian target of rapamycin (mTOR) was determined by Western blot. MDC staining was used to observe the number of autophagosomes. The cell viability was measured by CCK-8 assay, and the number of viable cells were counted by automated cell counter. RESULTS:Compared with control group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased, mTOR expression was increased (P<0.05), the viability of HepG2 cells was decreased, and the number of autophagosomes were reduced in MEG3 group and adenosine group. In MEG3+adenosine group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased significantly (P<0.01), mTOR expression was increased significantly (P<0.01), and the viability and autophagosomes of HepG2 cells were reduced markedly as compared with MEG3 group and adenosine group. After treated with MEG3 and adenosine for 24~72 h, the viable HepG2 cells reduced significantly in MEG3 group and adenosine group (P<0.01), especially in MEG3+adenosine group (P<0.01). CONCLUSION:MEG3 overexpression and low concentration of adenosine activate the mTOR pathway, and inhibit the autophagy and proli-feration of HepG2 cells. MEG3 enhances the effect of adenosine on HepG2 cells.  相似文献   

8.
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.  相似文献   

9.
AIM:To investigate the down-regulation of insulin-like growth factor tgpe 1 receptor(IGF-1R) on the migration and invasion abilities of human endometrial cancer cell HEC-1B. METHODS:The siRNAs targeting IGF-1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC-1B cells(HEC-1B-KD group). The control cells(without virus transfection, HEC-1B-CON group) and negative virus transfection control cells(HEC-1B-NC group) were also set up. The gene silencing effect of siRNA targeting IGF-1R was determined by real-time PCR and Western blotting at mRNA and protein levels,respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experiment. The mRNA levels of matrix metalloproteinase(MMP)-2 and MMP-9 were measured by real-time PCR. RESULTS:The mRNA and protein levels of IGF-1R in HEC-1B-KD cells were significantly reduced by 81% and 91.5%, respectively(P<0.05). In anchorage-dependent growth by colony formation assay, HEC-1B-KD cells showed much less colonies than HEC-1B-CON cells and HEC-1B-NC cells. Compared with the control cells, knockdown of IGF-1R in HEC-1B cells resulted in significant reduction of cell motility. Down-regulation of IGF-1R in HEC-1B cells also significantly reduced the invasion potential(P<0.05). Down-regulation of IGF-1R substantially reduced the expression of MMP-2 and MMP-9 compared with the control cells. CONCLUSION:Knockdown of IGF-1R reduces the migration and invasion abilities of human endometrial cancer cells in vitro accompanied with a decrease in MMP-2 and MMP-9 expression.  相似文献   

10.
AIM:To analyze the effects of outer membrane protein A (OmpA) from Acinetobacter baumannii ATCC 19606 on the autophagy of RAW264.7 cells. METHODS:The RAW264.7 cell model stimulated by OmpA was established. The effects of OmpA on the autophagy of RAW264.7 cells were detected by immunofluorescence, Western blot and transmission electron microscopy. RESULTS:The OmpA increased the expression of LC3B-Ⅱ and reduced the phosphorylation levels of Akt, mTOR and p70S6K. Rapamycin further reduced the phosphorylation levels of mTOR and p-70S6K, and increased the expression of LC3B-Ⅱ induced by OmpA. CONCLUSION:The OmpA of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in the RAW264.7 cells. This work provides a basis for further research on the molecular mechanism of autophagy induced by Acinetobacter baumannii to find a new method against the infection of Acinetobacter baumannii.  相似文献   

11.
AIM:To investigate the potential mechanism of interleukin-17 (IL-17) promoting the viability, migration and invasion of human endometrial carcinoma cells. METHODS:The expression of IL-17 and microRNA-195-5p (miR-195-5p) in the human endometrial carcinoma and benign uterine lesion samples were detected by RT-qPCR. The expression of miR-195-5p in human endometrial carcinoma HEC-1-B cells after treatment with IL-17 at different concentrations for 48 h was detected by RT-qPCR. The viability, migration and invasion of HEC-1-B cells after treatment with IL-17 at 100 μg/L or transfection of miR-195-5p mimics were detected by MTT assay and Transwell assays. The viability, migration and invasion of HEC-1-B cells after over-expression of miR-195-5p combined with 100 μg/L IL-17 intervention were also observed. RESULTS:The expression of IL-17 was increased while the expression of miR-195-5p was decreased in the human endometrial carcinoma samples (P<0.05). The expression of miR-195-5p in the HEC-1-B cells after treatment with IL-17 at 10,100 and 300 μg/L for 48 h was significantly decreased (P<0.05). The results of MTT assay and Transwell experiments indicated that IL-17 at 100 μg/L enhanced the viability, migration and invasion of HEC-1-B cells, while over-expression of miR-195-5p resulted in the opposite effect. CONCLUSION:Over-expression of miR-195-5p inhibits the enhancing effects of IL-17 on the viability, invasion and migration of HEC-1-B cells.  相似文献   

12.
ATM: To probe the effect and the mechanism of astragaloside IV and ginsenoside Rg1 on autophagy of PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The autophagy injury model of PC12 cells induced by OGD/R was established(PC12 cells were exposed to 2 h of OGD followed by 24 h of reoxygenation). The effects of astragaloside IV combined with ginsenoside Rg1 on autophagy of PC12 cells were observed, and the mechanism was studied through PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways. RESULTS: After OGD/R, LC3-Ⅱ/LC3-Ⅰin PC12 cells was increased. Astragaloside IV, ginsenoside Rg1 and astragaloside IV combined with ginsenoside Rg1 restrained the increase in LC3-Ⅱ/LC3-Ⅰ, the effect of the combination was greater than using the drug alone. Ginsenoside Rg1, astragaloside IV combined with ginsenoside Rg1 up-regulated the phosphorylation level of PI3K Ⅰ, Akt and mTOR. The effects of the combination were stronger than those of using the drug alone. Astragaloside IV, astragaloside IV combined with ginsenoside Rg1 inhibited the protein expression of PI3K Ⅲ and becline-1, the effects of the combination were better than those of single astragaloside IV and single ginsenoside Rg1. Meanwhile, the combination treatment increased Bcl-2 protein expression. CONCLUSION: The autophagy of PC12 cells induced by OGD/R is inhibited by astragaloside IV and ginsenoside Rg1. Furthermore, astragaloside IV combined with ginsenoside Rg1 plays synergitic inhibition on autophagy, the mechanism may be related to PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways.  相似文献   

13.
AIM: To investigate the mechanism of L-type calcium channel (L-Ca2+)/calpain signal transduction pathway in verapamil inversing resistance of papillary thyroid carcinoma to doxorubicin.METHODS: Human papillary thyroid carcinoma TPC-1 cells were cultured for 2 d. For determining the appropriate concentrations and treatment time of verapamil and doxorubicin, a compatibility test was conducted to detect the cell viability by CCK-8 assay. The cells were divided into control group, doxorubicin group, verapamil group and doxorubicin+verapamil group. The techniques of whole-cell patch-clamp was used to record L-Ca2+ currents. The protein expression levels of calpain 1 and LC3 were detected by Western blot.RESULTS: Compared with control group, the density of L-Ca2+ current decreased in doxorubicin group and verapamil group (P<0.05). Compared with verapamil group, the density of L-Ca2+ current decreased in doxorubicin+verapamil group (P<0.01). Compared with control group, the expression of calpain 1 decreased in doxorubicin group and verapamil group (P<0.05). Compared with doxorubicin group, the expression of calpain 1 decreased in doxorubicin+verapamil group (P<0.05). Compared with control group, the expression of LC3 increased in doxorubicin group and verapamil group (P<0.05). Compared with doxorubicin group, the expression of LC3 increased in doxorubicin+verapamil group (P<0.01).CONCLUSION: The drug resistance of TPC-1 cells to doxorubicin may be related to the increase in autophagic activity. Verapamil further increases autophagic activity of TPC-1 cells, resulting in autophagic death and inversing the resistance of TPC-1 cells to doxorubicin. The mechanism may be involved in L-Ca2+/calpain 1 signal transduction pathway of autophagy.  相似文献   

14.
YANG Yi  TANG Xiao-li  LIU Yue  FANG Fang 《园艺学报》2019,35(11):2028-2034
AIM: To explore whether the damage of neurons induced by amyloid β-protein (Aβ) is related to the regulation of autophagy and its mechanism based on Akt/mTOR pathway. METHODS: SH-SY5Y cells were incubated with Aβ25-35 (5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L and 25 μmol/L) for 24 h, and the cell viability was measured by MTT assay. The protein levels of LC3-I, LC3-II, Akt, p-Akt, mTOR and p-mTOR in the SH-SY5Y cells were determined by Western blot. After the SH-5Y5Y cells were incubated with autophagy inducer rapamycin (Rapa) or autophagy inhibitor 3-methyladenine (3-MA) combined with Aβ25-35 for 24 h, the cell viability and related protein expression were detected by the same methods above mentioned. RESULTS: Each concentration of Aβ25-35 damaged SH-SY5Y cells and decreased the viability of SH-SY5Y cells. Aβ25-35 increased the expression of autophagy marker protein LC3-II, increased the level of LC3-II/LC3-I, and down-regulated the phosphorylation level of Akt and mTOR proteins (P<0.05). When combined with autophagy inducer Rapa, the cell viability was not significantly affected, the expression of LC3-II protein was increased, LC3-II/LC3-I was increased significantly, and p-mTOR/mTOR level was decreased (P<0.05). When combined with autophagy inhibitor 3-MA, the protein expression of LC3-II and the level of LC3-II/LC3-I showed a downward trend, while the level of p-Akt/Akt was decreased (P<0.05). CONCLUSION: Aβ25-35 may induce SH-SY5Y cell autophagy and injury by down-regulating phosphorylation levels of Akt and mTOR proteins.  相似文献   

15.
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.  相似文献   

16.
AIM: To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process. METHODS: MSCs were isolated and cultured in vitro. The cells were divided into LiCl group and control group. MSCs were treated with β-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons. The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associated protein 1 light chain 3 (LC3) were measured by immunofluorescence method and Western blot. An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells. The protein expression of NSE and MAP-2 were determined by Western blot. RESULTS: After induction, the expression of NSE and MAP-2 were increased. The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05). After induction, the number of LC3-positive dots and the expression of LC3-Ⅱ in LiCl group were greater than those in control group (P<0.05). The expression of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated-cells, and on the contrary, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA. CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy.  相似文献   

17.
AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation; （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h; （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling, and the rest were treated the same as the H/R group; （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia; （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h, then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure, and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）, beclin 1, LC3, mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK, beclin 1, LC3-Ⅱ, p-mTOR and P62. RESULTS Compared with Con group, the cell viability in H/R, DMSO, 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK, beclin 1 and LC3 was significantly increased, and the protein levels of p-AMPK, beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased, and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R, DMSO and HX531 groups （P<0.05）, and no significant difference among H/R, DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R, and its mechanism may be related to the inhibition of autophagy.  相似文献   

18.
AIM：To observe the effect of MK-2206, an inhibitor of Akt, on the cell apoptosis and autophagy of U2OS cells. METHODS：The cell viability was detected by MTT assay. The cell apoptosis was analyzed by TdT-mediated dUTP nick end labeling assay. The expression of LC3-II was examined by Western blotting. RESULTS：MK-2206 inhibited the cell viability in a dose-dependent manner. MK-2206 induced the cell apoptosis via activation of caspase-3, caspase-9 and PARP. MK-2206 treatment substantially induced the U2OS cell autophagy by increasing in the levels of LC3-II. Blockage of autophagy using chloroquine magnified MK-2206-induced cell death in U2OS cells. CONCLUSION：The Akt inhibitor MK-2206 induces cell apoptosis and autophagy. Blocking autophagy magnifies MK-2206-induced the inhibition of the viability in U2OS cells.  相似文献   

19.
AIM：To examine the effects of high concentration of extracellular ATP on human neuroblastoma SH-SY5Y cell injury. METHODS：Cultured SH-SY5Y cells were grouped according to the concentrations of ATP and treatment time. The cell viability was detected by CCK-8 assay. The variation of autophagic vacuoles was observed with monodansylcadaverine staining. The cell apoptosis was analyzed by Hoechst 33258 staining. Meanwhile, apoptotic rate was detected by flow cytometry. The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) were determined by Western blotting. RESULTS：Compared with control group, the survival rate of SH-SY5Y cells was significantly reduced by ATP at different concentrations (3, 6, 9, 12 and 15 mmol/L for 3 h) and different treatment time (1, 2, 3 and 6 h with 6 mmol/L ATP, peaking at 3 h). The autophagic vacuoles of SH-SY5Y cells were significantly increased at 1 h with ATP treatment, trended to decrease over time and returned to control level at 6 h. The protein expression of LC3-Ⅱ was significantly increased at 1 h with ATP treatment, which was consistent with the time points of increasing autophagic vacuoles. LC3-Ⅱ expression level gradually decreased at 2~3 h with ATP treatment, and returned to control level at 6 h. Compared with control group, the apoptotic rate and the expression level of caspase-3 were enhanced synchronously. The peak of apoptotic rate occurred at 3 h, and kept until 6 h.The level of cleaved caspase-3 expression peaked at 6 h. CONCLUSION：High concentration of extracellular ATP induces the autophagy and apoptosis of SH-SY5Y cells. The increased autophagy shows up, followed by the climax of apoptosis until 6 h. With the prolonged duration of ATP, apoptosis is the main process in the cells.  相似文献   

20.
AIM: To establish an autophagy model of glutamic acid-induced rat gastric interstitial cells of Cajal (ICCs) in vitro. METHODS: Glutamic acid was added to the medium of cultured ICCs in vitro at different concentrations (high, medium and low concentrations were 10 mmol/L, 5 mmol/L and 2.5 mmol/L, respectively) for different periods (3 h, 6 h and 24 h). The cell viability was measured by CCK-8 assay. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) in the ICCs after cultured with glutamic acid at different concentrations for different periods was determined by Western blot. The ultrastructure and autophagic vacuoles of ICCs were observed under electron microscope, and immunofluorescence technique was used to measure the fluorescence intensity of autophagic protein LC3 after co-cultured with glutamic acid at medium concentration. RESULTS: Compared with control group, glutamic acid significantly inhibited the viability of ICCs (P<0.01). The ratios of LC3-Ⅱ/LC3-I in medium and high concentration groups at 3 h, 6 h and 24 h were significantly increased as compared with control group and low concentration group (P<0.01), and that in medium concentration group at 3 h was the best to increased the ratio of LC3-Ⅱ/LC3-I (P<0.01). Under electron microscope, the ICCs in glutamic acid group showed obvious autophagic vacuoles and cytoplasmic vacuoles. The fluorescence intensity of autophagic protein LC3 in the ICCs of glutamic acid group was significantly enhanced (P<0.01). CONCLUSION: Glutamic acid successfully induces autophagy in ICCs. The optimum condition of glutamic acid was 5 mmol/L for 3 h.  相似文献   

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