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Aim To investigate the expression of IncRNA AC079466. 1 in non-small cell lung cancer (NSCLC) tissues and cells, and the effect of its overexpression on the proliferation, apoptosis, migration and invasion of A549 and H1299 cells. Methods Cancer tissues and corresponding adjacent tissues from 20 NSCLC patients were collected, and the expression of IncRNA AC079466. 1 in tissue and cells was detected by qRT-PCR. AC079466. 1 group was transfected with overexpression plasmid, NC group was transfected with empty plasmid, and no transfection was used in the Blank group. MTT, flow cytometry and Transwell were used to detect the effects of IncRNA AC079466. 1 overexpression on the viability, apoptosis, migration and invasion of A549 and HI299 cells. Western blot was used to detect the effect of overexpression of IncRNA AC079466. 1 on the expression of endoplasmic reticulum stress-related factors GRP78, PERK, eIF2a, ATF4, CHOP, Bax and caspase-3. Results Compared with adjacent tissues, the expression of IncRNA AC079466. 1 in cancer tissues significantly decreased. Compared with HBE cells, the expression of IncRNA AC079466. 1 significantly decreased in A549 and H1299 cells. Compared with the Blank group and NC group, the viability, migration and invasion abilities of A549 and H1299 cells in AC079466. 1 group all markedly decreased, the apoptosis rate apparently increased, and the expressions of endoplasmic reticulum stress-related factors GRP78, p-PERK, eIF2a, ATF4, CHOP, Bax and caspase-3 were significantly up-regulated. Conclusion The overexpression of IncRNA AC079466. 1 significantly inhibits the viability, migration and invasion of A549 and HI299 cells, and promotes cell apoptosis. The mechanism may be related to the promotion of endoplasmic reticulum stress-mediated cell apoptosis.
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To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P<0.05 or P<0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P<0.05 or P<0.01), while Bcl-2 was significantly up-regulated(P<0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P<0.01), whereas Bcl-2 was significantly up-regulated(P<0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.
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Acetatos , Apoptosis , Bidens , Estrés del Retículo Endoplásmico , Hepatocitos , Factor de Transcripción CHOP/genética , eIF-2 Quinasa/genéticaRESUMEN
This study aimed to investigate the effect and possible mechanism of Bidens pilosa decoction on non-alcoholic fatty liver disease(NAFLD) induced by high fat and high glucose in mice. Bald/c mice were randomly divided into normal group, model group, metformin(200 mg·kg~(-1)) treatment group, Bidens pilosa decoction(10 g·kg~(-1)) treatment group, metformin and B. pilosa decoction(100 mg·kg~(-1)+5 g·kg~(-1)) treatment group. Except for the normal group, mice in the other four groups were fed with high-fat and high-glucose diet for 8 weeks to establish the non-alcoholic fatty liver model. After 4 weeks of treatment, blood was collected from the eyeballs, the mice were sacrificed, and relevant indicators were detected. The results showed that compared with the model group, blood lipid and blood glucose levels of each treatment group were significantly lower(P<0.05); HE staining results showed that liver pathological damage in each treatment group was significantly improved; oil red O staining results showed fat distribution in each treatment group significantly reduced(P<0.01); immunohistochemical staining showed that glucose regulated the protein expression of protein 78(GRP78) in liver tissues of each treatment group was also significantly reduced(P<0.01); Western blot results showed that endoplasmic reticulum stress signal pathway-related factors GRP78, phosphorylated-protein kinase R-like ER kinase(p-PERK), eukaryotic translation-initiation factor 2α(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(Chop), inositol requiring 1α(IRE1α), and cleaved-cysteinyl aspartate specific proteinase 12(cleaved-caspase-12) were significantly reduced(P<0.01). The results of the combined drug treatment group were better than those of the single drug treatment group. These results showed that B. pilosa decoction had the effect in improving non-alcoholic fatty liver, and its mechanism may be related to the down-regulation of the expression of endoplasmic reticulum stress(ERS)-related factors, and the reduction of the apoptosis of hepatocytes caused by ERS and the down-regulation of blood lipid and blood glucose levels.
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Animales , Ratones , Apoptosis , Bidens , Estrés del Retículo Endoplásmico , Endorribonucleasas , Glucosa , Enfermedad del Hígado Graso no Alcohólico , Proteínas Serina-Treonina QuinasasRESUMEN
The aim of this study was to investigate the preventive and therapeutic effects of Erzhi Pills on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine( MPTP)-induced Parkinson's disease( PD) in mice,and explore its possible mechanism of action. Mice were intraperitoneally injected with MPTP( 30 mg·kg-1,0. 01 m L·g-1) once daily to induce PD for 8 days. In the treatment group,Erzhi Pills were given by intragastric administration( 2. 5 g·kg-1,once daily for 30 days). The normal group received an equal volume of normalsaline. In terms of behavior,the limb movement coordination ability of the mice was detected by climbing,hanging and swimming experiments. The spatial learning and memory ability of the mice was detected by Morris water maze test. The content of MDA,as well as the activity of GSH-PX and SOD were determined in mice serum. Western blot was used to detect the protein expression levels of TH,MAOB and apoptosis-related factors CHOP and caspase-12 in brain tissues. Immunohistochemistry was used to detect the expression of TH in section of brain tissues in mice. The results showed that in behavioral aspects,as compared with the model group,the scores of limb movement ability as well as scores of spatial learning and memory ability were significantly improved in the treatment groups( P<0. 05). In terms of serological indicators,as compared with the model group,the activities of SOD and GSH-PX were significantly increased in the serum of treatment groups,and the content of MDA was significantly decreased( P<0. 05). The results of Western blot showed that as compared with the model group,the protein levels of TH in the brain tissues of the mice in treatment group were significantly up-regulated,while the protein levels of MAOB and apoptosis-related factors CHOP and caspase-12 were significantly down-regulated( P<0. 05). The results of immunohistochemistry showed that the number of TH positive cells in the brain tissues of the mice in the treatment group was significantly increased as compared with the model group( P<0. 05). In summary,Erzhi Pills have a certain preventive and therapeutic effect on MPTP-induced PD mice,which can significantly improve the limb motor coordination ability and spatial learning and memory ability of PD mice. Its mechanism may be related to down-regulating the expression of apoptosis-related factors CHOP and caspase-12,reducing the dopaminergic neuron damage and inhibiting dopaminergic neuronal apoptosis.
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Animales , Ratones , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/farmacología , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson , Sustancia NegraRESUMEN
This study focused on the protective effect of earthworm active ingredients (EWAs) on hepatocyte apoptosis induced by endoplasmic reticulum stress (ERS) in L-02 cells. The L-02 cells were cultured in vitro. The cell viability was measured with CCK-8, the apoptosis of L-02 cells was detected by flow cytometry, and the relevant protein and mRNA expressions were detected by Western blot and qPCR. According to the findings, tunicamycin (TM) could obviously reduce the survival rate of L-02 cells in a time-dependent and dose-dependent manner. Compared with normal group, the apoptosis rate in model group was significantly increased (<0.05 or <0.01). The protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, were significantly up-regulated (<0.05 or <0.01), while Bcl-2 was significantly down-regulated (<0.05 or <0.01). After the administration with different concentrations of EWAs, compared with model group, EWAs could significantly increase the survival rate ofL-02 hepatocyte and decrease the cell apoptosis rates. It could also reduce the protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, in a dose-dependent manner (<0.05 or <0.01) and increased the protein and mRNA expressions of Bcl-2(<0.05 or <0.01). These results showed that EWAs had a significantly protective effect on hepatocyte apoptosis induced by ERS in L-02 cells. Its mechanism may be related to the down-regulation of mRNA and protein expressions of GRP78, PERK, ATF4, eLF2α, CHOP and Bax, and the up-regulation, the relief of ERS and the promotion of the proliferation of impaired L-02 cells.
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OBEJECTIVE Gecko has been clinically used in China for many years. It has been proved that the gecko polypeptide mixture(GPM)extracted from gecko could inhibit the growth of multiple types of tumor cells.In order to investigate the possible anti-tumor molecular mechanisms of GPM,we used RNA-seq technology to identify the differentially expressed genes of human hepatocellular carci-noma(HCC)HepG2 cells treated with or without GPM.METHODS The HepG2 cells were treated with different concentration of GPM(0,0.1,0.2,0.3,0.4 mg·mL-1)for 6 h,12 h and 24 h,respectively.MTT assay was used to detect the viability of HepG2 cells. DAPI fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells.Western blot analysis was applied to observe the expres-sion of apoptosis- related proteins and endoplasmic reticulum stress (ERs)-related proteins in HepG2 cells.Flow cytometry was also applied to detect reactive oxygen species(ROS)generation.In this report, we showed that GPM could induce HepG2 cells apoptosis and influence HepG2 cells proliferation in a dose-dependent manner.We applied many analysis methods,including differentially expressed genes analysis,Gene Ontology(GO)enrichment analysis,KEGG pathway enrichment analysis,protein-protein interaction network analysis to screen out possible molecular mechanisms.RESULTS ER-nucleus signaling pathway, cellular response to stress and apoptotic processes were identified the potential anti-cancer molecular biological process of GPM.GPM may also induce apoptosis in HepG2 cells via endoplasmic reticulum stress pathway. The GPM could induce ROS generation and up-regulate ERs-related proteins. CONCLUSION The present study revealed the potential anti-tumor mechanism of GPM.
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To study the protective effect of earthworm active ingredients(EWAs) against endoplasmic reticulum stress(ERS)-induced acute liver injury in mice. The model of liver injury was induced through intraperitoneal injection of 10%CCl4. Serum glutamic-pyruvic transaminase(ALT), glutamic-oxaloacetic transaminase(AST), superoxide dismutase(SOD) and glutathione peroxidase(GSH-PX) activity and malondialdehyde(MDA) concentration were detected by colorimetric method. Histological examination was performed through hematoxylin-eosin staining and light microscopy, and apoptosis was detected using terminal transferase dUTP nick end labeling. The expressions of ERS related proteins, including glucose regulated protein 78(GRP78), protein kinase R-like ER kinase(PERK), eukaryotic transcription initiation factor 2α(eIF2α), active transcription factor-4(ATF4) and CCAAT/enhancer binding homologous protein(CHOP), were measured by immunohistochemistry and Western blot. According to the results, compared with the model group,serological indexes in the high, middle and low doses of EWAs were significantly improved (P<0.05 or P<0.01), the extent of liver lesion was decreased and the degree of injury was significantly reduced, and that the liver index and the spleen index of mice were significantly changed(P<0.05 or P<0.01). In liver tissue, the expressions of GRP78 and CHOP were significantly decreased(P<0.05 or P<0.01). The protein expressions of GRP78, CHOP and its upstream signaling pathway PERK-eIF2-ATF4 were significantly decreased in each dose group(P<0.05 or P<0.01). In summary, EWAs has a significant protective effect on ERS-induced acute liver injury, and its mechanism may be correlated with the inhibition of oxidative stress and ERS, and down-regulation of ERS marker protein CHOP expression, andinhibition of apoptosis.