Objective To investigate the effects and mechanism of 3-methyladenine （3-MA， an autophagy inhib-itor） on the apoptosis of hepatocellular carcinoma cell line SMMC7721 induced by 5-fluorouracil （5-FU）. Methods The autophagy was observed using fluorescent microscope by monodansyicadaverin （MDC） staining. The viability of SMMC-7721 cell induced by 5-FU was measured using CCK8 assay before and after autophagy inhibited by 3-MA， meanwhile the apoptosis of SMMC7721 cell was determined via AnnexinⅤ/PI assay. The light chain 3 protein （LC3， the autophagy specific protein） and caspase-3， PARP protein were detected by Western blot. Results The autophagy of SMMC7721 cell could be induced by 5-FU after treatment for 48 h， the cell survival rate was （60.73±2.65）%， and the apoptosis rate was （40.42±2.34）%. Compared with the group of 5-FU treatment， the survival rate of SMMC7721 cell in the combination of 5-FU and 3-MA after treatment for 48 h decreased to （42.31±1.32）% （P＜0.01）， and the apoptosis rate increased to （60.92±2.99）% （P＜0.01）， meanwhile the expressions of LC3-Ⅱ， activation fragment of caspase-3， and cleavage frag-ment of PARP significantly increased （P＜0.01）. Conclusions Autophagy is a protective phenomenon during the SMMC7721 cell line apoptosis induced by 5-FU， and autophagy inhibition may enhance the sensitivity of SMMC7721 cell line to 5-FU treatment， which is probably associated with the activation of caspase-3 and cleavage of PARP. Therefore， autophagy inhibition could be a promising strategy for adjuvant chemotherapy in hepatocellular carcinoma.
ObjectiveTo investigate the expression of autophagy-related genes and proteins in the lung tissues of patients with non-small cell lung cancer (NSCLC).MethodsPulmonary tissues were obtained from the surgically resected lung tissues of patients with NSCLC who were clinical diagnosed. The lung cancer tissues were derived from the pathologically diagnosed NSCLC and the normal tissues were from lung tissues 5 cm away from the lung lesions (29 cases in the lung cancer group and 32 cases in the normal group). The expression of autophagy-related proteins ATG5, LC3B, and p62 in lung tissues were measured by Western blot, and mRNA expression of ATG5 and p62 in the lung tissues were measured by real-time PCR.ResultsWestern blot analysis showed that the expression of ATG5 and p62 in lung cancer group were significantly higher than those in normal group (P<0.05). However, the expression of LC3B in lung cancer group was significantly lower than that in normal group (P<0.05). Real-time PCR analysis found that the mRNA expression of ATG5 and p62 in lung cancer group were significantly higher than those in normal group (P<0.05). The expression of ATG5, LC3B and p62 had no relationship with gender, age, smoking history, tumor location, tumor size, clinicopathological classification, differentiation or TNM stage. The expression of ATG5 had statistical significance in lymph node metastasis (P<0.05), but there was no difference for LC3B or p62 in lymph node metastasis (P>0.05).ConclusionsAutophagy plays a role in the tumorigenesis of lung cancer. If it’s possible to regulate and control autophagy-related genes and proteins effectively, it may supply new insights or targets into treatment for lung cancer patients.
ObjectiveTo investigate the role of autophagy-lysosomal system in skeletal muscle atrophy in rats with chronic obstructive pulmonary disease (COPD).
MethodsPassive cigarette smoking was used to establish COPD model. The mRNA and protein expression of FOXO transcription factor and autophagy-related genes Bnip3, Beclin1, p62, MAP-LC3Ⅱ/Ⅰ, Atg5 in extensor digitorum longus of rats were measured by real time PCR and Western blot. The changes of extensor digitorum longus tissue sections and lung tissue sections in the experimental group rats were observed under transmission electron microscopy.
ResultsCompared with the control group, the mRNA expression of FOXO transcription factor and autophagy-related genes Bnip3, Beclin1, p62, Atg5 in extensor digitorum longus of the experimental group group rats was significantly increased (all P<0.05, as for Bnip3, the P value between two groups <0.01); The mRNA expression of MAP-LC3Ⅱ/Ⅰwas not significantly different between two groups (P>0.05). The protein expression of FOXO, Bnip3, Beclin1, p62, MAP-LC3Ⅱ/Ⅰ, Atg5 significantly increased in the COPD group (all P<0.05, as for Bnip3, MAP-LC3Ⅱ/Ⅰ, Beclin1, the P values between two groups <0.01). Compared with the control group, autolysosome in extensor digitorum longus tissue sections of the experimental group rats increased and lung tissue fibrosis and more inflammatory cells were observed in lung tissue sections of the experimental group rats under transmission electron microscopy.
ConclusionThe mRNA and protein expressions of FOXO transcription factor and autophagy-related genes in extensor digitorum longus increase significantly in the experimental group rats, suggesting that the activity of autophagy-lysosomal system, which may be one mechanism of skeletal muscle atrophy in COPD.
The neuroretinal injuries of diabetic retinopathy (DR) include retinal neuronal damage and reactive gliosis, both of which are induced by hyperglycemia and presented as early features of DR. They promote to develop mutually and accelerate the progression of DR. The molecular mechanisms study of neuronal damage mainly focuses on the alterations of extracellular environment and related signaling pathways, include inflammation, oxidative stress, endoplasmic reticulum stress, the formation of advanced glycation end products, glutamate toxicity and so on. These alterations mainly result in neuronal apoptosis and autophagy. The damaged neurons activate the glial cells with apparent changes in morphology, cell counts and the level of intracellular protein expression. In non-proliferative DR, glial cells are moderately hypertrophic and slightly increased in numbers. In proliferative DR, there is a significant rise in glial cell number with enhanced level of inflammatory factors and vascular active substances which lead a further neuronal damage. Signaling pathways of extracellular signal-regulated kinase 1/2, c-Fos and p38 mitogen-activated protein kinase are associated with their activation. Researches on the molecular mechanisms and signaling pathways of the DR will promote controlling the DR progression at the cellular level.
Objective To realize the research progress of regulating the endoplasmic reticulum stress response to inhibit autophagy in tumor cells. Method The literatures about regulating the endoplasmic reticulum stress response to inhibit autophagy in tumor cells were reviewed. Result In the endoplasmic reticulum stress response induced by the release of calcium and accumulation of unfolded proteins, autophagy can be activated by several pathways, and to regulate physiological and pathological processes. Conclusion Further research about the endoplasmic reticulum stress response in tumor cells need to be done to regulate the response factors to inhibit autophagy.
ObjectiveTo summarize the autophagy and its research progress in gastric cancer.
MethodsIn combination with available literatures published in recent years involving the relationship between autophagy and gastric cancer, the characteristics of autophagy, molecular marker, control factors, and the significance and role in gastric cancer were reviewed.
ResultsAutophagy not only promotes cell death, but also can prolong the survival of cancer cells during the tumor formation. Reagents (including traditional Chinese medicine) regulating autophagy have broad prospect of application in cancer therapy, but anti-tumor therapeutic effect based on the regulation of autophagy depends on the actual level of intracellular autophagy.
ConclusionThe autophagy in the gastric cancer is still poorly understood, and to clarify the molecular mechanism of autophagy and kill cancer cells by reasonable regulation of autophagy still needs more further in-depth studies.
Objective To study the effect and mechanism of Xuebijing injection on sepsis-induced acute lung injury (ALI) in mice by regulating autophagy. Methods A total of 80 BALB/c male mice were randomly divided into control group, model group, Xuebijing group and Xuebijing+3-methyladenosine (3-MA) group, with 20 mice in each group. The control group received sham operation, while sepsis-induced ALI model was established in the later three groups by cecal ligation and puncture, on that basis, the Xuebijing group was given 10 mL/kg Xuebijing by intraperitoneal injection and the Xuebijing+3-MA group was given 10 mL/kg Xuebijing and 10 mg/kg autophagy inhibitor 3-mA by intraperitoneal injection. The cumulative survival rates of the four groups were observed 72 h after modeling, and the pathological changes of lung tissues, lung wet weight /dry weight ratios, inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1 β, and IL-18] contents, numbers of autophagosome, and the protein expression levels of autophagy genes [microtubule-associated protein light chain3 (LC3)-Ⅱ/LC3-Ⅰand Beclin-1] were detected. Results In the control group, the 72-hour cumulative survival rate was 100%, the lung wet weight /dry weight ratio was 3.89±0.85, the TNF-α, IL-1β, and IL-18 contents were (0.83±0.14) ng/mg, (0.74±0.15) ng/mg, and (84.51±13.25) pg/mg, respectively, the number of autophagosome was (0.41±0.09)/field, and the expression levels of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 were 0.20±0.04 and 0.17±0.03, respectively. In the model group, the lung tissue showed typical ALI pathological changes, the 72-hour cumulative survival rate (50%) was lower than that in the control group, and the lung wet weight /dry weight ratio (6.77±0.94), contents of TNF-α, IL-1β, and IL-18 [(3.15±0.76) ng/mg, (2.88±0.62) ng/mg, (274.62±45.58) pg/mg], autophagosome number [(3.14±0.55)/field], and expression levels of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 (0.69±0.09, 0.35±0.06) were higher than those in the control group (P<0.05). In the Xuebijing group, the ALI pathological changes alleviated, the 72-hour cumulative survival rate (75%), autophagosome number [(5.77±0.75)/field], and expression levels of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 (0.98±0.13, 0.62±0.08) were higher than those in the model group (P<0.05), and the lung wet weight /dry weight ratio (4.23±0.76) and contents of TNF-α, IL-1β, and IL-18 [(1.52±0.32) ng/mg, (1.29±0.30) ng/mg, (121.36±26.51) pg/mg] were lower than those in the model group (P<0.05). In the Xuebijing+3MA group, the ALI pathological changes aggravated, the 72-hour cumulative survival rate (55%), autophagosome number [(0.78±0.16)/field], and expression levels of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 (0.37±0.05, 0.32±0.05) were lower than those in the Xuebijing group (P<0.05), and the lung wet weight /dry weight ratio (6.31±0.91) and contents of TNF-α, IL-1β, and IL-18 [(2.88±0.56) ng/mg, (2.41±0.58) ng/mg, (252.35±37.65) pg/mg] were higher than those in the Xuebijing group (P<0.05). Conclusion Xuebijing injection can reduce ALI induced by sepsis in mice, and activation of autophagy is the molecular mechanism.
Age-related macular degeneration is one of the major causes of blindness in the elderly. As an important pathway of cell metabolism, autophagy maintains intracellular homeostasis through the degradation and recycle of damaged organelles and macromolecules. Understanding its mechanism may promote discoveries to delay aging process, reduce the incidence of age-related diseases. In mammals, silent information regulator protein 6 (SIRT6) plays its deacetylase and ribonucleotransferase activity in multiple signaling pathways, including inhibition of cellular senescence, tumorigenesis, metabolic diseases, regulating cellular lifespan. It has a significant impact on the structure and function of tissues and organs. SIRT6 regulates intracellular autophagy mainly through the insulin-like growth factor-protein kinase B-mammalian target of rapamycin, reducing the accumulation of toxic metabolites and cellular senescence. The function of SIRT6 in age-related macular degeneration need to be combined with the genetic background, pathogenesis, clinical manifestations and other aspects of the disease, and it is expected to be further studied in subsequent studies.
ObjectiveTo summarize the latest research progress of autophagy on regulation of tumor energy metabolism.
MethodThe latest research progresses of the regulation mechanism of autophagy on the energy metabolism of tumor cells and the influence on the biologic behavior of tumor in recent years were reviewed.
ResultThe autophagy could affect the biological behavior of the tumor by influence of the tumor cell glucose uptake, glycolysis, oxidative phosphorylation, lipid metabolism, and amino acid metabolism.
ConclusionsThe regulation of autophagy on tumor energy metabolism provides a theoretical basis for the survival mechanism of tumor under the stress condition. What's more, to research the mechanism of autophagy on regulation of energy metabolism and the effect on tumor biologic behavior will contribute to the development of new and more effective personalized anticancer therapy. As far as the research progress about tumor cells energy metabolism regulated by autophagy, what we must be revealed is whether other factors including oncogene, anti-oncogene, and signaling pathways take part in this regulation and what influence would be on the biological behavior of different tumors.
Objective To investigate the potential mechanism of cellular senescence-related mitochondrial autophagy genes in diabetic retinopathy (DR). MethodsThe DR gene datasets GSE53257 and GSE60436 from the GEO database and screened the differentially expressed genes (DEG) were downloaded. Cellular senescence-related genes and mitochondrial autophagy-related genes from the GeneCards database, and the intersection of the two to obtain the DR-related differentially expressed genes (CSRMRDEG) were collected. The obtained CSRMRDEG was subjected to Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis, protein-protein interaction network (PPI) analysis, and hub gene identification using Maximal Clique Centrality (MCC), Degree, Maximum Neighborhood Component (MNC)、Edge Percolated Component (EPC) and Closeness algorithms. Gene Set Enrichment Analysis (GSEA) was conducted to obtain the enriched pathways of DEG, and ssGSEA immune infiltration analysis was performed to screen the correlation between immune cells and DR. The diagnostic efficacy of hub genes for DR was evaluated by drawing the receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Meanwhile, the Wilcoxon rank sum test was used to compare the differences in the infiltration level of immune cells between the DR Group and the control group. Results23 DR-related CSRMRDEG were obtained. GO analysis showed that they were mainly enriched in the pathways of dicarboxylic acid, biosynthetic process of folate-containing compounds, tetrahydrofolate conversion, mitochondrial matrix, mitochondrial endomembrane, structural components of ribosomes, and glutamate transmembrane transporter protein activity. The results of KEGG pathway enrichment analysis showed that CSRMRDEG was highly enriched in pathways such as the folate carbon pool, biosynthesis of cofactors, and pyruvate metabolism. The PPI analysis results show that there are 16 related CSRMRDEG. Five algorithms (MCC, Degree, MNC, EPC, Closeness) obtained the nine Hub genes. The results of ROC curve analysis showed that the AUC of the expression levels of 9 hub genes for diagnosing DR ranged from 0.7-0.9. The ssGSEA results showed that there were statistically significant differences in Wilcoxon of central memory CD4+ T cells, macrophages, natural killer cells, and helper T cell 1 between the DR group and the control group (Z=?2.85, ?2.23, ?2.10, ?2.52; P＜0.05). ConclusionMitochondrial autophagy genes related to cellular senescence are potential diagnostic targets for DR.
