ObjectiveTo investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.MethodsThe skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.ResultsAfter identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased (P<0.05), and the level of ROS also significantly increased (P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased (P<0.05), the relative expression of Bcl-2 protein significantly increased (P<0.05), the cell apoptosis rate and level of ROS also significantly decreased (P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.ConclusionInhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.
ObjectiveTo evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).MethodsBMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10−5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.ResultsWith the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually (P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin (P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group (t=−10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups (P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β1/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group (P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups (P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group (P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group (P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group (P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group (P<0.05).ConclusionIcariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.
Immunoglobulin A nephropathy (IgAN) is an immune-mediated chronic inflammatory disease with a complex pathogenesis and diverse clinical manifestations. Currently, there is no specific treatment plan. Programmed cell death is an active and orderly way of cell death controlled by genes in the body, which maintains the homeostasis of the body and the development of organs and tissues by participating in various molecular signaling pathways. In recent years, programmed cell death has played an important regulatory role in the occurrence and development of IgAN, involving complex signaling pathways. Under pathological conditions, it may relieve kidney damage through various pathways such as reducing oxidative stress, inhibiting inflammation, and improving energy metabolism. This article provides a review of the research progress of IgAN in apoptosis, autophagy, pyroptosis, ferroptosis,and cuproptosis in order to provide new therapeutic targets for IgAN.
ObjectiveTo analyze the expression and significance of NF-κBp65 and autophagy-related proteins Beclin1 and p62 in patients with papillary thyroid carcinoma (PTC).MethodsOne hundred and sixty cases of PTC patients' tumor tissue specimens and paracancerous tissue specimens in our hospital from March 2013 to February 2015 were collected, and 90 cases of cervical lymph node metastasis tissue specimens of the above patients were collected. The expressions of NF-κBp65, Beclin1 and p62 in PTC tissues, metastatic lymph node tissues and paracancerous tissues were detected by immunohistochemical method, and the relationship between the above indexes and the clinicopathological characteristics and prognosis of PTC patients was analyzed.ResultsThe positive rates of expression of NF-kappa Bp65 and p62 in PTC tissues and metastatic lymph node tissues were higher than those in paracancerous tissues (P<0.05). The expression rate of Beclin1 in PTC tissues and metastatic lymph node tissues was lower than that in paracancerous tissues (P<0.05). The positive rate of NF-κBp65 expression in PTC tissues was not related to the clinicopathological characteristics of patients (P>0.05). The expression of p62 decreased with the increase of tumor differentiation (P<0.05). The expression of Beclin1 in patients with stage Ⅲ+Ⅳ and lymph node metastasis were lower than those in patients with stage Ⅰ+Ⅱ and without lymph node metastasis (P<0.05), while the expression of p62 was opposite. Spearman correlation analysis showed that the expression of Beclin1 and p62 in PTC tissues was negatively correlated (r=–0.656, P<0.01). In metastatic lymph node tissues, the expression of Beclin1 and p62 was also negatively correlated (r=–0.562, P<0.01). The 3-year survival rates of patients with positive expression of p62 and NF-κBp65 in PTC tissues were lower than that of patients with negative expression (P<0.05). The 3-year survival rate of patients with positive expression of Becrin1 was higher than that of negative expression (P<0.05). TNM stage, lymph node metastasis, NF-κBp65 and p62 were independent risk factors for PTC prognosis, and Beclin1 was protective factor.ConclusionsNF-κBp65 and p62 are highly expressed in PTC tissues and lymph node metastasis tissues, while Beclin1 is poorly expressed, which could be used as independent prognostic factors for PTC patients. In addition, Beclin1 and p62 are related to PTC biological behavior and may become potential indicators for PTC diagnosis.
Epilepsy is a heterogeneous disease with a very complex etiological mechanism, characterized by recurrent and unpredictable abnormal neuronal discharge. Epilepsy patients mainly rely on oral antiseizure medication (ASMs) the for treatment and control of disease progression. However, about 30% patients are resistance to ASMs, leading to the inability to alleviate and cure seizures, which gradually evolve into refractory epilepsy. The most common type of intractable epilepsy is temporal lobe epilepsy. Therefore, in-depth exploration of the causes and molecular mechanisms of seizures is the key to find new methods for treating refractory epilepsy. Mitochondria are important organelles within cells, providing abundant energy to neurons and continuously driving their activity. Neurons rely on mitochondria for complex neurotransmitter transmission, synaptic plasticity processes, and the establishment of membrane excitability. The process by which the autophagy system degrades and metabolizes damaged mitochondria through lysosomes is called mitophagy. Mitophagy is a specific autophagic pathway that maintains cellular structure and function. Mitochondrial dysfunction can produce harmful reactive oxygen species, damage cell proteins and DNA, or trigger programmed cell death. Mitophagy helps maintain mitochondrial quality control and quantity regulation in various cell types, and is closely related to the occurrence and development of epilepsy. The imbalance of mitophagy regulation is one of the causes of abnormal neuronal discharge and epileptic seizures. Understanding its related mechanisms is crucial for the treatment and control of the progression of epilepsy in patients.
Cell autophagy plays a key role in maintaining intracellular nutritional homeostasis during starvation through elimination of aberrant or obsolete cellular structures. The cellular cytoskeleton has a crucial role in multiple processes involving membrane rearrangements and vesicle-mediated events. Autophagy is mediated by both microtubules and actin networks: microtubules promote the synthesis of autophagosome and are related to the movement of autophagosome; actin networks have been implicated in structurally supporting the expanding of phagophore, moving autophagosomes and enabling their efficient fusion with the lysosome; non-muscle myosinⅡoperates in the early stages of autophagy during the initiation and expansion of the phagophore, whereas myosinⅥ and myosin 1C are involved in the late stages of autophagosome maturation and fusion with the lysosome, respectively. This review summarizes the multiple regulation of cytoskeleton on autophagy and focuses on the regulation of autophagy by actin and myosin, providing a new approach for the study of pathogenesis and innovative therapies of autophagy related diseases.
Objective To analyze the hotspots and development trends in the research field of tumor cell apoptosis and autophagy. Methods Relevant literature on tumor apoptosis and autophagy published between January 2012 and December 2021 were searched through the Web of Science core collection database, and CiteSpace 5.8.R3 software and VOSviewer version 1.6.10 software were used to analyze the country/region, institution, keywords and citation node information of the literature. Results A total of 6716 foreign-language articles were included in the study after searching and screening, and the number of papers showed a linear upward trend year by year. China published the largest number of articles and cooperated closely with other research institutions, but there were not many high-quality and influential articles. The two journals Autophagy and Cell were more authoritative in the field of tumor apoptosis and autophagy research. The signaling pathways and related proteins of apoptosis and autophagy, and the study of tumor suppressor mechanisms based on apoptosis/autophagy were current research hot topics. The migration, apoptosis and epithelial mesenchymal transformation of cancer cells would be the research focus and direction in the future. Conclusions In the past 10 years, the research on tumor apoptosis and autophagy has continued to develop. With the in-depth research on the molecular level, the study of its mechanism is expected to further reveal the mystery of tumor apoptosis and autophagy.
Objective To explore the influence and mechanism of mechanistic target of rapamycin kinase (mTOR)/ receptor of advanced glycation end products (RAGE) pathway mediated-ferritinophagy on high glucose consumption promoting invasion and migration of colorectal cancer (CRC). Methods① Patients and tissue samples. Clinical data and tissues were collected from CRC patients underwent surgery and completed the dietary questionnaire in the Second Affiliated Hospital of Harbin Medical University from October 2022 to October 2023. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to analyzed the expression of nuclear receptor coactivator 4 (NCOA4) and ferritin in CRC and para-carcinoma tissues. ② Cell culture and treatment. The HT29 and HCT116 cells were treated by RPMI1640 medium containing 0, 35, 70, 105, 140 mmol/L glucose, and cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) activity analysis were performed to confirm 105 mmol/L glucose was the optimal concentration in the current study. Then the HT29 and HCT116 cells were randomly divided into: control group, glucose group; control group, glucose group, si-RAGE group, and glucose+si-RAGE group; control group, glucose group, rapamycin group, and glucose+rapamycin group. Untreated HT29 and HCT116 cells were considered as control group. The cells in glucose group were treated with 105 mmol/L glucose for 48 h. The CRC cells in the si-RAGE group were transfected with si-RAGE for 6 h. The CRC cells in the rapamycin group were treated with 10 nmol/L rapamycin for 48 h. The CRC cells in the glucose+si-RAGE group were treated with 105 mmol/L glucose for 48 h combination transfected with si-RAGE for 6 h. The CRC cells in the glucose+rapamycin group were treated with 105 mmol/L glucose for 48 h combination treated with 10 nmol/L rapamycin for 48 h. Then electron microscopy and Western blot, wound healing assay and transwell assay were exhibited, respectively. ③ Azoxymethane (AOM)-induced CRC rat model. The effects of glucose consumption on malignant behavior and ferritinophagy mediated by mTOR/RAGE pathway were evaluated in AOM-induced CRC rat models. A total of 16 rats were randomly divided into control group and glucose group, the CRC number was recorded and HE staining of CRC tissues was further performed. The expression of RAGE, mTOR, NCOA4, and ferritin in colorectal tissues of rats from each group was detected by RT-qPCR. Results① More lymphatic node metastasis and TNM Ⅲ/Ⅳ stages were observed in CRC patients from high glucose consumption group (P=0.004, P=0.004). Moreover, we confirmed that NCOA4 expression was significantly decreased (P<0.001) while ferritin was significantly increased (P<0.001) in CRC tissues especially in the CRC tissues from patients with positive lymph nodes metastasis. ② High glucose treatment significantly decreased autophagosomes in HT29 and HCT116 cells while si-RAGE transfection increased autophagic vacuoles compared to the control group. When compared with the glucose group, autophagosomes were increased in the glucose+si-RAGE group. Moreover, compared to the control group, the expressions of RAGE, p-mTOR, and ferritin were increased (P<0.001) while the expression of NCOA4 was decreased (P<0.001) in glucose group, but the expressions of RAGE, p-mTOR, and ferritin were decreased (P<0.001) while the expression of NCOA4 was increased (P<0.001) in the si-RAGE group; when compared with the glucose group, the expressions of RAGE, p-mTOR, and ferritin were downregulated (P<0.001) while the expression of NCOA4 was upregulated (P<0.001) in HT29 and HCT116 cells from the glucose+si-RAGE group. Compared to the control group, the HT29 and HCT116 cells in the glucose group performed enhanced wound scratch healing , migration and invasion viabilities (P<0.05); but the HT29 and HCT116 cells in the si-RAGE group performed impaired wound scratch healing, migration and invasion viabilities (P<0.05). When compared with the glucose group, the HT29 and HCT116 cells in the glucose+si-RAGE group performed impaired wound scratch healing, migration and invasion viabilities (P<0.05). ③ Rapamycin treatment significantly inhibited the expression of ferritin (P<0.05) but induced the expression of NCOA4 (P<0.05) compared to the control group. When compared with the glucose group, the expression of ferritin was downregulated (P<0.05) while the expression of NCOA4 was upregulated (P<0.05) in HT29 and HCT116 cells from the glucose+rapamycin group. Additionally, compared to the control group, rapamycin treatment performed inhibited effect on wound scratch healing, migration and invasion viabilities in the HT29 and HCT116 cells (P<0.05); while the HT29 and HCT116 cells in the glucose+rapamycin group performed impaired wound scratch healing, migration and invasion viabilities (P<0.05) when compared with the glucose group. ④ In the AOM induced CRC rat models, we found the more CRCs, aggravated cellular pleomorphism and upregulated expressions of RAGE, mTOR, ferritin (P<0.05) while downregulated expression of NCOA4 (P<0.05) in the glucose group than those of the control group. ConclusionHigh glucose consumption promote invasion and migration in CRC through suppressing ferritinophagy via activating the mTOR/RAGE pathway.
ObjectiveTo summarize the role of chondrocyte mitochondrial homeostasis imbalance in the pathogenesis of osteoarthritis (OA) and analyze its application prospects. Methods The recent literature at home and abroad was reviewed to summarize the mechanism of mitochondrial homeostasis imbalance, the relationship between mitochondrial homeostasis imbalance and the pathogenesis of OA, and the application prospect in the treatment of OA. Results Recent studies have shown that mitochondrial homeostasis imbalance, which is caused by abnormal mitochondrial biogenesis, the imbalance of mitochondrial redox, the imbalance of mitochondrial dynamics, and damaged mitochondrial autophagy of chondrocytes, plays an important role in the pathogenesis of OA. Abnormal mitochondrial biogenesis can accelerate the catabolic reaction of OA chondrocytes and aggravate cartilage damage. The imbalance of mitochondrial redox can lead to the accumulation of reactive oxygen species (ROS), inhibit the synthesis of extracellular matrix, induce ferroptosis and eventually leads to cartilage degradation. The imbalance of mitochondrial dynamics can lead to mitochondrial DNA mutation, decreased adenosine triphosphate production, ROS accumulation, and accelerated apoptosis of chondrocytes. When mitochondrial autophagy is damaged, dysfunctional mitochondria cannot be cleared in time, leading to ROS accumulation, which leads to chondrocyte apoptosis. It has been found that substances such as puerarin, safflower yellow, and astaxanthin can inhibit the development of OA by regulating mitochondrial homeostasis, which proves the potential to be used in the treatment of OA. Conclusion The mitochondrial homeostasis imbalance in chondrocytes is one of the most important pathogeneses of OA, and further exploration of the mechanisms of mitochondrial homeostasis imbalance is of great significance for the prevention and treatment of OA.