Autophagy-related Proteins LC3B Research Articles

Immunohistochemistry analysis of autophagy-related proteins Beclin-1, p62/SQSTM1, and LC3B in breast carcinoma progression to bone metastasis

Autophagy is a catabolic pathway involved both in tissue homeostasis and in cellular response to stress. The precise role of autophagy in cancer is still undefined and seems to depend on the tumor stage, appearing tumor-suppressive in physiological conditions and helpful to tumor progression in the established tumor. Here we analyzed by immunohistochemistry Beclin-1, p62, and LC3B, autophagic markers, in human specimens of normal breast, bone metastasis together with pair-matched invasive breast carcinoma of no special type (IBC-NST) as well as non-metastatic breast carcinoma, to disclose the possibility that they could be early prognostic indicators of the evolution of the disease toward the worst outcome. Different regions of metastatic carcinomas, i.e., areas adjacent to the tumor without signs of neoplastic growth, dysplastic lesions, and areas with invasive growth were considered. The pattern of autophagic parameters showed differences among the stages of breast carcinoma progression with a trend that indicated the activation of autophagic process in normal breast (Beclin-1 more elevated than p62), a pattern that was maintained in non-metastatic carcinoma. As the neoplasia proceeds with malignancy, the modification of the pattern of expression of autophagic markers (low ratio between Beclin-1 and p62) in areas of invasive growth of carcinomas suggested inhibition of the process. Of note, the parameters showed a different pattern in bone metastasis with respect to bone metastatic (bm)-IBC-NST, suggesting the reactivation of the autophagic process in the new growth site, helpful to the colonization. The course of autophagy markers during tumor progression could have a prognostic value towards bone metastasis and reveal different roles of the process in different phases of neoplastic growth. The understanding of the role of autophagy in bone metastasis could disclose new therapeutic targets to improve the conditions of patients.

Induction of Peroxiredoxin 1 by Hypoxia Promotes Cellular Autophagy and Cell Proliferation in Oral Leukoplakia via HIF-1α/BNIP3 Pathway.

Hypoxia is a key trigger in the transformation of oral leukoplakia into oral cancer. However, it is still too early to determine the role of hypoxia in the development of oral leukoplakia. Prx1, an antioxidant protein, upregulated by hypoxia, regulates cellular autophagy in leukoplakia. This study aimed to understand the mechanisms by which hypoxia induces Prx1 expression during autophagy in oral leukoplakia. We used an experimental model of tongue epithelial hyperplasia induced by 4-nitroquinoline-1-oxide (4NQO) and dysplastic oral keratinocytes. Prx1 knockdown DOK cells, Leuk-1 cells and control cells were harvested, and cell proliferation was assayed using the Cell Counting Kit-8. Several hypoxia and autophagy-related proteins were examined using quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and western blotting in cells and mouse tongue tissues. In addition, the ultrastructure of the cells was observed by transmission electron microscopy. Hypoxia induces cell proliferation, autophagic vesicles and the expression of Prx1, BNIP3, LC3II/I and Beclin-1 in DOK and Leuk-1 cells. However, these effects were all attenuated by Prx1 knockdown. Histologically, 4NQO induced epithelial hyperplasia in the tongue mucosa. The expression of proliferation marker PCNA, autophagy-related proteins LC3B and Beclin-1, as well as HIF-1α/BNIP3 was significantly lower in the tongue tissues of Prx1flox/flox:Cre+ mice compared with Prx1flox/flox mice. In Prx1flox/flox:Cre+ mice, an increased expression of HIF-1α/BNIP3, LC3B and Beclin-1 was detected in epithelial hyperplasia tongue tissues compared to normal tissues. The current study suggests that Prx1 may promotes cell proliferation and autophagy in oral leukoplakia cells via the HIF-1α/BNIP3 pathway.

Intravitreal Injection of ZYAN1 Restored Autophagy and Alleviated Oxidative Stress in Degenerating Retina via the HIF-1α/BNIP3 Pathway.

Mitochondrial autophagy plays a contributary role in the pathogenesis of retina degeneration (RD). ZYAN1 is a novel proline hydroxylase domain (PHD) inhibitor that can enhance the expression of hypoxia-inducible factor 1-alpha (HIF-1α). This study investigated whether ZYAN1 could alleviate progressive photoreceptor loss and oxidative damage in a pharmacologically induced RD model via the modulation of mitophagy. ZYAN1 was injected into the vitreous body of the RD model, and the retinal autophagy level was analyzed. The therapeutic effects of ZYAN1 were evaluated via a function examination, a morphological assay, in situ reactive oxygen species (ROS) detection, and an immunofluorescence assay. It was shown that the thickness of the outer nuclear layer (ONL) increased significantly, and visual function was efficiently preserved via ZYAN1 treatment. The mitochondria structure of photoreceptors was more complete in the ZYAN1-treated mice, and the number of autophagosomes also increased significantly. Membrane disc shedding and ROS overproduction were alleviated after ZYAN1 treatment, and the axonal cilia were more structurally intact. A Western blot analysis showed that the expression levels of the autophagy-related proteins LC3-B, Beclin-1, and ATG5 increased significantly after ZYAN1 treatment, while the expression of P62 was down-regulated. Moreover, the expression levels of HIF-1α and BNIP3 were up-regulated after ZYAN1 treatment. Therefore, an intravitreal injection of ZYAN1 can act as part of the pharmacologic strategy to modulate mitophagy and alleviate oxidative stress in RD. These findings enrich our knowledge of RD pathology and provide insights for the discovery of a therapeutic molecule.

Effects of tension on mitochondrial autophagy and osteogenic differentiation of periodontal ligament stem cells.

This study aimed to explore the osteogenic ability and mitochondrial autophagy of periodontal ligament stem cells (PDLSCs) under cyclic tensile stress (CTS). Primary PDLSCs were isolated from the periodontal membrane and cultured by passage. Alizarin red staining, alkaline phosphatase detection, reverse transcription polymerase chain reaction (RT-PCR), and Western blotting were used to detect the osteogenic differentiation level of PDLSCs. Mitochondrial autophagy in PDLSCs after CTS was measured using a mitochondrial autophagy detection kit, and the expression levels of autophagy-related proteins LC3B, LAMP1 and Beclin1 were measured using cellular immunofluorescence technology, RT-PCR and Western blot. After applying CTS, the osteogenic differentiation ability of PDLSCs was significantly improved, and the expression of alkaline phosphatase on the surface of the cell membrane and the formation of calcium nodules in PDLSCs were significantly increased respectively. We also studied the relevant mechanism of action and found that applying CTS can promote the osteogenic differentiation of PDLSCs and is related to the activation of mitochondrial autophagy. This study provides new insights into the mechanism of increased osteogenic differentiation on the tension side of orthodontic teeth and provides new experimental evidence for the involvement of mitochondrial autophagy in the regulation of osteogenic differentiation.

Cold exposure protects against medial arterial calcification development via autophagy

Medial arterial calcification (MAC), a systemic vascular disease different from atherosclerosis, is associated with an increased incidence of cardiovascular events. Several studies have demonstrated that ambient temperature is one of the most important factors affecting cardiovascular events. However, there has been limited research on the effect of different ambient temperatures on MAC. In the present study, we showed that cold temperature exposure (CT) in mice slowed down the formation of vitamin D (VD)-induced vascular calcification compared with room temperature exposure (RT). To investigate the mechanism involved, we isolated plasma-derived exosomes from mice subjected to CT or RT for 30 days (CT-Exo or RT-Exo, respectively). Compared with RT-Exo, CT-Exo remarkably alleviated the calcification/senescence formation of vascular smooth muscle cells (VSMCs) and promoted autophagy by activating the phosphorylation of AMP-activated protein kinase (p-AMPK) and inhibiting phosphorylation of mammalian target of rapamycin (p-mTOR). At the same time, CT-Exo promoted autophagy in β-glycerophosphate (β-GP)-induced VSMCs. The number of autophagosomes and the expression of autophagy-related proteins ATG5 and LC3B increased, while the expression of p62 decreased. Based on a microRNA chip microarray assay and real-time polymerase chain reaction, miR-320a-3p was highly enriched in CT-Exo as well as thoracic aortic vessels in CT mice. miR-320a-3p downregulation in CT-Exo using AntagomiR-320a-3p inhibited autophagy and blunted its anti-calcification protective effect on VSMCs. Moreover, we identified that programmed cell death 4 (PDCD4) is a target of miR-320a-3p, and silencing PDCD4 increased autophagy and decreased calcification in VSMCs. Treatment with CT-Exo alleviated the formation of MAC in VD-treated mice, while these effects were partially reversed by GW4869. Furthermore, the anti-arterial calcification protective effects of CT-Exo were largely abolished by AntagomiR-320a-3p in VD-induced mice. In summary, we have highlighted that prolonged cold may be a good way to reduce the incidence of MAC. Specifically, miR-320a-3p from CT-Exo could protect against the initiation and progression of MAC via the AMPK/mTOR autophagy pathway.Graphic

Mechanisms of Xuefu Zhuyu Decoction in the treatment of coronary heart disease based on integrated metabolomics and network pharmacology approach

Coronary heart disease (CHD) has become the leading cause of mortality, morbidity, and disability worldwide. Though the therapeutic effect of Xuefu Zhuyu Decoction (XFZY) on CHD has been demonstrated in China, the active ingredients and molecular mechanisms of XFZY have not been elucidated. The purpose of the current study is to explore the molecular mechanisms of XFZY in the treatment of CHD via network pharmacology, metabolomics, and experimental validation. First, we established a CHD rat model by permanently ligating the left anterior descending coronary artery (LAD), and evaluated the therapeutic effect of XFZY by hemorheology and histopathology. Second, network pharmacology was employed to screen the active ingredients and potential targets of XFZY for the treatment of CHD. Metabolomic was applied to identify the molecules present in the serum after XFZY treatment. Third, the results of network pharmacology and metabolomics were further analyzed by Cytoscape to elucidate the core ingredients and pathways. Finally, the obtained key pathways were verified by transmission electron microscopy and immunofluorescence assay. The results showed that XFZY was effective in the treatment of CHD in the rat model, and the highest dose exerted the best effect. Network pharmacology analysis revealed 215 active ingredients and 129 key targets associated with XFZY treatment of CHD. These targets were enriched in pathways of cancer, lipid and atherosclerosis, fluid shear stress and atherosclerosis, proteoglycans in cancer, chemical carcinogenesis - receptor activation, HIF-1 signaling, et al. Serum metabolomic identified 1081 metabolites involved in the therapeutic effect of XFZY on CHD. These metabolites were enriched in taurine and hypotaurine metabolism, histidine metabolism, retrograde endocannabinoid signaling pathways, et al. Cytoscape analysis combining the data from serum metabolomic and network pharmacology revealed that energy metabolism as the core pathway for XFZY treatment of CHD. Electron microscope observation identified changes in the level of autophagy in the mitochondrial structure of cardiomyocytes. Immunofluorescence assay showed that the expression levels of autophagy-related proteins LC3-B and P62/SQSTM1 were consistent with the levels of autophagy observed in mitochondria. In conclusion, our findings suggest that the possible mechanisms of XFZY in the treatment of CHD are reducing the level of autophagy, improving energy metabolism, and maintaining mitochondrial homeostasis in cardiomyocytes. Our study also shows that the combined strategies of network pharmacology, metabolomics, and experimental validation may provide a powerful approach for TCM pharmacology study.

Activation of autophagy inhibits the activation of NLRP3 inflammasome and alleviates sevoflurane-induced cognitive dysfunction in elderly rats

Aims/introductionAs a common complication in elderly patients after surgery/anesthesia, postoperative cognitive dysfunction (POCD) is mainly characterized by memory, attention, motor, and intellectual retardation. Neuroinflammation is one of the most uncontroversial views in POCD. The sevoflurane-induced neurotoxicity has attracted widespread attention in recent years. However, its mechanism has not been determined. This study aimed to observe the effects of sevoflurane on cognitive function and the changes in inflammatory indices and autophagy protein expression in the prefrontal cortex in aged rats.MethodBefore the experiment, D-galactose was diluted with normal saline into a liquid with a concentration of 125 mg/kg and injected subcutaneously into the neck and back of rats for 42 days to establish the aging rat model. Morris water maze experiments were performed, including positioning navigation (5 days) and space exploration (1 day). The POCD model was established by 3.2% sevoflurane inhalation. The rats were treated with or without MCC950, a potent and selective nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3) inhibitor, followed by autophagy agonists and autophagy inhibitors. The expression levels of inflammasome-related protein NLRP3 and autophagy-related proteins LC3B and P62 were detected to test the behavior of rats with a water maze.ResultsWe found that sevoflurane exposure affected learning and working memory ability in aged rats. We also observed microglia activation in the prefrontal cortex. NLRP3 protein expression was significantly upregulated after sevoflurane inhalation. NLRP3 inflammasome activation induced increased expression and mRNA expression of cleaved Caspase-1 and inflammatory cytokines IL-1β and IL-18, and increased secretion of peripheral proinflammatory cytokines. The inhibitor MCC950 was used to improve cognitive ability and inflammation in rats and inhibit the secretion of cytokines. In addition, we demonstrated that significant inhibition of autophagy (decreased LC3-II/I and increased P62) was accompanied by increased activation of NLRP3 inflammasomes and more severe neural cell damage. However, autophagy inhibitor rapamycin administration to activate autophagy resulted in the inhibition of NLRP3 inflammasomes, ultimately attenuating neuronal injury.ConclusionsThe activation of autophagy suppressed the formation of NLRP3 inflammasomes. It also alleviated cognitive impairment in aged rats.

Eye acupuncture improves cerebral ischemia reperfusion injury by improving autophagy via ATF6 pathway in cerebral ischemia reperfusion injury rats

To explore the effect of eye acupuncture on autophagy and expressions of autophagy-related proteins Beclin1, LC3B, ATF6 and XBP1 in the infarction area of brain tissue in rats with acute cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. Male SD rats were randomly divided into sham operation, model and eye acupuncture groups (n=16 in each group). The CIRI model was prepared by occlusion of the middle cerebral artery. Eye acupuncture stimulation was applied to bilateral "Gan"(Liver), "Shangjiao"(Upper-energizer), "Xiajiao"(Lower-energizer) and "Shen"(Kidney) regions at 0, 12 and 24 h after CIRI, 30 min each time. The neurological deficit score was given by referring to Longa's method, and TTC staining used to determine the success of model replication. After the treatment, the pathological changes of the cerebral infarction area were observed under light microscope, and the autophagosomes were observed by electron microscope. The protein expression levels of LC3B, Beclin1, ATF6 and XBP1 in the infarction area of brain tissue were detected by Western blot. The immunoactivity of Beclin1 and the immunofluorescence density of ATF6 and XBP1 in the infarction area of brain tissue were determined by immunohistochemistry. The Longa's score, and the protein expression levels of LC3B, Beclin1, ATF6 and XBP1 and immunoactivity or immunofluorescence density of Beclin1, ATF6 and XBP1 were significantly higher in the model group than those in the sham operation group (P<0.01), and considerably lower in the eye acupuncture group than those in the model group (P<0.01). Under light microscope, the model group had typical ethmoidal reticular cerebral infarction, while the eye acupuncture group had significantly smaller areas and clearer edges. Under electron microscope, there were more autophagosomes in the cytoplasm of neurons in the model group, and fewer autophagosomes in the eye acupuncture group (in contrast to the model group). Eye acupuncture can improve the neurological function and mitigate cerebral injury in CIRI rats which may be associated with its function in inhibiting autophagy in the brain tissue by regulating ATF6 pathway.

Aerosol Inhalation of Heat-Killed Clostridium butyricum CGMCC0313-1 Alleviates Allergic Airway Inflammation in Mice.

Epidemiological studies have shown that exposure to beneficial microorganisms can reduce the risk of asthma, but the clinical use of live probiotics is controversial due to the risk of infection. As heat-killed probiotics can also exhibit immunomodulatory activity, this study is aimed at investigating whether heat-killed Clostridium butyricum (HKCB) CGMCC0313-1 could reduce allergic airway inflammation in an ovalbumin-induced mouse model. Mice received aerosol inhalation of HKCB, oral administration of HKCB, or oral administration of live Clostridium butyricum (CB) during sensitization. Bronchoalveolar lavage fluid cell number, histology, and levels of the cytokines interferon-gamma and IL-4, the autophagy-related proteins LC3B, Beclin1, and p62, and members of the nuclear factor kappa B (NF-κB)/NLRP3 inflammasome signaling pathway were examined. Our results demonstrated that aerosol inhalation of HKCB, oral HKCB administration, and oral live CB administration alleviated allergic airway inflammation and mucus secretion in allergic mice. Aerosol inhalation of HKCB was the most effective method; it restored the Th1/Th2 balance, ameliorated autophagy, and inhibited the NF-κB/NLRP3 inflammasome signaling pathway in the lungs of allergic mice. Thus, aerosol inhalation of HKCB could be a promising strategy for the prevention or treatment of asthma.

Inhibitory Effect of Ursolic Acid on Proliferation and Migration of Renal Carcinoma Cells and Its Mechanism.

Background Renal carcinoma is one of the most common malignant tumors in the urinary system. Autophagy can be both activated and inhibited in renal carcinoma, and it plays a double-edged role in the development of renal carcinoma. In the early stage of cancer, autophagy can suppress tumors. In the late stage, autophagy contributes to the survival of tumor cells in an unfavorable environment, and some autophagy-related proteins P62, LC3B, and beclin-1 have become indicators of the prognosis of patients with renal carcinoma. Aim To demonstrate that ursolic acid activates autophagy in renal carcinoma 786-O cells by inhibiting the hedgehog signaling pathway. Methods The effect of ursolic acid on the viability of 786-O cells was determined by the MTT method; the effect of ursolic acid on the proliferation and migration of 786-O cells was examined by crystalline violet staining and scratch assay, respectively. For the study of autophagy, we firstly screened the time points. Western blot assay was used to detect the expression level of autophagic protein P62 at different time points of ursolic acid on 786-O. Then, the Cell MeterTM Autophagy Assay Kit was used to detect the effect of different doses of ursolic acid on the autophagic fluorescence intensity of 786-O cells; the Western blot method was used to detect the effect of different doses of ursolic acid on the expression levels of LC3II and P62 proteins in 786-O cells. Further, AdPlus-mCherry-GFP-LC3B adenovirus transfection was used to detect the effect of ursolic acid on the autophagic flow of 786-O cells; ursolic acid was combined with the autophagy inhibitor chloroquine (CQ) to detect the expression level of autophagy protein LC3II by Western blot. In terms of mechanism, the effect of ursolic acid on hedgehog signaling pathway-related proteins in 786-O cells was detected by Western blot. Results Ursolic acid inhibited the activity, proliferation, and migration of 786-O cells, enhanced the fluorescence intensity of autophagosomes in 786-O cells, increased the expression level of autophagy marker protein LC3II, and inhibited the expression level of P62 in a time and dose-dependent manner; ursolic acid activated the autophagic flow in 786-O cells, which showed that ursolic acid caused the accumulation of autophagic fluorescent spots and enhanced the fluorescence intensity of autophagosomes. Ursolic acid activated the autophagic flow in 786-O cells, as evidenced by the accumulation of autophagic fluorescent spots and enhanced fluorescence intensity of autophagosomes, and the combined use of the autophagy inhibitor CQ increased the expression level of LC3II compared to ursolic acid alone; ursolic acid decreased the expression levels of PTCH1, GLI1, SMO, SHH, and c-Myc and increased the expression level of Sufu in the hedgehog signaling pathway. Conclusion Ursolic acid activates autophagy in renal carcinoma 786-O cells, probably by inhibiting hedgehog signaling pathway activity.

Low doses of niclosamide and quinacrine combination yields synergistic effect in melanoma via activating autophagy-mediated p53-dependent apoptosis

Malignant melanoma is a highly aggressive, malignant, and drug-resistant tumor. It lacks an efficient treatment approach. In this study, we developed a novel anti-melanoma strategy by using anti-tapeworm drug niclosamide and anti-malarial drug quinacrine, and investigated the molecular mechanism by in vitro and in vivo assays. Meanwhile, other types of tumor cells, immortalized epithelial cells and bone marrow mesenchymal stem cells were used to evaluate the universal role of anti-cancer and safety of the strategy. The results showed, briefly, an exposure to niclosamide and quinacrine led to an increased apoptosis-related protein p53, cleaved caspase-3 and cleaved PARP and autophagy-related protein LC3B expression, and a decreased expression of autophagy-related protein p62, finally leading to cell apoptosis and autophage. After inhibiting autophagy by Baf-A1, flow cytometry and western blot showed that the expression of apoptosis-related proteins was down-regulated and the number of apoptotic cells decreased. Subsequently, in the siRNA-mediated p53 knockdown cells, the expression of apoptosis-related proteins and the number of apoptotic cells were also reduced, while the expression of autophagy-related proteins including LC3B, p62 did not change significantly. To sum up, we developed a new, safe strategy for melanoma treatment by using low doses of niclosamide and quinacrine to treat melanoma; and found a novel mechanism by which the combination application of low doses of niclosamide and quinacrine exerts an efficient anti-melanoma effect through activation of autophagy-mediated p53-dependent apoptosis. The novel strategy was verified to exert a universal anti-cancer role in other types of cancer.

Asthma Susceptibility Gene ORMDL3 Promotes Autophagy in Human Bronchial Epithelium.

The genome-wide association study (GWAS)-identified asthma susceptibility risk alleles on chromosome 17q21 increase the expression of ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) in lung tissue. Given the importance of epithelial integrity in asthma, we hypothesized that ORMDL3 directly impacted bronchial epithelial function. To determine whether and how ORMDL3 expression impacts the bronchial epithelium, in studies using both primary human bronchial epithelial cells and human bronchial epithelial cell line, 16HBE (16HBE14o-), we assessed the impact of ORMDL3 on autophagy. Studies included: autophagosome detection by electron microscopy, RFP-GFP-LC3B to assess autophagic activity, and Western blot analysis of autophagy-related proteins. Mechanistic assessments included immunoprecipitation assays, intracellular calcium mobilization assessments, and cell viability assays. Coexpression of ORMDL3 and autophagy-related genes was measured in primary human bronchial epithelial cells derived from 44 subjects. Overexpressing ORMDL3 demonstrated increased numbers of autophagosomes and increased levels of autophagy-related proteins LC3B, ATG3, ATG7, and ATG16L1. ORMDL3 overexpression promotes autophagy and subsequent cell death by impairing intracellular calcium mobilization through interacting with SERCA2. Strong correlation was observed between expression of ORMDL3 and autophagy-related genes in patient-derived bronchial epithelial cells. Increased ORMDL3 expression induces autophagy, possibly through interacting with SERCA2, thereby inhibiting intracellular calcium influx, and induces cell death, impairing bronchial epithelial function in asthma.

Early Growth Response 1 Suppresses Macrophage Phagocytosis by Inhibiting NRF2 Activation Through Upregulation of Autophagy During Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa is an opportunistic pathogen that causes life-threatening infections in cystic fibrosis patients and immunocompromised individuals. A tightly regulated immune response possessed by healthy individuals can effectively control P. aeruginosa infections, whereas the patients with dysregulated immune response are susceptible to this bacterial pathogen. Early growth response 1 (Egr-1) is a zinc-finger transcription factor involved in regulation of various cellular functions, including immune responses. We previously identified that Egr-1 was deleterious to host in a mouse model of acute P. aeruginosa pneumonia by promoting systemic inflammation and impairing bacterial clearance in lung, which associated with reduced phagocytosis and bactericidal ability of leucocytes, including macrophages and neutrophils. However, the molecular mechanisms underlying the Egr-1-suppressed phagocytosis of P. aeruginosa are incompletely understood. Herein, we investigated whether the Egr-1-regulated autophagy play a role in macrophage phagocytosis during P. aeruginosa infection by overexpression or knockdown of Egr-1. We found that overexpression of Egr-1 inhibited the phagocytic activity of macrophages, and the autophagy activator rapamycin and inhibitor chloroquine could reverse the effects of Egr-1 knockdown and Egr-1 overexpression on phagocytosis of P. aeruginosa, respectively. Furthermore, the Egr-1-overexpressing macrophages displayed upregulated expression of autophagy-related proteins LC3A, LC3B and Atg5, and decreased levels of p62 in macrophages. Further studies revealed that the macrophages with Egr-1 knockdown displayed enhanced activation of transcription factor NRF2 and expression of scavenger receptors MACRO and MSR1. Altogether, these findings suggest that Egr-1 suppresses the phagocytosis of P. aeruginosa by macrophages through upregulation of autophagy and inhibition of NRF2 signaling.

Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein.

Mastitis is one of the most serious diseases that causes losses in the dairy industry, seriously impairing milk production and milk quality, and even affecting human health. Menthol is a cyclic monoterpene compound obtained from the stem and leaves of peppermint, which has a variety of biological activities, including anti-inflammatory and antioxidant activity. The purpose of this study was to investigate the preventive effect of menthol on the lipopolysaccharide-induced inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein cows by enzymatic hydrolysis. BMECs were treated with menthol (10, 50, 100, 200 μM) for 1h, followed by lipopolysaccharide (5μg/ml) for 12 h. Lipopolysaccharide treatment upregulated the protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS) and the mRNA abundance of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5μg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited by menthol. In addition, menthol (200 μM) treatment was able to significantly upregulate the expression level of autophagy-related protein LC3B, downregulate the expression level of P62, promote the expression abundance of autophagy-related gene mRNA, and enhance significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 μM) promoted the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1) and increased the nuclear localization of nuclear factor-E2 associated factor 2 (Nrf-2). When the AMPK pathway was blocked using compound C (10μg/ml), an inhibitor of AMPK, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking the Nrf-2 pathway using ML385 (5μg/ml), an inhibitor of Nrf-2. Overall, the data suggest that menthol activates the AMPK-ULK1 pathway to initiate the onset of autophagy and maintains the level of autophagy through the AMPK-Nrf-2 pathway. In conclusion, the findings suggest that menthol may alleviate the inflammatory response in BMECs via the AMPK/ULK1/Nrf-2/autophagy pathway.

Antitumor Activities of Co-loading Gemcitabine and Oxaliplatin into Oleic Acid-Based Solid Lipid Nanoparticle against Non-Small Cell Lung Cancer Cells

Lung cancer is a main global health problem with high incidence and case-fatality rates. The use of solid lipid nanoparticles (SLN) as a nanocarrier for chemotherapeutic agents has been suggested as an effective therapeutic approach. The current work objective was to investigate the antineoplastic activity of gemcitabine (GM) and oxaliplatin (OXA) co-loaded into oleic acid-based solid lipid nanoparticle (OA-SLN) in A549 non-small cell lung cancer cells. OA-SLN was synthesized using homogenization and physically characterized using the dynamic light scattering techniques. The anticancer properties of the combination of GM and OXA encapsulated in OA-SLN were evaluated using a series of cellular assays, such as cell viability using crystal violate, apoptosis using caspase-3 assay kit, and autophagy using human autophagy-related protein LC3-B ELISA kit. The z-average diameter of (GM+OXA) OA-SLN was (63.10 ± 1.53 nm). The (GM+OXA) OA-SLN formulation had significantly reduced cell growth in a dose-dependent manner on the A549 cells within 24 hours. The combination (GM+OXA) OA-SLN had more pronounced effects on autophagy (326.38 ± 4.21 pg/ml) than the untreated control cells (206.2 ± 6.69 pg/ml). Our findings indicate that co-encapsulation of GM and OXA into OA-SLN significantly improved their therapeutic efficacy against A549 cells.

MiR-222 regulates cell growth, apoptosis, and autophagy of interstitial cells of Cajal isolated from slow transit constipation rats by targeting c-kit

Excessive autophagy and apoptosis of the interstitial cells of Cajal (ICC) have been identified in gastrointestinal (GI) motility disorders including slow transit constipation (STC). MicroRNA 222 (miR-222) has been shown to affect GI motility. This study aimed to explore whether miR-222 influences apoptosis and excessive autophagy of isolated ICC. miR-222, c-kit, and stem cell factor (SCF) were evaluated in colon tissues in STC rats compared with normal control by qRT-PCR and western blot analysis. The condition of autophagy of colon tissue was observed by transmission electron microscope. ICC were isolated from the colon of STC rats. Cell Counting Kit-8 (CCK-8) assay and wound healing assay were carried out to examine the cell viability and migration rate. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and Annexin V-Flourescein Isothiocyanate/Propidine Iodide (FITC/PI) apoptosis detection kit. Western blot analysis was performed to detect the c-kit and SCF expression; apoptosis-related proteins Bcl-2, Bax, caspase-3, and pro-caspase-3; and autophagy-related proteins LC3B and Beclin-1. The connection between miR-222 and c-kit was detected by bioinformatics and luciferase activity analysis. miR-222 expression was significantly higher, whereas c-kit and SCF expressions were markedly lower in STC rats’ colon tissue compared with normal control. Meanwhile, STC rats exhibited excessive autophagy in colon tissue than normal control. Inhibition of miR-222 expression promoted cell proliferation as well as migration and inhibited autophagy, whereas upregulation of miR-222 had the opposite effect. In addition, miR-222 upregulation induced apoptosis and excessive autophagy compared with normal controls (NC). Western blot analysis showed that miR-222 overexpression caused decreased c-kit and SCF protein levels compared with NC. Bioinformatics and luciferase activity analysis revealed that miR-222 could be a predictive regulator of c-kit. miR-222 induces apoptosis and excessive autophagy of ICC and may serve as potential biomarker for ICC loss in STC.

The effects of autophagy in rat tracheal epithelial cells induced by silver nanoparticles.

The massive use of silver nanoparticles (AgNPs) is potentially harmful to exposed humans. Although previous studies have found that AgNPs can induce cell autophagy, few studies have focused on the toxic pathways and mechanisms of autophagy induced by AgNPs in rat respiratory epithelial (RTE) cells. In this study, RTE cells were exposed to two kinds of AgNPs in vitro to ascertain the influence of mTOR-autophagy pathway-associated protein expression, including Beclin1, LC3B, Atg5, and Atg7. After exposure to different sizes and concentrations of AgNPs for 12 h, the uptake of silver in RTE cells reached 0.45 μg/L to 1.11 μg/L, indicating that AgNPs can enter RTE cells, leading to toxic effects. Our study found that this toxic effect was related to autophagy caused by ROS accumulation that was mediated by the mTOR pathway. With increasing AgNP exposure concentrations, the expression of p-mTOR was significantly downregulated, and expression of the autophagy-related proteins Beclin1, LC3B, Atg5, and Atg7 was significantly increased in RTE cells in all exposed groups. At a concentration of 1000 μg/L, the expression of LC3BII/LC3BI in all exposed groups was 24.49 times and 12.71 times that of the control, and the expression of Atg7 in all exposed groups was 23.21 times and 13.21 times that of the control. The upregulation of autophagy-related proteins in the AgNP-10 nm exposure groups was greater than that of the AgNP-100 nm exposure group. In summary, the mTOR pathway mediates AgNP-induced autophagy in RTE cells, which leads to damage to the respiratory system barrier and human health risks. This study can facilitate the development of prevention and intervention policies against adverse consequences induced by AgNPs.

Icariin improves brain function decline in aging rats by enhancing neuronal autophagy through the AMPK/mTOR/ULK1 pathway

Context Icariin (ICA) is the main active ingredient of Epimedium brevicornu Maxim (Berberidaceae), which is used in the immune, reproductive, neuroendocrine systems, and anti-aging. Objective To evaluate the effect of ICA on natural aging rat. Materials and methods 16-month-old Sprague–Dawley (SD) rats were randomly divided into aging, low and high-dose ICA groups (n = 8); 6-month-old rats were taken as the adult control (n = 8). Rats were fed regular feed (aging and adult control) or feed containing ICA (ICA 2 and 6 mg/kg group) for 4 months. HE and Nissl staining were used to assess pathological changes. Western blot was used to test the expression of autophagy (LC3B, p62, Atg5, Beclin1) and p-AMPK, p-mTOR and p-ULK1 (ser 757). Immunofluorescence was used to detect the co-localization of LC3 and neurons. Results ICA improved neuronal degeneration associated with aging and increased the staining of Nissl bodies. Western blot showed that ICA up-regulated autophagy-related proteins LC3B (595%), Beclin1 (73.5%), p-AMPK (464%) protein (p < 0.05 vs. 20 M) in the cortex and hippocampus of aging rats, down-regulated the expression of p62 (56.9%), p-mTOR (53%) and p-ULK1 (ser 757) (65.4%) protein (p < 0.05 vs. 20 M). Immunofluorescence showed that the fluorescence intensity of LC3 decreased in the aging rat brain, but increased and mainly co-localized with neurons after ICA intervention. Conclusions Further research needs to verify the expression changes of AMPK/mTOR/ULK1 and the improvement effect of ICA in elderly. These results will further accelerate the applications of ICA and the treatment for senescence.

Saponins from Panax japonicus attenuate cognitive impairment in ageing rats through regulating microglial polarisation and autophagy

Context Panax japonicus is the dried rhizome of Panax japonicus C.A. Mey. (Araliaceae). Saponins from Panax japonicus (SPJ) exhibit anti-inflammatory and antioxidative effects. Objective To explore the neuroprotective effect of SPJ on natural ageing of rat. Materials and methods Sprague-Dawley (SD) rats 18-month-old were divided into ageing control, ageing treated with SPJ 10 or 30 mg/kg (n = 8). Five-month-old rats were taken as the adult control (n = 8). Rats were fed regular feed or feed containing SPJ for 4 months. Cognitive level was evaluated by Morris water maze (MWM) test. The mechanisms of SPJ’s neuroprotection were evaluated by transmission electron microscope, western blot analysis, and immunofluorescence in vivo and in vitro. Results SPJ attenuated ageing-induced cognitive impairment as indicated by elevated number of times crossing the target platform (from 1.63 to 3.5) and longer time spent in the target platform quadrant (from 1.33 to 1.98). Meanwhile, SPJ improved the morphology of microglia and synapse, and activated M2 microglia polarisation including increased hippocampus levels of CD206 (from 0.98 to 1.47) and YM-1 (from 0.67 to 1.1), and enhanced autophagy-related proteins LC3B (from 0.48 to 0.82), Beclin1 (from 0.32 to 0.51), Atg5 (from 0.22 to 0.89) whereas decreased p62 level (from 0.71 to 0.45) of ageing rats. In vitro study also showed that SPJ regulated the microglial polarisation and autophagy. Discussion and conclusions SPJ improved cognitive deficits of ageing rats through attenuating microglial inflammation and enhancing microglial autophagy, which could be used to treat neurodegenerative disorders.

Impact of Bupivacaine on malignant proliferation, apoptosis and autophagy of human colorectal cancer SW480 cells through regulating NF-κB signaling path

ABSTRACT To probe into the impact of Bupivacaine on colorectal cancer (CRC) proliferation, apoptosis, and autophagy through regulating the NF-κB signaling pathway. Our work treated CRC cells with Bupivacaine, detected cell vitality through MTT assay, apoptosis through flow cytometry, cell migration through wound healing assay, NF-κB activity through immunofluorescence, inflammatory factor level, including TNF-α, IL-1β as well as IL-6 through ESLIA, apoptosis factor mRNA expression, including Bcl-2, Bax and caspase-3q through qRT-PCR, and protein expression linking with NF-κB signaling pathway as well as autophagy-related proteins via western blot. In in vivo experiments, we explored the impact of Bupivacaine on tumor volume, tumor and NF-κB expression. The results showed that 1 mM Bupivacaine was available to signally inhibit CRC cell vitality, promoted apoptosis rate and apoptosis gene expression, like Bax, and caspase-3, inhibited Bcl-2 expression, inhibited cancer cell migration, promoted autophagy-related protein LC3B II/LC3B I ratio and beclin-1 expression, and inhibited p62 expression. Additionally, it could elevate inflammatory factor level and induce IKK and IκB phosphorylation as well as NF-κB proteins. In in vivo experiments, Bupivacaine inhibited tumor volume and tumor, as well as NF-κB expression. In short, bupivacaine is available to inhibit CRC proliferation through regulating NF-κB signaling pathway, promote apoptosis and autophagy, and can be used as a potential drug to treat CRC in the future.