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Cognitive impairment caused by chronic cerebral hypoperfusion (CCH) is associated with white matter injury (WMI), possibly through the alteration of autophagy. Here, the autophagy-lysosomal pathway (ALP) dysfunction in white matter (WM) and its relationship with cognitive impairment were investigated in rats subjected to two vessel occlusion (2VO). The results showed that cognitive impairment occurred by the 28th day after 2VO. Injury and autophagy activation of mature oligodendrocytes and neuronal axons sequentially occurred in WM by the 3rd day. By the 14th day, abnormal accumulation of autophagy substrate, lysosomal dysfunction, and the activation of mechanistic target of rapamycin (MTOR) pathway were observed in WM, paralleled with mature oligodendrocyte death. This indicates autophagy activation was followed by ALP dysfunction caused by autophagy inhibition or lysosomal dysfunction. To target the ALP dysfunction, enhanced autophagy by systemic rapamycin treatment or overexpression of Beclin1 (BECN1) in oligodendrocytes reduced mature oligodendrocyte death, and subsequently alleviated the WMI and cognitive impairment after CCH. These results reveal that early autophagy activation was followed by ALP dysfunction in WM after 2VO, which was associated with the aggravation of WMI and cognitive impairment. This study highlights that alleviating ALP dysfunction by enhancing oligodendrocyte autophagy has benefits for cognitive recovery after CCH.
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The global incidence rate of nonalcoholic steatohepatitis (NASH) continues to rise. The pathogenesis of NASH is complex, and there is no effective clinical treatment. Previous study has shown that DEAD box protein 5 (DDX5) can significantly alleviate the NASH process in mice. This study screened the natural product library of the research group and found that the active compound hypercalin B (HB) in Hypericum beanii N. Robson, a traditional Chinese medicine, can upregulate the expression of DDX5 protein in a dose-dependent manner. In this study, an in vitro model of NASH stimulated by palmitic acid (PA) and an animal model of NASH induced by the methionine- and choline-deficient diet (MCD) were constructed. Different concentrations of HB were used to investigate the effect and mechanism of HB in alleviating NASH progression. All animal experiments in this paper were approved by the Ethics Committee of China Pharmaceutical University (NO: 2021-02-003). In vitro model results showed that HB significantly reduced the intracellular lipid deposition induced by free fatty acid (FFA). Animal experiments showed that HB improved liver injury by significantly reducing lipid accumulation in the liver of NASH mice, and reducing serum aspartate transaminase (AST) and alanine transaminase (ALT) levels. Moreover, HB could inhibit liver inflammation by reducing the mRNA levels of liver pro-inflammatory cytokines including interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNFα). Further research showed that HB could reduce the phosphorylation level of the mechanical target of rapamycin (mTOR) and reduce the expression of sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN), thereby improving lipid metabolism and alleviating NASH progression, and the effects of HB against NASH were dependent on DDX5. In conclusion, HB can improve lipid metabolism and inhibit inflammatory activation by suppressing mTORC1 pathway via upregulating DDX5 protein, and showed promising anti-NASH activity in vitro and in vivo.
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Macrolide and corticosteroid resistance has been reported in patients with Mycoplasma pneumoniae (MP) pneumonia (MPP). MP clearance is difficult to achieve through antibiotic treatment in sensitive patients with severe MPP (SMPP). SMPP in children might progress to airway remodeling and even bronchiolitis/bronchitis obliterans. Therefore, identifying serum biomarkers that indicate MPP progression and exploring new targeted drugs for SMPP treatment require urgency. In this study, serum samples were collected from patients with general MPP (GMPP) and SMPP to conduct proteomics profiling. The Fc fragment of the IgG-binding protein (FCGBP) was identified as the most promising indicator of SMPP. Biological enrichment analysis indicated uncontrolled inflammation in SMPP. ELISA results proved that the FCGBP level in patients with SMPP was substantially higher than that in patients with GMPP. Furthermore, the FCGBP levels showed a decreasing trend in patients with GMPP but the opposite trend in patients with SMPP during disease progression. Connectivity map analyses identified 25 possible targeted drugs for SMPP treatment. Among them, a mechanistic target of rapamycin kinase (mTOR) inhibitor, which is a macrolide compound and a cell proliferation inhibitor, was the most promising candidate for targeting SMPP. To our knowledge, this study was the first proteomics-based characterization of patients with SMPP and GMPP.
Subject(s)
Child , Humans , Biomarkers , Carrier Proteins , Immunoglobulin Fc Fragments , Immunoglobulin G , Macrolides , Mycoplasma pneumoniae , Pneumonia, Mycoplasma/drug therapy , ProteomicsABSTRACT
Objective:To study the efficacy and mechanism of Shugan Jianpi Jiedu prescription （SJJ） in the treatment of triple-negative breast cancer through <italic>in vitro</italic> cell experiments. Method:The following groups were set up in this study： a normal serum group，a pirarubicin group，and low-，medium-， and high-dose SJJ-medicated serum groups. Twenty SD rats were randomly divided into four groups and administered with SJJ solution （16.8，8.2，4.05 g·kg<sup>-1</sup>） and normal saline （equal volume） according to the body surface area to prepare serum. MDA-MB-231 cells were treated separately. The proliferation， migration and invasion of MDA-MB-231 cells were detected by the cell counting kit-8（CCK-8），wound healing assay and transwell cell invasion assay. The phosphoinositide 3-kinase （PI3K），protein kinase B （Akt）， and mechanistic target of rapamycin （mTOR） protein expression levels in MDA-MB-231 cells were tested by the Western blot. Result:The cell proliferation in the three different doses of medicated serum groups and the pirarubicin positive control group was significantly inhibited as compared with that in the normal serum group（<italic>P</italic><0.01），and there was no statistical difference for this between the medium/high dose medicated serum group and the pirarubicin positive control group.The wound healing in the SJJ-medicated serum groups and the pirarubicin group was slowed down as compared with that in the normal serum group （<italic>P</italic><0.01），and the effect in the SJJ-medicated serum groups was weaker than that in the pirarubicin group （<italic>P</italic><0.05，<italic>P</italic><0.01）. The number of cells invading the lower transwell chamber was decreased as compared with that in the normal serum group （<italic>P</italic><0.01），and there was no statistical difference between the medium-/high-dose SJJ-medicated serum groups and the pirarubicin group. Western blot results showed that 48 h after treatment，the PI3K，Akt， and mTOR expression levels in the cells of SJJ-medicated serum groups and the pirarubicin group were lower than those of the normal serum group（<italic>P</italic><0.01）. Conclusion:The SJJ-medicated serum could inhibit the proliferation， migration and invasion of MDA-MB-231 cells presumedly by down-regulating the protein expression levels in the PI3K/Akt/mTOR signaling pathway.
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Objective:To explore the effects and mechanism of zedoary turmeric oil and its active components on the vascular endothelial growth factor A （VEGFA）， signal transducer and activator of transcription 3 （STAT3）， and mechanistic target of rapamycin （mTOR） in the ovarian cancer （OC）. Method:Network pharmacology technology was employed to analyze the mechanism of Curcumae Rhizoma on OC. Bioinformatics was used to analyze the expression of VEGFA， STAT3， and mTOR in OC and the effect on the prognosis of OC to explore the feasibility of zedoary turmeric oil in regulating VEGFA， STAT3， and mTOR in OC.The xenograft tumor model of nude mice was established， and the effects of zedoary turmeric oil and its active components on VEGFA， STAT3， and mTOR in OC were observed by hematoxylin-eosin （HE） staining， real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）， Western blot， and immunohistochemistry （IHC）. Result:Bioinformatics analysis and literature research showed that VEGFA， STAT3， and mTOR played a special regulatory role in the occurrence and development of OC， and were potential key targets for the proliferation of OC. Network pharmacology analysis revealed that Curcumae Rhizoma could regulate multiple disease targets of OC， and mediate VEGFA， STAT3， and mTOR in OC through these multiple targets. As demonstrated by HE staining， the tumor cells in the model group were densely arranged， with no erosion on the edge and no vesicles inside. Compared with the model group， the cell density in other treatment groups was reduced， and strip-shaped erosion on the edge and small empty vesicles were observed in the tumor tissue， especially in the zedoary turmeric oil group. According to the results of Real-time PCR and IHC， zedoary turmeric oil and its active components could inhibit the mRNA and protein expression of VEGFA， STAT3， and mTOR in the OC tissue （<italic>P</italic><0.05）. Conclusion:Zedoary turmeric oil and its active components could reduce the expression of VEGFA， STAT3， and mTOR in tumor tissue of nude mice， and inhibited the proliferation of OC through VEGFA， STAT3， and mTOR.
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Objective : To investigate the effect of mechanistic target of rapamycin complex 1 (mTORC1) on group 3 innate lymphoid cells (ILC3) function. Methods ¡¤ Intestinal lamina propria leukocytes (LPL) of C57BL/6 wild type mice were stimulated by rapamycin, the specific inhibitor of mTORC1 signaling pathway, in vitro, and then quantity and function of ILC3 were detected by flow cytometry. Next, purified ILC3 from mice intestinal LPL were sorted by flow cytometry. After the activation of ILC3 with IL-23, mRNA expression levels of Rorc (the gene encoding retinoic acid receptor related orphan receptor, i.e. RORγt), Il22 and Rptor (the gene encoding key component protein of mTORC1, i.e. Raptor) were detected by real-time qPCR. For further study, a genetically engineered mouse model specifically knocked out Raptor in ILC3 was constructed. Effects of mTORC1 loss on the quantity and function of ILC3 as well as gut structure were detected by flow cytometry, real-time qPCR and hematoxylin-eosin staining. Results ¡¤ The total ILC3 number had no change, but the secretion of IL-22 by ILC3 reduced after stim-ulation with rapamycin. Il22, Rorc and Rptor mRNA expression levels were upregulated simultaneously in ILC3 after activation with IL-23. In addition, there was no significant difference in the numbers and proportions of total ILC3 and ILC3 subsets as well as gut structure in Rap-tor-deficient mice, but the cytokine IL-22 secretion level of ILC3 significantly decreased in these mice. Conclusion ¡¤ Loss of mTORC1 func-tion inhibits ILC3 from secreting IL-22 but has no effect on the intestinal structure and intestinal ILC3 development, which reveals the positive regulation of mTORC1 signaling on intestinal ILC3 function.
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Objective: To investigate the protective effect of extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on vascular senescence induced by high glucose in mice from adenosine 5'-monophosphate (AMP)-activated protein kinase/mechanistic target of rapamycin (AMPK/mTOR) pathway. Method: A total of 130 male C57BL/6 mice were randomly divided into control group and high glucose group. The high glucose group was intraperitoneally injected with streptozocin(STZ) and fed with a high-fat diet continuously for seven months. Mice were divided into 4 groups:model group, low-dose extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma(0.819 g·kg-1) group, high-dose extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma group (1.638 g·kg-1) and metformin group (150 mg·kg-1), and intragastrically administered once a day for nine weeks. The changes in body weight and blood glucose were measured. At the end of the administration, htoxylin eosin(HE) was performed for the detection of aortic morphology, and the expressions of cyclin-dependent kinase inhibitor 2A (p16), cyclin-dependent kinase inhibitor 1A (p21), AMPK, p-AMPK, mTOR, p-mTOR, liver kinase B1 (LKB1), p-LKB1, Ribosomal protein s6 kinase (p70s6k) and p-p70s6k proteins in mouse aorta were detected by Western blot. Result: Compared with blank group, mice in model group had lower body weight and higher blood glucose (PPPPPPPPPPPConclusion: High glucose can induce vascular senescence, and extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma can improve vascular aging induced by high glucose through AMPK/mTOR pathway.
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The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to various environmental inputs, especially amino acids. In fact, the activity of mTORC1 is highly sensitive to changes in amino acid levels. Over past decades, a variety of proteins have been identified as participating in the mTORC1 pathway regulated by amino acids. Classically, the Rag guanosine triphosphatases (GTPases), which reside on the lysosome, transmit amino acid availability to the mTORC1 pathway and recruit mTORC1 to the lysosome upon amino acid sufficiency. Recently, several sensors of leucine, arginine, and S-adenosylmethionine for the amino acid-stimulated mTORC1 pathway have been coming to light. Characterization of these sensors is requisite for understanding how cells adjust amino acid sensing pathways to their different needs. In this review, we summarize recent advances in amino acid sensing mechanisms that regulate mTORC1 activity and highlight these identified sensors that accurately transmit specific amino acid signals to the mTORC1 pathway.
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Mechanistic target of rapamycin (mTOR) pathway has been associated to various neurological diseases,including epilepsy.As a newly discovered mTOR pathway disease,DEPDC5 (dishevelled,Egl-10,and pleckstrin domain-containing protein 5) gene related epilepsy covers a range of clinical phenotypes.Mutations of DEPDC5 gene have been detected in genetic focal epilepsies,focal cortical dysplasia type Ⅱ,and sporadic focal epilepsy cases.It has been reported that hyperactivation of mTOR signaling due to the loss of function of DEPDC5 contributes to epileptogenesis,however more detailed mechanisms remain to be elucidated.DEPDC5 gene-related epilepsy together with other neurodevelopmental diseases characterized by hyperactivation of mTOR signaling has formed a disease spectrum named mTORopathies.
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The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to various environmental inputs, especially amino acids. In fact, the activity of mTORC1 is highly sensitive to changes in amino acid levels. Over past decades, a variety of proteins have been identified as participating in the mTORC1 pathway regulated by amino acids. Classically, the Rag guanosine triphosphatases (GTPases), which reside on the lysosome, transmit amino acid availability to the mTORC1 pathway and recruit mTORC1 to the lysosome upon amino acid sufficiency. Recently, several sensors of leucine, arginine, and S-adenosylmethionine for the amino acid-stimulated mTORC1 pathway have been coming to light. Characterization of these sensors is requisite for understanding how cells adjust amino acid sensing pathways to their different needs. In this review, we summarize recent advances in amino acid sensing mechanisms that regulate mTORC1 activity and highlight these identified sensors that accurately transmit specific amino acid signals to the mTORC1 pathway.
Subject(s)
Animals , Humans , Amino Acids/chemistry , Arginine/chemistry , Cell Membrane/metabolism , GTP Phosphohydrolases/metabolism , Gene Expression Regulation , Golgi Apparatus/metabolism , Leucine/chemistry , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Methionine/chemistry , S-Adenosylmethionine/chemistry , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
Mast cells integrate innate and adaptive immunity and are implicated in pathophysiological conditions, including allergy, asthma, and anaphylaxis. Cross-linking of the high-affinity IgE receptor (FcεRI) initiates diverse signal transduction pathways and induces release of proinflammatory mediators by mast cells. In this study, we demonstrated that hyperactivation of mechanistic target of rapamycin (mTOR) signaling using the mTOR activator MHY1485 suppresses FcεRI-mediated mast cell degranulation and cytokine secretion. MHY1485 treatment increased ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation, which are downstream targets of mTOR complex 1 (mTORC1), but decreased phosphorylation of Akt on mTOR complex 2 (mTORC2) target site serine 473. In addition, this activator decreased β-hexosaminidase, IL-6, and tumor necrosis factor α (TNF-α) release in murine bone marrow-derived mast cells (BMMCs) after FcεRI stimulation. Furthermore, MHY1485-treated BMMCs showed significantly decreased proliferation when cultured with IL-3. These findings suggested hyperactivation of mTORC1 as a therapeutic strategy for mast cell-related diseases.
Subject(s)
Adaptive Immunity , Anaphylaxis , Asthma , Cell Degranulation , Cell Proliferation , Hypersensitivity , Immunoglobulin E , Interleukin-3 , Interleukin-6 , Mast Cells , Peptide Initiation Factors , Phosphorylation , Ribosomal Protein S6 Kinases , Serine , Signal Transduction , Sirolimus , Tumor Necrosis Factor-alphaABSTRACT
Objective To explore the potential mechanisms of low density lipoprotein receptor (LDLr) in high glucose peritoneal dialysis solution (PDS)-induced peritoneal fibrosis.Methods Human peritoneal mesothelial cells (PMCs) were applied.In pre-experiment,human PMCs were cultured with 1.5％ PDS,2.5％ PDS and 4.25％ PDS for 6 h,12 h and 24 h.4.25％ mannitol was used as high osmotic pressure control.In formal experiment,PMCs were divided into the control group (treated with phosphate buffer saline) and the high glucose PDS group (treated with 4.25％ PDS for 24 h).Morphological change of PMCs was observed by inverted microscope.The mRNA and protein expressions of extracellular matrix proteins such as α-smooth muscle actin (α-SMA),fibroblast specific protein-1 (FSP-1) and collagen Ⅰ in PMCs were respectively measured by real-time PCR and Western blotting.The lipid accumulation was observed by oil red O staining and filipin staining,and the content of intracellular cholesterol ester was detected by high-performance liquid chromatography.The co-expression of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) with golgin was observed with immunofluorescent staining.The mRNA and protein expressions of LDLr,SREBP-2 and SCAP were respectively detected by real-time PCR and Western blotting.The mRNA and protein expressions of mammalian target of rapamycin (mTOR),eukaryotic initiation factor 4E-binding protein 1 (4EBP1),and p70 S6 kinase (S6K1) were respectively detected by real-time PCR and Western blotting.Results (1) Compared with the 1.50％ PDS stimulation,4.25％ PDS for 24 h intervention significantly increased the expression of LDLr in PMCs (P ＜ 0.05),and high osmotic pressure control at 6 h,12 h and 24 h had no statistical difference (P ＞ 0.05).(2) Compared with those in the control group,in high glucose PDS group PMCs showed notable elongation consistent with the morphology of myofibroblasts,the expressions of α-SMA,FSP-1 and collagen Ⅰ were increased (all P ＜ 0.05),and the intracellular cholesterol were enhanced (P ＜ 0.05).Meanwhile,the co-expression of SCAP with golgin was enhanced,and the mRNA and protein expressions of LDLr,SREBP-2 and SCAP were up-regulated in high glucose PDS group (all P ＜ 0.05).Further,the mRNA and protein phosphorylation of mTOR,4EBP1 and S6K1 were increased (all P ＜ 0.05).Conclusions The disruption of LDLr feedback regulation is involved in high glucose PDS-mediated cholesterol accumulation in PMCs by mammalian target of rapamycin complex 1 (mTORC1) pathway,which promotes the accumulation of extracellular matrix and peritoneal fibrosis.
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Diabetic nephropathy is a leading cause of end stage renal disease and its occurance is increasing worldwide. The most effective treatment strategy for the condition is intensive treatment to strictly control glycemia and blood pressure using renin-angiotensin system inhibitors. However, a fraction of patients still go on to reach end stage renal disease even under such intensive care. New therapeutic targets for diabetic nephropathy are, therefore, urgently needed. Autophagy is a major catabolic pathway by which mammalian cells degrade macromolecules and organelles to maintain intracellular homeostasis. The accumulation of damaged proteins and organelles is associated with the pathogenesis of diabetic nephropathy. Autophagy in the kidney is activated under some stress conditions, such as oxidative stress and hypoxia in proximal tubular cells, and occurs even under normal conditions in podocytes. These and other accumulating findings have led to a hypothesis that autophagy is involved in the pathogenesis of diabetic nephropathy. Here, we review recent findings underpinning this hypothesis and discuss the advantages of targeting autophagy for the treatment of diabetic nephropathy.