Protective Effect Of Rapamycin Research Articles

Rapamycin Affects the Hippocampal SNARE Complex to Alleviate Cognitive Dysfunction Induced by Surgery in Aged Rats.

Delayed neurocognitive recovery (dNCR) is a common complication that occurs post-surgery, especially in elderly individuals. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex plays an essential role in various membrane fusion events, such as synaptic vesicle exocytosis and autophagosome-lysosome fusion. Although SNARE complex dysfunction has been observed in several neurodegenerative disorders, the causal link between SNARE-mediated membrane fusion and dNCR remains unclear. We previously demonstrated that surgical stimuli caused cognitive impairment in aged rats by inducing α-synuclein accumulation, inhibiting autophagy, and disrupting neurotransmitter release in hippocampal synaptosomes. Here, we evaluated the effects of propofol anesthesia plus surgery on learning and memory and investigated levels of SNARE proteins and chaperones in hippocampal synaptosomes. Aged rats that received propofol anesthesia and surgery exhibited learning and memory impairments in a Morris water maze test and decreased levels of synaptosome-associated protein 25, synaptobrevin/vesicle-associated membrane protein 2, and syntaxin 1. Levels of SNARE chaperones, including mammalian uncoordinated-18, complexins 1 and 2, cysteine string protein-α, and N-ethylmaleimide-sensitive factor, were all significantly decreased following anesthesia with surgical stress. However, the synaptic vesicle marker synaptophysin was unaffected. The autophagy-enhancer rapamycin attenuated structural and functional disturbances of the SNARE complex and ameliorated disrupted neurotransmitter release. Our results indicate that perturbations of SNARE proteins in hippocampal synaptosomes may underlie the occurrence of dNCR. Moreover, the protective effect of rapamycin may partially occur through recovery of SNARE structural and functional abnormalities. Our findings provide insight into the molecular mechanisms underlying dNCR.

DOP68 Autophagy induction in the intestinal epithelium protects against inflammation-induced barrier loss in vivo.

Abstract Background Loss of the paracellular tight junction (TJ) barrier and defective autophagy in the gut are pathogenic factors for inflammatory bowel disease (IBD). Earlier, we demonstrated the role of autophagy in the degradation of the pore-forming claudin-2 protein and the resultant TJ barrier enhancement. Here, we investigated the role of autophagy in the regulation of the TJ barrier in vivo and in Crohn’s disease (CD) patient-derived tissues and patient-derived organoids. Methods Wild type and autophagy-deficient Atg7-/- mice were given intraperitoneal rapamycin injection to induce autophagy along with LPS or TNF-α to induce inflammation. Surgically resected tissues from CD patients were incubated with rapamycin and the paracellular barrier was measured by Ussing chambers. Autophagy was induced in human colonic organoids by incubation with either rapamycin or by nutrient starvation. Results Intraperitoneal rapamycin injection in WT mice successfully induced autophagy as evidenced through the degradation of the autophagy substrate p62/SQSTM. Rapamycin injection increased the colonic occludin levels and enhanced the paracellular TJ barrier as evidenced by the decreasing paracellular flux of inulin. Rapamycin prevented LPS and TNF-a induced colonic paracellular barrier loss and reduction in occludin levels in WT mice. Rapamycin reduced the LPS and TNF-a-induced increase in occludin’s association with caveolin-1 thereby preventing its caveolae-mediated endocytosis and degradation. The protective effect of rapamycin was dependent on autophagy. Conditional knockout of Atg7 (cAtg7) in mouse colon decreased baseline occludin levels and increased inulin flux in mouse colon. Furthermore, rapamycin failed to prevent the LPS and TNF-a-induced colonic barrier loss in the cAtg7 mice. Additionally, these mice lacking the ability to undergo autophagy in the colon were more susceptible to Dextran Sodium Sulphate (DSS)-induced colitis. In normal and inflamed colonic tissues from CD patients, incubation with rapamycin induced autophagy, evidenced through the reduction in p62/SQSTM1 protein levels, increased occludin levels and enhanced the paracellular TJ barrier. Autophagy induction by rapamycin or nutrient starvation also increased occludin levels and its membrane localization in patient-derived organoids. Conclusion Our data show that rapamycin successfully induced autophagy in WT mice’s colon and protected these mice from the barrier disruptive effect of both LPS and TNF-α, indicating the importance of autophagy in regulating the intestinal TJ barrier. Our data also highlights the application of autophagy induction as a potential therapeutic approach in the treatment of IBD.

Rapamycin ameliorates brain damage and maintains mitochondrial dynamic balance in diabetic rats subjected to middle cerebral artery occlusion.

To investigate the effect of rapamycin on mitochondrial dynamic balance in diabetic rats subjected to cerebral ischemia-reperfusion injury. Male Sprague Dawley (SD) rats (n = 78) were treated with high fat diet combined with streptozotocin injection to construct diabetic model in rats. Transient middle cerebral artery occlusion (MCAO) of 2hours was induced and the brains were harvested after 1 and 3days of reperfusion. Rapamycin was injected intraperitoneally for 3days prior to and immediately after operation, once a day. The neurological function was assessed, infarct volumes were measured and HE staining as well as immunohistochemistry were performed. The protein of hippocampus was extracted and Western blotting were performed to detect the levels of mTOR, mitochondrial dynamin related proteins (DRP1, p-DRP1, OPA1), SIRT3, and Nix/BNIP3L. Diabetic hyperglycemia worsened the neurological function performance (p < 0.01), enlarged infarct size (p < 0.01) and increased ischemic neuronal cell death (p < 0.01). The increased damage was associated with elevations of p-mTOR, p-S6, and p-DRP1; and suppressions of SIRT3 and Nix/BNIP3L. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and reversed the biomarker alterations caused by diabetes. High glucose activated mTOR pathway and caused mitochondrial dynamics toward fission. The protective effect of rapamycin against diabetes-enhanced ischemic brain damage was associated with inhibiting mTOR pathway, redressing mitochondrial dynamic imbalance, and elevating SIRT3 and Nix/BNIP3L expression.

Rapamycin attenuated podocyte apoptosis via upregulation of nestin in Ang II-induced podocyte injury

BackgroundAngiotensin II (Ang II) contributes to the progression of glomerulosclerosis, mainly by inducing podocyte injury. Convincing evidence indicates that the mTOR inhibitor rapamycin could play a fundamental role in protection against podocyte injury. Nestin, a major cytoskeletal protein, is stably expressed in podocytes and correlates with podocyte damage. The purpose of this study was to investigate the effect of rapamycin on podocyte injury induced by Ang II and to clarify the role and mechanism of nestin in the protective effect of rapamycin of podocyte injury.Methods and resultsWe established an Ang II perfusion animal model, and the effects of rapamycin treatment on podocytes were investigated in vivo. In vitro, podocytes were stimulated with Ang II and rapamycin to observe podocyte injury, and nestin-siRNA was transfected to investigate the underlying mechanisms. We observed that Ang II induced podocyte injury both in vivo and in vitro, whereas rapamycin treatment relieved Ang II-induced podocyte injury. We further found that nestin co-localized with p-mTOR in glomeruli, and the protective effect of rapamycin was reduced by nestin-siRNA in podocytes. Moreover, co-IP indicated the interaction between nestin and p-mTOR, and nestin could affect podocyte injury via the mTOR/P70S6K signaling pathway.ConclusionWe demonstrated that rapamycin attenuated podocyte apoptosis via upregulation of nestin expression through the mTOR/P70S6K signaling pathway in an Ang II-induced podocyte injury.

Mammalian Target of Rapamycin Inhibitor Rapamycin Alleviates 7-Ketocholesterol Induced Inflammatory Responses and Vascular Endothelial Growth Factor Elevation by Regulating MAPK Pathway in Human Retinal Pigment Epithelium Cells.

Purpose: To validate the protective effect of a mammalian target of rapamycin (mTOR) inhibitor on human retinal pigment epithelium (RPE) cells challenged with 7-ketocholesterol (7-KC) and explored the underlying mechanisms. Methods: Human primary RPE (hRPE) cells and ARPE-19 cells were cultured with or without 10 nM of rapamycin for 6 h before being exposed to 10 μM of 7-KC for 24 h. The transcriptome of 7-KC challenged ARPE-19 cells was investigated by RNA sequencing (RNA-seq). The effects of 7-KC and rapamycin on the viability of ARPE-19 cells were measured with CCK-8. Gene expression was verified by real-time PCR, and protein levels were determined by ELISA or Western blotting. Results: The expression of IL-6, IL-8, and vascular endothelial growth factor (VEGF) in RPE cells was markedly increased after stimulation with 7-KC for 12/24 h compared with the controls. RNA-seq showed that a total of 10,243 genes were differentially expressed, with 5,518 genes upregulated and 4,725 genes downregulated between the 7-KC treated and the control group. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that 7-KC stimulation activated mTOR signaling and other pathways, including adherent junction, MAPK, and Wnt signalings. mTOR inhibitor rapamycin significantly suppressed the elevation of IL-6, IL-8, and VEGF stimulated by 7-KC. Rapamycin not only decreased the level of phosphorylated mTOR, P70S6K, 4EBP1 but also inhibited the activation of MAPK pathway. Conclusions: Inhibition of mTOR signaling pathway suppressed the elevation of inflammatory cytokines IL-6, IL-8, and the angiogenic agent VEGF induced by 7-KC. The protective effect of rapamycin was associated with its downregulation on MAPK pathway.

Aversive Learning Deficits and Depressive-Like Behaviors Are Accompanied by an Increase in Oxidative Stress in a Rat Model of Fetal Alcohol Spectrum Disorders: The Protective Effect of Rapamycin.

Fetal alcohol spectrum disorders (FASDs) are one of the most common consequences of ethanol exposure during pregnancy. In adulthood, these disorders can be manifested by learning and memory deficits and depressive-like behavior. Ethanol-induced oxidative stress may be one of the factors that induces FASD development. The mammalian target of the Rapamycin (mTOR) signaling pathway that acts via two distinct multiprotein complexes, mTORC1 and mTORC2, can affect oxidative stress. We investigated whether mTOR-dependent or mTOR-independent mechanisms are engaged in this phenomenon. Thus, Rapamycin—a selective inhibitor of mTORC1, Torin-2—a non-selective mTORC1/mTORC2 inhibitor, and FK-506—a drug that impacts oxidative stress in an mTOR-independent manner were used. Behavioral tests were performed in adult (PND60-65) rats using a passive avoidance (PA) task (aversive learning and memory) and forced swimming test (FST) (depressive-like behaviors). In addition, the biochemical parameters of oxidative stress, such as lipid peroxidation (LPO), as well as apurinic/apyrimidinic (AP)-sites were determined in the hippocampus and prefrontal cortex in adult (PND65) rats. The rat FASD model was induced by intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4–9 (an equivalent to the third trimester of human pregnancy). All substances (3 mg/kg) were given 30 min before ethanol. Our results show that neonatal ethanol exposure leads to deficits in context-dependent fear learning and depressive-like behavior in adult rats that were associated with increased oxidative stress parameters in the hippocampus and prefrontal cortex. Because these effects were completely reversed by Rapamycin, an mTORC1 inhibitor, this outcome suggests its usefulness as a preventive therapy in disorders connected with prenatal ethanol exposure.

Protective effect of rapamycin against acrylamide-induced hepatotoxicity: The associations between autophagy, apoptosis, and necroptosis.

Acrylamide (ACRL) was demonstrated to induce hepatotoxicity and programmed cell death (PCD). Rapamycin (RAPA)-induced autophagy had been reported to limit the progression of hepatocellular injury in experimental models. This research was designed to study two death pathways involved in ACRL-induced hepatotoxicity and the modulating effect of RAPA on the resulting hepatic injury. Thirty-six adult male rats were divided into three groups: control group, ACRL-treated group (20 mg kg/day), and the last group co-treated with ACRL plus RAPA (0.5mg kg/day). Drugs were administered for 21 days via oral gavage. Blood samples were collected to assess alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Livers were dissected; parts were used for detection of superoxide dismutase (SOD) and malondialdehyde (MDA) tissue levels. Other parts were processed for hematoxylin and eosin, Masson's trichrome staining, immunostaining for microtubule-associated proteins 1A/1B light chain 3B (LC3), ubiquitin-binding protein (p62), caspase-3, and receptor-interacting protein kinase 1 (RIPK1). ACRL induced a significant elevation in ALT, AST, MDA levels, and reduction in the SOD level. ACRL also induced hepatocellular injury, fibrosis, and defective autophagy indicated by elevation of LC3 and p62 and increased p62/LC3 ratio. Moreover, it increased the apoptotic (caspase-3) and necroptotic (RIPK1) markers expression. RAPA significantly reduced liver enzymes, oxidative stress, fibrosis, and improved liver histology. Moreover, RAPA decreased p62/LC3 ratio indicated enhanced autophagy, and significantly reduced caspase-3 and RIPK1 expression. In conclusion, RAPA maintained autophagic activity which may save the hepatocytes from PCD and enhance cell viability.

Protective effects of rapamycin on the retinal vascular bed during the vaso-obliteration phase in mouse oxygen-induced retinopathy model.

Retinopathy of prematurity (ROP) is a vision-threatening disorder characterized with retinal vaso-obliteration in phase 1 and pathological neovascularization (NV) in phase 2. However, there has been no effective and safe treatment for ROP. Current management is mainly focused on the reduction of abnormal NV in phase 2, and anti-vascular endothelial growth factor (VEGF) therapy is the first-line treatment, yet, with great risks of late recurrence and systemic side effects. It has been reported that the severity of vaso-obliteration in phase 1 largely influences subsequent NV, suggesting that it may be a promising target to develop novel treatments for ROP. Here, we investigated the therapeutic potential and safety of early rapamycin intervention in treating phase 1 ROP and possible underlying mechanisms using the mouse model of oxygen-induced retinopathy (OIR). We found that intravitreal injection of rapamycin at postnatal day 7 (P7) significantly reduced retinal avascular area, increased vascular density, and reversed the suppression of deep capillaries development in phase 1 of OIR mice. Rapamycin treatment not only reduced vascular apoptosis, but also promoted proliferation and tip cell functions. Additionally, rapamycin did not interfere with normal retinal vascular development. Further investigation showed that Ang1/Tie2 pathway might be involved in rapamycin's vascular protection in phase 1 OIR retinas. Moreover, early rapamycin treatment at P7 had long-term protective effects of reducing retinal NV and avascular area, as well as enhancing vascular maturity in phase 2 of OIR mice. Together, our data suggest that rapamycin may be a safe and promising strategy for early intervention of ROP.

Rapamycin improves renal injury induced by Iodixanol in diabetic rats by deactivating the mTOR/p70S6K signaling pathway

AimsTo study how to effectively prevent or reduce renal injury caused by contrast agents in diabetic patients. Main methodsSprague Dawley (SD) rats were bred with a high-fat diet for eight weeks, then intraperitoneally injected with Streptozotocin (STZ) to prepare the diabetes model. Rats were treated with Iodixanol to prepare a contrast-induced acute kidney injury (CIAKI) model. Moreover, 3-methyladenine (3-MA), an autophagy inhibitor, was administrated to diabetic rats with or without Rapamycin treatment. Serum creatinine (SCr) and blood urea nitrogen (BUN) were examined using Biochemical detector. Kidney injury molecule-1 (KIM-1), N-acetyl-β-D-amino glycosidase (NAG) in urine, inflammatory and oxidative stress factors in serum were determined by ELISA. The expression level of ROS was quantified by immunofluorescence (IF). The protein expressions of Bax, BCl-2, LC3, Beclin1, mTOR and p70S6K in renal tissue were detected by Western blot. Key findingsRapamycin was demonstrated to improve renal injury induced by Iodixanol diabetic rats, decrease the levels of SCr, BUN, KIM-1, NAG, improve renal functions, reduce inflammatory response and oxidative stress injury, down-regulate Bax, while up-regulate BCl-2 and inhibit apoptosis. Moreover, Rapamycin could inhibit the phosphorylation of mTOR/p70S6K pathway-associated proteins, activate autophagy and increase the levels of LC3 and Beclin1. After treatment with 3MA, an inhibitor of mTOR/p70S6K signaling pathway, the protective effects of Rapamycin on CIAKI were weakened. SignificanceRapamycin can alleviate renal injury induced by Iodixanol diabetic rats, and its regulatory mechanisms may be related to the regulation of mTOR/p70S6K signaling pathway and the activating autophagy.

P6273Inhibitory phosphorylation of RIP1 at Ser320 induces nuclear translocation of TFEB, leading to suppression of necroptosis in cardiomyocytes

Abstract Background Necroptosis, a form of programmed necrosis, has been shown to contribute to the pathogenesis of various diseases including ischemia/reperfusion injury and heart failure. We recently reported that necroptotic signals suppresses autophagy in cardiomyocytes and that rapamycin, an mTORC1 inhibitor, not only promotes autophagy but also protect the cells from necroptosis. Purpose We examined the mechanism by which rapamycin suppresses necroptosis of cardiomyocytes, focusing on regulation of RIP1 activity and autophagic flux. Methods and results In H9c2 cardiomyoblasts, necroptosis was induced by treatment with TNF and z-VAD-fmk (zVAD) for 24 h, and cell death was determined by LDH release (as % of total). The treatment with TNF/zVAD increased LDH release from 3.4±1.3% to 46.1±2.3%, and LDH release was suppressed by necrostatin-1 (5.9±0.9%), a RIP1 inhibitor, and by rapamycin (23.5±1.4%). The protective effect of rapamycin was mimicked by Ku-0063794, an mTORC1/2 inhibitor. TNF/zVAD induced RIP1-RIP3 complex formation, together with suppression of TNF-induced RIP1 cleavage, which was mitigated by rapamycin. In addition, rapamycin not only suppressed TNF/zVAD-induced phosphorylation of RIP1-Ser166, an index of RIP1 activation, but also increased phosphorylation of RIP1-Ser320, an inhibitory phosphorylation site. In cells transfected with RIP1-S320A, which lack Ser320 for inhibitory phosphorylation, rapamycin failed to suppress TNF/zVAD-induced RIP1-RIP3 binding and cell death. Immunoblot analyses showed that TNF/zVAD significantly increased level of LC3-II. The accumulation of LC3-II protein was not further increased by bafilomycin A1 (100 nM), an inhibitor of lysosomal protein degradation, indicating that accumulation of LC3-II by TNF/zVAD reflected suppression of autophagic flux. Inhibition of RIP1 by necrostatin-1 attenuated TNF/zVAD-induced accumulation of LC3 II. The restoration of autophagic flux in TNF/zVAD-treated cells by necrostatin-1 was confirmed by monitoring tandem RFP-GFP-LC3 transfected cells; necrostatin-1 increased a ratio of RFP-LC3-puncta (autolysosomes) to RFP-GFP-LC3-puncta (autophagosomes) in TNF/zVAD-treated cells. In addition, necrostatin-1 and rapamycin induced nuclear translocation of TFEB, a regulator of lysosome biogenesis, which was associated with upregulation of MCOLN1 mRNA, a downstream target of TFEB. Restoration of autophagic flux in TNF/zVAD-treated cells by necrostatin-1 was inhibited by siRNA-mediated knockdown of TFEB. Conclusion Activation of TFEB by inhibitiory phosphorylation of RIP1-Ser320 is a primary mechanism of cytoprotection afforded by mTORC1 inhibition against necroptosis.

MTORC1 inhibition attenuates necroptosis through RIP1 inhibition-mediated TFEB activation

Accumulating evidence indicates that necroptosis contributes to cardiovascular diseases. We recently reported suppression of autophagy by necroptotic signals in cardiomyocytes and protective action of rapamycin. Here we examined the mechanism by which mTORC1 inhibition protects cardiomyocytes from necroptosis. Necroptosis of H9c2 cells was induced by treatment with tumor necrotic factor-α (TNF) and z-VAD-fmk (zVAD), and the extent of necroptosis was determined as the level of LDH release (as % of total). TNF/zVAD increased RIP1-RIP3 interaction and LDH release from 3.4 ± 1.3% to 46.1 ± 2.3%. The effects of TNF/zVAD were suppressed by an mTORC1 inhibitor, rapamycin, and an mTORC1/2 inhibitor, Ku-0063794, but not by a p70s6K inhibitor, PF-4708671. Protection by rapamycin was not abolished by inhibitors of TAK1, IKKα/β, and cIAP, endogenous necroptosis suppressors upstream of RIP1. Rapamycin and Ku-0063794 suppressed TNF/zVAD-induced RIP1-Ser166 phosphorylation and increased phosphorylation of RIP1-Ser320, an inhibitory phosphorylation site, though such an effect on RIP1-Ser320 was not observed for PF-4708671. Protective effects of rapamycin on TNF/zVAD-induced RIP1-RIP3 binding and necroptosis were undetected in cells transfected with RIP1-S320A. In TNF/zVAD-treated cells, rapamycin and a RIP1 inhibitor, necrostatin-1, increased nuclear localization of transcriptional factor EB (TFEB) and promoted autolysosome formation from autophagosomes in a TFEB-dependent manner. Knockdown of TFEB expression attenuated rapamycin-induced protection from necroptosis in TNF/zVAD-treated cells. The results suggest that mTORC1 inhibition promotes autophagy and protects cardiomyocytes from necroptosis by a TFEB-dependent mechanism and that inhibition of RIP1 by increased phosphorylation at Ser320 is crucial in the cardiomyocyte protection afforded by mTORC1 inhibition.

Rapamycin attenuates the paraquat‐induced pulmonary fibrosis through activating Nrf2 pathway

Oxidative stress is a key regulator of idiopathic pulmonary fibrosis. Paraquat (PQ)‐induced pulmonary fibrosis seriously endangers people's health. Rapamycin has been reported to alleviate PQ‐induced pulmonary fibrosis, but its underlying mechanism is unclear. The nuclear factor E2‐related factor 2 (Nrf2) plays an important regulatory role in the antioxidant therapy of PQ‐induced pulmonary fibrosis. In this study, we tried to confirm that rapamycin attenuates PQ‐induced pulmonary fibrosis by regulating Nrf2 pathway. In vivo, we proved that rapamycin could inhibit the degree of PQ‐induced oxidant stress as well as enhanced the expression of Nrf2. In vitro, rapamycin decreased the upregulated effects of cell death and apoptosis, fibrosis‐related factors expression and fibroblast‐to‐myofibroblast transformation by PQ treatment. In vivo, rapamycin treatment reduced fibrosis degree and the expression of fibrosis‐related factors in lung tissues of rat treated PQ. Furthermore, we also found that Nrf2 knockdown reduced the inhibitory effect of rapamycin on PQ‐induced pulmonary fibrosis, as well as decreased Nrf2 transfer from the cytoplasm into the nucleus. Our findings demonstrated that the protective effect of rapamycin is associated with the activation of the Nrf2 pathway in pulmonary fibrosis induced by PQ poisoning.

Rapamycin Removes Damaged Mitochondria and Protects Human Trabecular Meshwork (TM-1) Cells from Chronic Oxidative Stress.

Glaucoma is a chronic optic neuropathy that could lead to permanent vision loss. Primary open-angle glaucoma (POAG) is the most common type of glaucoma, with elevated intraocular pressure (IOP) as a major risk factor. IOP is mainly regulated by trabecular meshwork (TM), an important component of the conventional aqueous humor (AH) outflow pathway. TM cells are constantly subjected to oxidative stress. Long-term exposure to oxidative stress has been shown to cause elevation of AH outflow resistance, leading to higher IOP. In this study, we induced chronic oxidative stress in human trabecular meshwork (TM-1) cells with 1μM rotenone and investigated the levels of reactive oxygen species (ROS), autophagy, and mitochondrial functions. Protective effects of rapamycin, an inducer of autophagy, were also investigated. Our data indicated that rotenone significantly increased oxidative stress, but not autophagy, in TM-1 cells. Rapamycin at 10nM effectively suppressed the rotenone-induced cell apoptosis, as well as the ROS elevation. The protective effects of rapamycin could be associated to the induction of autophagy and removal of damaged mitochondria in TM-1 cells. Our results suggest autophagy has important roles in protecting TM-1 cells from oxidative stress, which could be further developed into a novel treatment to POAG.

The protective effect and mechanism of rapamycin in the rat model of IgA nephropathy

Background: The pathogenesis of the development of IgA nephropathy has not been clear up to now. At present, some studies revealed that the mTOR pathway may participate in IgA nephropathy; however, the mechanism has not been systematically studied. In this study, we established an IgAN rat model to investigate the protective effects of rapamycin as a new type of immunosuppressant, as well as its therapeutic mechanisms.Methods: After the establishment of IgA nephropathy model, rats were treated with different concentrations of rapamycin, and the protective effect of different concentrations of rapamycin on renal function of the rats was observed. The deposition of IgA was observed by immunofluorescence. The kidney expression of Akt and p70S6k proteins in mTOR pathway was examined using the western blot assay after rapamycin treatment.Results: Morphology and immunofluorescence confirmed that the rat model of IgA nephropathy was successfully established. In particular, the level of proteinuria decreased with the increase of the dose of rapamycin, as well as the deposition of IgA in glomeruli. Moreover, the western blot analysis indicated that the expression of p70S6K in the downstream of mTOR pathway decreased and the upstream protein AKT of the mTOR pathway was overexpressed in the rats model.Conclusion: We found that rapamycin has protective effects in the IgA nephropathy rat model in a dose-dependent manner. In addition, the result of western blot assay suggested that rapamycin may display its therapeutic effects through interfering the AKT-mTOR-p70S6K signaling pathway.

Protective effect of rapamycin on endothelial-to-mesenchymal transition in HUVECs through the Notch signaling pathway

Neointima hyperplasia is one of the predominant features of cardiovascular diseases such as atherosclerosis, and is also responsible for the restenosis of vascular surgery including arteriovenous fistula and stent implantation. Endothelial-to-mesenchymal transition (EndMT) contributes to neointima hyperplasia by activation of the Notch or TGF-β signaling pathway. Rapamycin has been utilized as anti-restenosis drug due to its anti-proliferative activity. However, its effects on the EndMT have not been investigated yet. Thus, we examined the biological effects of rapamycin on the EndMT and its potential mechanisms. We showed that rapamycin significantly reversed TGF-β1 stimulated EndMT by upregulating endothelial marker CD31 expression and downregulating mesenchymal marker SMA-α expression in human umbilical vein endothelial cells (HUVECs). Rapamycin also inhibited TGF-β1 induced expression of the Notch signaling pathway components expression, such as Notch-1, Jagged-1, RBP-jκ and Hes-5. Among the different Notch receptors and ligands, Jagged-1/Notch-1 cascade was most remarkably blocked by rapamycin. Finally, consistently with the results from Notch inhibitor DAPT treatment, rapamycin suppressed the migration of HUVECs in vitro. Together, these findings indicate that rapamycin may function as an effective inhibitor of the EndMT in HUVECs by suppressing targeting the Notch signaling pathway.

The protective effects of rapamycin on cell autophagy in the renal tissues of rats with diabetic nephropathy via mTOR-S6K1-LC3II signaling pathway

Background: Previous studies have shown that podocyte autophagy is an important trigger for proteinuria and glomerulosclerosis. The mammalian rapamycin target protein (mTOR) occupies a pivotal position in the autophagy pathway. In this study, we planned to clarify the mechanism of mTOR regulation of podocyte autophagy and the effect of rapamycin (RAPA).Methods: All rats were randomly divided into normal control group (n = 8), DN group (n = 8), and RAPA group (n = 8). Blood and urine samples were collected at the 4th, 8th, and 12th weeks of the experiment. The serum creatinine (Scr), urine volume levels, and the 24 h urine protein (UP) levels were examined. The nephrin, podocin, mTOR, ribosomal S6 kinase 1 (S6K1), and autophagy marker light chain 3 (LC3II) expression levels were evaluated by immunohistochemistry, quantitative PCR, and immunoblotting.Results: The urine volume, 24 h UP, and Scr of the DN and RAPA groups increased significantly compared with the NC group (p < .05). Nephrin and podocin expression was decreased in the kidney tissues of the DN and RAPA groups compared with the NC group (p < .05). The expression levels of mTOR and S6K1 increased and LC3II expression decreased in the renal tissues of the DN and RAPA groups compared with the NC group (p < .05). After RAPA treatment, all the above indexes were improved compared with the DN group (p < .05), but were significantly abnormal compared with the NC group (p < .05).Conclusion: The proteinuria and kidney function had improved after RAPA treatment. These results confirmed that RAPA specifically binds to mTOR kinase, and inhibits mTOR activity, thereby regulating the pathological autophagic process.

Negative feedback loop of autophagy and endoplasmic reticulum stress in rapamycin protection against renal ischemia-reperfusion injury during initial reperfusion phase.

Rapamycin, an immunosuppressant, is widely used in patients with kidney transplant. However, the therapeutic effects of rapamycin remain controversial. Additionally, previous studies have revealed deleterious effects of rapamycin predominantly when administered for ≥24 h. Few studies, however, have focused on the short-term effects of rapamycin administered only during the initial reperfusion phase. As such, we designed this study to explore the potential effects and mechanisms of rapamycin under a specific therapeutic regimen in which rapamycin is mixed in the perfusate during the initial reperfusion phase (within 24 h). Interestingly, we found that rapamycin maintained renal function and attenuated ischemia-reperfusion (I/R)-induced apoptosis in vivo and in vitro during the initial reperfusion phase, especially at 8 h after reperfusion. Simultaneously, rapamycin activated autophagy and inhibited endoplasmic reticulum (ER) stress and 3 pathways of unfolding protein response: ATF6, PERK, and IRE1α. Interestingly, we further found that the protective effects of rapamycin were suppressed when autophagy was inhibited by chloroquine and 3-methyladenine or when ER stress was induced by thapsigargin. Moreover, in terms of the regulatory effects of rapamycin, a negative-feedback loop between autophagy and ER stress occurred, with autophagy inhibiting ER stress and increased ER stress promoting autophagy during the initial reperfusion phase of renal I/R injury. Our study provides evidence that immediate reperfusion with rapamycin during the initial reperfusion phase repairs renal function and reduces apoptosis via activating autophagy, which could further inhibit ER stress. These results suggest a novel treatment modality for application during the initial reperfusion phase of renal I/R injury caused by kidney transplantation.-Li, X., Zhu, G., Gou, X., He, W., Yin, H., Yang, X., Li, J. Negative feedback loop of autophagy and endoplasmic reticulum stress in rapamycin protection against renal ischemia-reperfusion injury during initial reperfusion phase.

Activation of autophagy attenuates EtOH-LPS-induced hepatic steatosis and injury through MD2 associated TLR4 signaling

Autophagy serves as a protective mechanism to degrade damaged organelles and proteins. Acute alcohol exposure is known to activate the hepatic autophagy response, whereas chronic alcohol exposure slows autophagosome formation along with an elevation of gut-derived endotoxin. In the current study, we examined whether lipopolysaccharide (LPS) administration decreased autophagic response in the liver of mice treated by short-term alcohol and whether activation of autophagy by rapamycin attenuates EtOH-LPS-induced liver steatosis and injury. We demonstrated that ten-day alcohol feeding primed the liver to LPS-induced lipid accumulation and liver injury with significantly increased hepatic steatosis and serum AST level as well as hepatic cellular NF-κB activation. LPS increased alcohol-mediated reactive oxygen species (ROS) formation while reducing autophagy activation. These deleterious effects were attenuated by rapamycin administration in mice. The protective effects of rapamycin are associated with decreased cellular MD2/TLR4 expression and interaction in Raw264.7 cells. Taken together, our results demonstrated that enhanced gut-derived LPS decreases the hepatic autophagosome numbers in response to alcohol exposure, and activation of autophagy by rapamycin protects from EtOH-LPS-induced liver injury, probably through reduced macrophage expression and interaction of TLR4/MD2 signaling complex.

Protective effect of rapamycin on hypoxia-injuried retinal ganglion cells and its mechanism

Background Studies showed that rapamycin (Rapa) plays a protective effect on central nervous system by improving the aging of human brain tissue and ameliorating rat cerebral metabolism after ischemia.Optical nerve and retinal ganglion cells (RGCs) are central nerve, however, whether Rapa can protect hypoxia-injuried RGCs is still unclear. Objective This study was to explore the protective role of Rapa on hypoxia RGC-5, a rat RGC line, and its underlying mechanism in order to provide a new strategy for the treatment of traumatic optic neuropathy. Methods Rat RGC-5 cells were cultured using DMEM with 10% fetal bovine serum, and the cells were observed under the inverted phase contrast microscope.The cells were treated by 50, 100, 200, 400 and 600 μmol/L CoCl2 for 24 hours and 48 hours, respectively, and Clone Select Imager was employed to assess the survival rate.The CoCl2-induced hypoxia cell models were established by adding 200 μmol/L CoCl2 in the medium for 24 hours, and then the 0.1, 0.4, 1.6, 6.4 μmol/L Rapa was used to treat the models for 24 hours in the Rapa intervention group, respectively.The cells cultured by DMEM with 10% fetal bovine serum served as the normal control group.The survival rate of the cells was evaluated by Clone Select Imager; the apoptotic rate of the cells was assayed by AnnexinV-FITC/PI double-staining flow cytometry; JC-1 probe was used to detect the mitochondrial trans-membrane potential, and the expression of bax mRNA in the cells was detected by real-time fluorescence quantitative PCR. Results The survival rate of the cells was (70.51±5.00)% in the 200 μmol/L CoCl2-treated group, which was significantly lower than (100.00±3.29)% in the normal control group (P<0.01). The survival rate of the cells was significantly different among the normal control group and 0.1, 0.4, 1.6, 6.4 μmol/L Rapa intervention groups (F=167.904, P=0.000), and the survival rate was evidently higher in the 0.1 μmol/L Rapa intervention group than that in the model control group (P<0.05). The apoptotic rate was 25.4%, 37.7% and 25.3%, while mitochondrial trans-membrane potential reduced by 0.4%, 6.3% and 1.4% in the normal control group, model control group and 0.1 μmol/L Rapa intervention group, respectively.The relative expression of bax mRNA in the cells was 1.01±0.21, 3.52±0.30 and 1.66±0.20 in the normal control group, model control group and 0.1 μmol/L Rapa intervention group, showing a significant difference among the groups (F=88.034, P=0.000), and the relative expression of bax mRNA in the model control group was considerably elavated in comparison with the normal control group and 0.1 μmol/L Rapa intervention group (both at P<0.05). Conclusions Rapa protects the RGC-5 cells against CoCl2-induced hypoxic damage primarily by down-regulating the expression of bax in the cells and improving the survival rate of RGC-5 cells in vitro. Key words: Rapamycin; Retinal ganglion cells; Mammalian target of rapamycin; Apoptosis

Neuroprotective effects of intrastriatal injection of rapamycin in a mouse model of excitotoxicity induced by quinolinic acid

BackgroundThe mammalian target of rapamycin (mTOR) is a kinase involved in a variety of physiological and pathological functions. However, the exact role of mTOR in excitotoxicity is poorly understood. Here, we investigated the effects of mTOR inhibition with rapamycin against neurodegeneration, and motor impairment, as well as inflammatory profile caused by an excitotoxic stimulus.MethodsA single and unilateral striatal injection of quinolinic acid (QA) was used to induce excitotoxicity in mice. Rapamycin (250 nL of 0.2, 2, or 20 μM; intrastriatal route) was administered 15 min before QA injection. Forty-eight hours after QA administration, rotarod test was performed to evaluate motor coordination and balance. Fluoro-Jade C, Iba-1, and GFAP staining were used to evaluate neuronal cell death, microglia morphology, and astrocytes density, respectively, at this time point. Levels of cytokines and neurotrophic factors were measured by ELISA and Cytometric Bead Array 8 h after QA injection. Striatal synaptosomes were used to evaluate the release of glutamate.ResultsWe first demonstrated that rapamycin prevented the motor impairment induced by QA. Moreover, mTOR inhibition also reduced the neurodegeneration and the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α induced by excitotoxic stimulus. The lowest dose of rapamycin also increased the production of IL-10 and prevented the reduction of astrocyte density induced by QA. By using an in vitro approach, we demonstrated that rapamycin differently alters the release of glutamate from striatal synaptosomes induced by QA, reducing or enhancing the release of this neurotransmitter at low or high concentrations, respectively.ConclusionTaken together, these data demonstrated a protective effect of rapamycin against an excitotoxic stimulus. Therefore, this study provides new evidence of the detrimental role of mTOR in neurodegeneration, which might represent an important target for the treatment of neurodegenerative diseases.