ABSTRACT
Dry eye disease (DED) is caused by inflammation and damage to the corneal surface due to tear film instability and hyperosmolarity. Various eye drops are used to treat this condition. Each eye drop has different properties and mechanisms of action, so the appropriate drug should be used according to clinical phenotypes. This study aims to compare the therapeutic mechanisms of cyclosporine A (CsA) and diquafosol tetrasodium (DQS). An experimental in vivo/in vitro model of DED using hyperosmolarity showed decreased cell viability, inhibited wound healing, and corneal damage compared to controls. Treatment with cyclosporine or diquafosol restored cell viability and wound healing and reduced corneal damage by hyperosmolarity. The expression of the inflammation-related genes il-1ß, il-1α, and il-6 was reduced by cyclosporine and diquafosol, and the expression of Tnf-α, c1q, and il-17a was reduced by cyclosporine. Increased apoptosis in the DED model was confirmed by increased Bax and decreased Bcl-2 and Bcl-xl expression, but treatment with cyclosporine or diquafosol resulted in decreased apoptosis. Diquafosol increased NGF expression and translocation into the extracellular space. DED has different damage patterns depending on the progression of the lesion. Thus, depending on the type of lesion, eye drops should be selected according to the therapeutic target, focusing on repairing cellular damage when cellular repair is needed or reducing inflammation when inflammation is high and cellular damage is severe.
Subject(s)
Cornea , Cyclosporine , Disease Models, Animal , Dry Eye Syndromes , Nerve Growth Factor , Uracil Nucleotides , Wound Healing , Uracil Nucleotides/pharmacology , Nerve Growth Factor/metabolism , Nerve Growth Factor/genetics , Wound Healing/drug effects , Animals , Dry Eye Syndromes/drug therapy , Dry Eye Syndromes/metabolism , Dry Eye Syndromes/pathology , Cornea/drug effects , Cornea/pathology , Cornea/metabolism , Cyclosporine/pharmacology , Humans , Cell Survival/drug effects , Apoptosis/drug effects , Polyphosphates/pharmacology , MiceABSTRACT
INTRODUCTION: The comorbidities of ischemic heart disease (IHD) and diabetes mellitus (DM) compromise the protection of the diabetic heart from ischemia/reperfusion (I/R) injury. We hypothesized that manipulation of reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways might protect the diabetic heart, and intervention of these pathways could be a new avenue for potentially protecting the diabetic heart. METHODS: All hearts were subjected to 30-min ischemia and 30-min reperfusion. During reperfusion, hearts were exposed to molecules proven to protect the heart from I/R injury. The hemodynamic data were collected using suitable software. The infarct size, troponin T levels, and protein levels in hearts were evaluated. RESULTS: Both cyclosporine-A and nitric oxide donor (SNAP) infusion at reperfusion protected 4-week diabetic hearts from I/R injury. However, 6-week diabetic hearts were protected only by SNAP, but not cyclosporin-A. These treatments significantly (p < 0.05) improved cardiac hemodynamics and decreased infarct size. CONCLUSIONS: The administration of SNAP to diabetic hearts protected both 4- and 6-week diabetic hearts; however, cyclosporine-A protected only the 4-week diabetic hearts. The eNOS/GLUT-4 pathway executed the SNAP-mediated cardioprotection.
Subject(s)
Cyclosporine , Diabetes Mellitus, Experimental , Myocardial Reperfusion Injury , Myocardium , Nitric Oxide Donors , Nitric Oxide , Signal Transduction , Animals , Myocardial Reperfusion Injury/prevention & control , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/physiopathology , Nitric Oxide/metabolism , Diabetes Mellitus, Experimental/complications , Male , Cyclosporine/pharmacology , Nitric Oxide Donors/pharmacology , Myocardium/metabolism , Myocardium/pathology , Myocardial Infarction/pathology , Myocardial Infarction/metabolism , Myocardial Infarction/prevention & control , Blood Glucose/metabolism , Blood Glucose/drug effects , Time Factors , Rats, Sprague-Dawley , Troponin T/metabolism , Hyperglycemia/metabolism , Hyperglycemia/complications , Glucose Transporter Type 4ABSTRACT
The recent study delves into the role of both liraglutide and/or resveratrol on the nephropathic affection in rats treated with cyclosporine A (CsA). Rats were intoxicated with CsA (25 mg/kg) orally for 21 days and were supplemented with liraglutide (30 µg/kg) s/c daily and 20 mg/kg of resveratrol (20 mg/kg) orally. At the end of the experiment, serum samples and renal tissues were collected to determine renal damage markers, apoptotic markers, proinflammatory markers, and antioxidant status markers. Kidney function tests and antioxidant activity notably improved in the treated rats (CsA + Lir/CsA + Res/CsA + Lir + Res). Moreover, both Lir and/or Res enhanced Bcl-2 levels while down-regulating the Bax levels in rats treated with CsA. Interestingly, the immune-staining for tumor necrosis factor (TNF-α) was tested negative and mild positive in renal tissue of rats given Lir and/or Res while being treated with Cs A which indicated their anti-inflammatory effect that reduced the renal damage. The findings of this investigation revealed the ameliorative anti-inflammatory in addition to the antioxidant role of both liraglutide and resveratrol against the kidney damage caused due to CsA administration.
Subject(s)
Antioxidants , Apoptosis , Cyclosporine , Kidney , Liraglutide , Resveratrol , Animals , Liraglutide/pharmacology , Liraglutide/therapeutic use , Resveratrol/pharmacology , Resveratrol/therapeutic use , Cyclosporine/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Male , Rats , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Biomarkers/metabolism , Biomarkers/blood , Rats, Wistar , Kidney Diseases/metabolism , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Kidney Diseases/drug therapy , Oxidative Stress/drug effects , Inflammation/drug therapy , Inflammation/metabolismABSTRACT
BACKGROUND: Long-term immunosuppressive maintenance therapy is necessary to prevent the rejection of xenografts. However, it is still unclear which oral immunosuppressant is most suitable for pig-to-human xenotransplantation . METHODS: A xenogeneic mixed lymphocyte reaction (MLR) system was established using peripheral blood mononuclear cells (PBMCs) isolated from wildtype (WT) or GTKO/CMAHKO/ß4GalNT2KO (TKO) pigs as stimulator cells and human PBMCs as responder cells. Various concentrations of tacrolimus (Tac), cyclosporine (CsA), or rapamycin (Rapa) were added to the MLR system as interventions. The inhibitory effects of the three immunosuppressants on the proliferation and cytokine production of human T cells were studied and compared. The inhibitory effect of anti-CD154 mAb alone or in combination with Tac/CsA/Rapa on xenoreactive MLR was also investigated. RESULTS: PBMCs from both WT and TKO pigs stimulated significant proliferation of human T cells. Tac had a strong inhibitory effect on human T-cell proliferation stimulated by pig PBMCs. CsA inhibited human T-cell proliferation in a typical dose-dependent manner. When Tac and CsA concentrations reached 5 and 200 ng/mL, respectively, the proliferation rates of CD3+/CD4+/CD8+ T cells were reduced almost to a negative level. Even at high concentrations, Rapa had only a moderate inhibitory effect on xenogeneic MLR. The inhibitory effects of these three immunosuppressants on xenogeneic T-cell responses were further confirmed by the detection of CD25 expression and supernatant cytokines (IL-2, IL-6, IFN-γ, TNF-α, IL-4, IL-10, and IL-17). Although anti-CD154 mAb monotherapy showed only moderate inhibitory effects on xenoreactive T-cell proliferation, low-dose anti-CD154 mAb combined with low-dose Tac, CSA, or Rapa could produce significant synergistic inhibitory effects. CONCLUSION: Tac is more efficient than CsA or Rapa in inhibiting xenogeneic T-cell responses in vitro. If used in combination with anti-CD154 mAb, all the three immunosuppressants can achieve satisfactory synergistic inhibitory effects.
Subject(s)
Cell Proliferation , Cyclosporine , Immunosuppressive Agents , Lymphocyte Culture Test, Mixed , Sirolimus , Tacrolimus , Transplantation, Heterologous , Animals , Sirolimus/pharmacology , Humans , Tacrolimus/pharmacology , Immunosuppressive Agents/pharmacology , Cyclosporine/pharmacology , Transplantation, Heterologous/methods , Swine , Cell Proliferation/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/drug effects , Graft Rejection/immunology , Graft Rejection/prevention & control , Cytokines/metabolism , Cytokines/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/drug effects , Animals, Genetically ModifiedABSTRACT
INTRODUCTION: The calcineurin inhibitor cyclosporine A (CsA) has been shown to effectively reduce proteinuria. However, its precise mechanism is still not fully understood. Our previous study showed that CsA reduced proteinuria by directly stabilizing the foot process (FP) cytoskeletal structure via cofilin-1, suggesting that synaptopodin, a podocyte-specific actin protein, is not the sole target of CsA in podocytes. METHODS: In this study, we established an adriamycin (ADR)-induced nephropathy rat model and a cultured podocyte injury model. We employed Western blotting and immunofluorescence techniques to assess the expression and distribution of transgelin, Krüppel-like factor-4 (KLF-4), nephrin, and synaptopodin. RESULTS: We observed a significant increase in proteinuria levels accompanied by loss of normal FP structure in the ADR-induced nephropathy rat model. The levels of the actin cross-linking protein transgelin were increased significantly, while those of the podocyte-specific molecules nephrin and synaptopodin were decreased in vivo. Treatment with CsA effectively reduced proteinuria while restoring FP effacement stability in ADR-induced nephropathy models and restoring the expression of transgelin, nephrin, and synaptopodin both in vivo and in vitro. Furthermore, CsA treatment dose-dependently decreased transgelin levels while significantly increasing KLF-4 expression in injured podocytes. In addition, CsA failed to downregulate transgelin when KLF-4 was specifically knocked down. CONCLUSION: Our findings suggest that CsA protects against podocyte injury by downregulating abnormally high levels of transgelin via upregulation of KLF-4 expression.
Subject(s)
Cyclosporine , Doxorubicin , Kruppel-Like Factor 4 , Microfilament Proteins , Muscle Proteins , Podocytes , Podocytes/drug effects , Podocytes/pathology , Podocytes/metabolism , Animals , Microfilament Proteins/metabolism , Rats , Cyclosporine/pharmacology , Kruppel-Like Factor 4/metabolism , Muscle Proteins/metabolism , Muscle Proteins/biosynthesis , Male , Membrane Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/genetics , Rats, Sprague-Dawley , Kidney Diseases/chemically induced , Kidney Diseases/prevention & control , Kidney Diseases/metabolism , Kidney Diseases/pathology , ProteinuriaABSTRACT
PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.
Subject(s)
Insulysin , Plasmodium falciparum , Protozoan Proteins , Plasmodium falciparum/enzymology , Plasmodium falciparum/genetics , Insulysin/genetics , Insulysin/chemistry , Insulysin/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Substrate Specificity , Insulin/chemistry , Insulin/metabolism , Insulin/genetics , Zinc/chemistry , Zinc/metabolism , Genome, Protozoan , Recombinant Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/isolation & purification , Gene Expression , Cloning, Molecular , Antimalarials/chemistry , Antimalarials/pharmacology , Cyclosporine/chemistry , Cyclosporine/pharmacologyABSTRACT
BACKGROUND: Identifying drug-binding targets and their corresponding sites is crucial for drug discovery and mechanism studies. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a sophisticated method used for the detection of compound and protein interactions. However, in some cases, LiP-MS cannot identify the target proteins due to the small structure changes or the lack of enrichment of low-abundant protein. To overcome this drawback, we developed a thermostability-assisted limited proteolysis-coupled mass spectrometry (TALiP-MS) approach for efficient drug target discovery. RESULTS: We proved that the novel strategy, TALiP-MS, could efficiently identify target proteins of various ligands, including cyclosporin A (a calcineurin inhibitor), geldanamycin (an HSP90 inhibitor), and staurosporine (a kinase inhibitor), with accurately recognizing drug-binding domains. The TALiP protocol increased the number of target peptides detected in LiP-MS experiments by 2- to 8-fold. Meanwhile, the TALiP-MS approach can not only identify both ligand-binding stability and destabilization proteins but also shows high complementarity with the thermal proteome profiling (TPP) and machine learning-based limited proteolysis (LiP-Quant) methods. The developed TALiP-MS approach was applied to identify the target proteins of celastrol (CEL), a natural product known for its strong antioxidant and anti-cancer angiogenesis effect. Among them, four proteins, MTHFD1, UBA1, ACLY, and SND1 were further validated for their strong affinity to CEL by using cellular thermal shift assay. Additionally, the destabilized proteins induced by CEL such as TAGLN2 and CFL1 were also validated. SIGNIFICANCE: Collectively, these findings underscore the efficacy of the TALiP-MS method for identifying drug targets, elucidating binding sites, and even detecting drug-induced conformational changes in target proteins in complex proteomes.
Subject(s)
Proteolysis , Humans , Mass Spectrometry/methods , Lactams, Macrocyclic/pharmacology , Lactams, Macrocyclic/chemistry , Benzoquinones/chemistry , Benzoquinones/pharmacology , Temperature , Pentacyclic Triterpenes/chemistry , Cyclosporine/pharmacology , Cyclosporine/chemistry , Cyclosporine/metabolism , Staurosporine/pharmacology , Staurosporine/metabolism , Ligands , Drug Discovery , Binding SitesABSTRACT
Mucosal-delivered drugs have to pass through the mucus layer before absorption through the epithelial cell membrane. Although there has been increasing interest in polymeric mucins, a major structural component of mucus, potentially acting as important physiological regulators of mucosal drug absorption, there are no reports that have systematically evaluated the interaction between mucins and drugs. In this study, we assessed the potential interaction between human polymeric mucins (MUC2, MUC5B, and MUC5AC) and various drugs with different chemical profiles by simple centrifugal method and fluorescence analysis. We found that paclitaxel, rifampicin, and theophylline likely induce the aggregation of MUC5B and/or MUC2. In addition, we showed that the binding affinity of drugs for polymeric mucins varied, not only between individual drugs but also among mucin subtypes. Furthermore, we demonstrated that deletion of MUC5AC and MUC5B in A549 cells increased the cytotoxic effects of cyclosporin A and paclitaxel, likely due to loss of mucin-drug interaction. In conclusion, our results indicate the necessity to determine the binding of drugs to mucins and their potential impact on the mucin network property.
Subject(s)
Mucin 5AC , Paclitaxel , Humans , Paclitaxel/pharmacology , Paclitaxel/metabolism , Mucin 5AC/metabolism , Mucin 5AC/genetics , A549 Cells , Drug Interactions , Mucin-5B/metabolism , Mucin-5B/genetics , Mucins/metabolism , Mucin-2/metabolism , Mucin-2/genetics , Rifampin/pharmacology , Cyclosporine/pharmacology , Protein BindingABSTRACT
Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.
Subject(s)
Cyclophilin A , Cyclosporine , Lymphocyte Activation , Proteolysis , T-Lymphocytes , Humans , Cyclophilin A/metabolism , Cyclosporine/pharmacology , Proteolysis/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Lymphocyte Activation/drug effects , HeLa Cells , Cell Proliferation/drug effects , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/chemistry , Proteolysis Targeting ChimeraABSTRACT
Mucosal-associated invariant T (MAIT) cells, a subset of Vα7.2+ T cells, are a crucial link between innate and adaptive immunity, responding to various stimuli through TCR-dependent and independent pathways. We investigated the responses of MAIT cells and Vα7.2+/CD161- T cells to different stimuli and evaluated the effects of Cyclosporin A (CsA) and Vitamin D3 (VitD). Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with various agents (PMA/Ionomycin, 5-OP-RU, 5-OP-RU/IL-12/IL-33) with or without CsA and VitD. Flow cytometric analysis assessed surface markers and intracellular cytokine production. Under steady-state conditions, MAIT cells displayed elevated expression of CCR6 and IL-13. They showed upregulated activation and exhaustion markers after activation, producing IFNγ, TNFα, and TNFα/GzB. CsA significantly inhibited MAIT cell activation and cytokine production. Conversely, Vα7.2+/CD161- T cells exhibited distinct responses, showing negligible responses to 5-OP-RU ligand but increased cytokine production upon PMA stimulation. Our study underscores the distinct nature of MAIT cells compared to Vα7.2+/CD161- T cells, which resemble conventional T cells. CsA emerges as a potent immunosuppressive agent, inhibiting proinflammatory cytokine production in MAIT cells. At the same time, VitD supports MAIT cell activation and IL-13 production, shedding light on potential therapeutic avenues for immune modulation.
Subject(s)
Mucosal-Associated Invariant T Cells , NK Cell Lectin-Like Receptor Subfamily B , Humans , Mucosal-Associated Invariant T Cells/immunology , Mucosal-Associated Invariant T Cells/metabolism , Mucosal-Associated Invariant T Cells/drug effects , NK Cell Lectin-Like Receptor Subfamily B/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Immunologic Factors/pharmacology , Cytokines/metabolism , Cyclosporine/pharmacology , Cholecalciferol/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunologyABSTRACT
Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.
Subject(s)
Calcineurin Inhibitors , Graft vs Host Disease , Isoantigens , Memory T Cells , Graft vs Host Disease/immunology , Graft vs Host Disease/prevention & control , Graft vs Host Disease/pathology , Animals , Mice , Isoantigens/immunology , Calcineurin Inhibitors/pharmacology , Chronic Disease , Memory T Cells/immunology , Tacrolimus/pharmacology , CD4-Positive T-Lymphocytes/immunology , Cyclosporine/pharmacology , Female , CD8-Positive T-Lymphocytes/immunology , T-Lymphocyte Subsets/immunologyABSTRACT
Chronic hepatitis E mostly occurs in organ transplant recipients and can lead to rapid liver fibrosis and cirrhosis. Previous studies found that the development of chronic hepatitis E virus (HEV) infection is linked to the type of immunosuppressant used. Animal models are crucial for the study of pathogenesis of chronic hepatitis E. We previously established a stable chronic HEV infection rabbit model using cyclosporine A (CsA), a calcineurin inhibitor (CNI)-based immunosuppressant. However, the immunosuppression strategy and timing may be optimized, and how different types of immunosuppressants affect the establishment of chronic HEV infection in this model is still unknown. Here, we showed that chronic HEV infection can be established in 100% of rabbits when CsA treatment was started at HEV challenge or even 4 weeks after. Tacrolimus or prednisolone treatment alone also contributed to chronic HEV infection, resulting in 100% and 77.8% chronicity rates, respectively, while mycophenolate mofetil (MMF) only led to a 28.6% chronicity rate. Chronic HEV infection was accompanied with a persistent activation of innate immune response evidenced by transcriptome analysis. The suppressed adaptive immune response evidenced by low expression of genes related to cytotoxicity (like perforin and FasL) and low anti-HEV seroconversion rates may play important roles in causing chronic HEV infection. By analyzing HEV antigen concentrations with different infection outcomes, we also found that HEV antigen levels could indicate chronic HEV infection development. This study optimized the immunosuppression strategies for establishing chronic HEV infection in rabbits and highlighted the potential association between the development of chronic HEV infection and immunosuppressants.IMPORTANCEOrgan transplant recipients are at high risk of chronic hepatitis E and generally receive a CNI-based immunosuppression regimen containing CNI (tacrolimus or CsA), MMF, and/or corticosteroids. Previously, we established stable chronic HEV infection in a rabbit model by using CsA before HEV challenge. In this study, we further optimized the immunosuppression strategies for establishing chronic HEV infection in rabbits. Chronic HEV infection can also be established when CsA treatment was started at the same time or even 4 weeks after HEV challenge, clearly indicating the risk of progression to chronic infection under these circumstances and the necessity of HEV screening for both the recipient and the donor preoperatively. CsA, tacrolimus, or prednisolone instead of MMF significantly contributed to chronic HEV infection. HEV antigen in acute infection phase indicates the development of chronic infection. Our results have important implications for understanding the potential association between chronic HEV infection and immunosuppressants.
Subject(s)
Cyclosporine , Disease Models, Animal , Hepatitis E virus , Hepatitis E , Immunosuppression Therapy , Immunosuppressive Agents , Tacrolimus , Animals , Rabbits , Hepatitis E/immunology , Hepatitis E/virology , Hepatitis E/drug therapy , Hepatitis E virus/immunology , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Cyclosporine/pharmacology , Cyclosporine/therapeutic use , Tacrolimus/pharmacology , Tacrolimus/therapeutic use , Prednisolone/therapeutic use , Prednisolone/pharmacology , Male , Immunity, Innate/drug effects , Mycophenolic Acid/pharmacology , Hepatitis, Chronic/drug therapy , Hepatitis, Chronic/immunology , Hepatitis, Chronic/virology , Chronic Disease , Calcineurin Inhibitors/pharmacology , Calcineurin Inhibitors/therapeutic useABSTRACT
Sirtuin3 (SIRT3), a mitochondrial deacetylase, has been shown to be involved in various kidney diseases. In this study, we aimed to clarify the role of SIRT3 in cyclosporine-induced nephrotoxicity and the associated mitochondrial dysfunction. Madin-Darby canine kidney (MDCK) cells were transfected with Flag-tagged SIRT3 for SIRT3 overexpression or SIRT3 siRNA for the inhibition of SIRT3. Subsequently, the cells were treated with cyclosporine A (CsA) or vehicle. Wild-type and SIRT3 knockout (KO) mice were randomly assigned to receive cyclosporine A or olive oil. Furthermore, SIRT3 activator, honokiol, was treated alongside CsA to wild type mice. Our results revealed that CsA treatment inhibited mitochondrial SIRT3 expression in MDCK cells. Inhibition of SIRT3 through siRNA transfection exacerbated apoptosis, impaired the expression of the AMP-activated protein kinase-peroxisome proliferator-activated receptor gamma coactivator 1 alpha (AMPK-PGC1α) pathway, and worsened mitochondrial dysfunction induced by CsA treatment. Conversely, overexpression of SIRT3 through Flag-tagged SIRT3 transfection ameliorated apoptosis, increased the expression of mitochondrial superoxide dismutase 2, and restored the mitochondrial regulator pathway, AMPK-PGC1α. In SIRT3 KO mice, CsA treatment led to aggravated kidney dysfunction, increased kidney tubular injury, and accumulation of oxidative end products indicative of oxidative stress injury. Meanwhile, SIRT3 activation in vivo significantly mitigated these adverse effects, improving kidney function, reducing oxidative stress markers, and enhancing mitochondrial health following CsA treatment. Overall, our findings suggest that SIRT3 plays a protective role in alleviating mitochondrial dysfunction caused by CsA through the activation of the AMPK-PGC1α pathway, thereby preventing further kidney injury.
Subject(s)
Apoptosis , Cyclosporine , Mice, Knockout , Mitochondria , Oxidative Stress , Sirtuin 3 , Animals , Sirtuin 3/metabolism , Sirtuin 3/genetics , Cyclosporine/adverse effects , Cyclosporine/toxicity , Cyclosporine/pharmacology , Mitochondria/metabolism , Mitochondria/drug effects , Mice , Dogs , Apoptosis/drug effects , Oxidative Stress/drug effects , AMP-Activated Protein Kinases/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Madin Darby Canine Kidney Cells , Kidney Diseases/chemically induced , Kidney Diseases/metabolism , Kidney Diseases/prevention & control , Kidney Diseases/pathology , Kidney Diseases/genetics , Kidney/pathology , Kidney/drug effects , Kidney/metabolism , Mice, Inbred C57BL , Male , Signal Transduction/drug effectsABSTRACT
Strong precorneal clearance mechanisms including reflex blink, constant tear drainage, and rapid mucus turnover constitute great challenges for eye drops for effective drug delivery to the ocular epithelium. In this study, cyclosporine A (CsA) for the treatment of dry eye disease (DED) was selected as the model drug. Two strategies, PEGylation for mucus penetration and cationization for potent cellular uptake, were combined to construct a novel CsA nanosuspension (NS@lipid-PEG/CKC) by coating nanoscale drug particles with a mixture of lipids, DSPE-PEG2000, and a cationic surfactant, cetalkonium chloride (CKC). NS@lipid-PEG/CKC with the mean size â¼173 nm and positive zeta potential â¼+40 mV showed promoted mucus penetration, good cytocompatibility, more cellular uptake, and prolonged precorneal retention without obvious ocular irritation. More importantly, NS@lipid-PEG/CKC recovered tear production and goblet cell density more efficiently than the commercial cationic nanoemulsion on a dry eye disease rat model. All results indicated that a combination of PEGylation and cationization might provide a promising strategy to coordinate mucus penetration and cellular uptake for enhanced drug delivery to the ocular epithelium for nanomedicine-based eye drops.
Subject(s)
Cyclosporine , Dry Eye Syndromes , Phospholipids , Polyethylene Glycols , Animals , Cyclosporine/chemistry , Cyclosporine/pharmacology , Cyclosporine/pharmacokinetics , Cyclosporine/administration & dosage , Polyethylene Glycols/chemistry , Rats , Dry Eye Syndromes/drug therapy , Dry Eye Syndromes/pathology , Phospholipids/chemistry , Rats, Sprague-Dawley , Nanoparticles/chemistry , Drug Delivery Systems , Cations/chemistry , Ophthalmic Solutions/chemistry , Ophthalmic Solutions/pharmacology , Humans , Male , Cornea/metabolism , Cornea/drug effectsABSTRACT
Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 µM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.
Subject(s)
Becaplermin , Cell Proliferation , Cyclosporine , Fibroblasts , Glucuronosyltransferase , Graves Ophthalmopathy , Hyaluronan Synthases , Hyaluronic Acid , Hyaluronoglucosaminidase , Proto-Oncogene Proteins c-sis , Hyaluronic Acid/biosynthesis , Hyaluronic Acid/pharmacology , Humans , Becaplermin/metabolism , Becaplermin/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Hyaluronan Synthases/metabolism , Hyaluronan Synthases/genetics , Cyclosporine/pharmacology , Hyaluronoglucosaminidase/metabolism , Hyaluronoglucosaminidase/antagonists & inhibitors , Cell Proliferation/drug effects , Proto-Oncogene Proteins c-sis/metabolism , Glucuronosyltransferase/metabolism , Glucuronosyltransferase/genetics , Graves Ophthalmopathy/metabolism , Graves Ophthalmopathy/pathology , Graves Ophthalmopathy/drug therapy , Cells, Cultured , Orbit/metabolism , Orbit/drug effects , Orbit/pathology , RNA, Messenger/metabolism , RNA, Messenger/genetics , Cell Adhesion Molecules/metabolism , GPI-Linked ProteinsABSTRACT
Increasing shortage of donor organs leads to the acceptance of less than optimal grafts for transplantation, up to and including organs donated after circulatory standstill of the donor. Therefore, protective strategies and pharmacological interventions destined to reduce ischemia induced tissue injury are considered a worthwhile focus of research. The present study evaluates the potential of a multidrug pharmacological approach as single flush at the end of static preservation to protect the liver from reperfusion injury. Livers were retrieved from male Wistar rats 20 min after cardiac standstill. The organs were cold stored for 18 h, flushed with 20 ml of saline, kept at room temperature for 20 min, and reperfused at 37 °C with oxygenated Williams E solution. In half of the cases, the flush solution was supplemented with a cocktail containing metformin, bucladesine and cyclosporin A. Upon reperfusion, treated livers disclosed a massive mitigation of hepatic release of alanine aminotransferase and aspartate aminotransferase, along with a significant approximately 50 % reduction of radical mediated lipid peroxidation, caspase activation and release of TNF-alpha. Even after preceding cold preservation, a pharmacological cocktail given as single flush is capable to mitigate manifestations of reperfusion injury in the present model.
Subject(s)
Cyclosporine , Lipid Peroxidation , Liver , Organ Preservation , Rats, Wistar , Reperfusion Injury , Tumor Necrosis Factor-alpha , Animals , Reperfusion Injury/prevention & control , Reperfusion Injury/drug therapy , Male , Rats , Liver/drug effects , Liver/metabolism , Liver/blood supply , Organ Preservation/methods , Cyclosporine/pharmacology , Lipid Peroxidation/drug effects , Tumor Necrosis Factor-alpha/metabolism , Metformin/pharmacology , Metformin/therapeutic use , Alanine Transaminase/metabolism , Alanine Transaminase/blood , Aspartate Aminotransferases/metabolism , Rewarming/methods , Organ Preservation Solutions/pharmacologyABSTRACT
Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.
Subject(s)
Calcium , Mitochondria, Heart , Mitochondria, Liver , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Rats, Wistar , Animals , Mitochondrial Permeability Transition Pore/metabolism , Male , Calcium/metabolism , Mitochondria, Heart/metabolism , Mitochondria, Liver/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Rats , Oxygen Consumption , Liver/metabolism , Mitochondrial Swelling/drug effects , Cyclosporine/pharmacologyABSTRACT
Cyclosporin A, an immunosuppressive agent, is extensively utilized for the prevention of transplant rejection and treat autoimmune disease in the clinic, despite its association with a high risk of hypertension development among patients. Resveratrol is a kind of non-flavonoid phenolic compound that widely exists in many plants. The aim of the present study was to investigate the mechanism by which resveratrol ameliorates cyclosporin A-induced hypertension. The arterial rings of the mesentery were incubated with cyclosporin A and resveratrol in vitro. Rats were administered cyclosporin A and/or resveratrol for 3 weeks in vivo. Blood pressure was measured via the tail arteries. Vasoconstriction curves were recorded using a sensitive myograph. The protein expression was evaluated through Western blotting. This study demonstrated that resveratrol mitigated the cyclosporin A-induced increase in blood pressure in rats. Furthermore, resveratrol markedly inhibited the cyclosporin A-induced upregulation of thromboxane A2 receptor-mediated vasoconstriction in the rat mesenteric artery both in vitro and in vivo. Moreover, resveratrol activated AMPK/SIRT1 and inhibited the MAPK/NF-κB signaling pathway. In conclusion, resveratrol restored the cyclosporin A-induced upregulation of the thromboxane A2 receptor and hypertension via the AMPK/SIRT1 and MAPK/NF-κB pathways in rats.
Subject(s)
AMP-Activated Protein Kinases , Cyclosporine , Hypertension , Mesenteric Arteries , NF-kappa B , Rats, Sprague-Dawley , Resveratrol , Sirtuin 1 , Up-Regulation , Animals , Resveratrol/pharmacology , Cyclosporine/pharmacology , Sirtuin 1/metabolism , Mesenteric Arteries/drug effects , Mesenteric Arteries/metabolism , Hypertension/drug therapy , Hypertension/metabolism , Hypertension/physiopathology , Male , NF-kappa B/metabolism , Up-Regulation/drug effects , Rats , AMP-Activated Protein Kinases/metabolism , Vasoconstriction/drug effects , Blood Pressure/drug effects , Signal Transduction/drug effects , Mitogen-Activated Protein Kinases/metabolismABSTRACT
Cyclosporin A is a well-established immunosuppressive drug used to treat or prevent graft-versus-host disease, the rejection of organ transplants, autoimmune disorders, and leukemia. It exerts its immunosuppressive effects by inhibiting calcineurin-mediated dephosphorylation of the nuclear factor of activated T cells (NFAT), thus preventing its nuclear entry and suppressing T cell activation. Here we report an unexpected immunostimulatory effect of cyclosporin A in activating the mammalian target of rapamycin complex 1 (mTORC1), a crucial metabolic hub required for T cell activation. Through screening a panel of tool compounds known to regulate mTORC1 activation, we found that cyclosporin A activated mTORC1 in CD8+ T cells in a 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB/AKT)-dependent manner. Mechanistically, cyclosporin A inhibited the calcineurin-mediated AKT dephosphorylation, thereby stabilizing mTORC1 signaling. Cyclosporin A synergized with mTORC1 pathway inhibitors, leading to potent suppression of proliferation and cytokine production in CD8+ T cells and an increase in the killing of acute T cell leukemia cells. Consequently, relying solely on CsA is insufficient to achieve optimal therapeutic outcomes. It is necessary to simultaneously target both the calcineurin-NFAT pathway and the mTORC1 pathway to maximize therapeutic efficacy.
Subject(s)
CD8-Positive T-Lymphocytes , Cyclosporine , Immunosuppressive Agents , Lymphocyte Activation , Mechanistic Target of Rapamycin Complex 1 , Signal Transduction , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Cyclosporine/pharmacology , Mechanistic Target of Rapamycin Complex 1/metabolism , Animals , Immunosuppressive Agents/pharmacology , Mice , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Signal Transduction/drug effects , Proto-Oncogene Proteins c-akt/metabolism , NFATC Transcription Factors/metabolism , Humans , TOR Serine-Threonine Kinases/metabolism , Calcineurin/metabolism , Mice, Inbred C57BL , Phosphorylation/drug effects , 3-Phosphoinositide-Dependent Protein Kinases/metabolism , Cell Proliferation/drug effects , Multiprotein Complexes/metabolismABSTRACT
Dry eye disease (DED) affects a substantial worldwide population with increasing frequency. Current single-targeting DED management is severely hindered by the existence of an oxidative stress-inflammation vicious cycle and complicated intercellular crosstalk within the ocular microenvironment. Here, a nanozyme-based eye drop, namely nanoceria loading cyclosporin A (Cs@P/CeO2), is developed, which possesses long-term antioxidative and anti-inflammatory capacities due to its regenerative antioxidative activity and sustained release of cyclosporin A (CsA). In vitro studies showed that the dual-functional Cs@P/CeO2 not only inhibits cellular reactive oxygen species production, sequentially maintaining mitochondrial integrity, but also downregulates inflammatory processes and repolarizes macrophages. Moreover, using flow cytometric and single-cell sequencing data, the in vivo therapeutic effect of Cs@P/CeO2 was systemically demonstrated, which rebalances the immune-epithelial communication in the corneal microenvironment with less inflammatory macrophage polarization, restrained oxidative stress, and enhanced epithelium regeneration. Collectively, our data proved that the antioxidative and anti-inflammatory Cs@P/CeO2 may provide therapeutic insights into DED management.