Tetracyclines enhance antitumor T-cell immunity via the Zap70 signaling pathway.

BACKGROUND: Cancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we demonstrated that tetracyclines enhanced T-cell responses. Therefore, we herein investigated the efficacy of tetracyclines on antitumor T-cell responses by human peripheral T cells, murine models, and the lung tumor tissues of patients with non-small cell lung cancer (NSCLC), with a focus on signaling pathways in T cells. METHODS: The cytotoxicity of peripheral and lung tumor-infiltrated human T cells against tumor cells was assessed by using bispecific T-cell engager (BiTE) technology (BiTE-assay system). The effects of tetracyclines on T cells in the peripheral blood of healthy donors and the tumor tissues of patients with NSCLC were examined using the BiTE-assay system in comparison with anti-programmed cell death-1 (PD-1) antibody, nivolumab. T-cell signaling molecules were analyzed by flow cytometry, ELISA, and qRT-PCR. To investigate the in vivo antitumor effects of tetracyclines, tetracyclines were administered orally to BALB/c mice engrafted with murine tumor cell lines, either in the presence or absence of anti-mouse CD8 inhibitors. RESULTS: The results obtained revealed that tetracyclines enhanced antitumor T-cell cytotoxicity with the upregulation of granzyme B and increased secretion of interferon-γ in human peripheral T cells and the lung tumor tissues of patients with NSCLC. The analysis of T-cell signaling showed that CD69 in both CD4+ and CD8+ T cells was upregulated by minocycline. Downstream of T-cell receptor signaling, Zap70 phosphorylation and Nur77 were also upregulated by minocycline in the early phase after T-cell activation. These changes were not observed in T cells treated with anti-PD-1 antibodies under the same conditions. The administration of tetracyclines exhibited antitumor efficacy with the upregulation of CD69 and increases in tumor antigen-specific T cells in murine tumor models. These changes were canceled by the administration of anti-mouse CD8 inhibitors. CONCLUSIONS: In conclusion, tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. These results will contribute to the development of novel cancer immunotherapy.

Minocycline and photodynamic priming significantly improve chemotherapy efficacy in heterotypic spheroids of pancreatic ductal adenocarcinoma.

The prognosis for patients with advanced-stage pancreatic ductal adenocarcinoma (PDAC) remains dismal. It is generally accepted that combination cancer therapies offer the most promise, such as Folforinox, despite their associated high toxicity. This study addresses the issue of chemoresistance by introducing a complementary dual priming approach to attenuate the DNA repair mechanism and to improve the efficacy of a type 1 topoisomerase (Top1) inhibitor. The result is a regimen that integrates drug-repurposing and nanotechnology using 3 clinically relevant FDA-approved agents (1) Top1 inhibitor (irinotecan) at subcytotoxic doses (2) benzoporphyrin derivative (BPD) as a photoactive molecule for photodynamic priming (PDP) to improve the delivery of irinotecan within the cancer cell and (3) minocycline priming (MNP) to modulate DNA repair enzyme Tdp1 (tyrosyl-DNA phosphodiesterase) activity. We demonstrate in heterotypic 3D cancer models that incorporate cancer cells and pancreatic cancer-associated fibroblasts that simultaneous targeting of Tdp1 and Top1 were significantly more effective by employing MNP and photoactivatable multi-inhibitor liposomes encapsulating BPD and irinotecan compared to monotherapies or a cocktail of dual or triple-agents. These data are encouraging and warrant further work in appropriate animal models to evolve improved therapeutic regimens.

Gramine sensitizes Klebsiella pneumoniae to tigecycline killing.

BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters. METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect. RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1). CONCLUSION: This study provides the first evidence of gramine's therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.

Minocycline-loaded nHAP/PLGA microspheres for prevention of injury-related corneal angiogenesis.

BACKGROUND: Corneal neovascularization (CoNV) threatens vision by disrupting corneal avascularity, however, current treatments, including pharmacotherapy and surgery, are hindered by limitations in efficacy and adverse effects. Minocycline, known for its anti-inflammatory properties, could suppress CoNV but faces challenges in effective delivery due to the cornea's unique structure. Therefore, in this study a novel drug delivery system using minocycline-loaded nano-hydroxyapatite/poly (lactic-co-glycolic acid) (nHAP/PLGA) nanoparticles was developed to improve treatment outcomes for CoNV. RESULTS: Ultra-small nHAP was synthesized using high gravity technology, then encapsulated in PLGA by a double emulsion method to form nHAP/PLGA microspheres, attenuating the acidic by-products of PLGA degradation. The MINO@PLGA nanocomplex, featuring sustained release and permeation properties, demonstrated an efficient delivery system for minocycline that significantly inhibited the CoNV area in an alkali-burn model without exhibiting apparent cytotoxicity. On day 14, the in vivo microscope examination and ex vivo CD31 staining corroborated the inhibition of neovascularization, with the significantly smaller CoNV area (29.40% ± 6.55%) in the MINO@PLGA Tid group (three times daily) than that of the control group (86.81% ± 15.71%), the MINO group (72.42% ± 30.15%), and the PLGA group (86.87% ± 14.94%) (p < 0.05). Fluorescein sodium staining show MINO@PLGA treatments, administered once daily (Qd) and three times daily (Tid) demonstrated rapid corneal epithelial healing while the Alkali injury group and the DEX group showed longer healing times (p < 0.05). Additionally, compared to the control group, treatments with dexamethasone, MINO, and MINO@PLGA were associated with an increased expression of TGF-ß as evidenced by immunofluorescence, while the levels of pro-inflammatory cytokines IL-1ß and TNF-α demonstrated a significant decrease following alkali burn. Safety evaluations, including assessments of renal and hepatic biomarkers, along with H&E staining of major organs, revealed no significant cytotoxicity of the MINO@PLGA nanocomplex in vivo. CONCLUSIONS: The novel MINO@PLGA nanocomplex, comprising minocycline-loaded nHAP/PLGA microspheres, has shown a substantial capacity for preventing CoNV. This study confirms the complex's ability to downregulate inflammatory pathways, significantly reducing CoNV with minimal cytotoxicity and high biosafety in vivo. Given these findings, MINO@PLGA stands as a highly promising candidate for ocular conditions characterized by CoNV.

Minocycline induced apoptosis and suppressed expression of matrix metalloproteinases 2 and 9 in the breast cancer MCF-7 cells.

BACKGROUND: In women, breast cancer is the second most frequent type of cancer. Looking for new and effective cancer-specific therapies with little to no adverse effects on healthy cells is critical. OBJECTIVE: Minocycline, a second-generation tetracycline, has shown anticancer effects by targeting multiple pathways in various cancers. This study aimed to determine minocycline effects on the cell proliferation, apoptosis, and invasion of the human MCF-7 cells. METHODS: MTT assay was used to evaluate the cytotoxicity of minocycline on the cells. Flow cytometry was performed to investigate the induction of apoptosis and the cell cycle progression. The expression levels of apoptotic and migration proteins and genes were assessed by western blotting and qRT-PCR. The scratch test was performed to evaluate the anti-migration effect of the drug. RESULTS: The results indicated that the IC50 value of minocycline for MCF-7 cells was 36.10 µM. Minocycline treatment caused sub-G1 cell accumulation, indicating a significant apoptotic effect on the MCF-7 cells. Annexin-V/PI staining revealed a significant rise in early and late apoptotic cell percentages. Minocycline up-regulated Bax and Caspase-3 expression and down-regulated Bcl-2 and Pro-Cas3. The scratch test revealed significant anti-migration effects for minocycline. Furthermore, it caused down-regulation of MMP-2 and MMP-9 in a concentration-dependent method. CONCLUSION: These findings further confirmed the anticancer effect of minocycline and highlighted that minocycline maybe considered as potential therapeutic agent for breast cancer treatment.

In vitro evaluation of BMSCs early proliferation on minocycline-loaded electrospun nanofibers membrane.

BACKGROUND: Electrospun nanofibers could simulate the natural extracellular matrix (ECM) of the host bone, while minocycline (MINO) is a broad-spectrum tetracycline antibiotic which has been found to have multiple non-antibiotics biological effects that promotes osteogenesis in vitro and in vivo. OBJECTIVE: The present study aims at constructing a polylactic acid (PLA) electrospun nanofiber membrane loaded with MINO to enhance Bone marrow mesenchymal stem cells (BMSCs) adhesion and proliferation for early clinical treatment. METHODS: The MINO-PLA membrane were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR) and in vitro drug release study. The antibacterial ability was also investigated. In addition, in vitro cellular proliferation experiment was performed to verify whether the PLA electrospun nanofibers membrane loaded with MINO enhance BMSCs adhesion and proliferation. RESULTS: Analyzing the drug release and cell growth results, it was found that only the effective concentration of MINO-PLA could help the growth of BMSCs in the short term. This is related to the drug release rate of MINO-PLA and the initial concentration of MINO. CONCLUSION: This study shows that by controlling the concentration and release rate of MINO with electrospinning PLA, BMSCs could proliferate on it, and a new bone repair material had been made in this study.

Minocycline mitigates tau pathology via modulating the TLR-4/NF-Ðºß signalling pathway in the hippocampus of neuroinflammation rat model.

INTRODUCTION: The neuroinflammatory response was seen to impact the formation of phosphorylated tau protein in Alzheimer's disease (AD). This study aims to investigate the molecular mechanism of minocycline in reducing phosphorylated tau protein formation in the hippocampus of lipopolysaccharide (LPS)-induced rats. METHODS: Fifty adult male Sprague Dawley (SD) rats were randomly allocated to 1 of 5 groups: control, LPS (5 mg/kg), LPS + minocycline (25 mg/kg), LPS + minocycline (50 mg/kg) and LPS + memantine (10 mg/kg). Minocycline and memantine were administered intraperitoneally (i.p) for two weeks, and LPS was injected i.p. once on day 5. ELISA was used to determine the level of phosphorylated tau protein in SD rats' hippocampal tissue. The density and expression of Toll-like receptor-4 (TLR-4), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кß), tumour necrosis factor-alpha (TNF-α), and cyclooxygenase (COX)-2 were determined using Western blot and immunohistochemistry. RESULTS: Minocycline, like memantine, prevented LPS-induced increasein phosphorylated tau protein level suggested via reduced density and expression of TLR-4, NF-кß, TNF-αand COX-2 proteins in rat hippocampal tissue. Interestingly, higher doses were shown to be more neuroprotective than lower doses. CONCLUSION: This study suggests that minocycline suppresses the neuroinflammation signalling pathway and decreased phosphorylated tau protein formation induced by LPS in a dose-dependent manner. Minocycline can be used as a preventative and therapeutic drug for neuroinflammatory diseases such as AD.

Tetracycline antibiotics: Potential anticancer drugs.

In recent years, research on tetracycline antibiotics has gradually shifted from their antibacterial effects to anticancer effects. Doxycycline, minocycline, and tigecycline as the US Food and Drug Administration (FDA) approved tetracycline antibiotics have been the main subjects of studies. Evidence indicated that they have anticancer properties and are able to control cancer progression through different mechanisms, such as anti-proliferation, anti-metastasis, and promotion of autophagy or apoptosis. In addition, studies have shown that these three tetracycline antibiotics can be utilized in conjunction with chemotherapeutic and targeted drugs to inhibit cancer progression and improve the quality of patient survival. Therefore, doxycycline, minocycline, and tigecycline are taken as examples in this work. Their mechanisms of action in different cancers and related combination therapies are introduced. Their current roles in alleviating the suffering of patients undergoing chemotherapy when used as adjuvant drugs in clinical treatment are also described. Finally, the research gaps and potential research directions at this stage are briefly summarized.

Minocycline attenuates hydrocephalus and inhibits iron accumulation, ependymal damage and epiplexus cell activation after intraventricular hemorrhage in aged rats.

Intracerebral hemorrhage is primarily a disease of the elderly and it is frequently accompanied by intraventricular hemorrhage (IVH) which can lead to posthemorrhagic hydrocephalus and poor prognosis. Red blood cell iron has been implicated in brain injury after cerebral hemorrhage. The current study examined using T2* magnetic resonance imaging (MRI) to detect periventricular iron deposition after IVH and investigated the effects of minocycline on hydrocephalus in an aged rat IVH model. It had three parts. In part 1, male aged rats received a 200 µl injection of saline or autologous blood into the lateral ventricle and were euthanized at day 14. In parts 2 and 3, aged IVH rats were treated with vehicle or minocycline and euthanized at day 7 or 14. Rats underwent MRI to quantify hydrocephalus and iron deposition followed by brain histology and immunohistochemistry. Periventricular iron overload was found after IVH using T2* MRI and confirmed by histology. IVH also caused ventricular wall damage and increased the number of CD68(+) choroid plexus epiplexus cells. Minocycline administration reduced iron deposition and ventricular volume at days 7 and 14 after IVH, as well as ventricle wall damage and epiplexus cell activation. In summary, IVH-induced hydrocephalus is associated with periventricular iron deposition, ependymal damage and choroid plexus epiplexus cell activation in aged rats. Minocycline attenuated those effects and might be a potential treatment for posthemorrhagic hydrocephalus in the elderly.

Minocycline and antipsychotics inhibit inflammatory responses in BV-2 microglia activated by LPS via regulating the MAPKs/ JAK-STAT signaling pathway.

BACKGROUND: Abnormal activation of microglia is involved in the pathogenesis of schizophrenia. Minocycline and antipsychotics have been reported to be effective in inhibiting the activation of microglia and thus alleviating the negative symptoms of patients with schizophrenia. However, the specific molecular mechanism by which minocycline and antipsychotics inhibit microglial activation is not clear. In this study, we aimed to explore the molecular mechanism of treatment effect of minocycline and antipsychotics on schizophrenia. METHODS: Microglia cells were activated by lipopolysaccharide (LPS) and further treated with minocycline, haloperidol, and risperidone. Then cell morphology, specific marker, cytokines, and nitric oxide production process, and the proteins in related molecular signaling pathways in LPS-activated microglia were compared among groups. RESULTS: The study found that minocycline, risperidone, and haloperidol significantly inhibited morphological changes and reduced the expression of OX-42 protein induced by LPS. Minocycline significantly decreased the production of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1ß). Risperidone also showed significant decrease in the production of IL-6 and TNF-α, while haloperidol only showed significant decrease in the production of IL-6. Minocycline, risperidone, and haloperidol were found to significantly inhibit nitric oxide (NO) expression, but had no effect on inducible nitric oxide synthase (iNOS) expression. Both minocycline and risperidone were effective in decreasing the activity of c­Jun N­terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinases (MAPKs) signal pathway. Additionally, minocycline and risperidone were found to increase the activity of phosphorylated-p38. In contrast, haloperidol only suppressed the activity of ERK. Minocycline also suppressed the activation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), while risperidone and haloperidol only suppressed the activation of STAT3. CONCLUSIONS: The results demonstrated that minocycline and risperidone exert stronger anti-inflammatory and neuroprotective effects stronger than haloperidol, through MAPKs and Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathways in BV2 cells stimulated with LPS, revealing the underlying mechanisms of minocycline and atypical antipsychotics in the treatment of negative schizophrenia symptoms.

After Ischemic Stroke, Minocycline Promotes a Protective Response in Neurons via the RNA-Binding Protein HuR, with a Positive Impact on Motor Performance.

Ischemic stroke is the most common cause of adult disability and one of the leading causes of death worldwide, with a serious socio-economic impact. In the present work, we used a new thromboembolic model, recently developed in our lab, to induce focal cerebral ischemic (FCI) stroke in rats without reperfusion. We analyzed selected proteins implicated in the inflammatory response (such as the RNA-binding protein HuR, TNFα, and HSP70) via immunohistochemistry and western blotting techniques. The main goal of the study was to evaluate the beneficial effects of a single administration of minocycline at a low dose (1 mg/kg intravenously administered 10 min after FCI) on the neurons localized in the penumbra area after an ischemic stroke. Furthermore, given the importance of understanding the crosstalk between molecular parameters and motor functions following FCI, motor tests were also performed, such as the Horizontal Runway Elevated test, CatWalk™ XT, and Grip Strength test. Our results indicate that a single administration of a low dose of minocycline increased the viability of neurons and reduced the neurodegeneration caused by ischemia, resulting in a significant reduction in the infarct volume. At the molecular level, minocycline resulted in a reduction in TNFα content coupled with an increase in the levels of both HSP70 and HuR proteins in the penumbra area. Considering that both HSP70 and TNF-α transcripts are targeted by HuR, the obtained results suggest that, following FCI, this RNA-binding protein promotes a protective response by shifting its binding towards HSP70 instead of TNF-α. Most importantly, motor tests showed that reduced inflammation in the brain damaged area after minocycline treatment directly translated into a better motor performance, which is a fundamental outcome when searching for new therapeutic options for clinical practice.

Minocycline induces tolerance to dendritic cell production probably by targeting the SOCS1/ TLR4/NF-κB signaling pathway.

OBJECTIVE: Dendritic cells (DCs) are professional antigen-presenting cells that play a key role in maintaining peripheral immune tolerance. The use of tolerogenic DCs (tolDCs), i.e., semi-mature DCs that express co-stimulatory molecules but not pro-inflammatory cytokines, has been proposed. However, the mechanism of tolDCs induced by minocycline is still unclear. Our previous bioinformatics analyses based on multiple databases suggested that the suppressor of cytokine signaling 1/Toll-like receptor 4/NF-κB (SOCS1/TLR4/NF-κB) signal pathway was associated with DCs maturation. Thus, we studied whether minocycline could induce DC tolerance through this pathway. METHODS: A search for potential targets was carried out through public databases, and pathway analysis was performed on these potential targets to obtain pathways relevant to the experiment. Flow cytometry was used to detect the expression of DC surface markers CD11c, CD86, and CD80, and major histocompatibility complex II. The secretion of interleukin (IL)-12p70, tumor necrosis factor alpha (TNF- α), and IL-10 in the DC supernatant was detected by enzyme-linked immunoassay. The ability of three groups (Ctrl-DCs, Mino-DCs, and LPS-DCs) of DCs to stimulate allogeneic CD4+ T cells was analyzed using a mixed lymphocyte reaction assay. Western blotting was used to detect the expression of TLR4, NF-κB-p65, NF-κB-p-p65, IκB-α, and SOCS1 proteins. RESULTS: The hub gene plays a vital role in biological processes; in related pathways, the regulation of other genes is often affected by it. The SOCS1/TLR4/NF-κB signaling pathway was further validated by searching for potential targets through public databases to obtain relevant pathways. The minocycline-induced tolDCs showed characteristics of semi-mature DCs. Moreover, the IL-12p70 and TNF-α levels in the minocycline-stimulated DC group (Mino-DC group) were lower than those in the lipopolysaccharide (LPS)-DC group, and the IL-10 levels were higher in the Mino-DC group than in the LPS-DC and control DC groups. In addition, the Mino-DC group had decreased protein expression levels of TLR4 and NF-κB-p65 and upregulated protein levels of NF-κB-p-p65, IκB-α, and SOCS1 compared with the other groups. CONCLUSION: The results of this study indicate that minocycline could improve the tolerance of DCs probably by blocking the SOCS1/TLR4/NF-κB signaling pathway.

The interaction between kynurenine pathway, suicidal ideation and augmentation therapy with minocycline in patients with treatment-resistant depression.

BACKGROUND AND AIMS: We investigated kynurenine pathway (KP) metabolites levels and their association with suicidal ideation in patients with treatment-resistant depression (TRD) and elevated peripheral inflammation. The effect of antidepressant augmentation with minocycline on KP metabolites was tested. METHODS: We analysed data from MINocycline in DEPression, a 4-week, randomized, placebo controlled (1:1) trial of minocycline added to antidepressant treatment in 39 TRD patients (n = 18 minocycline; n = 21 placebo) with C-reactive protein (CRP) ⩾1 mg/L. At baseline and at week 4, we collected data on suicidality (Beck Depression Inventory) and blood samples to measure inflammatory markers and KP metabolites. We tested (1) the association of KP metabolites ratios with inflammatory markers and suicidal ideation at baseline and (2) the role of suicidality and treatment (minocycline vs placebo) in affecting KP changes over time. RESULTS: At baseline, kynurenine/tryptophan (KYN/TRP) ratio positively correlated with high-sensitivity CRP (Spearman's ρ = 0.35, p = 0.02) and IL-10, (ρ = 0.41, p = 0.009); and tumour necrosis factor was positively correlated with quinolinic acid/3-hydroxykynurenine ratio (ρ = 0.55, p < 0.001). Moreover, participants with suicidal ideation showed higher levels of KYN/TRP (U = 143.000, p = 0.02) than those without suicidal ideation. There was no significant effect of minocycline on KP metabolites changes from baseline to week 4. However, in the minocycline group, the number of participants with suicidal thoughts decreased from 44.4% (8/18) to 22.2% (4/18). CONCLUSION: Increased KP neurotoxic metabolites are associated with elevated peripheral inflammation in depressed individuals, particularly in those with suicidal ideation. Targeting KP in this population could be a potential effective personalized approach. Whether this includes minocycline should be investigated in future larger trials.

Therapeutic effects of minocycline on oleic acid-induced acute respiratory distress syndrome (ARDS) in rats.

Acute respiratory distress syndrome (ARDS) is a serious intensive care condition. Despite advances in treatment over the previous few decades, ARDS patients still have high fatality rates. Thus, more research is needed to improve the outcomes for people with ARDS. Minocycline is an antibiotic with antioxidant, anti-inflammatory, and anti-apoptotic effects. In the current investigation, the therapeutic effects of minocycline on oleic acid-induced ARDS were evaluated. Male rats were classified into 6 groups, 1. control (normal saline), 2. oleic acid (100 µL, i.v.), 3-5. oleic acid + minocycline (50, 100, 200 mg/kg, i.p.), and 6. minocycline (200 mg/kg, i.p.) alone. Twenty-four hours after the oleic acid injection, the lung tissue is isolated, weighed, and the middle part of the right lung is immediately placed in the freezer, while the middle part of the left lung is placed in formalin and sent to the laboratory for pathology testing. Then, the amounts of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), cytokines (interleukin-1 beta (IL-1ß), tumor necrosis factor-α (TNF-α)), B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), and cleaved caspase-3 were determined in lung tissue. Administration of oleic acid increased emphysema, inflammation, vascular congestion, hemorrhage, MDA amount, Bax/Bcl-2 ratio, cleaved caspase-3, IL-1ß, TNF-α levels, and decreased GSH, SOD, and CAT levels in comparison with the control group. The administration of minocycline could significantly reduce pathological and biochemical alterations induced by oleic acid. Minocycline has a therapeutic effect on oleic acid-induced ARDS through antioxidant, anti-inflammatory, and anti-apoptotic properties.

Minocycline protects against microgliopathy in a Csf1r haplo-insufficient mouse model of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

BACKGROUND: Mutations in colony-stimulating factor 1 receptor (CSF1R) are known to cause adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), which has been recently demonstrated as a primary microgliopathy characterized by cognitive impairment. Although the molecular mechanism underlying CSF1R-mediated microgliopathy remains unclear, therapeutic strategies have generally targeted modulation of microglial function. In particular, the microglial inhibitor, minocycline, has been shown to attenuate learning and memory deficits in several neurodegenerative diseases. The objectives of this study were to investigate the pathogenic mechanisms underlying ALSP and to explore the therapeutic effects of minocycline in an in vivo model of ALSP. We hypothesized that inhibiting microglial activation via minocycline could reverse the behavior and pathological defects in ALSP model mice. METHODS: We generated a Csf1r haploinsufficiency mouse model of ALSP using CRISPR/Cas9 genome editing and conducted electrophysiological recordings of long-term potentiation (LTP) and behavioral tests to validate the recapitulation of clinical ALSP characteristics in 8- to 11-month-old mice. RNA-sequencing was used to explore enriched gene expression in the molecular pathogenesis of ALSP. Microglial activation was assessed by immunofluorescent detection of Iba1 and CD68 in brain sections of male ALSP mice and pro-inflammatory activation and phagocytosis were assessed in Csf1r+/- microglia. Therapeutic effects were assessed by behavioral tests, histological analysis, and morphological examination after four weeks of intraperitoneal injection with minocycline or vehicle control in Csf1r+/- mice and wild-type control littermates. RESULTS: We found that synaptic function was reduced in LTP recordings of neurons in the hippocampal CA1 region, while behavioral tests showed impaired spatial and cognitive memory specifically in male Csf1r+/- mice. Increased activation, pro-inflammatory cytokine production, and enhanced phagocytic capacity were also observed in Csf1r+/- microglia. Treatment with minocycline could suppress the activation of Csf1r+/- microglia both in vitro and in vivo. Notably, the behavioral and pathological deficits in Csf1r+/- mice were partially rescued by minocycline administration, potentially due to inhibition of microglial inflammation and phagocytosis in Csf1r+/- mice. CONCLUSIONS: Our study shows that CSF1R deficiency results in aberrant microglial activation, characterized by a pro-inflammatory phenotype and enhanced phagocytosis of myelin. Our results also indicate that microglial inhibition by minocycline can ameliorate behavioral impairment and ALSP pathogenesis in CSF1R-deficient male mice, suggesting a potential therapeutic target for CSF1R-related leukoencephalopathy. Collectively, these data support that minocycline confers protective effects against CSF1R-related microgliopathy in male ALSP model mice.

Minocycline protects against neuronal mitochondrial dysfunction and cognition impairment.

The potential of minocycline to protect against methylphenidate­induced neurodegeneration has been extensively reported in the literature but the mechanism of action is still unknown. This study aims to determine the role of mitochondrial chain enzymes and redox homeostasis on the neuroprotective effects of minocycline in methylphenidate­induced neurodegeneration. Wistar adult male rats were randomly assigned to the seven experimental groups: Group 1 received saline solution; Group 2 received methylphenidate (10 mg/kg, i.p.); Groups 3, 4, 5, and 6 received methylphenidate and minocycline for 21 days; Group 7 received minocycline alone. Cognition was evaluated with the Morris water maze test. Activity of the hippocampal mitochondrial quadruple complexes I, II, III and IV, mitochondrial membrane potential, adenosine triphosphate (ATP) levels, total antioxidant capacity, and reactive oxygen species were determined. Treatment with minocycline inhibited methylphenidate­induced cognitive dysfunction. Minocycline treatment increased mitochondrial quadruple complex activities, mitochondrial membrane potential, total antioxidant capacity, and ATP levels in the dentate gyrus and cornu ammonis­1 (CA1) areas of the hippocampus. Minocycline is likely to confer neuroprotection against methylphenidate­induced neurodegeneration and cognition impairment by regulating mitochondrial activity and oxidative stress.

Minocycline pre-treatment up-regulates antioxidant enzymes and enhances the regenerative potential of MSCs in rat myocardial infarction model.

Objectives: To determine the effect of the pre-treatment of mesenchymal stem cells (MSCs) with minocycline on the expression of antioxidant genes and cardiac repair post myocardial infarction (MI) in rats. METHODS: Rat bone marrow derived MSCs were used in the study. Cytotoxicity of minocycline in MSCs was determined using JC1 assay to identify a safe drug dose for further experiments. The MSCs were pre-treated with 1.0 µM minocycline for 24 hours and then treated with hydrogen peroxide (H2O2), after that mRNA was isolated and the expression levels of antioxidant genes including peroxiredoxin, glutathione peroxidase, and superoxide dismutase were determined. Finally, minocycline pre-treated MSCs were used to treat rats induced with MI by the ligation of left anterior descending coronary artery. The cardiac function was evaluated at two and four weeks post MI using echocardiography. RESULTS: At 1.0 µM concentration, minocycline was found to be safe for MSCs and used for subsequent experiments. Minocycline pre-treatment was found to up regulate several antioxidant genes in oxidatively stressed MSCs. Furthermore, minocycline pre-treated MSCs displayed greater improvement in cardiac left ventricular function at two and four-weeks post MI as compared to untreated rats. CONCLUSIONS: Pre-treatment of MSCs with minocycline enhances the expression of antioxidant genes and promotes their capability to repair cardiac function after MI.

Microglial voltage-dependent anion channel 1 signaling modulates sleep deprivation-induced transition to chronic postsurgical pain.

STUDY OBJECTIVES: This study verified that sleep deprivation before and after skin/muscle incision and retraction (SMIR) surgery increased the risk of chronic pain and investigated the underlying roles of microglial voltage-dependent anion channel 1 (VDAC1) signaling. METHODS: Adult mice received 6 hours of total sleep deprivation from 1 day prior to SMIR until the third day after surgery. Mechanical and heat-evoked pain was assessed before and within 21 days after surgery. Microglial activation and changes in VDAC1 expression and oligomerization were measured. Minocycline was injected to observe the effects of inhibiting microglial activation on pain maintenance. The VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and oligomerization inhibitor VBIT-4 were used to determine the roles of VDAC1 signaling on microglial adenosine 5' triphosphate (ATP) release, inflammation (IL-1ß and CCL2), and chronicity of pain. RESULTS: Sleep deprivation significantly increased the pain duration after SMIR surgery, activated microglia, and enhanced VDAC1 signaling in the spinal cord. Minocycline inhibited microglial activation and alleviated sleep deprivation-induced pain maintenance. Lipopolysaccharide (LPS)-induced microglial activation was accompanied by increased VDAC1 expression and oligomerization, and more VDAC1 was observed on the cell membrane surface compared with control. DIDS and VBIT-4 rescued LPS-induced microglial ATP release and IL-1ß and CCL2 expression. DIDS and VBIT-4 reversed sleep loss-induced microglial activation and pain chronicity in mice, similar to the effects of minocycline. No synergistic effects were found for minocycline plus VBIT-4 or DIDS. CONCLUSIONS: Perioperative sleep deprivation activated spinal microglia and increases the risk of chronic postsurgical pain in mice. VDAC1 signaling regulates microglial activation-related ATP release, inflammation, and chronicity of pain.

Microglia participate in postoperative cognitive dysfunction by mediating the loss of inhibitory synapse through the complement pathway.

BACKGROUND: Elderly patients after surgery are prone to cognitive decline known as postoperative cognitive dysfunction (POCD). Several studies have shown that the microglial activation and the increase of complement protein expression in hippocampus induced by surgery may be related to the pathogenesis of POCD. The purpose of this study was to determine whether microglia and complement system were involved in cognitive dysfunction in aged mice. METHODS: The POCD model was established by exploratory laparotomy in 15-month-old male C57BL/6J mice and animal behavioral tests were performed to test hippocampal-dependent memory capacity. Minocycline was used to suppress the activation of microglia, and complement 3 receptor inhibitor was used to suppress the association between microglia and complement 3. Western blot and immunofluorescence were used to detect the microglial activation, complement protein, and synaptic protein expressions. RESULTS: Operation induced hippocampal-dependent memory impairment (P < 0.01), which was accompanied by microglial activation (P < 0.01). There was also a significant reduction in inhibitory synaptic protein expression in the hippocampus of mice in the surgery group (P < 0.01). However, minocycline, a microglia inhibitor, rescued all the above changes. In addition, C3RI intervention inhibited the phagocytosis of inhibitory synapses by microglia (P < 0.05) and improved the cognitive function of mice (P < 0.01). CONCLUSION: Microglia participate in postoperative cognitive dysfunction by mediating inhibitory synaptic loss through the complement pathway.

Minocycline Protects Against Lipopolysaccharide-Induced Cognitive Impairment and Oxidative Stress: Possible Role of the CREB-BDNF Signaling Pathway.

The oxidative stress-induced dysregulation of the cyclic AMP response element-binding protein- brain-derived neurotrophic factor (CREB-BDNF) cascade has been linked to cognitive impairment in several studies. This study aimed to investigate the effect of minocycline on the levels of oxidative stress markers, CREB, and BDNF in lipopolysaccharide (LPS)-induced cognitive impairment. Fifty adult male Sprague Dawley rats were divided randomly into five groups. Group 1 was an untreated control group. Groups 2, 3, 4 and 5 were treated concurrently with LPS (5 mg/kg, i.p) once on day 5 and normal saline (0.7 ml/rat, i.p) or minocycline (25 and 50 mg/kg, i.p) or memantine (10 mg/kg, i.p) once daily from day 1 until day 14, respectively. From day 15 to day 22 of the experiment, Morris Water Maze (MWM) was used to evaluate learning and reference memory in rats. The levels of protein carbonyl (PCO), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were determined by enzyme-linked immunosorbent assay (ELISA). CREB and BDNF expression and density were measured by immunohistochemistry and western blot analysis, respectively. LPS administration significantly increased escape latency to the hidden platform with decreased travelled distance, swimming speed, target crossings and time spent in the target quadrant. Besides, the hippocampal tissue of LPS rats showed increased levels of PCO and MDA, decreased levels of CAT and SOD, and reduced expression and density of BDNF and CREB. Treatment with minocycline reversed these effects in a dose-dependent manner, comparable to the effects of memantine. Both doses of minocycline treatment protect against LPS-induced cognitive impairment by reducing oxidative stress and upregulating the CREB-BDNF signalling pathway in the rat hippocampus.