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.

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.

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 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.

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.

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.

The immunomodulatory effect of minocycline on gene expression of inflammation related cytokines in lipopolysaccharide-treated human peripheral blood mononuclear cells.

To evaluate the immunomodulatory effect of minocycline, the present study was carried out on the gene expression of toll-like receptor type-4 (TLR4) and some pro-inflammatory (IL-1ß, IL-6) and anti-inflammatory cytokines (IL-10) associated with lipopolysaccharide (LPS) -induced inflammation in human peripheral blood mononuclear cells (PBMCs). The PBMCs were collected and then 5.4 × 106 PBMCs/mL were used in eight groups as follows: control group (only media), LPS group (only LPS), methylprednisolone (Pred) group (LPS plus Pred), meloxicam (Melo) group (LPS plus Melo), three minocycline groups [M1, M5 and M25] (LPS plus 1, 5, and 25 µg/mL minocycline, respectively) and minocycline control (MC) group (5 µg/mL minocycline). After incubation for 24 h, the PBMCs were subjected to quantitative PCR assays. Gene expression levels of TLR4 were not changed in any groups. The IL-1ß levels were increased in the LPS group but the increases were much more intense in the other groups except Pred group. Compared with control group, IL-6 levels increased significantly in Melo, M1 and M25 groups. Significant increases of IL-10 levels were also observed in Melo, M25 and MC groups. It can be concluded that minocycline had dual pro- and anti-inflammatory activities with potential clinical immunomodulatory effects.

Characterization of Astrocytes in the Minocycline-Administered Mouse Photothrombotic Ischemic Stroke Model.

Astrocytes, together with microglia, play important roles in the non-infectious inflammation and scar formation at the brain infarct during ischemic stroke. After ischemia occurs, these become highly reactive, accumulate at the infarction, and release various inflammatory signaling molecules. The regulation of astrocyte reactivity and function surrounding the infarction largely depends on intercellular communication with microglia. However, the mechanisms involved remain unclear. Furthermore, recent molecular biological studies have revealed that astrocytes are highly divergent under both resting and reactive states, whereas it has not been well reported how the communication between microglia and astrocytes affects astrocyte divergency during ischemic stroke. Minocycline, an antibiotic that reduces microglial activity, has been used to examine the functional roles of microglia in mice. In this study, we used a mouse photothrombotic ischemic stroke model to examine the characteristics of astrocytes after the administration of minocycline during ischemic stroke. Minocycline increased astrocyte reactivity and affected the localization of astrocytes in the penumbra region. Molecular characterization revealed that the induced expression of mRNA encoding the fatty acid binding protein 7 (FABP7) by photothrombosis was enhanced by the minocycline administration. Meanwhile, minocycline did not significantly affect the phenotype or class of astrocytes. The expression of Fabp7 mRNA was well correlated with that of tumor-necrosis factor α (TNFα)-encoding Tnf mRNA, indicating that a correlated expression of FABP7 from astrocytes and TNFα is suppressed by microglial activity.

Effects of Minocycline on Cognitive Impairment, Hippocampal Inflammatory Response, and Hippocampal Alzheimer's Related Proteins in Aged Rats after Propofol Anesthesia.

The aim of this study was to evaluate the effect of minocycline preadministration on cognitive dysfunction, hippocampal inflammatory response, and hippocampal senile dementia-related proteins induced by propofol anesthesia in aged rats. Sixty male SD rats, aged 20 months and weighing 340-410 g, were randomly divided into three groups: normal saline (NC) group, propofol group (prop), and minocycline (M) group. Prop group rats were injected intraperitoneally with 100 mg/kg propofol. The rats in group M were injected intraperitoneally with 50 mg/kg minocycline 30 minutes before injection of 100 mg/kg propofol, and the rest were the same as prop group. The rats in NC group were received intraperitoneal injection of the same amount of normal saline. The results indicated that compared with group C, the expressions of GSK-3ß, acetyl-NF-κB (Lys310), Tau, and Amlyoid-beta were upregulated, the levels of TNF-α, IL-1ß, and IL-6 were increased, the escape incubation period was prolonged, and the exploration time was shortened in prop group, while the expression of GSK-3ß, acetyl-NF-κB (Lys310), Tau, and Amlyoid-beta in minocycline group was downregulated, the levels of TNF-α, IL-1ß, and IL-6 were decreased, the escape incubation period was shortened, and the exploration time was shortened. In conclusion, preadministration of minocycline can improve cognitive impairment induced by propofol anesthesia in aged rats, and its mechanism of action may be related to minocycline inhibiting hippocampal inflammatory reaction and downregulating the expression of GSK-3ß, acetyl-NF-κB (Lys310), Tau, and Amlyoid-beta proteins in hippocampus.

Minocycline Pretreatment Prevents Blood-Brain Barrier Disruption in Septic Rats.

INTRODUCTION: The aim of the study was to explore the mechanism by which minocycline protects the blood-brain barrier (BBB) in septic rats. METHODS: A sepsis rat model was generated in healthy, male Sprague-Dawley rats by cecal ligation and puncture (CLP). The rats were randomly divided into four groups and treated as follows: sham-operated plus normal saline (Sham + S group), CLP plus normal saline (CLP + S group), CLP plus minocycline pretreatment (CLP + M1 group), and CLP plus minocycline treatment (CLP + M2 group). Rats in the CLP + M1 group received 45 mg/kg minocycline by intraperitoneal injection every 12 h for 72 h. Rats in the Sham + S and CLP + S groups were injected with the same volume of normal saline every 12 h for 72 h. Rats in the CLP + M2 group were intraperitoneally injected with 45 mg/kg minocycline immediately after CLP and once every 12 h for 72 h. All rats were sacrificed at 72 h after operation. Tumor necrosis factor α and interleukin 6 levels, the expression of ionized calcium-binding adaptor molecule-1 (Iba-1), and the permeability of the BBB were measured. The expression of matrix metalloproteinases-9 (MMP-9) and the tight junction proteins zonula occludens-1 (ZO-1) and occludin was detected by Western blot. In addition, Evans blue (EB) staining, immunohistochemistry, and ELISA analysis were carried out. RESULTS: Minocycline pretreatment significantly inhibited microglial activation, decreased the sepsis-induced expression of MMP-9, increased the expression of ZO-1 and occludin, and improved the permeability of the BBB. Minocycline treatment failed to inhibit microglial activation, decrease the sepsis-induced expression of MMP-9, increase the expression of ZO-1 or occluding, or improve the permeability of the BBB. CONCLUSIONS: Minocycline pretreatment can effectively improve the altered permeability of the BBB caused by sepsis. The mechanism may be related to the inhibition of microglial activation and MMP-9 expression and increased expression of ZO-1 and occludin.

Neuroprotective effects of minocycline and KML29, a potent inhibitor of monoacylglycerol lipase, in an experimental stroke model: a small-animal positron emission tomography study.

Hypoxia caused by ischemia induces acidosis and neuroexcitotoxicity, resulting in neuronal death in the central nervous system (CNS). Monoacylglycerol lipase (MAGL) is a modulator of 2-arachidonoylglycerol (2-AG), which is involved in retrograde inhibition of glutamate release in the endocannabinoid system. In the present study, we used positron emission tomography (PET) to monitor MAGL-positive neurons and neuroinflammation in the brains of ischemic rats. Additionally, we performed PET imaging to evaluate the neuroprotective effects of an MAGL inhibitor in an ischemic injury model. Methods: Ischemic-injury rat models were induced by intraluminal right middle cerebral artery occlusion (MCAO). PET studies of the brains of the ischemic rats were performed at several experimental time points (pre-occlusion, days 2, 4, and 7 after the MCAO surgery) using [11C]SAR127303 for MAGL and [18F]FEBMP for 18 kDa translocator protein (TSPO, a hall-mark of neuroinflammation). Medication using minocycline (a well-known neuroprotective agent) or KML29 (a potent MAGL inhibitor) was given immediately after the MCAO surgery and then daily over the subsequent three days. Results: PET imaging of the ischemic rats using [11C]SAR127303 showed an acute decline of radioactive accumulation in the ipsilateral side at two days after MCAO surgery (ratio of the area under the curve between the ipsilateral and contralateral sides: 0.49 ± 0.04 in the cortex and 0.73 ± 0.02 in the striatum). PET imaging with [18F]FEBMP, however, showed a moderate increase in accumulation of radioactivity in the ipsilateral hemisphere on day 2 (1.36 ± 0.11), and further increases on day 4 (1.72 ± 0.15) and day 7 (1.99 ± 0.06). Treatment with minocycline or KML29 eased the decline in radioactive accumulation of [11C]SAR127303 for MAGL (minocycline-treated group: 0.82 ± 0.06 in the cortex and 0.81 ± 0.05 in the striatum; KML29-treated group: 0.72 ± 0.07 in the cortex and 0.88 ± 0.04 in the striatum) and increased uptake of [18F]FEBMP for TSPO (minocycline-treated group: 1.52 ± 0.21 in the cortex and 1.56 ± 0.11 in the striatum; KML29-treated group: 1.63 ± 0.09 in the cortex and 1.50 ± 0.17 in the striatum). In MCAO rats, minocycline treatment showed a neuroprotective effect in the sensorimotor cortex suffering from severe hypoxic injury, whereas KML29 treatment saved neurons in the striatum, including bundles of myelinated axons. Conclusions: PET imaging allowed visualization of the different neuroprotective effects of minocycline and KML29, and indicated that combination pharmacotherapy using these drugs may be an effective therapy in acute ischemia.

Minocycline prevents depression-like behavior in streptozotocin-induced diabetic mice.

Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia. Diabetic patients are known to have a higher prevalence and a higher risk of depression compared with the general population. The pathogenesis of diabetes-related depression is unclear, and the treatment is not well-established. Therefore, the prevention of diabetes-related depression is important for improving the quality of life of diabetic patients. Minocycline, a second-generation tetracycline antibiotic, has recently gained attention as a new agent for depression. In this study, we investigated the effect of minocycline on diabetes-related depression in a streptozotocin-induced mouse model of diabetes. Eight-week-old male C57BL/6 mice were injected with streptozotocin (200 mg/kg, i.p.). Seven days after injection, the mice received minocycline treatment through drinking water. We compared these mice with vehicle-treated control mice and diabetic mice not receiving minocycline treatment. On day 34, depression-like behavior was investigated using the forced swim test. On the following day, brain samples were collected, and formalin-fixed, paraffin-embedded specimens were prepared for immunohistochemistry. Compared with the control group, the diabetic mice not receiving minocycline treatment showed a prolonged duration of immobility in the forced swim test, the observation being interpreted as a depression-like behavior. Immunohistochemistry revealed an increase in microglia with an activated morphology in the diabetic mice without minocycline treatment. The expression of tumor necrosis factor alpha in microglia was increased. In addition, a decrease in the number of doublecortin-positive immature neurons was found in the hippocampus of diabetic mice. Minocycline treatment of diabetic animals prevented the depression-like behavior and microglial activation; however, minocycline did not reverse impaired hippocampal neurogenesis. These results indicate that minocycline has a preventive effect on diabetes-related depression. Inhibition of microglial activation would be a critical target for the antidepressant mechanism of minocycline. Impaired hippocampal neurogenesis was observed in diabetic mice; however, this may not be involved in the pathogenesis of depression.

Molecular, histological and behavioral evidences for neuroprotective effects of minocycline against nicotine-induced neurodegeneration and cognition impairment: Possible role of CREB-BDNF signaling pathway.

AIM: Neurodegeneration is one of the serious adverse effects of stimulant agents such as nicotine. Minocycline possess established neuroprotective properties. The role of CREB-BDNF signaling pathway in mediating the neuroprotective effects of minocycline against nicotine-induced neurodegeneration in rats was evaluated in current study. METHODS: Seventy adult male rats were divided randomly into seven groups. Group 1 and 2, received 0.7 ml/rat of normal saline (i.p) and nicotine (10 mg/kg, s.c) respectively. Groups 3, 4, 5 and 6, treated concurrently with nicotine (10 mg/kg) and minocycline (10, 20, 30 and 40 mg/kg, i.p, respectively) for 21 days. Group 7 received minocycline alone (40 mg/kg, i.p) for 21 days. From 17th to 21 st days of experiment, Morris water maze (MWM) was used to evaluate learning and spatial memory in rats treated in different groups. According to the critical role of hippocampus in cognitive behavior, hippocampal neurodegenerative parameters (oxidative stress and inflammatory biomarkers) and also cyclic AMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) levels were evaluated in isolated hippocampus in day 22 of experiment and after drug treatment. Also hippocampal cell density and tissue changes were evaluated by hematoxylin and eosin staining. RESULT: Nicotine administration impaired the learning and spatial memory in rats and simultaneous treatment with various doses of minocycline attenuated the nicotine-induced cognition disturbances. In addition, nicotine treatment increased lipid peroxidation and the levels of oxidized form of glutathione (GSSG), interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and Bax protein, while decreasing reduced form of glutathione (GSH), Bcl-2 protein, P-CREB and BDNF levels in the hippocampus of experimental animals. Nicotine also reduced the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) in the hippocampus. Minocycline attenuated nicotine-induced neurodegeneration and elevating CREB (both forms) and BDNF levels. Also minocycline treatment alone increases the cognitive activity and increased CREB (both forms) and BDNF levels and decreased oxidative stress, inflammation and apoptotic biomarkers. Minocycline at high doses cause inhibition of nicotine induced cell density and changes in both area of dentate gyrus (DG) and CA1 in hippocampus. CONCLUSION: It can be concluded that minocycline, probably through activation of P-CREB/BDNF signaling pathway, confers neuroprotection against nicotine-induced neurodegeneration in rat hippocampus.

Minocycline protects neurons against glial cells-mediated bilirubin neurotoxicity.

Unconjugated bilirubin, the end product of heme catabolism and antioxidant, induced brain damage in human neonates is a well-recognized clinical syndrome. However, the cellular and molecular mechanisms underlying bilirubin neurotoxicity remain unclear. To characterize the sequence of events leading to bilirubin-induced neurotoxicity, we investigated whether bilirubin-induced glial activation was involved in bilirubin neurotoxicity by exposing co-cultured rat glial cells and cerebellar granule neurons (CGN) to bilirubin. We found that bilirubin could markedly induce the expression of TNF-α and iNOS in glial cells, and even at low concentrations, the co-culture of glial cells with neurons significantly enhances neurotoxicity of bilirubin. Pretreatment of the co-cultured cells with minocycline protected CGN from glia-mediated bilirubin neurotoxicity and inhibited overexpression of TNF-α and iNOS in glia. Furthermore, we found that high doses of bilirubin were able to induce glial injury, and minocycline attenuated bilirubin-induced glial cell death. Our data suggest that glial cells play an important role in brain damage caused by bilirubin, and minocycline blocks bilirubin-induced encephalopathy possibly by directly and indirectly inhibiting neuronal death pathways.

The allosteric modulation effects of doxycycline, minocycline, and their derivatives on the neuropeptide receptor PAC1-R.

Class B G-protein coupled receptors (GPCR) PAC1-R is a neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptor that mediates the effective neuroprotective activity. Based on our previous data showing that doxycycline and minocycline work as the positive allosteric modulator (PAM) of PAC1-R, we used computer molecular docking and isothermal titration calorimetry assay to further determine the bindings of doxycycline/minocycline's derivatives including tetracycline/tigecycline with the N-terminal extracellular domain of PAC1-R (PAC1-EC1). Then the cAMP assay combined with the PAC1-R natural agonist PACAP27 was used to confirm the possible PAM roles of the small-molecule antibiotics. The results showed that tetracycline/tigecycline had significant lower affinity to PAC1-EC1 than doxycycline/minocycline, which was consistent with their non-positive allosteric modulation activity on PAC1-R. Furthermore, by comparing the key residues contributing to the PAM binding with the predicted allosteric site in PAC1-EC1, we characterized four motifs contributing to PAM binding in PAC1-EC1. The site-directed mutation results showed that ASN60 played the most important role in the PAM binding of the small-molecule antibiotics, while ASP116 played a sensitive marginal role in the PAM binding. These results not only help to explain the clinical and experimental neuroprotective effects of doxycycline/minocycline, but also help to characterize the PAM binding site in PAC1-EC1, which will promote the screening and characterization of novel small-molecule PAMs targeting PAC1-EC1 with drug development potency in nerve system disease.

Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation.

Brain metastases are prevalent in various types of cancer and are often terminal, given the low efficacy of available therapies. Therefore, preventing them is of utmost clinical relevance, and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a toll-like receptor (TLR) 9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, natural killer (NK) cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier (BBB) integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG- C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than nonactivated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the antitumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.

Morphine increases macrophages at the lesion site following spinal cord injury: Protective effects of minocycline.

Opioids are among the most effective and widely prescribed medications for the treatment of pain following spinal cord injury (SCI). Spinally-injured patients receive opioids within hours of arrival at the emergency room, and prolonged opioid regimens are often employed for the management of post-SCI chronic pain. However, previous studies in our laboratory suggest that the effects of opioids such as morphine may be altered in the pathophysiological context of neurotrauma. Specifically, we have shown that morphine administration in a rodent model of SCI increases mortality and tissue loss at the injury site, and decreases recovery of motor and sensory function, and overall health, even weeks after treatment. The literature suggests that opioids may produce these adverse effects by acting as endotoxins and increasing glial activation and inflammation. To better understand the effects of morphine following SCI, in this study we used flow cytometry to assess immune-competent cells at the lesion site. We observed a morphine-induced increase in the overall number of CD11b+ cells, with marked effects on microglia, in SCI subjects. Next, to investigate whether this increase in the inflammatory profile is necessary to produce morphine's effects, we challenged morphine treatment with minocycline. We found that pre-treatment with minocycline reduced the morphine-induced increase in microglia at the lesion site. More importantly, minocycline also blocked the adverse effects of morphine on recovery of function without disrupting the analgesic efficacy of this opioid. Together, our findings suggest that following SCI, morphine may exacerbate the inflammatory response, increasing cell death at the lesion site and negatively affecting functional recovery.

Kinetic analysis of the inhibition of the drug efflux protein AcrB using surface plasmon resonance.

Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl ß-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Neuroprotective effect of minocycline on cognitive impairments induced by transient cerebral ischemia/reperfusion through its anti-inflammatory and anti-oxidant properties in male rat.

Memory deficit is the most visible symptom of cerebral ischemia that is associated with loss of pyramidal cells in CA1 region of the hippocampus. Oxidative stress and inflammation may be involved in the pathogenesis of ischemia/reperfusion (I/R) damage. Minocycline, a semi-synthetic tetracycline derived antibiotic, has anti-inflammatory and antioxidant properties. We evaluated the neuroprotective effect of minocycline on memory deficit induced by cerebral I/R in rat. I/R was induced by occlusion of common carotid arteries for 20min. Minocycline (40mg/kg, i.p.) was administered once daily for 7days after I/R. Learning and memory were assessed using the Morris water maze test. Nissl staining was used to evaluate the viability of CA1 pyramidal cells. The effects of minocycline on the microglial activation was also investigated by Iba1 (Ionized calcium binding adapter molecule 1) immunostaining. The content of malondialdehyde (MDA) and pro-inflammatory cytokines (IL-1ß and TNF-α) in the hippocampus were measured by thiobarbituric acid reaction substances method and ELISA, respectively. Minocycline reduced the increase in escape latency time and in swimming path length induced by cerebral I/R. Furthermore, the ischemia-induced reduction in time spent in the target quadrant during the probe trial was increased by treatment with minocycline. Histopathological results indicated that minocycline prevented pyramidal cells death and microglial activation induced by I/R. Minocycline also reduced the levels of MDA and pro-inflammatory cytokines in the hippocampus in rats subjected to I/R. Minocycline has neuroprotective effects on memory deficit induced by cerebral I/R in rat, probably via its anti-inflammatory and antioxidant properties.