Mushfiquddin Khan

Cytokine-mediated Induction of Ceramide Production Is Redox-sensitive IMPLICATIONS TO PROINFLAMMATORY CYTOKINE-MEDIATED APOPTOSIS IN DEMYELINATING DISEASES

The present study underlines the importance of reactive oxygen species in cytokine-mediated degradation of sphingomyelin (SM) to ceramide. Treatment of rat primary astrocytes with tumor necrosis factor-α (TNF-α) or interleukin-1β led to marked alteration in cellular redox (decrease in intracellular GSH) and rapid degradation of SM to ceramide. Interestingly, pretreatment of astrocytes with N-acetylcysteine (NAC), an antioxidant and efficient thiol source for glutathione, prevented cytokine-induced decrease in GSH and degradation of sphingomyelin to ceramide, whereas treatment of astrocytes with diamide, a thiol-depleting agent, alone caused degradation of SM to ceramide. Moreover, potent activation of SM hydrolysis and ceramide generation were observed by direct addition of an oxidant like hydrogen peroxide or a prooxidant like aminotriazole. Similar to NAC, pyrrolidinedithiocarbamate, another antioxidant, was also found to be a potent inhibitor of cytokine-induced degradation of SM to ceramide indicating that cytokine-induced hydrolysis of sphingomyelin is redox-sensitive. Besides astrocytes, NAC also blocked cytokine-mediated ceramide production in rat primary oligodendrocytes, microglia, and C6 glial cells. Inhibition of TNF-α- and diamide-mediated depletion of GSH, elevation of ceramide level, and DNA fragmentation (apoptosis) in primary oligodendrocytes by NAC, and observed depletion of GSH, elevation of ceramide level, and apoptosis in banked human brains from patients with neuroinflammatory diseases (e.g. X-adrenoleukodystrophy and multiple sclerosis) suggest that the intracellular level of GSH may play a critical role in the regulation of cytokine-induced generation of ceramide leading to apoptosis of brain cells in these diseases.

S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke

Preservation of endothelial functions with low-dose nitric oxide (NO) and inhibition of excessive production of NO from inducible NO synthase (iNOS) is a potential therapeutic approach for acute stroke. Based on this hypothesis, an NO modulator, S-nitrosoglutathione (GSNO) was used, which provided neuroprotection in a rat model of focal cerebral ischemia. Administration of GSNO after the onset of ischemia reduced infarction and improved cerebral blood flow. To understand the mechanism of protection, the involvement of inflammation in ischemic brain injury was examined. Treatment with GSNO reduced the expression of tumor necrosis factor-α, interleukin-1β, and iNOS; inhibited the activation of microglia/macrophage (ED1, CD11-b); and downregulated the expression of leukocyte function-associated antigen-1 and intercellular adhesion molecule-1 in the ischemic brain. The number of apoptotic cells (including neurons) and the activity of caspase-3 were also decreased after GSNO treatment. Further, the antiinflammatory effect of GSNO on expression of iNOS and activation of NF-κB machinery in rat primary astrocytes and in the murine microglial cell line BV2 was tested. Cytokine-mediated expression of iNOS and activation of NF-κB were inhibited by GSNO treatment. That GSNO protects the brain against ischemia/reperfusion injury by modulating NO systems, resulting in a reduction in inflammation and neuronal cell death was documented by the results.

N-Acetyl cysteine protects against injury in a rat model of focal cerebral ischemia.

Ischemic cerebrovascular disease (stroke) is one of the leading causes of death and long-time disability. Ischemia/reperfusion to any organ triggers a complex series of biochemical events, which affect the structure and function of every organelle and subcellular system of the affected cells. The purpose of this study was to investigate the therapeutic efficacy of N-acetyl cysteine (NAC), a precursor of glutathione and a potent antioxidant, to attenuate ischemia/reperfusion injury to brain tissue caused by a focal cerebral ischemia model in rats. A total of 27 male Sprague-Dawley rats weighing 250-300 g were used in this study. Focal cerebral ischemia (45 min) was induced in anesthetized rats by occluding the middle cerebral artery (MCA) with an intra-luminal suture through the internal carotid artery. The rats were scored post-reperfusion for neurological deficits. They were then sacrificed after 24 h of reperfusion and infarct volume in the brain was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC). Brain sections were immunostained for tumor necrosis factor (TNF-alpha) and inducible nitric oxide synthase (iNOS). Animals treated with NAC showed a 49.7% (S.E.M.=1.25) reduction in brain infarct volume and 50% (S.E.M.=0.48) reduction in the neurological evaluation score as compared to the untreated animals. NAC treatment also blocked the ischemia/reperfusion-induced expression of tumor necrosis factor and inducible nitric oxide synthase. The data suggest that pre-administration of NAC attenuates cerebral ischemia and reperfusion injury in this brain ischemia model. This protective effect may be as a result of suppression of TNF-alpha and iNOS.

AICAR inhibits adipocyte differentiation in 3T3L1 and restores metabolic alterations in diet-induced obesity mice model

BackgroundObesity is one of the principal causative factors involved in the development of metabolic syndrome. AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. The role of AMP-activated protein kinase in adipocyte differentiation is not completely understood, therefore, we examined the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK) on adipocyte differentiation in 3T3L1 cells and in a mouse D iet i nduced o besity (DIO) model.MethodsTo examine the effect of AICAR on adipocyte differentiation in 3T3L1 cells and in a mouse D iet i nduced o besity (DIO) model, 3T3L1 cells were differentiatied in the presence or absence of different concentration of AICAR and neutral lipid content and expression of various adipocyte-specific transcription factors were examined. In vivo study, treated and untreated mice with AICAR (0.1–0.5 mg/g body weight) were fed high-fat diet (60% kcal% fat) to induce DIO and several parameters were studied.ResultsAICAR blocked adipogenic conversion in 3T3L1 cells along with significant decrease in the neutral lipid content by downregulating several adipocyte-specific transcription factors including peroxisome proliferators-activated receptor γ (PPARγ), C/EBPα and ADD1/SREBP1, which are critical for adipogenesis in vitro. Moreover, intraperitoneal administration of AICAR (0.5 mg g/body weight) to mice fed with high-fat diet (60% kcal% fat) to induce DIO, significantly blocked the body weight gain and total content of epididymal fat in these mice over a period of 6 weeks. AICAR treatment also restored normal adipokine levels and resulted in significant improvement in glucose tolerance and insulin sensitivity. The reduction in adipose tissue content in AICAR treated DIO mice was due to reduction in lipid accumulation in the pre-existing adipocytes. However, no change was observed in the expression of PPARγ, C/EBPα and ADD1/SREBP1 transcription factors in vivo though PGC1α expression was significantly induced.ConclusionThis study suggests that AICAR inhibits adipocyte differentiation via downregulation of expression of adipogenic factors in vitro and reduces adipose tissue content in DIO mice by activating expression of PGC1α without inhibiting adipocyte-specific transcription factors in DIO mice.

Administration of S-nitrosoglutathione after traumatic brain injury protects the neurovascular unit and reduces secondary injury in a rat model of controlled cortical impact.

BackgroundTraumatic brain injury (TBI) is a major cause of preventable death and serious morbidity in young adults. This complex pathological condition is characterized by significant blood brain barrier (BBB) leakage that stems from cerebral ischemia, inflammation, and redox imbalances in the traumatic penumbra of the injured brain. Once trauma has occurred, combating these exacerbations is the keystone of an effective TBI therapy. Following other brain injuries, nitric oxide modulators such as S-nitrosoglutathione (GSNO) maintain not only redox balance but also inhibit the mechanisms of secondary injury. Therefore, we tested whether GSNO shows efficacy in a rat model of experimental TBI.MethodsTBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO (50 μg/kg body weight) was administered at two hours after CCI. GSNO-treated injured animals (CCI+GSNO group) were compared with vehicle-treated injured animals (CCI+VEH group) in terms of tissue morphology, BBB leakage, edema, inflammation, cell death, and neurological deficit.ResultsTreatment of the TBI animals with GSNO reduced BBB disruption as evidenced by decreased Evans blue extravasation across brain, infiltration/activation of macrophages (ED1 positive cells), and reduced expression of ICAM-1 and MMP-9. The GSNO treatment also restored CCI-mediated reduced expression of BBB integrity proteins ZO-1 and occludin. GSNO-mediated improvements in tissue histology shown by reduction of lesion size and decreased loss of both myelin (measured by LFB staining) and neurons (assayed by TUNEL) further support the efficacy of GSNO therapy. GSNO-mediated reduced expression of iNOS in macrophages as well as decreased neuronal cell death may be responsible for the histological improvement and reduced exacerbations. In addition to these biochemical and histological improvements, GSNO-treated injured animals recovered neurobehavioral functions as evaluated by the rotarod task and neurological score measurements.ConclusionGSNO is a promising candidate to be evaluated in humans after brain trauma because it not only protects the traumatic penumbra from secondary injury and improves overall tissue structure but also maintains the integrity of BBB and reduces neurologic deficits following CCI in a rat model of experimental TBI.

HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis

Impaired remyelination due to degeneration of both postmitotic oligodendrocytes and oligodendrocyte progenitors (OPs) is the major hallmark of inflammatory demyelination in multiple sclerosis (MS) lesions and experimental autoimmune encephalomyelitis (EAE). Here, we have demonstrated the potential of lovastatin, a HMG‐CoA reductase inhibitor, for the restoration of impaired remyelination mediated through enhanced survival and differentiation of OPs in the spinal cord of treated EAE animals. Lovastatin treatment significantly increased the level of myelin lipids in the spinal cord of treated EAE animals, coinciding with the attenuation of disease severity and inflammatory demyelination as compared with untreated EAE animals. The increased expression of myelin proteins and transcription factors associated with differentiating oligodendrocytes along with the increase in number of NG2+/BrdU− and NG2+/BrdU+ cells, and the expression of proliferating OP‐specific proteins, demonstrated the restoration of remyelination in the spinal cord of lovastatin‐treated EAE animals. Corresponding to this, in vitro studies further corroborated the increased survival and differentiation of OPs in lovastatin‐treated activated mixed glial cells suggesting that lovastatin protects against the degeneration of OPs and enhances their differentiation through induction of a pro‐remyelinating environment in the spinal cord of treated EAE animals. Together, these data demonstrate that lovastatin has the potential to augment remyelination in MS lesions and other neuroinflammatory diseases. Paintlia, A. S., Paintlia, M. K., Khan, M., Vollmer, T., Singh, A. K., Singh, I. HMG‐CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis. FASEB J. 19, 1407–1421 (2005)

Correlation of very long chain fatty acid accumulation and inflammatory disease progression in childhood X-ALD:: implications for potential therapies

This study was designed to understand the role of inflammatory mediators involved in the neurobiology of childhood adrenoleukodystrophy (cALD) by comparing the differential expression of the inflammatory mediators with metabolite very long chain fatty acids that accumulate in this disease. Histopathological examinations indicated extensive demyelination and accumulation of infiltrates in perivascular cuffs in plaque area (PA) and inflammatory area (IA) compared to normal looking area (NLA) of the cALD brain and controls. The PA had excessive accumulation of cholesterol ester (25-30-fold), VLC fatty acids (8-12-fold), and exhaustive depletion of cholesterol (60-70%) and sphingomyelin (50-55%) in comparison to controls. The mRNA expression of cytokines (IL-1alpha, IL-2, IL-3, IL-6, TNF-alpha, and GM-CSF), chemokines (CCL2, -4, -7, -11, -16, -21, -22, CXCL1, CX3CL1, and SDF-2) and iNOS in IA was significantly increased compared to NLA of the cALD and controls determined by gene array, semiquantitative RT-PCR, and immunohistochemistry. These results indicate that accumulation of VLC fatty acid contents in membrane domains associated with signal transduction pathways may trigger the inflammatory process through activation of resident glial cells (microglia and astrocytes) resulting in loss of myelin and oligodendrocytes.

Lovastatin and sodium phenylacetate normalize the levels of very long chain fatty acids in skin fibroblasts of X‐ adrenoleukodystrophy

The present study underlines the importance of lovastatin, an inhibitor of 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase, and the sodium salt of phenylacetic acid (NaPA), an inhibitor of mevalonate pyrophosphate decarboxylase, in normalizing the pathognomonic accumulation of saturated very long chain fatty acids (VLCFA) in cultured skin fibroblasts of X‐adrenoleukodystrophy (X‐ALD) in which the ALD gene is either mutated or deleted. Lovastatin or NaPA alone or in combination stimulated the β‐oxidation of lignoceric acid (C24:0) and normalized the elevated levels of VLCFA in skin fibroblasts of X‐ALD. Ability of lovastatin and NaPA to normalize the pathognomonic accumulation of VLCFA in skin fibroblasts of X‐ALD may identify these drugs as possible therapeutics for X‐ALD.

Krabbe disease: psychosine-mediated activation of phospholipase A2 in oligodendrocyte cell death

Globoid cell leukodystrophy (Krabbe disease) is an inherited neurological disorder caused by the pathogenomic accumulation of psychosine (galactosylsphingosine), a substrate for the deficient enzyme galactocerebroside β-galactosidase. This study underscores the mechanism of action of psychosine in the regulation of oligodendrocyte cell death via the generation of lysophosphatidylcholine (LPC) and arachidonic acid (AA) by the activation of secretory phospholipase A2 (sPLA2). There was a significant increase in the level of LPC, indicating a phospholipase A2 (PLA2)-dependent pathobiology, in the brains of Krabbe disease patients and those of twitcher mice, an animal model of Krabbe disease. In vitro studies of the treatment of primary oligodendrocytes and the oligodendrocyte MO3.13 cell line with psychosine also showed the generation of LPC and the release of AA in a dose- and time-dependent manner, indicating psychosine-induced activation of PLA2. Studies with various pharmacological inhibitors of cytosolic phospholipase A2 and sPLA2 and psychosine-mediated induction of sPLA2 enzymatic activity in media supernatant suggest that psychosine-induced release of AA and generation of LPC is mainly contributed by sPLA2. An inhibitor of sPLA2, 7,7-dimethyl eicosadienoic acid, completely attenuated the psychosine-mediated accumulation of LPC levels, release of AA, and generation of reactive oxygen species, and blocked oligodendroyte cell death, as evident from cell survival, DNA fragmentation, and caspase 3 activity assays. This study documents for the first time that psychosine-induced cell death is mediated via the sPLA2 signaling pathway and that inhibitors of sPLA2 may hold a therapeutic potential for protection against oligodendrocyte cell death and resulting demyelination in Krabbe disease.

Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia

Ischemic stroke is a neurovascular disease treatable by thrombolytic therapy, but the therapy has to be initiated within 3 h of the incident. This therapeutic limitation stems from the secondary injury which results mainly from oxidative stress and inflammation. A potent antioxidant/anti‐inflammatory agent, caffeic acid phenethyl ester (CAPE) has potential to mitigate stroke’s secondary injury, and thereby widening the therapeutic window. We observed that CAPE protected the brain in a dose‐dependent manner (1–10 mg/kg body weight) and showed a wide therapeutic window (about 18 h) in a rat model of transient focal cerebral ischemia and reperfusion. The treatment also increased nitric oxide and glutathione levels, decreased lipid peroxidation and nitrotyrosine levels, and enhanced cerebral blood flow. CAPE down‐regulated inflammation by blocking nuclear factor kappa B activity. The affected mediators included adhesion molecules (intercellular adhesion molecule‐1 and E‐selectin), cytokines (tumor necrosis factor‐α and interleukin‐1β) and inducible nitric oxide synthase. Anti‐inflammatory action of CAPE was further documented through reduction of ED1 (marker of activated macrophage/microglia) expression. The treatment inhibited apoptotic cell death by down‐regulating caspase 3 and up‐regulating anti‐apoptotic protein Bcl‐xL. Conclusively, CAPE is a promising drug candidate for ischemic stroke treatment due to its inhibition of oxidative stress and inflammation, and its clinically relevant wide therapeutic window.