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【摘要】
Human immunodeficiency virus (HIV)-1 Tat protein is an important pathogenic factor in HIV-associated neuropathogenesis. Despite recent progress, the molecular mechanisms underlying Tat neurotoxicity are still not completely understood. However, few therapeutics have been developed to specifically target HIV infection in the brain. Recent development of an inducible brain-specific Tat transgenic mouse model has made it possible to define the mechanisms of Tat neurotoxicity and evaluate anti-neuroAIDS therapeutic candidates in the context of a whole organism. Herein, we demonstrate that administration of EGb 761, a standardized formulation of Ginkgo biloba extract, markedly protected Tat transgenic mice from Tat-induced developmental retardation, inflammation, death, astrocytosis, and neuron loss. EGb 761 directly down-regulated glial fibrillary acidic protein (GFAP) expression at both protein and mRNA levels. This down-regulation was, at least in part, attributable to direct effects of EGb 761 on the interactions of the AP1 and NF-B transcription factors with the GFAP promoter. Most strikingly, Tat-induced neuropathological phenotypes including macrophage/microglia activation, central nervous system infiltration of T lymphocytes, and oxidative stress were significantly alleviated in GFAP-null/Tat transgenic mice. Taken together, these results provide the first evidence to support the potential for clinical use of EGb 761 to treat HIV-associated neurological diseases. Moreover, these findings suggest for the first time that GFAP activation is directly involved in Tat neurotoxicity, supporting the notion that astrocyte activation or astrocytosis may directly contribute to HIV-associated neurological disorders.
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Human immunodeficiency virus type 1 (HIV-1) infects the central nervous system, causing a variety of neuropathologies and neurobehavioral deficits. Common HIV-1 neuropathologies include astrocytosis, multinuclear giant cell formation, increased permeability of the blood-brain barrier, and neuron loss.1 Memory loss, loss of motor control, and cognitive deficiencies often ensue.2,3 A number of studies have shown that HIV-1 Tat protein is an important neuropathogenic factor that contributes to HIV-associated neurological diseases including dementia. The proposed mechanisms for Tat neurotoxicity include direct depolarization of neurons, increased levels of intracellular calcium, increased production/release of proinflammatory cytokines, increased infiltration of macrophages/monocytes, activation of excitatory amino acid receptors, and increased apoptosis.4 Despite the significant progress made during the last few years, it is evident that our understanding of the molecular mechanisms underlying Tat neurotoxicity is still rapidly evolving.
Currently, no therapeutics have been developed to specifically target HIV-associated neurological disorders. Since introduction of highly active antiretroviral therapy in 1995, highly active antiretroviral therapy has dramatically improved the outlook for HIV-positive patients. With increased life expectancy, the prevalence of HIV-associated cognitive and neurological impairment is actually rising despite highly active antiretroviral therapy.5,6 A number of therapeutic agents have been tried to target pathological sequelae of HIV neurological infection, ranging from the pain associated with peripheral neuropathology to neuron dysfunction and death, but few have been approved for clinical use. Thus, it is necessary to explore alternative strategies for treating HIV-associated neurological diseases.
Herbal products account for a substantial portion of the current interest in alternative treatments, and Ginkgo biloba extract (EGb) figures prominently in this interest. EGb possesses neuroprotective activity in animal models of neurodegenerative diseases7 and ischemia.8 EGb has been considered as a polyvalent therapeutic agent in the treatment of disturbances of multifactorial origin, including cerebral insufficiency,9 mild cognitive impairments in elderly patients,10 Alzheimer??s disease, and vascular dementia.11,12 Patients have displayed good tolerance for EGb, with no verified adverse drug interactions.11 EGb has become the most widely sold phytomedicine in Europe and 1 of the 10 best-selling herbal medications in the United States.13 One of the proposed mechanisms for the neuroprotective functions of EGb is that it protects neurons from LRP ligands such as occur in ??-amyloid peptide-induced neurotoxicity.14,15 Our recent studies suggest that interaction of HIV-1 Tat protein with LRP, with resulting disruption of the normal metabolic balance of LRP ligands, may contribute to AIDS-associated neuropathology including dementia.16 These findings raise the possibility of using EGb as an alternative strategy to treat HIV-induced neurological disorders.
With recent development of a doxycycline (Dox)-inducible and brain-targeted HIV-1 Tat transgenic mouse model, we have shown that Tat expression in the brain resulted in neuropathologies reminiscent of several hallmarks noted in the brain of AIDS patients.17 The small rodent model not only offers an opportunity to define further the molecular mechanisms of Tat neurotoxicity but also provides a platform to develop and validate therapeutic candidates targeted at HIV-associated neurological diseases. Therefore, in the present study, we determined the effects of EGb 761 against Tat-induced neurotoxicity in this unique neuroAIDS model.
【关键词】 protection immunodeficiency neurotoxicity involving fibrillary
Materials and Methods
Cell Cultures, Transfection, EGb 761 Treatment, and Reporter Gene Assay
Human astrocytoma U373.MG cells were purchased from the American Type Culture Collection (Manassas, VA). U373.MG cells stably expressing HIV Tat protein (U373.Tat) have been described elsewhere.18,19 These cells were maintained in Dulbecco??s modified Eagle??s medium, supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin, in a 37??C, 5% CO2 incubator. For transfection, cells were plated in a six-well plate at a density of 3 x 105 cells/well, and the cells were then transfected with a luciferase reporter plasmid using Lipofectamine (Invitrogen, Carlsbad, CA). A CMV??Gal plasmid (Clontech, Mountain View, CA) was included as a control to normalize variations among transfections. Transfected cells were then cultured for 2 days in the presence of EGb 761 at 0 to 200 µg/ml, or its purified components terpene bilobalide and ginkgolide B at equivalent composition percentages (Ipsen Laboratories, Paris, France),20,21 and harvested for the luciferase reporter gene assay using a luciferase assay system (Promega, Madison, WI). EGb 761 is extracted from green leaves of the Ginkgo biloba tree to a formulation of 24% flavonoids, 6% terpenes (eg, ginkgolides and bilobalide), 5 to 10% organic acids, and <0.5% proanthocyanidins.22 A human glial fibrillary acidic protein (GFAP) promoter-driven luciferase reporter plasmid was obtained from Dr. Michael Brenner of the University of Alabama at Birmingham, Birmingham, AL,23 and pNFB-luc and pAP1-luc reporter plasmids were purchased from Clontech.
Animals and Treatments
The animals were housed in the Laboratory Animal Care Center of Indiana University School of Medicine with a 12-hour light and 12-hour dark photoperiod. Water and food were provided ad libitum. All animal procedures were approved by the institutional biosafety committee. Tat transgenic mice and GFAP-null mice were previously generated.17,24 The GFAP-null mice used for these experiments are congenic in a C57BL/6J background. Combination GFAP-null/Tat mice were obtained by standard cross-breeding using a higher Tat-expressing line Tg 271.17 Progeny carrying both the GFAP-null alleles and Tat transgene were identified by PCR analysis of genomic DNA, which was extracted from mouse tail clippings (0.5 to 1 cm long) using the Wizard genomic DNA isolation kit (Promega). For amplification of the GFAP-Tet-on transgene, the primers 5'-GCTCCACCCCCTCAGGCTATTCAA-3' and 5'-TAAAGGGCAAAAGTGAGTATGGTG-3' were used, whereas the TRE-Tat86 transgene was amplified with the primers 5'-GTCGAGCTCGGTACCCGGGTC-3' and 5'-CGGGATCCCTATTCCTTCGGGCCTGT-3'. For the GFAP-null and wild-type alleles, the primers were 5'-CGAGAACCGAGCTGGAGTCT-3', 5'-TGGGCAAGACTGGTCATCTA-3' and 5'-AAGCGCATGCTCCAGACTGC-3'. Fifty ng of genomic DNA was used in the PCR reactions, with a program of 1 cycle of 94??C for 3 minutes, 35 cycles of 94??C for 1 minute, 60??C for 1 minute, and 72??C for 1 minute, and 1 cycle of 72??C for 7 minutes for TRE-Tat and GFAP-tet-on transgene amplification and a program of 1 cycle of 95??C for 3 minutes, 31 cycles of 95??C for 40 seconds, 61??C for 30 seconds, and 72??C for 2 minutes, and 1 cycle of 72??C for 5 minutes for GFAP-null and wild-type allele amplification. The expected sizes of the amplified fragments for GFAP-tet-on and TRE-Tat86 transgenes and GFAP-null and wild-type GFAP allele were 467 bp, 424 bp, 80 bp, and 310 bp, respectively. Mice at postnatal day 21 (P21) were given Dox (Sigma, Louis, MO) once a day via intraperitoneal injection in a volume of 100 µl at a dosage of 80 mg/kg/day for 7 days.17 In case of EGb 761 treatment, unless stated otherwise, mice were given EGb 761 once a day intraperitoneally for an additional 7 days at a dosage of 100 mg/kg/day, which has been widely used in similar studies, or its purified components terpene bilobalide and ginkgolide B at equivalent composition percentages.25 All animals were assigned to each experimental group in a random manner. Mice were monitored on a daily basis for growth (weight) and survival. Mice were sacrificed at the last day of the treatment, and the brains were harvested and divided saggitally. The hemi-brain was fixed at least 3 days in phosphate-buffered saline (PBS)-buffered 4% paraformaldehyde and processed for paraffin embedding. In all experiments, unless stated otherwise, comparisons were made between mice treated with Dox or vehicle control (water) and between mice treated with EGb 761 or vehicle control (PBS).
Hematoxylin and Eosin (H&E) Staining and Immunohistochemical Staining
To ensure objective assessments and reliability of results, brain sections from mice to be compared in any given experiment were processed in parallel and examined by three independent individuals. H&E and immunohistochemical staining were performed as previously described.17 For immunohistochemical staining, 10-µm paraffin sections were cut on a microtome and mounted directly on glass slides. The sections were then deparaffinized in xylene and rehydrated and then stained using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) or a Dakocytomation ARK kit (for MAP-2 staining; DAKO, Carpinteria, CA) according to the manufacturer??s instructions. The sources of antibodies were rabbit polyclonal anti-GFAP (1:500) and rabbit anti-CD3 (1:600) from DAKO, mouse anti-MAP-2 (1:100) from Santa Cruz Biotechnologies (Santa Cruz, CA), rabbit anti-Iba-1 (1:1000) from Wako Chemicals (Richmond, VA), and rabbit anti-S100?? (1:1000) from Swant Biotech (Bellinzona, Switzerland). Omission of the primary antibodies was included as a control to evaluate nonspecific staining. Sections were examined, and bright-field microscopic images were captured with a Zeiss digital color camera mounted on an Axiovert M200 microscope (Zeiss, Thornwood, NY) using a x10 or x40 plan apochromat objective.
In Situ Apoptosis TUNEL Staining
Ten-µm paraffin brain sections were deparaffinized and rehydrated. Apoptosis was evaluated with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL)-based TdT-FragEL DNA fragmentation detection kit (EMD Biosciences, La Jolla, CA). Briefly, deparaffinized sections were treated in 10 mmol/L of Tris-HCl, pH 8.0, containing 20 µg/ml of proteinase K at room temperature for 20 minutes, and in 3% H2O2 at room temperature for 5 minutes. Sections were then equilibrated with 1x TdT buffer, and incubated with the TdT-labeling reaction mixture at 37??C for 1.5 hours. The labeling reaction was terminated by addition of the stop solution supplied in the kit and incubated at room temperature for an additional 5 minutes. Sections were rinsed with TBS (20 mmol/L Tris-HCl, pH 7.6, and 140 mmol/L NaCl) between steps. Finally, apoptosis was visualized via incubation of the sections in 3, 3'-diaminobenzidine at room temperature for 15 minutes. Counterstaining was performed in 0.3% methyl green solution after a thorough rinse with distilled water.
Western Blot Analysis and Semiquantitative Reverse Transcriptase (RT)-PCR
For Western blot analysis, U373.MG cells or U373.Tat cells were treated with EGb 761 at a concentration between 0 and 200 µg/ml for 3 days and then washed twice to remove EGb 761 in ice-cold PBS, pelleted, and lysed in RIPA buffer (150 mmol/L NaCl, 1.0% Nonidet P-40, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris-HCl, pH 8.0). Protein concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA). Whole-cell lysates of 25 µg of protein were electrophoretically separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to the HyBond-P membrane (Amersham, Piscataway, NJ). Proteins on the membrane were detected with primary antibodies and appropriate peroxidase-labeled secondary antibodies followed by the ECL chemiluminescence reagents (Amersham). Anti-mouse-GFAP antibody and anti-mouse ??-actin antibody were both from Sigma. To determine GFAP mRNA levels, total RNA was isolated using the Trizol reagents (Invitrogen) according to the manufacturer??s instructions, and subjected to RT-PCR using a Titan one-tube RT-PCR system kit (Boehringer Mannheim, Indianapolis, IN) with GFAP-specific primers (5'-AAGCAGATGAAGCCACCCTG-3' and 5'-GTCTGCACG-GGAATGGTGAT-3'). RT-PCR was performed on a PE Thermocycler 9700 (PE Applied Biosystems, Foster City, CA) with a program of 50??C for 30 minutes, 94??C for 3 minutes, followed by 25 cycles of 94??C for 1 minute, 52??C for 1 minute and 68??C for 1 minute, and 1 cycle of 68??C for 7 minutes. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the RT-PCR as a loading control with GAPDH-specific primers (5'-CTCAGTGTAGCCCAGGATGC-3' and 5'-ACCACCATGGA-GAAGGCTGG-3'). The sizes of RT-PCR products for GFAP and GAPDH were 625 bp and 500 bp, respectively.
Electrophoretic Mobility Shift Assay
U373 cells were treated with EGb 761 at a concentration between 0 and 200 µg/ml for 3 days and then washed twice to remove EGb 761 before they were lysed in a high-salt buffer containing 20 mmol/L HEPES, pH 7.5, 400 mmol/L KCl, 0.5 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol, 20% glycerol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 µg/ml each of aprotinin, pepstatin A, leupeptin, and soybean trypsin inhibitor, and 1% Nonidet P-40, on ice for 30 minutes. The cell lysates were centrifuged at 20,000 x g at 4??C for 5 minutes, and the supernatants were saved as whole cell lysates for the electrophoretic mobility shift assay. Protein concentration was determined using a DC protein assay kit (Bio-Rad). Oligonucleotides containing respective AP1, AP2, NF-B, and CREB consensus binding sites were synthesized, annealed, and end-labeled with -32P-ATP using T4 polynucleotide kinase, and free -32P-ATP was removed by phenol:chloroform extraction and ethanol precipitation. 32P-labeled oligonucleotides of 250,000 cpm were then incubated with 300 ng of protein equivalent whole cell lysates made from each set of EGb 761-treated cells in a volume of 10 µl of binding buffer containing 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.5, and 50 µg/ml poly(dI-dC)?
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作者单位：From the Department of Microbiology and Immunology,* the Center for Acquired Immune Deficiency Syndrome Research, and the Walther Oncology Center,¶ Indiana University School of Medicine, Indianapolis, Indiana; the Walther Cancer Institute,|| Indianapolis, Indiana; the Department of Comparative