Triptolide protects astrocytes from hypoxia/reoxygenation injury＊＊★

Minfang Guo, Hongcui Fan, Jiezhong Yu, Ning Ji, Yongsheng Sun, Liyun Liang,Baoguo Xiao, , Cungen Ma

1Institute of Brain Science, Department of Neurology, Medical School, Shanxi Datong University, Datong 037008, Shanxi Province, China

2Institute of Neurology, Huashan Hospital, Fudan University, Shanghai 200025, China

lNTRODUCTlON

Astrocytes, the most abundant cell type in the central nervous system, play an important role in the maintenance of neuronal functions, including regulation of ionic/metabolic milieu, inflammatory response, antioxidant defense, the establishment and maintenance of blood-brain barrier (BBB), transportation of neurotransmitters, as well as neurotrophic effects. Several lines of evidence indicate that astrocytes are damaged and their death occurs prior to neuronal death in vivo after cerebral ischemia[1-2]. Experiments performed in vitro with primary cultures of cortical astrocytes exposed to oxygen and glucose deprivation (OGD) demonstrate that astrocytes can be protected from ischemic injury by elevating antioxidant capacity[3-4].

However, the mechanisms underlying ischemic astrocyte damage are poorly understood.

Matrix metalloproteinase (MMP) can degrade the extracellular matrix and carry out key functions during brain development.

MMP is expressed at low levels under physiological conditions. However,gelatinolytic MMP, such as MMP-9, is significantly increased and is associated with pathological changes in various neurological disorders[5-6]. There is a significant increase in MMP-2 and MMP-9 activity in hypoxic injury[7-8], which suggests MMP may play an important role in neuronal death related to ischemic injury.

Tripterygium wilfordii Hook F, a vine-like plant that grows in southern China, has been used to treat many diseases for hundreds of years. Its efficacy has been recently recognized by modern medicine[9].

Triptolide (molecular formula: C20H24O6,molecular weight: 360.4, lipid soluble and may go through the BBB), a major active component isolated from Tripterygium wilfordii Hook F, is a diterpenoid triepoxide that shows multiple pharmacological activities, such as anti-inflammatory,immunosuppressive, male anti-fertility, and antitumor activities[10-11]. Anti-inflammatory function and induction of the BBB change have been shown in rats with experimental autoimmune encephalomyelitis treated with triptolide in our earlier reports[12-13].

Furthermore, triptolide has been demonstrated to inhibit collagen degradation by down-regulating the production of MMP[14]. Therefore, we hypothesized that triptolide has the astrocyte-protective activity and this protection effect is correlated with the inhibition of MMP-9 expression and anti-inflammatory activity.

RESULTS

Triptolide treatment improved astrocyte viability after exposure to H/R

3-(4, 5) Dimethylthiazol-2-yl)-2, 5-Diphenyl Tetrazolium Bromide (MTT) assay showed that the viability of astrocytes in the hypoxia/reoxygenation (H/R) group was significantly reduced compared with controls (P ＜ 0.01).Astrocyte viability was significantly increased in the triptolide treated group (P ＜ 0.01), the difference was apparent at the concentration of 500 ng/mL compared with that of the H/R group (Table 1).

Table 1 Astrocyte viability in H/R injury, triptolide treated at different concentrations and control groups by 3-(4, 5)Dimethylthiazol-2-yl)-2, 5-Diphenyl Tetrazolium Bromide assay

Triptolide treatment suppressed inflammatory response in astrocytes after exposure to H/R

ELISA showed that the elevated expressions of interleukin(IL) 6, IL-1β and tumor necrosis factor α (TNF-α) in the H/R group were significantly different from those in control and triptolide treated groups (P ＜ 0.05). There was no significant difference in IL-10 secretion although it was detectable in both control and H/R groups. The secretion levels of IL-6, IL-1β and TNF-α were shown to be significantly lower,while IL-10 level was significantly higher after triptolide treatment compared with control and H/R groups (P ＜ 0.05;Figure 1, supplementary Table 1 online).

Figure 1 Secretions of IL-6, IL-1β, TNF-α and IL-10 in the supernatant of astrocytes in the H/R injury, triptolide treated at different concentrations and control groups by ELISA.aP ＜ 0.05, vs. H/R group. Data are expressed as mean ± SD of 8 independent experiments performed in duplicate (unpaired Student’s t test). H/R: Hypoxia/reoxygenation; IL: interleukin; TNF: tumor necrosis factor.

Triptolide treatment inhibited MMP-9 mRNA expression in astrocytes after exposure to H/R

Reverse transcription (RT)-PCR revealed that the mRNA expression of MMP-9 in the H/R group was significantly increased compared with normoxic controls (P ＜ 0.01),while in the triptolide treated group it was significantly inhibited, especially at the concentration of 500 ng/mL(P ＜ 0.01; Figure 2).

Figure 2 The expression of MMP-9 mRNA in the supernatant of astrocytes in the H/R injury, triptolide treated at different concentrations and control groups by reverse transcription-PCR. The production size of MMP-9 was 489 bp and β-actin was 228 bp (A). aP ＜ 0.01, vs.control group; bP ＜ 0.01, vs. H/R group. Data are expressed as mean ± SD of 10 independent experiments performed in triplicate (B) (unpaired Student’s t test). H/R:Hypoxia/reoxygenation; MPP: matrix metalloproteinase.

Triptolide treatment inhibited MMP-9 protein expression in astrocytes after exposure to H/R

The MMP-9 protein expression in astrocytes in the H/R group was significantly increased compared with that of the normoxic controls (P ＜ 0.01), and was significantly inhibited in the triptolide treated group (P ＜ 0.05 or P ＜0.01; Figure 3). At the same time, astrocytes in the H/R group exhibited higher expression of MMP-9 in the cytoplasm detected by immunofluorescence compared with normoxic controls, while the expression of MMP-9 in the triptolide treated group was obviously inhibited (Figure 4).

Figure 3 The expression of MMP-9 protein in the astrocytes in the H/R injury, triptolide treated at different concentrations and control groups by western blot assay.The resulting bands of MMP-9 and β-actin were visualized by chemiluminescence (A). aP ＜ 0.01, vs. control group;bP ＜ 0.05, cP ＜ 0.01, vs. H/R group. Data are expressed as mean ± SD of 10 independent experiments performed in triplicate (B) (unpaired Student’s t test). H/R: Hypoxia/reoxygenation; MPP: matrix metalloproteinase.

Figure 4 The expression of MMP-9 protein in the astrocytes in the H/R injury, triptolide treated at different concentrations and control groups by immunofluorescence staining (Hoechst 33342 for DNA and Rhodamineconjugated secondary antibody for MMP-9 protein(inverted fluorescence microscope, scale bar: 1 μm).Normoxic astrocytes as control (A), astrocyte exposure to H/R (B), astrocyte exposure to H/R and treated with 250 ng/mL triptolide (C), astrocyte exposure to H/R and treated with 500 ng/mL triptolide (D), astrocyte exposure to H/R and treated with 1 000 ng/mL triptolide (E), negative control (F). The fluorescent intensity of H/R group increased compared with control, and that of triptolide treated groups obviously weakened. H/R: Hypoxia/reoxygenation; MPP: matrix metalloproteinase.

DlSCUSSlON

Astrocytes possess the potential to play a central role in tissue destruction processes. For example, astrocytes are a source of MMP-9 secretion in ischemic injury[15].

During hypoxia/ischemia, abnormal expression and activation of MMP results in the opening of the BBB, prevents normal cell signaling, and eventually leads to cell death. Neuroprotection after inhibition of MMP (MMP-2,MMP-9) activation has previously been demonstrated in the adult brain after focal cerebral acute and chronic ischemia in both mice and rats[16].

Triptolide can protect dopaminergic neurons from damage by inhibiting the expression of MMP in chondrocytes and subepithelial myofibroblasts[17], indicating that triptolide is a potent anti-inflammatory and neuroprotective reagent. Therefore, we established an in vitro model of cerebral I/R related to the influence of astrocyte injury and death as described by Ginis[18]. Our study proved that astrocytes are integrally involved in inflammatory responses and MMP-9 production, and triptolide has an anti-inflammatory effect and inhibits the production of MMP-9.

The present results demonstrate that astrocyte viability is greatly decreased by hyperoxia compared with normoxic controls in an in vitro model of H/R. Triptolide treatment at three concentrations increased the viability of cultured astrocytes exposed to H/R injury, suggesting that triptolide may raise the degree of astrocyte viability in the H/R condition. Three cytokines, TNF-α, IL-1 and IL-6, which have been implicated in the regulation of barrier function in inflammatory states, are increased in blood and edema fluid after tissue injury[19]. IL-1β and TNF-α could induce the expression of MMP-9 probably through the activation of the transcription factors nuclear factor-κB and protein-1[20-21]. IL-1β and TNF-α are secreted by Th1 cells and have a higher activity of MMP-2/9 in Th1 cells than in Th2 cells.

Anti-inflammatory mediators, such as IL-10 and TGF-β,can reduce IL-6, IL-1β, TNF-α and MMP-9 expression and weaken their response to inflammation[22]. But at present, the role of IL-6 in inflammatory response remains controversial. IL-6 mediates the acute phase response and is a marker of inflammation[23]. However,endogenous IL-6 plays a crucial anti-inflammatory role in both local and systemic acute inflammatory responses[24]. Furthermore, triptolide can suppress the production of pro-inflammatory cytokines, such as TNF-α and IL-1β, in microglia[25]. The present study shows that the activity of IL-6, IL-1β and TNF-α was increased in H/R cells. On the contrary, IL-10 was not obviously increased compared with the control group.

And triptolide could inhibit the secretion of IL-6, IL-1β and TNF-α in astrocytes exposed to H/R and increase IL-10 production. This suggests that triptolide may directly inhibit the expression of Th1 type cytokines and promote the expression of Th2 type cytokines. Therefore, inflammatory mechanisms may be involved in astrocyte injury.

The effects of hypoxia on the expression and activity of MMP-9 have scarcely been studied, and conflicting results have been reported in different cell types and different oxygen concentrations[11,26]. The present study shows that reoxygenation after hypoxia leads to the increase in the amounts of soluble MMP-9 by causing the accumulation of pro-inflammatory cytokines, which in turn disturbs the strict regulation of the proteinase and leads to uncontrolled activation. These experimental findings suggest that the interaction between MMP-9 and pro-inflammatory cytokines may be of particular importance in astrocyte exposure to H/R injury. The number of astrocytes in the H/R group was decreased but the amount of MMP-9 was increased. Thus, the amount of MMP-9 was not a result of a number of cells, but possibly an actual consequence of adaptation to the oxidative stress and an induction of pro-inflammatory cytokines.

Triptolide may suppress the expression of nuclear factor-κB and reduce the infiltration of inflammatory factors by anti-oxidative stress, therefore depress the expression of MMP-9 and reduce the activation of MMP-9. The data, as described in the present study, shows that triptolide treatment can decrease the levels of the steady-state MMP-9 mRNA and even decrease the intracellular accumulation of the protein, indicating that triptolide not only affects MMP-9 production, but rather inhibits its secretion or activation via a post-translational effect.

In summary, the expression of MMP-9 and pro-inflammatory cytokines was increased in astrocytes exposed to H/R, and triptolide treatment could inhibit their production. These results suggest a possible mechanism of underlying triptolide to protect astrocytes from H/R injury.

MATERlALS AND METHODS

Design

A comparative observation pertaining to the cytology.

Time and setting

The experiment was performed at Institute of Neurology,Huashan Hospital, Fudan University, China, from May to October 2008.

Materials

Kunming mice at postnatal days 1-2 were obtained from the Animal Laboratory of Shanxi Datong University.

Methods

Isolation, culture and identification of astrocytes

Astrocytes were prepared from cortices of 1-2-day-old Kunming mice, as previously described[27]. The pure passaged astrocytes were used in the following experiments when their purity coefficient reached 98% assessed by glial fibrillary acidic protein (supplementary Figure 1 online).

Establishment of H/R models and triptolide treatment

The cultures were incubated in a glucose-free DMEM base medium (pH 6.4, Sigma) and then subjected to hypoxia for 3 hours in a mixture of 5% CO2, 10% H2and 85% N2(1025 Forma Anaerobic System, Thermo,Barrington, IL, USA). After the 3-hour hypoxic treatment,the astrocytes were subjected to reoxygenation by changing the medium for a high- glucose DMEM base medium supplemented with 10% fetal bovine serum.The drug groups were treated with triptolide (Institute of Dermatology, Chinese Academy of Medical Science,China; purity ＞ 98% HPLC) at different concentrations(250, 500, 1 000 ng/mL), followed by incubation at 95% room air, 5% CO2at 37°C for 12 hours.

MTT assay for the viability of astrocytes in different groups

Viability of astrocytes was assessed using MTT (Sigma)colorimetric assay. The MTT assay was carried out as described by Mosmann[28]. Absorbance was measured with an automatic ELISA reader at a wavelength of 570 nm. The percentage of the dehydrogenase activity was calculated from the absorbance values.

ELISA determination for the IL-1β, TNF-α, IL-6, IL-10 in the supernatants of astrocytes

Astrocyte supernatants were harvested from every group and stored at -80°C for cytokine determination.

Astrocytes were used for RT-PCR, western blot and immunofluorescence analysis. The levels of IL-1β,TNF-α, IL-6, IL-10 were measured with ELISA kits(eBioscience, San Diego, CA, USA) in accordance with the manufacturer’s instructions. Absorbance values were obtained with an automatic ELISA reader(Flow Laboratories, Irvine, United Kingdom) at a wavelength of 450 nm. Determinations were performed in duplicate.

RT-PCR analysis for the MMP-9 mRNA expression in the astrocytes

Comparability of astrocytes total RNAs was evaluated by RT-PCR of a house-keeping gene, β-actin. The primers of MMP-9 were 5’-GAG ATG CGT GGA GAG TCG AA-3’and 5’-CCG AGT TGG AAC CAC GAC GC-3’, length:489 bp. PCR was performed for 5 minutes at 95°C, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute and extension at 72°C for 1 minute, with an autoextension at 72°C for 7 minutes after completion of the last cycle. The primers of β-actin were 5’-AGC CAT GTA CGT AGC CAT CC-3’ and 5’-CTC TCA GCT GTG GTG GTG AA-3’, length: 228 bp.

PCR was performed for 3 minutes at 94°C followed by 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, with an autoextension at 72°C for 7 minutes after completion of the last cycle. Bands were quantified by densitometry using the software of Image-Pro Plus (IPP, Media Cybernetics, Inc., Bethesda,MD, USA). Each experiment was performed in triplicate.

Western blot analysis for the MMP-9 protein expression in the astrocytes

The samples (1.5-2 mg total protein) were boiled and reduced with 1% β-mercaptoethanol. After that the proteins were loaded on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a cellulose nitrate membrane. The membrane was blocked with 5% non-fat milk in 0.05% Tween/PBS for 2 hours. The membrane was subsequently incubated with rabbit anti-MMP-9 polyclonal antibody (1: 200;Santa Cruz, Santa Cruz, CA, USA) overnight at 4°C,then washed as described above and incubated with the appropriate horseradish peroxidase-conjugated anti-rabbit IgG (1: 10 000; Sigma) for 1 hour at room temperature.

The membrane was washed according to the same procedure as described above and the resulting bands were visualized by chemiluminescence (Sigma). Bands were quantified by densitometry using the software of IPP (n = 3). β-actin was used as internal reference.

Immunofluorescence for the MMP-9 protein expression in the astrocytes

The astrocytes were fixed with 4% paraformaldehyde for 20 minutes at room temperature. After being blocked with 5% normal goat serum in 0.3% Triton X-100/PBS for 30 minutes, the astrocytes were incubated with rabbit polyclonal anti-mouse MMP-9 (1: 200) at 4°C overnight.

The astrocytes were incubated further with Rhodamine-conjugated goat anti-rabbit IgG (1: 1 000; Santa Cruz) as the secondary antibody for 45 minutes at room temperature in the dark. Hoechst 33342 was added for staining DNA. Negative control experiments were done by omitting the primary antibody. Stained cells were observed with an inverted fluorescence microscope(Olympus, Tokyo, Japan).

Statistical Analysis

Data are expressed as mean ± SD for continuous variables and the results were analyzed by one-way variance analysis between groups. Differences between two groups were evaluated statistically by using the unpaired Student’s t-test. All statistical analyses were carried out using SPSS 13.0 statistical software (SPSS, Chicago, IL,USA). A level of P ＜ 0.05 was considered statistically significant.

Author contributions:Minfang Guo completed the majority of the experiments, wrote the manuscript, provided data and performed data analysis. Hongcui Fan completed part of the experiments, analyzed data and statistical management. Baoguo Xiao was responsible for the study proposal and design. Cungen Ma was the study proposer and designer, the paper validator, and the fund header. Jiezhong Yu, Ning Ji, Yongsheng Sun and Liyun Liang were responsible for the data integration and analysis.

Conflicts of interest:None declared.

Funding:This work was supported by the National Natural Science Foundation of China, No. 81070957 and Natural Science Foundation of Shanxi Province, No. 2008011082-1.

Ethical approval:All animal protocols were approved by the Animal Ethics Committee of Shanxi Datong University, China.

Supplementary information:Supplementary data associated with this article can be found in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 21, 2011 after selecting the “NRR Current Issue” button on the page.

[1]Swanson RA, Ying W, Kauppinen TM. Astrocyte influences on ischemic neuronal death. Curr Mol Med. 2004;4(2):193-205.

[2]Ouyang YB, Voloboueva LA, Xu LJ, et al. Selective dysfunction of hippocampal CA1 astrocytes contributes to delayed neuronal damage after transient forebrain ischemia. J Neurosci. 2007;27(16):4253-4260.

[3]Wang J, Ma JH, Giffard RG. Overexpression of copper/zinc superoxide dismutase decreases ischemia-like astrocyte injury.Free Radic Biol Med. 2005;38(8):1112-1118.

[4]Li Y, Bao Y, Jiang B, et al. Catalpol protects primary cultured astrocytes from in vitro ischemia-induced damage. Int J Dev Neurosci. 2008;26(3-4):309-317.

[5]Yong VW, Power C, Forsyth P, et al. Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci. 2001;2(7):502-511.

[6]Rosell A, Cuadrado E, Ortega-Aznar A, et al. MMP-9-positive neutrophil infiltration is associated to blood-brain barrier breakdown and basal lamina type IV collagen degradation during hemorrhagic transformation after human ischemic stroke. Stroke.2008;39(4):1121-1126.

[7]Ben-Yosef Y, Lahat N, Shapiro S, et al. Regulation of endothelial matrix metalloproteinase-2 by hypoxia/reoxygenation. Circ Res.2002;90(7):784-791.

[8]Dragun P, Makarewicz D, Wójcik L, et al. Matrix metaloproteinases activity during the evolution of hypoxic-ischemic brain damage in the immature rat. The effect of 1-methylnicotinamide (MNA). J Physiol Pharmacol. 2008;59(3):441-455.

[9]Chen BJ. Triptolide, a novel immunosuppressive and anti-inflammatory agent purified from a Chinese herb Tripterygium wilfordii Hook F. Leuk Lymphoma. 2001;42(3):253-265.

[10]Qiu D, Kao PN. Immunosuppressive and anti-inflammatory mechanisms of triptolide, the principal active diterpenoid from the Chinese medicinal herb Tripterygium wilfordii Hook. f. Drugs R D.2003;4(1):1-18.

[11]Wang W, Yang S, Su Y, et al. Enhanced antitumor effect of combined triptolide and ionizing radiation. Clin Cancer Res. 2007;13(16):4891-4899.

[12]Ma CG, Liu Y, Ma TH, et al. Monocyte chemokine protein-1 mRNA expression in experimental allergic encephalomyelitis rats treated with or without triptolide. J Neurol Sci. 2005;238(s):234.

[13]Ma CG, Yu JZ, Ji N, et al. The kinetic changes of BBB and expression of ICAM-1 in rats with EAE treated with or without Triptolide. J Neuroimmunol. 2008;203:145-146.

[14]Lu Y, Fukuda K, Seki K, et al. Inhibition by triptolide of IL-1-induced collagen degradation by corneal fibroblasts. Invest Ophthalmol Vis Sci. 2003;44(12):5082-5088.

[15]Zhang X, Cheng M, Chintala SK. Optic nerve ligation leads to astrocyte-associated matrix metalloproteinase-9 induction in the mouse retina. Neurosci Lett. 2004;356(2):140-144.

[16]Fujimoto M, Takagi Y, Aoki T, et al. Tissue inhibitor of metalloproteinases protects blood-brain barrier disruption in focal cerebral ischemia. J Cereb Blood Flow Metab. 2008;28(10):1674-1685.

[17]Tao QS, Ren JA, Li JS. Triptolide suppresses IL-1beta-induced chemokine and stromelysin-1 gene expression in human colonic subepithelial myofibroblasts. Acta Pharmacol Sin. 2007;28(1):81-88.

[18]Ginis I, Schweizer U, Brenner M, et al. TNF-alpha pretreatment prevents subsequent activation of cultured brain cells with TNF-alpha and hypoxia via ceramide. Am J Physiol. 1999;276(5 Pt 1):C1171-1183.

[19]Ertel W, Morrison MH, Ayala A, et al. Hypoxemia in the absence of blood loss or significant hypotension causes inflammatory cytokine release. Am J Physiol. 1995;269 (1 Pt 2):R160-166.

[20]Moon SK, Cha BY, Kim CH. ERK1/2 mediates TNF-alpha-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-kappaB and AP-1:Involvement of the ras dependent pathway. J Cell Physiol. 2004;198(3):417-427.

[21]Lee CW, Lin CC, Lin WN, et al. TNF-alpha induces MMP-9 expression via activation of Src/EGFR, PDGFR/PI3K/Akt cascade and promotion of NF-kappaB/p300 binding in human tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol.2007;292(3):L799-812.

[22]Meador BM, Krzyszton CP, Johnson RW, et al. Effects of IL-10 and age on IL-6, IL-1beta, and TNF-alpha responses in mouse skeletal and cardiac muscle to an acute inflammatory insult. J Appl Physiol. 2008;104(4):991-997.

[23]Benveniste EN. Cytokine actions in the central nervous system.Cytokine Growth Factor Rev. 1998;9(3-4):259-275.

[24]Xing Z, Gauldie J, Cox G, et al. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest. 1998;101(2):311-320.

[25]Jiao J, Xue B, Zhang L, et al. Triptolide inhibits amyloid-beta1-42-induced TNF-alpha and IL-1beta production in cultured rat microglia. J Neuroimmunol. 2008;205(1-2):32-36.

[26]Kolev K, Skopal J, Simon L, et al. Matrix metalloproteinase-9 expression in post-hypoxic human brain capillary endothelial cells:H2O2 as a trigger and NF-kB as a signal transducer. Thromb Haemost. 2003;90(3):528-537.

[27]Frangakis MV, Kimelberg HK. Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures. Neurochem Res. 1984;9(12):1689-1698.

[28]Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63.