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ROCK mediates the inflammatory response in thrombin induced microglia
Neuroscience Letters 554 (2013) 82–87
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
ROCK mediates the inflammatory response in thrombin induced microglia Guiyun Cui a , Tao Zuo b , Qiuchen Zhao c , Jinxia Hu a , Peisheng Jin d , Hui Zhao e , Jia Jing a , Jienan Zhu a , Hao Chen a , Bin Liu a , Fang Hua a , Xinchun Ye a,∗ a
Department of Neurology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province, China Department of Clinical Medicine, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China c Department of Clinical Medicine, Xuzhou Medical College, Xuzhou, Jiangsu Province, China d Department of Plastic Surgery, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province, China e Department of Neurology, Xuzhou Central Hospital, Xuzhou, Jiangsu Province, China b
h i g h l i g h t s • • • • •
Increased expression of ROCK, NO and TNF-␣ in thrombin induced microglia was found. Enhanced phagocytosis in thrombin induced microglia was also found. Argatroban or Y-27632 pretreatment decreased ROCK, NO and TNF-␣ expression. Argatroban or Y-27632 pretreatment reduced phagocytosis in thrombin induced microglia. ROCK may regulate the inflammatory response in thrombin induced microglia.
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Article history: Received 24 May 2013 Received in revised form 25 August 2013 Accepted 27 August 2013 Keywords: Inflammation ROCK Microglia Argatroban Y-27632
a b s t r a c t To investigate whether the ROCK pathway is involved in thrombin-induced microglial inflammatory response, thrombin-induced microglia were pretreated with the thrombin inhibitor argatroban or a ROCK inhibitor Y-27632. Microglial inflammatory response was evaluated by phagocytosis of fluorescein labeled latex beads analyses and inflammatory mediators’ expression such as nitric oxide (NO) and tumor necrosis factor-alpha (TNF-ɑ). Compared to non-induced microglia, thrombin-induced microglia show significantly enhanced phagocytotic capacity and increased ROCK, NO and TNF-ɑ expression. Pretreatment of thrombin-induced microglia with argatroban or Y-27632 significantly decreased phagocytotic capacity and reduced ROCK, NO and TNF-␣ expression. Therefore, the ROCK pathway may play a vital role in the mechanisms by which thrombin induces microglia in the inflammatory response. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Intracerebral hemorrhage (ICH) is a devastating disease accounting for 10–15% of all strokes. Clinical advances in ICH have been restricted by a poor understanding of the mechanisms underlying brain injury after hemorrhage [4]. Inflammatory activation plays a crucial role in the pathophysiological mechanisms of ICH, exerting deleterious effects on the progression of ICH-induced secondary brain injury [35]. Therefore, investigation of inflammatory activation under conditions of ICH is warranted.
∗ Corresponding author at: Department of Neurology, The Affiliated Hospital of Xuzhou Medical College, No. 99 West Huaihai Road, Xuzhou 221006, Jiangsu Province, China. Tel.: +86 516 85802129; fax: +86 516 85802129. E-mail address: [email protected] (X. Ye). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.08.065
Thrombin, a blood-derived serine protease, is essential to blood coagulation and is abundantly present in hematoma. Uncontrolled activation of thrombin-induced inflammation after ICH has been reported to be a central mechanism for secondary injury in ICH [15]. Our previous data also indicated that both PKC alpha and PKC delta play important roles in thrombin-induced brain injury after ICH [8]. However, the mechanisms by which thrombin activates microglia in the progression of inflammatory response remain unclear. Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC family of serine–threonine kinases [23]. The ROCK pathway has been implicated in numerous inflammatory diseases such as rheumatoid arthritis [18], atherosclerosis [10,25], asthma [17], multiple sclerosis [31], and disorders of the central nervous system [24,29]. ROCK appears to also serve as a mediator of thrombin-induced endothelial barrier dysfunction [3,6,16]. However, whether the ROCK pathway is involved in thrombin-induced microglial inflammatory response has not been investigated.
G. Cui et al. / Neuroscience Letters 554 (2013) 82–87
In the present study, we investigated whether the ROCK pathway is involved in thrombin-induced microglial inflammatory response. To this end, we explored the influence of pretreatment with the specific thrombin inhibitor, argatroban or the ROCK inhibitor Y-27632, in the response of microglia to thrombin. The inflammatory response, induced by thrombin, was evaluated by assessing the phagocytosis of fluorescein labeled latex beads and the expression of the inflammatory mediators, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a). Molecular mechanisms underlying activated microglia induced by thrombin are described.
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were subjected to Western blot analysis, as described previously [7,40]. The following primary antibodies were used: anti--actin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-ROCK (1:500; Abcam, Cambridge, MA). For immunofluorescence [41], cells growing on coverslips were fixed and treated with 0.1% TritonX100, incubated with anti-ROCK (1:500; Abcam, Cambridge, MA) followed by a FITC-conjugated secondary antibody. The absence of primary antibody was the negative control. Samples were examined by a laser scanning confocal microscope. The experiments were done with 3 different cell culture preparations. The data were calculated as a percentage of fluorescence area in each field.
2. Materials and methods All experiments were conducted in accordance with the standards and procedures of the American Council on Animal Care and Institutional Animal Care and Use Committee of Xuzhou Medical College. 2.1. Primary microglial cultures Primary microglial cultures were isolated from the cortex of neonatal SD rats less than 72 h old, as described previously [36]. Briefly, the cortical brain tissue was isolated from neonatal SD rats and the isolated cells were plated on poly-l-lysine-coated culture flasks at the density of 1.0 × 105 cells/cm2 . After 14 days, the mixed microglia/astrocyte cultures were digested with trypsin at 37 ◦ C. After digestion for 20 min, the remaining adherent cells in the flask are microglia.
2.6. Quantification of TNF-˛ release The release of TNF-␣ in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). Supernatants (detection limit: 5 pg/ml) were placed on ELISA kit strips (Boster, Wuhan). Sandwich ELISA was then performed according to the manufacturer’s instructions. 2.7. Nitrite assay The level of NO was evaluated by Nitrite assay as described previously [13]. In brief, cell culture supernatant was centrifuged at 1000 rpm for 5 min, aliquots of 100 l were incubated with 100 l Griess reagent (0.1% naphthylthylene, 1% sulfanilamide in 2.5% phosphoric acid), and the absorption at 450 nm was measured using a MR5000 ELISA reader.
2.2. The purity of the primary microglial cultures 2.8. Fluorescein labeled latex beads analysis To determine the purity of the primary microglial cultures, microglia were plated on 8-chamber poly-d-lysine coated glass slides. After blocking with 10% BSA in PBS for 1 h, the cells were incubated overnight at 4 ◦ C with IBA-1 goat polyclonal antibody (1:100, Abcam, Cambridge, MA), followed by incubation with FITCconjugated secondary antibody (1:200, ZSGB-Bio, Beijing) for 2 h at 37 ◦ C [34], then we randomly chose 5 visual fields (nearly 20 cells per visual field) to count the percentage of the positive cells by confocal microscopy. 2.3. The optimal dose and time for thrombin to activate microglia To figure out the optimal dose and time for thrombin to activate microglia, microglia were treated with thrombin with different concentrations (0, 10, 20, 40, and 60 U/ml) for 6 h and with different time (3, 6, 12, and 24 h). ROCK expression was evaluated among them. 2.4. Experiment groups To investigate whether ROCK mediates the inflammatory response in thrombin induced microglia. Microglia were randomly assigned to different groups and were treated with thrombin with optimal concentration for the optimal time (n = 3/group): (1) thrombin group: microglia were incubated with thrombin; (2) argatroban group (25 U/ml): microglial cells were pretreated with argatroban (Mitsubishi Pharma, GuangZhou) for 30 min and then treated with thrombin; (3) Y-27632 group (10 mol/L): microglial cells were pretreated with Y-27632 (≥95% by HPLC, Merck, USA) for 30 min and then treated with thrombin. 2.5. Western blotting and immunofluorescence ROCK expression levels in the microglia were analyzed by Western blotting and immunofluorescence. Equal amounts of cell lysate
Microglial phagocytotic activity was measured by the uptake of FITC (fluorescein isothiocyanate) labeled latex beads (Nanozymics, Wuxi) [33]. In brief, culture specimens were exposed to FITC labeled latex beads for 2 h at 37 ◦ C. Surface bound beads were removed by vigorous washing with PBS, then cells were stained with DiI (Molecular probe Inc., USA) for 5 min. The experiments were done with 3 different cell culture preparations. The data were calculated as number of positive cells in each field. 2.9. Statistical analysis The homogeneity of variances was assumed to hold for all measurements by using Levene test. One-way analysis of variance (ANOVA) was performed for ROCK expression, NO and TNF-␣ expression, fluorescein labeled latex beads analyses; the analyses started with the testing of overall group effect, followed by a post hoc test of least significant difference (LSD) for multiple comparison if the overall group effect was significant. The significance level was set to 0.05 in the paper and all the data analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago IL). The data are presented as mean ± SD. 3. Results 3.1. Purity of primary microglial cells isolation To determine the purity of the cultures, a subset of cells was seeded onto multi-chamber glass slides for characterization via immunofluorescence (Fig. 1). Using commonly employed cell-type specific antibodies, the percentage of microglia (Iba-1+) present in the microglial cultures was determined. Quantification showed that primary microglial cell cultures were over 95% Iba-1+ microglia with few astrocytes remaining (Fig. 1).
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Fig. 1. Shows microglia were identified using anti-Iba-1 primary and FITCconjugated secondary antibodies (green, DAPI, blue; scale bar = 100 m). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.2. The optimal dose and time for thrombin to induce microglia To investigate the optimal concentration and induction time of thrombin induced activation of microglial cells, Rho-kinase expression was assessed by Western blot analysis. Fig. 2A shows that various concentrations of thrombin can significantly increase ROCK expression in microglial cells when compared to non-induced microglial cells (p < 0.05, n = 3/group). It also shows that the optimal concentration of thrombin for induction is 20 U/ml. Fig. 2B shows that thrombin induction at various time points can significantly increase ROCK expression in microglial cells when compared to non-induced microglial cells (p < 0.05, n = 3/group), though the optimal induction time is 24 h.
Fig. 3. Shows that ROCK expression was significantly increased in the thrombin treated group, and that argatroban or Y-27632 pretreatment significantly decreased ROCK expression (p < 0.01, F = 84.360, n = 3/group) (T = Thrombin, A = argatroban).
n = 3/group). Fig. 4 shows that immunofluorescence data were consistent with Western blotting analysis (p < 0.05, n = 3/group).
3.3. ROCK expression
3.4. NO and TNF-˛ expression
To elucidate whether ROCK is involved with thrombin-induced microglia in the progression of inflammation, a thrombin inhibitor argatroban or a ROCK inhibitor Y-27632 was employed to assess the microglial ROCK expression by means of Western blotting analysis. Fig. 3 shows that ROCK expression was significantly increased in thrombin-treated group compared with control group. Argatroban or Y-27632 pretreatment significantly decreased ROCK expression in microglia compared to non-pretreated microglial cells (p < 0.05,
To investigate the inflammatory activation of microglia, TNF-␣ expression was assessed by ELISA and NO expression was evaluated by Nitrite assay. Fig. 5A and B shows that microglia exhibit significantly increased NO and TNF-␣ expression in thrombintreated group compared to cells in the control group (p < 0.05, n = 3/group). Argatroban or Y-27632 pretreatment significantly decreased NO and TNF-␣ expression in microglial cells compared to non-pretreated microglial cells (p < 0.05, n = 3/group).
Fig. 2. A shows the dose dependence of ROCK expression in thrombin-induced microglia (p < 0.01, F = 78.153, n = 3/group). B shows the time dependence of ROCK expression in thrombin-induced microglia (p < 0.01, F = 32.462, n = 3/group).
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Fig. 4. Shows ROCK expression was evaluated by immunofluorescence. Thrombin-induced microglia exhibited enhanced ROCK expression, and argatroban or Y-27632 pretreatment significantly decreased ROCK expression (p < 0.01, F = 109.801, n = 3/group).
3.5. Phagocytosis of fluorescein labeled latex beads analyses To evaluate phagocytosis of thrombin-activated microglia, phagocytosis of fluorescein labeled latex beads analyses was performed. Fig. 6 shows that phagocytosis was significantly increased in the thrombin-induced microglia compared with non-induced microglia. Argatroban or Y-27632 pretreatment exhibited significantly decreased phagocytosis in the thrombin-induced cells (p < 0.05, n = 3/group). 4. Discussion In this study, we found that ROCK expression increased significantly in thrombin-activated microglia when compared to non-activated microglia. Inflammation mediators NO and TNF-␣ expression were also significantly increased in activated microglia. In addition, phagocytosis of fluorescein labeled latex beads were significantly enhanced in thrombin-induced microglia. Pretreatment of thrombin-induced microglia with argatroban or Y-27632 significantly reduced ROCK, NO and TNF-␣ expression. Pretreatment also significantly reduced microglial phagocytosis. Therefore, ROCK may play a vital role in the mechanisms by which thrombin activates microglia. NO is also reported to be implicated in the pathogenesis of inflammation. It exerts either an anti-inflammatory effect under normal physiological conditions or a pro-inflammatory role under abnormal situations [28]. TNF-␣ is a proinflammatory cytokine which acts as a central regulator of inflammation [21]. It has been reported that NO and TNF-␣ are involved in the progression of inflammation by activated macrophages, which release them in high concentrations [28,39]. Phagocytosis is the cellular process of engulfment and destruction of invading microorganisms. It is also required for the clearance of apoptotic cells, which is essential for tissue homeostasis and remodeling [5]. Calcium influx plays a positive role in the regulation of phagocytic activity [30]. We found that thrombin-induced microglia significantly increased NO
and TNF-␣ expression as well as enhanced microglial phagocytosis compared to non-induced microglia. Thus, NO and TNF-␣ may also be implicated in the progression of inflammation in microglia which is activated by thrombin. In addition, thrombin may also enhance microglial phagocytic capacity. ROCK is a kinase belonging to the AGC family of serine–threonine kinases. The ROCK pathway is implicated in many cellular functions and up-regulates various molecules that accelerate inflammation [20,27]. Abnormal activation of the ROCK pathway has been observed in various disorders of the central nervous system [2,12,14,26,37]; lesions of the brain and spinal cord activate ROCKs, which have been revealed as major mediators of neuroinflammatory responses [24,29]. It has also been shown that during activation of inflammatory cells ROCK induces changes in the actin cytoskeleton that results in process retraction, cell spreading as well as changes in cell motility characteristics of activation [1,32]. In the present study, we found that ROCK expression was significantly enhanced in thrombin-induced microglia compared to non-induced microglia. Thus, the ROCK pathway may be involved in the thrombin induced inflammatory response of microglia. Y-27632, as a specific inhibitor of ROCK which specifically binds to the ROCK family of kinases and inhibits their kinase activity, has been reported to be involved in cell motility [9], cellular differentiation [38] and cell death [19]. In this study, we found that Y-27632 can decrease ROCK expression in thrombin-induced microglia. The decreased ROCK expression in microglia may be attributed to Y27632 induced cell death. Therefore, Y-27632 may also curtail ROCK’s expression more than inhibit its activity. Argatroban, as a synthetic small molecule derived from larginine, is a direct thrombin inhibitor that binds reversibly to, and inhibits both soluble and clot-bound thrombin [11]. Due to its anticoagulant effect, argatroban has been used in the treatment of ischemic stroke, acute coronary syndrome, and hemodialysis [22]. In this study, we found that either argatroban or Y-27632 pretreatment inhibits inflammation, as demonstrated by reduced
Fig. 5. A and B show that microglia significantly increased NO and TNF-␣ expression in thrombin treated group compared to cells in the control group. Argatroban or Y-27632 pretreatment significantly decreased NO and TNF-␣ expression (p < 0.01, n = 3/group. A, F = 27.395; B, F = 122.480).
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Fig. 6. Shows that thrombin-activated microglia exhibited significantly increased phagocytosis of fluorescein labeled latex beads. Argatroban or Y-27632 pretreatment significantly decreased phagocytosis of fluorescein labeled latex beads (p < 0.01, F = 57.075, n = 3/group).
inflammatory mediators such as NO and TNF-␣ expression and decreased phagocytosis in thrombin induced microglial cells compared to non-pretreated ones. These data indicated that both thrombin and ROCK inhibitors can curtail inflammatory response in thrombin-induced activated microglia in vitro. The ROCK pathway may play a vital role in thrombin induced inflammatory response of microglia. In conclusion, in thrombin induced microglia, we observed increased expression of NO and TNF-␣, as well as enhanced phagocytic capacity. Pretreatment with argatroban or Y-27632 reduces NO and TNF-␣ expression as well as microglial phagocytic capacity. Therefore, the ROCK pathway may play a vital role in the inflammatory response in microglia activated by thrombin. This study may offer new ideas to effectively treat patients who have suffered from ICH. Further studies of the pathophysiological functions of ROCK in activated microglia may be proved to be beneficial for clinical applications.
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Disclosures
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None. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81201025, 81271344, and 81271268), Natural Science Foundation of Jiangsu Province (No. BK20131118), the Summit of Six Top Talents Program of Jiangsu Province to Dr. Cui, the Mayor Program of Public Health of Jiangsu Province (No. Z201208), and Jiangsu SpeciallyAppointed Professor Program to Dr. Hua. The authors wish to thank Ping Zeng for statistical analysis assistance and Alex Zacharek for revision assistance. References [1] E. Bernhart, M. Kollroser, G. Rechberger, H. Reicher, A. Heinemann, P. Schratl, S. Hallstrom, A. Wintersperger, C. Nusshold, T. DeVaney, K. Zorn-Pauly, R. Malli, W. Graier, E. Malle, W. Sattler, Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture, Proteomics 10 (2010) 141–158. [2] S.D. Boomkamp, M.O. Riehle, J. Wood, M.F. Olson, S.C. Barnett, The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of
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ROCK mediates the inflammatory response in thrombin induced microglia Guiyun Cui a , Tao Zuo b , Qiuchen Zhao c , Jinxia Hu a , Peisheng Jin d , Hui Zhao e , Jia Jing a , Jienan Zhu a , Hao Chen a , Bin Liu a , Fang Hua a , Xinchun Ye a,∗ a
Department of Neurology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province, China Department of Clinical Medicine, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China c Department of Clinical Medicine, Xuzhou Medical College, Xuzhou, Jiangsu Province, China d Department of Plastic Surgery, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province, China e Department of Neurology, Xuzhou Central Hospital, Xuzhou, Jiangsu Province, China b
h i g h l i g h t s • • • • •
Increased expression of ROCK, NO and TNF-␣ in thrombin induced microglia was found. Enhanced phagocytosis in thrombin induced microglia was also found. Argatroban or Y-27632 pretreatment decreased ROCK, NO and TNF-␣ expression. Argatroban or Y-27632 pretreatment reduced phagocytosis in thrombin induced microglia. ROCK may regulate the inflammatory response in thrombin induced microglia.
a r t i c l e
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Article history: Received 24 May 2013 Received in revised form 25 August 2013 Accepted 27 August 2013 Keywords: Inflammation ROCK Microglia Argatroban Y-27632
a b s t r a c t To investigate whether the ROCK pathway is involved in thrombin-induced microglial inflammatory response, thrombin-induced microglia were pretreated with the thrombin inhibitor argatroban or a ROCK inhibitor Y-27632. Microglial inflammatory response was evaluated by phagocytosis of fluorescein labeled latex beads analyses and inflammatory mediators’ expression such as nitric oxide (NO) and tumor necrosis factor-alpha (TNF-ɑ). Compared to non-induced microglia, thrombin-induced microglia show significantly enhanced phagocytotic capacity and increased ROCK, NO and TNF-ɑ expression. Pretreatment of thrombin-induced microglia with argatroban or Y-27632 significantly decreased phagocytotic capacity and reduced ROCK, NO and TNF-␣ expression. Therefore, the ROCK pathway may play a vital role in the mechanisms by which thrombin induces microglia in the inflammatory response. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Intracerebral hemorrhage (ICH) is a devastating disease accounting for 10–15% of all strokes. Clinical advances in ICH have been restricted by a poor understanding of the mechanisms underlying brain injury after hemorrhage [4]. Inflammatory activation plays a crucial role in the pathophysiological mechanisms of ICH, exerting deleterious effects on the progression of ICH-induced secondary brain injury [35]. Therefore, investigation of inflammatory activation under conditions of ICH is warranted.
∗ Corresponding author at: Department of Neurology, The Affiliated Hospital of Xuzhou Medical College, No. 99 West Huaihai Road, Xuzhou 221006, Jiangsu Province, China. Tel.: +86 516 85802129; fax: +86 516 85802129. E-mail address: [email protected] (X. Ye). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.08.065
Thrombin, a blood-derived serine protease, is essential to blood coagulation and is abundantly present in hematoma. Uncontrolled activation of thrombin-induced inflammation after ICH has been reported to be a central mechanism for secondary injury in ICH [15]. Our previous data also indicated that both PKC alpha and PKC delta play important roles in thrombin-induced brain injury after ICH [8]. However, the mechanisms by which thrombin activates microglia in the progression of inflammatory response remain unclear. Rho-associated protein kinase (ROCK) is a kinase belonging to the AGC family of serine–threonine kinases [23]. The ROCK pathway has been implicated in numerous inflammatory diseases such as rheumatoid arthritis [18], atherosclerosis [10,25], asthma [17], multiple sclerosis [31], and disorders of the central nervous system [24,29]. ROCK appears to also serve as a mediator of thrombin-induced endothelial barrier dysfunction [3,6,16]. However, whether the ROCK pathway is involved in thrombin-induced microglial inflammatory response has not been investigated.
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In the present study, we investigated whether the ROCK pathway is involved in thrombin-induced microglial inflammatory response. To this end, we explored the influence of pretreatment with the specific thrombin inhibitor, argatroban or the ROCK inhibitor Y-27632, in the response of microglia to thrombin. The inflammatory response, induced by thrombin, was evaluated by assessing the phagocytosis of fluorescein labeled latex beads and the expression of the inflammatory mediators, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-a). Molecular mechanisms underlying activated microglia induced by thrombin are described.
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were subjected to Western blot analysis, as described previously [7,40]. The following primary antibodies were used: anti--actin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-ROCK (1:500; Abcam, Cambridge, MA). For immunofluorescence [41], cells growing on coverslips were fixed and treated with 0.1% TritonX100, incubated with anti-ROCK (1:500; Abcam, Cambridge, MA) followed by a FITC-conjugated secondary antibody. The absence of primary antibody was the negative control. Samples were examined by a laser scanning confocal microscope. The experiments were done with 3 different cell culture preparations. The data were calculated as a percentage of fluorescence area in each field.
2. Materials and methods All experiments were conducted in accordance with the standards and procedures of the American Council on Animal Care and Institutional Animal Care and Use Committee of Xuzhou Medical College. 2.1. Primary microglial cultures Primary microglial cultures were isolated from the cortex of neonatal SD rats less than 72 h old, as described previously [36]. Briefly, the cortical brain tissue was isolated from neonatal SD rats and the isolated cells were plated on poly-l-lysine-coated culture flasks at the density of 1.0 × 105 cells/cm2 . After 14 days, the mixed microglia/astrocyte cultures were digested with trypsin at 37 ◦ C. After digestion for 20 min, the remaining adherent cells in the flask are microglia.
2.6. Quantification of TNF-˛ release The release of TNF-␣ in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). Supernatants (detection limit: 5 pg/ml) were placed on ELISA kit strips (Boster, Wuhan). Sandwich ELISA was then performed according to the manufacturer’s instructions. 2.7. Nitrite assay The level of NO was evaluated by Nitrite assay as described previously [13]. In brief, cell culture supernatant was centrifuged at 1000 rpm for 5 min, aliquots of 100 l were incubated with 100 l Griess reagent (0.1% naphthylthylene, 1% sulfanilamide in 2.5% phosphoric acid), and the absorption at 450 nm was measured using a MR5000 ELISA reader.
2.2. The purity of the primary microglial cultures 2.8. Fluorescein labeled latex beads analysis To determine the purity of the primary microglial cultures, microglia were plated on 8-chamber poly-d-lysine coated glass slides. After blocking with 10% BSA in PBS for 1 h, the cells were incubated overnight at 4 ◦ C with IBA-1 goat polyclonal antibody (1:100, Abcam, Cambridge, MA), followed by incubation with FITCconjugated secondary antibody (1:200, ZSGB-Bio, Beijing) for 2 h at 37 ◦ C [34], then we randomly chose 5 visual fields (nearly 20 cells per visual field) to count the percentage of the positive cells by confocal microscopy. 2.3. The optimal dose and time for thrombin to activate microglia To figure out the optimal dose and time for thrombin to activate microglia, microglia were treated with thrombin with different concentrations (0, 10, 20, 40, and 60 U/ml) for 6 h and with different time (3, 6, 12, and 24 h). ROCK expression was evaluated among them. 2.4. Experiment groups To investigate whether ROCK mediates the inflammatory response in thrombin induced microglia. Microglia were randomly assigned to different groups and were treated with thrombin with optimal concentration for the optimal time (n = 3/group): (1) thrombin group: microglia were incubated with thrombin; (2) argatroban group (25 U/ml): microglial cells were pretreated with argatroban (Mitsubishi Pharma, GuangZhou) for 30 min and then treated with thrombin; (3) Y-27632 group (10 mol/L): microglial cells were pretreated with Y-27632 (≥95% by HPLC, Merck, USA) for 30 min and then treated with thrombin. 2.5. Western blotting and immunofluorescence ROCK expression levels in the microglia were analyzed by Western blotting and immunofluorescence. Equal amounts of cell lysate
Microglial phagocytotic activity was measured by the uptake of FITC (fluorescein isothiocyanate) labeled latex beads (Nanozymics, Wuxi) [33]. In brief, culture specimens were exposed to FITC labeled latex beads for 2 h at 37 ◦ C. Surface bound beads were removed by vigorous washing with PBS, then cells were stained with DiI (Molecular probe Inc., USA) for 5 min. The experiments were done with 3 different cell culture preparations. The data were calculated as number of positive cells in each field. 2.9. Statistical analysis The homogeneity of variances was assumed to hold for all measurements by using Levene test. One-way analysis of variance (ANOVA) was performed for ROCK expression, NO and TNF-␣ expression, fluorescein labeled latex beads analyses; the analyses started with the testing of overall group effect, followed by a post hoc test of least significant difference (LSD) for multiple comparison if the overall group effect was significant. The significance level was set to 0.05 in the paper and all the data analyses were performed using SPSS version 13.0 (SPSS Inc., Chicago IL). The data are presented as mean ± SD. 3. Results 3.1. Purity of primary microglial cells isolation To determine the purity of the cultures, a subset of cells was seeded onto multi-chamber glass slides for characterization via immunofluorescence (Fig. 1). Using commonly employed cell-type specific antibodies, the percentage of microglia (Iba-1+) present in the microglial cultures was determined. Quantification showed that primary microglial cell cultures were over 95% Iba-1+ microglia with few astrocytes remaining (Fig. 1).
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Fig. 1. Shows microglia were identified using anti-Iba-1 primary and FITCconjugated secondary antibodies (green, DAPI, blue; scale bar = 100 m). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.2. The optimal dose and time for thrombin to induce microglia To investigate the optimal concentration and induction time of thrombin induced activation of microglial cells, Rho-kinase expression was assessed by Western blot analysis. Fig. 2A shows that various concentrations of thrombin can significantly increase ROCK expression in microglial cells when compared to non-induced microglial cells (p < 0.05, n = 3/group). It also shows that the optimal concentration of thrombin for induction is 20 U/ml. Fig. 2B shows that thrombin induction at various time points can significantly increase ROCK expression in microglial cells when compared to non-induced microglial cells (p < 0.05, n = 3/group), though the optimal induction time is 24 h.
Fig. 3. Shows that ROCK expression was significantly increased in the thrombin treated group, and that argatroban or Y-27632 pretreatment significantly decreased ROCK expression (p < 0.01, F = 84.360, n = 3/group) (T = Thrombin, A = argatroban).
n = 3/group). Fig. 4 shows that immunofluorescence data were consistent with Western blotting analysis (p < 0.05, n = 3/group).
3.3. ROCK expression
3.4. NO and TNF-˛ expression
To elucidate whether ROCK is involved with thrombin-induced microglia in the progression of inflammation, a thrombin inhibitor argatroban or a ROCK inhibitor Y-27632 was employed to assess the microglial ROCK expression by means of Western blotting analysis. Fig. 3 shows that ROCK expression was significantly increased in thrombin-treated group compared with control group. Argatroban or Y-27632 pretreatment significantly decreased ROCK expression in microglia compared to non-pretreated microglial cells (p < 0.05,
To investigate the inflammatory activation of microglia, TNF-␣ expression was assessed by ELISA and NO expression was evaluated by Nitrite assay. Fig. 5A and B shows that microglia exhibit significantly increased NO and TNF-␣ expression in thrombintreated group compared to cells in the control group (p < 0.05, n = 3/group). Argatroban or Y-27632 pretreatment significantly decreased NO and TNF-␣ expression in microglial cells compared to non-pretreated microglial cells (p < 0.05, n = 3/group).
Fig. 2. A shows the dose dependence of ROCK expression in thrombin-induced microglia (p < 0.01, F = 78.153, n = 3/group). B shows the time dependence of ROCK expression in thrombin-induced microglia (p < 0.01, F = 32.462, n = 3/group).
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Fig. 4. Shows ROCK expression was evaluated by immunofluorescence. Thrombin-induced microglia exhibited enhanced ROCK expression, and argatroban or Y-27632 pretreatment significantly decreased ROCK expression (p < 0.01, F = 109.801, n = 3/group).
3.5. Phagocytosis of fluorescein labeled latex beads analyses To evaluate phagocytosis of thrombin-activated microglia, phagocytosis of fluorescein labeled latex beads analyses was performed. Fig. 6 shows that phagocytosis was significantly increased in the thrombin-induced microglia compared with non-induced microglia. Argatroban or Y-27632 pretreatment exhibited significantly decreased phagocytosis in the thrombin-induced cells (p < 0.05, n = 3/group). 4. Discussion In this study, we found that ROCK expression increased significantly in thrombin-activated microglia when compared to non-activated microglia. Inflammation mediators NO and TNF-␣ expression were also significantly increased in activated microglia. In addition, phagocytosis of fluorescein labeled latex beads were significantly enhanced in thrombin-induced microglia. Pretreatment of thrombin-induced microglia with argatroban or Y-27632 significantly reduced ROCK, NO and TNF-␣ expression. Pretreatment also significantly reduced microglial phagocytosis. Therefore, ROCK may play a vital role in the mechanisms by which thrombin activates microglia. NO is also reported to be implicated in the pathogenesis of inflammation. It exerts either an anti-inflammatory effect under normal physiological conditions or a pro-inflammatory role under abnormal situations [28]. TNF-␣ is a proinflammatory cytokine which acts as a central regulator of inflammation [21]. It has been reported that NO and TNF-␣ are involved in the progression of inflammation by activated macrophages, which release them in high concentrations [28,39]. Phagocytosis is the cellular process of engulfment and destruction of invading microorganisms. It is also required for the clearance of apoptotic cells, which is essential for tissue homeostasis and remodeling [5]. Calcium influx plays a positive role in the regulation of phagocytic activity [30]. We found that thrombin-induced microglia significantly increased NO
and TNF-␣ expression as well as enhanced microglial phagocytosis compared to non-induced microglia. Thus, NO and TNF-␣ may also be implicated in the progression of inflammation in microglia which is activated by thrombin. In addition, thrombin may also enhance microglial phagocytic capacity. ROCK is a kinase belonging to the AGC family of serine–threonine kinases. The ROCK pathway is implicated in many cellular functions and up-regulates various molecules that accelerate inflammation [20,27]. Abnormal activation of the ROCK pathway has been observed in various disorders of the central nervous system [2,12,14,26,37]; lesions of the brain and spinal cord activate ROCKs, which have been revealed as major mediators of neuroinflammatory responses [24,29]. It has also been shown that during activation of inflammatory cells ROCK induces changes in the actin cytoskeleton that results in process retraction, cell spreading as well as changes in cell motility characteristics of activation [1,32]. In the present study, we found that ROCK expression was significantly enhanced in thrombin-induced microglia compared to non-induced microglia. Thus, the ROCK pathway may be involved in the thrombin induced inflammatory response of microglia. Y-27632, as a specific inhibitor of ROCK which specifically binds to the ROCK family of kinases and inhibits their kinase activity, has been reported to be involved in cell motility [9], cellular differentiation [38] and cell death [19]. In this study, we found that Y-27632 can decrease ROCK expression in thrombin-induced microglia. The decreased ROCK expression in microglia may be attributed to Y27632 induced cell death. Therefore, Y-27632 may also curtail ROCK’s expression more than inhibit its activity. Argatroban, as a synthetic small molecule derived from larginine, is a direct thrombin inhibitor that binds reversibly to, and inhibits both soluble and clot-bound thrombin [11]. Due to its anticoagulant effect, argatroban has been used in the treatment of ischemic stroke, acute coronary syndrome, and hemodialysis [22]. In this study, we found that either argatroban or Y-27632 pretreatment inhibits inflammation, as demonstrated by reduced
Fig. 5. A and B show that microglia significantly increased NO and TNF-␣ expression in thrombin treated group compared to cells in the control group. Argatroban or Y-27632 pretreatment significantly decreased NO and TNF-␣ expression (p < 0.01, n = 3/group. A, F = 27.395; B, F = 122.480).
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Fig. 6. Shows that thrombin-activated microglia exhibited significantly increased phagocytosis of fluorescein labeled latex beads. Argatroban or Y-27632 pretreatment significantly decreased phagocytosis of fluorescein labeled latex beads (p < 0.01, F = 57.075, n = 3/group).
inflammatory mediators such as NO and TNF-␣ expression and decreased phagocytosis in thrombin induced microglial cells compared to non-pretreated ones. These data indicated that both thrombin and ROCK inhibitors can curtail inflammatory response in thrombin-induced activated microglia in vitro. The ROCK pathway may play a vital role in thrombin induced inflammatory response of microglia. In conclusion, in thrombin induced microglia, we observed increased expression of NO and TNF-␣, as well as enhanced phagocytic capacity. Pretreatment with argatroban or Y-27632 reduces NO and TNF-␣ expression as well as microglial phagocytic capacity. Therefore, the ROCK pathway may play a vital role in the inflammatory response in microglia activated by thrombin. This study may offer new ideas to effectively treat patients who have suffered from ICH. Further studies of the pathophysiological functions of ROCK in activated microglia may be proved to be beneficial for clinical applications.
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Disclosures
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None. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81201025, 81271344, and 81271268), Natural Science Foundation of Jiangsu Province (No. BK20131118), the Summit of Six Top Talents Program of Jiangsu Province to Dr. Cui, the Mayor Program of Public Health of Jiangsu Province (No. Z201208), and Jiangsu SpeciallyAppointed Professor Program to Dr. Hua. The authors wish to thank Ping Zeng for statistical analysis assistance and Alex Zacharek for revision assistance. References [1] E. Bernhart, M. Kollroser, G. Rechberger, H. Reicher, A. Heinemann, P. Schratl, S. Hallstrom, A. Wintersperger, C. Nusshold, T. DeVaney, K. Zorn-Pauly, R. Malli, W. Graier, E. Malle, W. Sattler, Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture, Proteomics 10 (2010) 141–158. [2] S.D. Boomkamp, M.O. Riehle, J. Wood, M.F. Olson, S.C. Barnett, The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of
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