ANTIPYRETIC ACTION OF DEXAMETHASONE ON
EGTAZIC ACID-INDUCED FEVER IN RABBITS*

WANG　HUA-DONG^**,　WANG　YAN-PING,　LI　CHU-JIE,
LU　DA-XIANG,　YAN　LIANG,　QI　REN-BIN
(Department of Pathophysiology, Medical College of Jinan University, Guangzhou, 510632)

ABSTRACT　　The purpose of the present study was to investigate whether the antipyretic effect of dexamethasone (DEX) delivered by intravenous injection (iv) on intracerebroventricularly (icv) administered egtazic acid-induced febrile response is relevant to the changes in cytosolic free calcium concentration of the hypothalamus. The colon temperatures were measured by a thermistor and the cytosolic free calcium concentration ([Ca²⁺]_i) in dissociated brain cells was measured by Fura 2-AM. The results demonstrated that the pyretic action of egtazic acid (0.6 μmol, icv) was markedly inhibited by DEX (5 mg/kg,iv), but DEX (60～120 μmol/L) did′t affect [Ca²⁺]_i in dissociated hypothalamus cells. Actinomycin D, which interferes with gene transcription (3 nmol, icv), completely abolished the antipyretic action of DEX on egtazic acid-induced fever. These findings suggest that the antipyretic action of DEX on egtazic acid-induced fever is related to the activation of certain gene expression in the brain,but not to the changes of transmembrane calcium ion current in hypothalamus neurons.
Key words: fever; egtazic acid; glucocorticoid; actinomycin D; calcium; dexamethasone

地塞米松对家兔乙二醇双(2-氨基乙醚)
四乙酸性发热的解热作用^*

王华东　王彦平　李楚杰　陆大祥　颜　亮　戚仁斌

摘　　要

    本文用脑室灌注和Fura-2 测定细胞内游离钙技术观察了地塞米松 (dexamethasone, DEX) 对家兔乙二醇双(2-氨基乙醚)四乙酸性发热效应和下丘脑细胞内游离钙浓度([Ca²⁺]_i)的影响, 借此深入探讨地塞米松解热作用的中枢机制。 结果发现: 脑室灌注乙二醇双(2-氨基乙醚)四乙酸(0.6 nmol)引起家兔结肠温度明显升高, 静脉注射地塞米松(5 mg/kg)显著抑制家兔乙二醇双(2-氨基乙醚)四乙酸性发热, 地塞米松(60～120 μmol/L)并不影响下丘脑细胞内[Ca²⁺]_i, 而事先脑室灌注抑制基因转录的放线菌素D(3 nmol)则完全取消了地塞米松对乙二醇双(2-氨基乙醚)四乙酸性发热的解热作用。 这些结果提示: 地塞米松显著抑制家兔乙二醇双(2-氨基乙醚)四乙酸性发热, 其机制与地塞米松激活脑内某些基因的表达有关, 而与下丘脑神经细胞跨膜钙离子流无关。
关键词: 发热； 糖皮质激素； 钙； 放线菌素; 地塞米松
学科分类号: R364.6

　　It is well established that glucocorticoids have an antipyretic action on fever induced by a variety of pyrogens^[1～3]. However, cellular and molecular mechanisms by which glucocorticoids inhibit fever have not been precisely defined. Although reduction of fever by glucocorticoids has generally been attributed to inhibition of cytokine and prostaglandin (PG) synthesis^[2～5], the antipyretic effects of these glucocorticoids on the responses to PGF_2α and interleukin-8 (IL-8)^[3,6] and IL-8-induced fever is independent of the action of PGs^[7]. It has been demonstrated that corticotropin releasing hormone (CRH) mediates the febrile responses to IL-6, IL-8, IL-1_β and PGF_2α ^[8] and glucocorticoids effectively inhibit the synthesis and release of CRH^[9], so that antipyretic action of glucocorticoid may result from inhibition of synthesis and /or release of CRH^[3]. However, this hypothesis is unable to explain why lipocortin, which mediates many of the glucocorticoid actions, significantly attenuates the pyrogenic effect of CRH^[7]. Furthermore, Coelho et al. showed that dexamethasone (DEX) has a significant antipyretic action even when this glucocorticoid is administered 1 h after the pyrogenic stimulation^[3]. It is inferred that other mechanisms may be involved in DEX antipyresis. Previous studies indicate that the ratio of Ca²⁺ and Na⁺ ions in the hypothalamus is the physiological basis for maintenance of the level of body temperature^[10], but the effect of DEX on egtazic acid-induced fever has not been investigated. Therefore, the present study was undertaken to investigate the effect of DEX on egtazic acid-induced fever and cytosolic free calcium concentration ([Ca²⁺]_i) in dissociated hypothalamus cells in rabbits.

1　MATERIALS AND METHODS
1.1　Animals　　New Zealand white rabbits of both sexes weighing 2.5±0.5 kg were used. Animals were caged individually and had access to food and water ad libitum. To minimize the stress response caused by handling during experimentation, animals were confined in stocks at room temperature of 20～22℃. At the same time, a catheter mimicking the thermistor probe was inserted 10 cm into the colon of the rabbit and fixed on the root of the tail for 6 h/d of 3 d.
1.2　Intracerebroventricular cannula　　At least 5 d before experimentation, guide cannulae were stereotaxically implanted into the lateral ventricle of rabbits anesthetized with pentobarbitone sodium (iv, 30 mg/kg) as previously described^[11]. The lateral ventricle infusion was controlled by a syringe pump (HL-2 Type, Shanghai Huxi Instruments Factory).
1.3　Body temperature measurement　　Colon temperatures were measured in lightly restrained rabbits by insertion of catheter-mounted thermistor probe 10 cm beyond the anal sphincter, the thermistors were fed into a telethermometer (ST-1 Type, Shanghai Medical Instruments Factory). Temperature records were taken at 10 min intervals until the experiment was over.
1.4　Chemicals　　The artificial cerebrospinal fluid (ACSF) was prepared routinely^[12]. Ethylene glycol bis (2-aminoethyl-ether) tetraacetic acid (Egtazic acid, Feinbiochemica Heidelberg, USA) was dissolved in sterile ACSF at a concentration of 4 mmol/L (pH 7.35). Dexamethasone 21-phosphate disodium (DEX) was dissolved in sterile pyrogen-free 0.9% sodium chloride (normal saline, NS) for injection. Actinomycin D (AM) was dissolved in ACSF at a concentration of 0.03 mmol/L. Fura 2-acetoxymethyl (Fura 2-AM), triton X-100, trypan blue, DEX and AM were purchased from Sigma Chemical.
1.5　Measurement of [Ca²⁺]_i in dissociated hypothalamus cells 　　The procedure was similar to that of previous studies^[12,13]. Rabbits were decapitated rapidly, the hypothalamus was removed and placed in 10 ml sterile ice-cold HEPES-buffered Hanks, pH 7.4, containing in mmol/L: HEPES 20, NaCl 137, KCl 5.0, KH₂PO₄ 0.4, Na₂HPO₄ 0.6, NaHCO₃ 3.0, CaCl₂ 1.3, MgSO₄ 0.4, MgCl₂ 0.5, and glucose 5.6. Meninges and blood vessels were meticulously removed. The hypothalamus was transferred to a glass beaker and minced. The minced tissue was placed into glass tubes containig 1 ml of 0.125% trypsin in D-Hanks and incubated at 37℃ for 20 min. After trypsinization, 1 ml of Dulbecco′s modified Eagle′s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum was added to the tubes. The dissociated cells were collected with filters, and were washed with Hanks and then centrifuged at 1?500 r/min for 5 min. Supernatants were decanted and the cells resuspended in a volume of DMEM to give a concentration of approximately 2×10⁹ cells/L. The trypan blue exclusion showed more than 90% cellular viability, which was not altered when samples were tested at random for trypan blue staining at the end of an experiment. The cell suspensions with Fura 2-AM at a final concentration of 5 μmol/L were kept in a humidified incubator with 95% air and 5% CO₂ at 37℃ for 50 min. The fluorescence spectrophotometer (RF-5000, Japan) was used for fluorescence determinations. Basal and drugs-stimulated peak values were obtained, 0.1% triton X-100 was added to determine the maximun fluorescence, and the minimun fluorescence was calculated using 4 mmol/L egtazic acid.
1.6　Experimental protocols
1.6.1　Determination of the effect of DEX on egtazic acid-induced fever in the rabbit. New Zealand white rabbits were divided into 3 groups, ACSF+NS, EGTA+NS and EGTA+DEX groups. 20 min after animals were infused intracerebroventricularly (icv) with 150 μl of ACSF or egtazic acid (EGTA), NS (1 ml/kg) or DEX (5 g/L, 1 ml/kg) was injected via marginal vein.
1.6.2.　Examination of the effect of DEX on [Ca²⁺]_i in dissociated hypothalamus cell.
1.6.3.　Determination of the effect of actinomycin D on DEX antipyresis. 15 min after the separate groups of animals were infused (icv) 150 μl of ACSF or egtazic acid, rabbits were injected (icv) with 100 μl of actinomycin D or ACSF, and DEX (5 mg/kg) or NS was given intravenously (iv) 5 min after treatment with actinomycin D or ACSF.
1.7　Procedures for prevention of contamination　　The laboratory and all materials were routinely sterilized using the method previously described^[11,12], experiments were performed under aseptic conditions.
1.8　Data analysis　　For statistical analysis of body temperature response, the febrile response was represented by the mean thermal response curve and the 6 h thermal response index (TRI₆). All values were expressed as ±s. Statistical differences were determined by an unpaired student′s t　test and differences were considered to be significant if P<0.05.

2　RESULTS
　　As can be seen in Fig.1 and Table 1, the ranges of colon temperature changes in the ACSF+NS group were less than 0.3℃.Infusion (icv) of egtazic acid caused a rapid rise in colon temperature compared with the ACSF group (P<0.001). Rabbits treated with DEX (5 mg/kg, iv) 20 min after infusion of egtazic acid developed fever, which was significantly milder compared with rabbits treated with an equal volume of NS (iv).

Table　1　Thermal response index between 0 and 360 min (TRI₆) after infusion (icv) shown in Fig.1

Group	n	TRI6/℃^.h
ACSF+NS	5	0.43±0.39
EGTA+NS	8	9.54±1.75^*
EGTA+DEX	9	7.52±1.53^**

^*　P<0.001 vs ACSF+NS group. ^**　P<0.05 vs EGTA+NS group. (±s).

Fig.1　Changes in colon temperature of rabbits in ACSF+NS, EGTA+NS and EGTA+DEX groups
DEX was administered intravenously 20 min after the infusion (icv) of egtazic acid (arrow). ^*P<0.05 vs EGTA+NS group.

　　Resting [Ca²⁺]_i in these dissociated hypothalamus cells was 140.1±14.0 nmol (n=6), and 50 mmol KCl stimulated a rapid increase from 140.1±14.0 nmol to 210.0±14.0 nmol. As shown in Fig.2, DEX did not induce any increase in [Ca²⁺]_i in dissociated hypothalamus cells.

Fig.2　Effect of DEX on the fluorescence ratios (340 nm/380 nm) in Fura-2-filled hypothalamus cells
↓: addition of DEX (60 nmol); : addition of triton X-100.

No significant differences were found between TRI₆ of rabbits infused with actinomycin D and those with ACSF. Rabbits in EGTA+ACSF group developed fever, and actinomycin D (icv, 15 min after infusion of egtazic acid) did not markedly affect egtazic acid-induced fever (Table 2).

　

Table　2　TRI₆ between 0 and 360 min of rabbits after infusion (icv) of ACSF, ACSF+AM, EGTA+ACSF and EGTA+AM respectively

Group	n	TRI6/℃^.h
ACSF	4	0.58±0.22
ACSF+AM	4	0.76±0.15^*
EGTA+ACSF	7	9.05±1.17
EGTA+AM	6	8.54±0.93^**

^*P>0.05 vs ACSF group. ^**　P>0.05 vs EGTA+ACSF group. (±s).

　　As demonstrated in Table 3, DEX (iv) markedly attenuated egtazic acid-induced fever. Actinomycin D (3 nmol, icv) administered 5 min before treatment with DEX completely abolished antipyretic action of DEX, actinomycin D took effect 225 min after icv administration. There was no significant difference between TRI₆ of EGTA+ACSF+NS group and that of EGTA+AM+DEX group.

Table　3　TRI₆ between 0 and 360 min of rabbits after infusion (icv) in EGTA+ACSF+NS, EGTA+AM+DEX and EGTA+ACSF+DEX groups

Group	n	TRI6/℃^.h
EGTA+ACSF+NS	7	9.84±1.65*
EGTA+AM+DEX	6	10.27±2.84^*
EGTA+ACSF+DEX	8	7.31±1.49

*P<0.05 vs EGTA+ACSF+DEX group. (±s).

3　DISCUSSION
　　The hypothesis that the antipyretic effect of glucocorticoids results from inhibition of cytokine, PG and CRH synthesis can not explain an antipyretic action even after delayed treatment with glucocorticoids. Coelho et al. speculated that very rapid actions of glucocorticoids are probably the result not of the changes in the synthesis of protein, but rather of other mechanisms such as the proposed direct interactions of glucocorticoids with membrane phospholipid, or changes in transmembrane ion currents^[3].
　　In the present work, it was first demonstrated that DEX markedly inhibited the febrile response induced by administration (icv) of egtazic acid. Although some studies suggested that the effect of Ca²⁺ ions on thermoregulation appears to be due to a non-selective depression of neuronal activity^[10], the results in our earlier study show that egtazic acid-induced decrease in concentration of cellular Ca²⁺ in the hypothalamus and the subsequent increase in hypothalamus cAMP content, which in turn alters the “set-point”, may be an important link in the pathogenesis of egtazic acid-induced fever^[12]. Previous studies have shown that glucocorticoid binding specifically in the anterior hypothalamus acts to downregulate or inhibit endotoxin-induced fever^[14,15]. Therefore, inhibition of egtazic acid-induced fever by DEX may be related to a direct action between DEX and neurons within the thermoregulatory center.
　　An increasing amount of evidence shows that some of the effects of steroids can not be explained by a genomic mode of action on the target cells^[16,17]. Shao-Ying HUA and Yi-Zhang CHEN reported that glucocorticoid can hyperpolarize the membrane potential of guinea pig ganglion neurons through its neuronal membrane receptor in vitro with a latency of less than 2 min, and the steroid-induced hyperpolarization was accompanied by a change in the input resistance of the cell, indicating an involvement of some kinds of ion channels in the action of glucocorticoid^[17]. However, the results presented in Fig.2 demonstrate DEX did not influence [Ca²⁺]_i in the dissociated hypothalamus cells. Thus, the possibility that the suppression of egtazic acid-induced fever by DEX is ascribed to the changes in transmembrane calcium ion currents may be excluded. On the other hand, the antipyretic action of DEX on egtazic acid-induced fever took place 220 min after administration of DEX (Fig.1), thus it appears that the antipyretic action of DEX may be related to some genomic mechanisms. Furthermore, pretreatment (icv) with actinomycin D, which interferes with gene transcription, completely abolished the antipyretic action of DEX on egtazic acid-induced fever, and neither normal body temperature nor egtazic acid-induced fever were influenced by administration (icv) of actinomycin D. These results indicate that DEX inhibits centrally egtazic acid-induced fever via activating certain gene expressions.
　　In conclusion, the present results support the hypothesis that the antipyretic action of glucocorticoids on egtazic acid-induced fever is the result of its direct effect on the function of neurons within the brain via activating certain gene expressions, but may be independent of the changes in transmembrane calcium ion currents in hypothalamus neurons. Therefore, together with the data of the previous studies^[3], it may be likely inferred that the antipyretic action of glucocorticoids on pyrogen-induced fever results from its direct action on the thermoregulatory center via gene transcription in addition to the inhibition of cytokine, PG and CRH synthesis.

?  The authors wish to thank Prof. HU Chao-Feng for her excellent technical assistance.

*　Work supported by the National Natural Science Foundation of China (No.39700055)
**　To whom correspondence should be addressed. Tel: 020-85220253.
^*国家自然科学基金资助 (No.39700055)

作者单位:王华东　王彦平　李楚杰　陆大祥　颜　亮　戚仁斌　暨南大学医学院病理生理教研室，广州　510632

REFERENCES

　[1]　Abul H, Davidson J, Milton AS, et al. Dexamethason pre-treatment is antipyretic toward pol～y～inosinic: polycytidylic acid, lipopolysaccharide and interleukin-1/endogenous mechanisms mediate antipyretic action of pyrogen. Naunyn-Schmiederbergs Arch Pharmacol, 1987, 335: 305～309.
　[2]　Davidson J, Milton AS， Rotondo D. A study of the pyrogenic actions of interleukin-1α and interleukin-1β: interactions with a steroidal and a non-steroidal anti-inflammatory agent. Br J Pharmacol, 1990, 100: 542～546.
　[3]　　Coelho MM, Luheshi G, Hopkins SJ, et al. Multiple mechanisms mediate antipyretic action of glucocorticoid. Am J Physiol, 1995, 269: R527～R535.
　[4]　　Staruch MJ, Wood DD. Reduction of serum interleukin-1-like activity after treatment with dexamethasone. J Leukocyte Biol, 1985, 37: 193～207.
　[5]　　Flower RJ. Lipocortin and the mechanism of action of the glucocorticoids. Br J Pharmacol, 1988, 65: 987～1015.
　[6]　　Coelho MM, Pela ZR, Rothwell NJ. Dexamethasone inhibits the pyrogenic activity of prostaglandin F_2α, but not prostaglandin E₂. Eur J Pharmacol, 1993, 283: 391～394.
　[7]　Strijbos PJ, Hardwick AJ, Relton JK, et al. Inhibition of central actions of cytokines on fever and thermogenesis by lipocortin-1 involves in CRF. Am　J Physiol, 1992, 263: E632～E636.
　[8]　　Rothwell NJ, Cooper A. Cytokines, CRF and BAT in fever. In: Bartfai T, Ottoson D, eds. Neuro-Immunology of Fever. New York: Pergamon Press, 1992, 257～265.
　[9]　　Harbuz MS, Lightman SL. Stress and the hypothalamo-pituitary-adrenal axis : acute, chronic and immunological activation. J Endocrinol, 1992, 134: 327～339.
　[10]　　Dascombe MJ. The pharmacology of fever. Prog Neurobiol, 1985, 25: 327～373．
　[11]　　Zhang Y (张 怡), Li CJ (李楚杰). The effects of perfusion of lateral ventricle with CaCl₂ on the febrile response and cAMP content in plasma and cerebrospinal fluid during LP-induced fever. Sci Chin (B)(中国科学), 1991, 34 (3): 317～326.
　[12]　　Wang HD (王华东), Li CJ (李楚杰), Qu Y (屈 洋)， et al. The role of hypothalamus [Ca²⁺]_i and cAMP in EGTA-induced fever of rabbits. Chin J Pathophysiol　(中国病理生理学杂志), 1996, 12 (4): 402～405.
　[13]　　Dildy JE, Leslie SW. Ethanol inhibits NMDA-induced increases in free intracellular Ca²⁺ in dissociated brain cells. Brain Res, 1989, 499: 383～387.
　[14]　　Morrow LE, Mcclellan JL, Klir JJ, et al. The CNS site of glucocorticoid negative feedback during LPS- and psychological stress-induced fevers. Am J Physiol, 1996, 271: R732～R737.
　[15]　　Willies GH, Woolf CJ. The site of action of corticosteroid antipyresis in the rabbit. J Physiol, 1980, 300: 1～6.
　[16]　　Feldman S, Dafny N. Changes in single cell responsiveness in the hypothalamus in cat following cortisol administration. Brain Res, 1970, 20: 369～371．
　[17]　Hua SY　(华少莹), Chen YZ　(陈宜张). Membrane receptor-mediated electrophysiological effects of glucocorticoid on mammalian neurons. Endocrinology, 1989, 124: 687～691.

Received 1998-06-11　　Revised 1998-09-16