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Progesterone Administration Modulates TLRs/NF-κB Signaling Pathway in Rat Brain after Cortical Contusion
Gang Chen,1 Jixin Shi,1 Wei Jin,1 Lin Wang,1 Weiying Xie,2 Jie Sun,2 and Chunhua Hang1
Departments of 1Neurosurgery and 2Anesthesiology, Jinling Hospital, School of Medicine,Nanjing University, Nanjing, Jiangsu Province, China
Abstract. This study investigated whether progesterone administration modulates toll-like receptors (TLRs) and the nuclear factor-kappa B (NF-κB) signaling pathway in the injured rat brain following traumatic brain injury (TBI). Right parietal cortical contusion was made by a weight-dropping method. Male rats were given 0 or 16 mg/kg injections of progesterone at postinjury hr 1 and 6 and on days 1, 2, 3, 4, and 5. Brain samples were extracted at 5 days after trauma. We measured mRNA expression of TLR2 and TLR4 by reverse-transcriptase polymerase chain reaction (RT-PCR), NF-κB binding activity by electrophoretic mobility shift assay (EMSA), concentrations of interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA), intercellular adhesion molecule-1 (ICAM-1) expression by immunohistochemistry, and brain damage by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The results showed that TBI induces strong up-regulation of TLR2, TLR4, NF-κB, pro-inflammatory cytokines, and ICAM-1 in the pericontusional area. Administration of progesterone following TBI down-regulates the cortical levels of these agents related to the TLRs/NF-κB signaling pathway. After progesterone administration, apoptotic TUNEL-positive cells in the injured brain were significantly decreased. In summary, post-TBI progesterone administration attenuates the TLRs/NF-κB signaling pathway in injured rat brain, and this may be a mechanism whereby progesterone improves the outcome following TBI.
Keywords: progesterone; traumatic brain injury; toll-like receptors; nuclear factor-kappaB
Although traumatic brain injury (TBI) represents a significant public health problem in the world, there are currently no treatments that improve clinical outcome measures [1,2]. Recently, several clinical and experimental studies have shown that progesterone plays neuroprotective roles in TBI, which include reducing cerebral edema, preventing neuronal loss, and improving cerebral function [3,4]. Following the demonstration that proges-terone is effective in attenuating neurological abnormalities, the next step is to determine how
progesterone mediates its neuroprotective effects. Because progesterone reduces edema after TBI and is considered an immune suppressant due to its role in maintaining pregnancy (eg, preventing immune-mediated rejection of the fetus) , we hypothesized that progesterone might help to promote recovery from TBI by modulating the pathophysiological pathways and signal transcription factors that are related to cerebral inflammation after TBI. Ten mammalian toll-like receptors (TRLs) have been identified by sequence analysis . Among them, TRL2 and TRL4, which are widely expressed in brain, can detect endogenous agonists, such as the degradation products of macromolecules, heat shock protein 60 and 70, products of proteolytic cascades, intracellular components of ruptured cells, and products of genes that are activated by
Address correspondence to Chunhua Hang, M.D., Dept. of Neurosurgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, P.R.China; tel 86 25 8197 3916; fax 86 25 8481 7581; email [email protected].
0091-7370/08/0100-0065. $3.00. © 2008 by the Association of Clinical Scientists, Inc.
Available online at www.annclinlabsci.org
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inflammation . Furthermore, TLR2 and TLR4 have been demonstrated to play an important role in initiating the cerebral inflammation related to stroke, Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease . All TLRs activate a common signaling pathway that culminates in the activation of NF-κB transcription factors and the mitogen-activated protein kinases (MAPKs). As one of the most important downstream molecules in the TRLs signaling pathway, NF-κB is a trans-criptional factor required for the gene expression of many inflammatory mediators, such as interleukin-b (IL-1b), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1) . The aim of the current study was to see if progesterone attenuates the TBI induced activation of TRLs/NF-κB signaling pathway in the peri-contusional area. We hypothesize that the effect of progesterone on modulating the TRLs/NF-κB signaling pathway is a mechanism whereby proges-terone protects neurons, reduces cerebral edema, and promotes behavioral recovery after TBI.
Materials and Methods
Animals. Male Wistar rats (250 to 300 g) were purchased from the Animal Center of the Chinese Academy of Sciences, Shanghai, China. The rats were housed in temperature- and humidity-controlled animal quarters with a 12-hr light/dark cycle. All experimental protocols complied with the Laboratory Animal Care and Use Guidelines of the Medical School of Nanjing University.
Cortical contusion trauma. Following anesthesia with urethane (1000 mg/kg, ip), the rat’s head was fixed in the stereotactic frame. A right parietal craniotomy (diameter 5 mm) was drilled under aseptic conditions 1 mm posterior and 2 mm lateral to the bregma. Trauma was induced by a modification of the Feeney’s weight-drop model  in which a freefalling weight drops onto the exposed intact cranial dura to produce a standardized parietal contusion. A steel rod (weight 40 g) with a flat end diameter of 4 mm was allowed to fall from a height of 25 cm onto a piston resting on the dura. The piston was allowed to compress the brain tissue a maximum of 5 mm. After this procedure, the rats were returned to their cages and the room temperature kept at 23±1°C. Heart rate, arterial blood pressure, and rectal temperature were monitored, and the rectal temperature was kept at 37±0.5°C by physical cooling (ice bag) when required throughout the experiment. Sham-operated control rats were anesthetized and mounted in the stereotaxic apparatus, and their scalps were cut and sutured, but were not trephinated.
Experimental protocol. The experimental groups consisted of sham+vehicle (SV; n = 6), lesion+vehicle (LV; n = 6), and lesion+progesterone (LP; n = 6). Progesterone (4-pregnene-3, 20-dione, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 2-hydroxypropyl-b-cyclodextrin (Sigma-Aldrich); the pro-portion was 20 mg (progesterone):4 ml (2-hydroxypropyl-b-cyclodextrin). Rats of the LP group received injections of 16 mg/kg progesterone (dose volume 3.2 ml/kg) at 1 and 6 hr and 1, 2, 3, 4, and 5 days after the surgery (ip for the first injection and sc for the remaining six). Rats of the SV and LV groups received equal volumes (3.2 ml/kg) of 2-hydroxypropyl-b-cyclodextrin [10,11]. The rats were decapitated 5 days after injury for tissue assays. The surrounding brain tissue of the injured cortex (Fig. 1) was dissected on ice as described in our previous study ; a portion of the tissue was fixed in 10% buffered formalin, the remainder was immediately stored in liquid nitrogen until analysis.
RNA extraction and RT-PCR. The levels of TLR2 and TLR4 mRNA expression were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Total RNA was extracted with TriPure Reagent (Roche Diagnostics, Indiana-polis, IN, USA) according to the manufacturer’s instructions. The cDNA synthesis from the isolated RNA was performed using a reverse transcriptional system. Briefly, 4 µg of total brain RNA was reversely transcribed using 0.5 µg oligo(dT)15 and incubated with 15 U Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) (all from Promega, Madison, WI, USA). The cDNA was amplified by PCR using oligonucleotide primers (Table 1), which amplified rat TLR2, TLR4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA in preliminary experiments. Thereafter, the amplified frag-ments were detected by agarose gel electrophoresis and
Fig. 1. Schematic representation of the cortical contusion area induced by weight-dropping trauma and the studied region surrounding the injured brain.
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visualized by ethidium bromide staining. The intensity of the bands was quantified using Glyko Bandscan software. As a control, GAPDH mRNA was detected in all samples, and TLR2/GAPDH and TLR4/GAPDH product ratios were used as indices of TLR2 and TLR4 mRNA expression.
Nuclear protein extract and electrophoretic mobility shift assay (EMSA). Nuclear protein was extracted and quantified as described previously [13,14]. EMSA was performed using a gel shift assay system (Promega, Madison, WI). NF-κB oligo-nucleotide probe (5’-AGTTGAGGGGACTTTCCCAGGC-3’) was end-labeled with [γ-32P]ATP (Free Biotech, Beijing, China). EMSA was performed as in previous studies [13,14].
Enzyme-linked immunosorbent assay (ELISA). The levels of inflammatory mediators were quantified using specific ELISA kits for rats according to the manufacturers’ instructions (TNF-a from Diaclone Research, France; IL-1b and IL-6 from Biosource Europe SA, Belgium) and previous studies [13,14]. Results were expressed as ng/g protein.
Immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded sections. Rabbit-anti-rat monoclonal antibody to ICAM-1 (diluted 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. Immunohistochemical assays were performed according to our previous study . The number of positive microvessels in each section was counted in 10 microscopic fields (at ×100 magnification) and averaged to determine the number of positively immunostained vessels per field.
TUNEL staining. Formalin-fixed tissue was embedded in paraffin and sectioned at 4 µm with a microtome. The sections were scored for apoptotic cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). An in situ cell death detection kit POD (ISCDD, Boehringer Mannheim, Manheim, Germany) was used according to the kit protocol and our previous study . The extent of brain damage was evaluated by the apoptotic index, which was the average number of TUNEL-positive cells in each section counted in 10 microscopic fields (at ×200 magnification).
Statistics. Data were expressed as mean ± SD. SPSS 12.0 was used for statistical analyses. All data were subjected to one-way ANOVA. Differences between experimental groups were determined by the Fisher’s LSD post-test. Statistical significance was inferred at p <0.05.
RT-PCR for TLR2 and TLR4 mRNA expression. TLR2 and TLR4 mRNAs were expressed at low levels in the rat brains of the SV group. The mRNA expression of TLR2 and TLR4 in the cortex adjacent to the injured site was significantly increased in the LV group compared with that of the SV group (p <0.01). The levels of TLR2 and TLR4 mRNA in the injured brains of the LP group were significantly lower than those of the LV group (p <0.01) (Fig. 2).
EMSA for NF-κB. EMSA autoradiography of NF-κB DNA binding activity of the injured brain samples is shown in Fig. 3. Low NF-κB binding activity (weak EMSA autoradiography) was found in the SV group. Compared to the SV group, NF-κB binding activity in the injured brain was significantly increased (p <0.01) in the LV group. In the LP group, the NF-κB binding activity was significantly down-regulated (p <0.01) in the brain area surrounding the injury site after TBI.
IL-b, TNF-a, and IL-6 in the injured brains. Concentrations of IL-b, TNF-a, and IL-6 were low in rat brains of the SV group (5.47±2.23, 0.53 ±0.22, and 0.20±0.08 ng/g protein, respectively) (Fig. 4). Compared to the SV group, cortical levels of the 3 inflammatory cytokines were greatly induced after TBI. As shown in Fig. 4, progesterone administration after TBI leads to decreased IL-1b, TNF-a, and IL-6 concentrations.
ICAM-1 expression in vessels of injured brain. As shown in Fig. 5A, few ICAM-1-immunostained cerebral microvessels were seen in the SV group. In the LV group, the number of ICAM-1-positive
Table 1. PCR primer sequences.
Target Sense primer Antisense primer Anneaing Cycles Size gene (5’ to 3’) (5’ to 3’) (°C) (N) (bp) TLR2 GCTGTTGCGTTACATCTTGGA GGCTCCGTATTGTTACCGTTT 58 40 137 TLR4 TTGCCTTCATTACAGGGACTT CAGAGCGGCTACTCAGAAACT 58 40 179 GAPDH AAGAAGGTGGTGAAGCAGGC TCCACCACCCTGTTGCTGTA 58 40 203
Progesterone inhibits TLRs/NF-kappaB pathway in traumatic brain injury 67
Fig. 2. TLR2 and TLR4 mRNA expression in the injured brains in the SV group (sham+vehicle) (n = 6), the LV group (lesion+ vehicle) (n = 6), and the LP group (lesion+progesterone) (n = 6). TBI induced a marked increase of TLR2 and TLR4 mRNA expression in the rat brain surrounding the injury site compared to the SV group. After progesterone injections, the TLR2 and TLR4 expression was significantly down-regu-lated. compared to the LV group. **p <0.01 vs the SV group, ##p <0.01 vs the LV group.
Fig. 3. NF-κB activity in the brain area surrounding the injury site in the SV group (sham+vehicle) (n = 6), the LV group (lesion+vehicle) (n = 6), and the LP group (lesion+ progesterone) (n = 6). Upper panel: EMSA autoradiography of NF-κB DNA binding activity. Lower panel: levels of NF-κB DNA binding activity quantified by computer-assisted densitometric scanning and expressed as arbitrary densito-metric units (ADU). Compared to the SV group, NF-κB binding activity measured by EMSA was significantly increased in the LV group. Compared to the LV group, progesterone sup-pressed NF-κB activation in the LP group. **p <0.01 vs the SV group, ##p <0.01 vs the LV group.
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vessels was significantly increased compared to that in the SV group (p <0.01) (Figs. 5B, 5D). In the LP group, when compared to the LV group, the number of ICAM-1-positive vessels was significantly decreased (p <0.05) (Figs. 5C, 5D). These results show that systemic injections of progesterone significantly down-regulate ICAM-1 immuno-reactivity in the injured cerebral vessels (Fig. 5D).
Apoptosis in the injured cortex. Few TUNEL-positive apoptotic cells were found in brains of the SV group (Fig. 6A). In the LV group, the apoptotic index in the cortex surrounding the injured site was significantly increased compared to the SV group (p <0.01) (Figs. 6B, 6D). In the LP group, when compared to the LV group, the apoptotic index in the injured cortex was significantly decreased (p <0.05) (Figs. 6C, 6D). These results show that progesterone administration following TBI leads to less cell death in the brain tissue surrounding the cortical contusion.
The main findings of this study are: (a) TLR2 and TLR4 mRNA and NF-κB binding activity are up-regulated remarkably following TBI; (b) the levels
of IL-b, TNF-a, IL-6, and ICAM-1 in the injured brain are significantly increased after brain contusion; (c) the cortical levels of these agents related to the TLRs/NF-κB signaling pathway are suppressed by treatment with progesterone; and (d) after progesterone administration, the number of TUNEL-positive apoptotic cells in the cortex surrounding the injured site are significantly decreased. These findings suggest that progesterone attenuates the TBI-induced TLRs/NF-κB signaling pathway activation that may facilitate development of secondary brain damage following primary trauma in the rat TBI model.
Progesterone as a CNS protector following TBI.The possibility that progesterone might be beneficial as a therapeutic agent in TBI was derived from observations that females sometimes recover better from TBI compared with males, based on an animal study by Attella et al  and a clinical study by Groswasser et al . Several animal studies have suggested that progesterone modulates excitoxicity , reconstitutes the blood brain barrier [19,20], reduces cerebral edema , decreases neuronal loss, and enhances recovery from TBI . Clinical trials [4,23] have shown that moderate TBI survivors who received
Fig. 4 .Changes of inflam-matory mediators in the injured brains as determined by ELISA in the SV group (sham+vehicle) (n = 6), the LV group (lesion+vehicle) (n = 6), and the LP group (lesion+prog-esterone) (n = 6). TBI induced increased concentrations of IL-1b, TNF-a, and IL-6 in the rat brain surrounding the injury site. In the LP group, the cortical concentrations of IL-1b, TNF-a, and IL-6 were down-regulated compared to the LV group. **p <0.01 and * p <0.05 vs the SV group; #p <0.05 and ##p <0.01 vs the LV group.
Progesterone inhibits TLRs/NF-kappaB pathway in traumatic brain injury 69
progesterone were more likely to have a moderate to good outcome than controls who received a placebo; no serious adverse events were attributed to progesterone. In the current study, we found that progesterone administration following TBI reduces apoptotic cell death in the brain tissue surrounding the cortical contusion, which was also
observed by previous investigators [24,25]. However, despite the demonstrated role of progesterone in neuroprotection, none of the previous studies have focused on the TLRs/NF-κB signaling pathway in relation to cerebral inflam-mation after TBI.
Fig. 5. ICAM-1 immunohistochemistry in the injured cortex in the SV group (sham+vehicle) (n = 6), the LV group (lesion+vehicle) (n = 6), and the LP group (lesion+progesterone) (n = 6). (Panel A) SV rats show few ICAM-1 positive vessels; (Panel B) LV rats show strong ICAM-1 positive vessels stained as brown; (Panel C) LP rats show less ICAM-1 positive vessels than the LV rats (scale bar, 50 µm). (Panel D) Administration of progesterone remarkably inhibited TBI-induced up-regulation of ICAM-1 expression in cerebrovascular endothelia. **p <0.01 vs the SV group; #p <0.05 vs the LV group.
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Progesterone and TBI-induced cerebral inflam-mation. Inflammation plays an important role in the pathophysiology of TBI. Although the CNS differs from other organs because of the almost complete isolation from the blood stream mediated by the blood-brain barrier, the main steps of the immune activation within the brain follow a scenario similar to that in other organs . TBI
initiates the inflammatory response by disrupting the blood-brain barrier, creating edema and infiltration of inflammatory cells . Numerous immune mediators such as IL-1b, TNF-a, and IL-6, which are released within minutes of the primary injury, can initiate the infiltration of inflammatory cells into the brain by activating ICAM-1 and other adhesion molecules.
Fig. 6. TUNEL immunohistochemical staining of the injured cortex in the SV group (sham+vehicle) (n = 6), the LV group (lesion+vehicle) (n = 6), and the LP group (lesion+progesterone) (n = 6). (Panel A) SV group rats show few TUNEL apoptotic cells; (Panel B) LV group rats show more TUNEL apoptotic cells (stained brown). (Panel C) LP group rats show less TUNEL apoptotic cells than the LV group (scale bar, 50 µm). (Panel D) Administration of progesterone significantly decreases the apoptotic index in injured brain following TBI. **p <0.01 vs the SV group; #p <0.05 vs the LV group.
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He et al  studied the changes in expression of IL-1b and TNF-a after progesterone treatment in brain-injured rats. They found that progesterone reduces both IL-1b and TNF-a at 3 hr post-injury, when the expression of these cytokines peaks. At 8 and 12 hr post-injury, IL-1b and TNF-a gene expression in injured rats was still elevated compared to sham controls. Conversely, Jones et al  reported that progesterone did not alter expression of the pro-inflammatory genes IL-1b and TNF-a at 48 hr following aseptic cryogenic cerebral injury. In this study, we found that progesterone treatment inhibited the inflammatory cytokines (IL-1b, TNF-a, and IL-6) that accompanied TBI, and we observed that ICAM-1 expression in the injured rat brain after cortical contusion trauma was suppressed by progesterone administration.
TLRs/NF-κB signaling pathway in TBI. Until now, no study has investigated TLRs expression in the brain after TBI. The family of TLRs plays a key role in controlling innate immunity in response to a wide variety of pathogen-associated molecules . Several TLRs have been identified in human cells and in brains of some animals [31,32]. Several studies have suggested that TLR2 and TLR4 are critical for lipopolysaccharide-induced injury in the central nervous system [31,33]. Both endothelial and smooth muscle cells of blood vessels in the brain also express TLRs and can respond to stimulation [34,35]. Thus TLR2 and TLR4 are well positioned in the central nervous system and may initiate inflammation following TBI. The TLRs-mediated intracellular signaling pathways converge to activate NF-κB and c-Jun N-terminal kinases (JNKs), which induce the transcription of a series of cytokine/chemokine genes that are involved in the initiation or regulation of the inflammatory response. Although the results of the present study indicate that the levels of TLR2 and TLR4 mRNA in the contused brain are increased following TBI and can be suppressed by progesterone administration, the mechanism of the initial effect on the TLRs signaling pathway following TBI remains unclear. As mentioned in the introduction section, certain endogenous stimuli of TLR2 and TLR4, such as heat shock protein 60 and 70, fibrinogens, and fibronectin,
have been reported to become present or elevated in the CSF and brain after TBI [36,37]. However, the principal TLRs ligands in the brain after TBI and the mechanisms responsible for the beneficial effects of progesterone call for further research. The functional importance of NF-κB in acute inflammation is based on its ability to regulate the promoters of a variety of genes whose products, such as IL-1b, TNF-a, IL-6, ICAM-1, and acute phase proteins, are critical to inflammatory processes . The inhibition of NF-κB activation by corticosteroid hormones, antioxidants, protease inhibitors, and other compounds may provide a pharmacological basis for interference with pathological inflammatory conditions . Pettus et al  suggested that progesterone given after TBI could reduce the initial cytotoxic surge of inflammatory factors including complement factor C3, glial fibrillary acidic protein, and NF-κB. In the current research, we found that NF-κB binding activity is significantly down-regulated at 5 days postinjury after progesterone administration, which is in agreement with the report by Pettus et al  who used Western blot techniques to analyze NF-κB expression in the total protein of brain samples. In this study, we detected the NF-κB binding activity in the nuclear protein, and it is in the nucleus that NF-κB binds to the κB consensus sequence and induces the expression of NF-κB-regulated genes. In summary, to the best of our knowledge, this is the first study to demonstrate an effect of progesterone on the TLRs/NF-κB signaling pathway in the injured brain after TBI. We found that TBI upregulates mRNA expression of TLR2 and TLR4, NF-κB binding activity, levels of IL-b, TNF-a, and IL-6, and ICAM-1 expression in the brain surrounding the injured site, which are markedly inhibited by progesterone administration. These results suggest that TBI induces the activation of the TLRs/NF-κB signaling pathway in the injured rat brain, and that this activation plays a central role in the inflammatory response that leads to secondary insults after TBI. The therapeutic benefit of progesterone administration after TBI may be due to its salutary effect on modulating the TLRs/NF-κB signaling pathway.
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The authors thank Dr. Bo Wu and Dr. Geng Bao Feng for their technical assistance.
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