Hum. Reprod. Advance Access published online on September 10, 2008
Human Reproduction, doi:10.1093/humrep/den318
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Hemoglobin and its derived peptides may play a role in the antibacterial mechanism of the vagina
1 Research Unit of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China 2 Department of Gynecology and Obstetrics, West China Woman and Children Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China
3 Correspondence address. Tel: +86-28-85503132; Fax: +86-28-85503204; E-mail: huangpanxiao{at}sina.com
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Abstract |
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BACKGROUND: Hemoglobin is a precursor of antibacterial peptides. Our aim was to identify an antibacterial peptide in human endometrium. We tested the antimicrobial activities of hemoglobin and a derived peptide in vitro and in vivo in rats.
METHODS: Samples (n = 3) were scraped from the surface of endometrium. Acid-soluble proteins underwent electrophoresis followed by gel overlay assay and reversed-phase high-performance liquid chromatography. Antibacterial activities were determined by agar radial diffusion assay. Purified peptides were further characterized by electrophoresis, mass spectrometry, N-terminal amino acid (AA) sequencing and protein structure analysis. A rat model was used to test the inhibitory activity of human hemoglobin on vaginal infection with Escherichia coli, using one experimental group (intravaginal hemoglobin, n = 9) and three control groups (n = 14). Vaginal histology was studied.
RESULTS: The purified peptide exhibited potent antibacterial activities against E. coli ML-35P. The N-terminal AA sequence was F L S F P T T K T Y, identical to AA 32–41 of the human hemoglobin 
chain, and it had the same mass (m/z = 6776.8) as the chain 32–93 AA fragment, with at least three -helices. Histology indicated that the hemoglobin group changed significantly compared with the matrix control group (no treatment after infection): the surface layer of stratified squamous epithelium was smoother, inflammatory cell infiltration was relieved in the lamina propria and congestion pattern was decreased.
CONCLUSIONS: These results suggest that erythrocytes from endometrium are another source of the antimicrobial molecules. Hemoglobin and its derived peptides may play a role in the host defense against pathogens in human vagina.
Key words: endometrial scraping samples/antibacterial polypeptide/hemoglobin-derived peptide/vagina
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Introduction |
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Various strategies are generally utilized to maintain homeostasis of the female reproductive tract. For example, the vaginal normal flora and uterine cervix are an ecological or biochemical barrier against ascending infections from lower genital tract (Sciarra, 1994
; Eggert-kruse et al., 2000; McDonald and Chambers, 2000; Hein et al., 2002; Goncalves et al., 2002). Several key mediators of innate immune response in female reproductive tract contained antimicrobial peptides or proteins originating from epithelial cells or leucocytes, such as human β-defensins (King et al., 2002; King et al., 2003a,b), secretary leukocyte protease inhibitor (King et al., 2000; King et al., 2002), WAP (whey acidic proteins) motif proteins, -defensins, lysozyme (Eggert-Kruse et al., 2000), lactoferrin, granulysin, etc. (King et al., 2003a,b). However, the antibacterial molecules of female genital tract have not yet been completely clarified (Bouyer et al., 2003; Bergstrom et al., 2003; King et al., 2003).
Recently, the experiments in vitro demonstrated that the hemoglobin is a precursor of antibacterial peptides (Parish et al., 2001; Liepke et al., 2003; Mak et al., 2004). This growing evidence suggests that erythrocytes from endometrium may be another source of endogenous antibacterial molecules, besides epithelial cells and leukocytes. However, hemoglobin generated in some conditions, such as vaginal discharge throughout the menstrual cycle, uterine lochia, uterine fibroid, etc. had been regarded as waste, therefore the physiological role of hemoglobin was ignored.
In the present study, we have purified a potent antibacterial peptide derived from human endometrial scraping samples, which was characterized as a fragment of human hemoglobin chain. The purified peptide exhibited potent antibacterial activity against certain gram-negative bacteria. The antibacterial activities of hemoglobin were investigated in the rat model of vaginal infection with Escherichia coli.
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Materials and Methods |
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Acid-soluble protein extraction from endometrial scraping samples
The study involved three uteri obtained from women with myoma (aged from 25
40 years, in non-menstrual period, without antibiotic or hormonal therapy prior to operation) undergoing abdominal hysterectomy at the Department of Gynecology and Obstetrics, West China Woman and Children Hospital, Sichuan University, Chengdu, Sichuan, China. The samples were scraped gently from the surface of endometrium with a glass slide, immediately dissolved in 5% acetic acid solution containing 1 mmol phenylmethylsulfonyl fluoride, 10 µmol leupeptin and 10 µmol pepstatin (Sigma Aldrich Com, USA), homogenized and centrifuged at 15 000g for 20 min at 4°C. The supernatant was dialyzed in a 3500 molecular weight cut off tube (Spectrum Medical Industrial Co., USA) against water at 4°C for 48 h, lyophilized, redissolved in 5% acetic acid and stored at –80°C for future use.
Gel overlay antibacterial assay
To demonstrate antibacterial components, the acid soluble protein (30 µg) from the endometrial scraping samples was subjected to acid-urea polyacrylamide gel electrophoresis (AU-PAGE) on a mini-gel, running at 150 V for 90 min. After electrophoresis, the gel was washed by phosphate-buffered saline (PBS, 10 mmol/l, pH 7.4) three times. Gel overlay technique was applied, according to the protocol of Panyim et al. (1969) and E. coli ML-35p, E. coli ATCC 25922, clinical isolate of E. coli ATCC 54080, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 90028 were used as the test micro-organisms to determine the antibacterial activities. Bacteria were grown overnight in Luria–Bertani broth, centrifuged at 12 000g for 10 min at 4°C and the pellet was washed twice using 20 mM PBS (pH 7.2). The bacterial concentration was adjusted to 1 x 106 colony-forming units (CFU)/ml using a colorimeter (Bio-Rad Inc., Hercules, CA, USA) and mixed with underlay gel solution, kept molten at 43°C, to a final concentration of 1 x 105 CFU/ml. The underlay bacterial gel (10 ml) was composed of 1% agar (Sigma A-6013), 0.3 mg/ml trypticase soy broth, 10 mM sodium citrate-phosphate buffer (pH 5.5) and 1 x 105 mid-logarithmic phase bacteria. Immediately, the mixture was poured into an integrid Petri dish (Fisher scientific, USA) to form a uniform layer 1–2 mm deep. The PAGE gel was later loaded on top of the underlay bacterial gel, incubated for 3 h at 37°C and replaced by overlay nutrient agar (10 ml) containing 30 g/l trypticase soy broth. After overnight incubation at 37°C, which allowed the colony to develop, the clearing zones of inhibition were observed.
Purification techniques
The gel overlay assay identified antibacterial peptides which were isolated via non-denaturing preparative AU-PAGE elution and subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). The preparative AU-PAGE elution was performed according to the method of Harwig et al. (1993). The elutes were lyophilized, and reconstituted in 0.01% acetic acid. For HPLC purification, 2 ml of lysate was diluted with 200 ml deionized water and filtered (0.45 mm, Schleicher and Schuell, Dassel, Germany). RP-HPLC was performed using Vydac C18 columns (4.6 x 250 mm, Agilent Inc., USA) on an Agilent 1100 system and eluted with a 0–60% linear gradient of solvent B (0.1% trifluoroacetic acid, 60% acetonitrile and 40% water) over 60 min at a flow rate of 1.0 ml/min. The elution profile was monitored at 214 nm. The column effluents collected every minute were lyophilized, reconstituted with 0.01% acetic acid and stored at –80°C.
Agar radial diffusion antimicrobial assay
Protein concentrations were determined with BCA protein assay reagent (Bicinchoninic acid, Pierce, Rockford, IL, USA) according to the manufacturer's instructions.
In the agar radial diffusion assay system, antimicrobial activities were determined as a clear zone of inhibition around the well according to the protocol of Lehrer et al. (1991). Lyophilized HPLC fractions were dissolved in sterile water and aliquoted. The underlay gel containing bacteria was prepared as described above for the gel overlay assay (1 x 105 CFU/ml). The bacteria were maintained on trypticase soy plates and grown to stationary phase overnight at 37°C in 10 ml of well-shaken trypticase soy broth. The next morning, 10 µl of this culture was transferred to 10 ml of fresh trypticase soy broth and incubated for 2.5 h at 37°C in a shaking incubator to obtained mid-phase organisms for antibacterial testing. Bacteria were washed with 10 mmol phosphate buffer (pH 7.4) and then adjusted to the desired concentrations by measuring their A620. A series of wells (3 mm diameter) were punched on the underlay gel and 5 µl of samples (200 ng) or positive control [0.01% acetic acid and lysozyme (10 µg)] were added to designated wells. After incubation at 37°C for 3 h to allow the peptide to diffuse into the underlay gel from the wells, the overlay gel was poured, the dishes were incubated overnight at 37°C and the diameters of inhibition zones were recorded. This assay was repeated three times.
The antimicrobial assays by Blastomyces albican were followed. All procedures were same as above, expect that Sabouraud's Agar was used as growth medium.
Determining lysozyme activity of purified peptide
The lysozyme activity of purified peptide was determined by the method of Gupta et al. (1987). Agarose plates (1%) were prepared with 0.5 mg/ml Micrococcus lysodeikticus in 10 mM sodium phosphate buffer (pH 7.0), and perforated with 3 mm diameter wells. Then 5 µl of samples (200 ng) were added to wells, while the control wells contained serial dilutions of lysozyme. The plates were incubated overnight at 37°C.
Electrophoresis and electrotransfer
Tricine sodium dodecyl sulphate (SDS)–PAGE was performed in minigel formats, using Modular Mini-PROTEAN II electrophoresis System (Bio-Rad Inc.). All reagents were obtained from Sigma unless otherwise noted. Molecular weight standards were from Bethesda Research Laboratories. The gel was subjected to electrophoresis for 3 h at 80 V and stained with 0.1% Coomassie Brilliant Blue R-250. The peptides were electrotransfered (20 V for 40 min) to ProBlott polyvinylidene difluoride membranes (0.45 mm, Invitrogen Inc., USA) before staining with silver. Nitrocellulose membranes (0.22 mm, Invitrogen Inc.) stained with Amido black were used for mass spectrometry.
N-terminal sequencing
N-terminal amino acid (AA) sequences were determined by sequential Edman degradation method in an Applied Biosystems Procise PROCISE Instrument using RP-HPLC for phenythiohydantion detection (Life Sciences Institution of Beijing University, Beijing, China).
Mass spectrometry
Mass spectrometry analysis was performed at Chengdu Branch of Chinese Academy of Sciences, Chengdu, China. The molecular weight of the isolated antimicrobial factor was determined using a Bruker high-resolution mass spectrometer (Bio TOF-Q, USA). The liquid molecules detected by electrospray ionization were stimulated into gas plasma, and the molecular weight determined through Quadrupole/time-of-flight (Q-Tof) detection. According to the N-terminal sequences and mass spectrometric measurement, the full corresponding AA sequences of the purified peptide were identified in GeneBank. The pI (isoelectric point) of the identified antibacterial peptide was determined using the Expert Protein Analysis System (ExPASY software, produced by Molecular Biology of the University of Geneva service station), whereas two-dimensional structure was analyzed using OMIGA software (Oxford Molecular Group).
Rat model of persistent vaginal infection with E. coli
The experiments were conducted with 23 female Wistar rats (6 weeks old on arrival) obtained from laboratory animal center in Sichuan University and maintained in our animal facility for 5 days after arrival. Both ovaries were removed and the rats were administered estradiol benzoate (10 mg/kg) and hydrocortisone sodium succinate (60 mg/kg) by s.c. injection every other day to maintain a state of estrus (confirmed by vaginal smear) and induce hypoimmunity, respectively.
In attempt to establish persistent infection over a 1 week period with an E. coli clinical isolated strain ATCC 54080 (kindly offered by Department of Gynecology and Obstetrics) burden similar to that found in women with mucosal colonization, we changed the pH of the rat vagina by washing daily with PBS 7-8 times, for 3 days. Observing vaginal secretions and local inflammation, 60 µl liquid of E. coli clinical isolated strain ATCC 54080 (2.1 x 109 CFU/ml) were applied intravaginally, three times. After 5 days, all rats were still alive. The persistence of vaginal colonization by E. coli was determined by examining pathological sections, which suggested vaginal hyperemia, dropsy and infiltration with leucocytes. The animals were divided randomly into four groups: the hemoglobin group received an injection of 5 mg/kg hemoglobin (in distilled water, once daily for 5 days) after infection (n = 9), the matrix control group, with no treatment after infection, but injected daily with 100 µl distilled water for 5 days (n = 7), the blank control group, ovariectomized but with no infection (n = 5) and non-ovariectomized rats as the normal control group (n = 2).
Human hemoglobin was isolated from peripheral blood of healthy volunteers (aged 20 years) or from outdated blood. The erythrocytes cell membrane were disrupted by mixing erythrocytes with CH2Cl2, and hemoglobin was then purified by differential centrifugation.
Histological analysis of vaginal tissues
Pathological sections of vaginal tissues were used to evaluate the degree of inflammation. Rats were killed and the uterus, cervix and vagina were removed and fixed in 10% neutral buffered formalin 24 h after the last administration. Standard procedures were used for microtome sectioning (4 µm) and staining with hematoxylin and eosin-Y. Finally, the sections were dehydrated, cleared in propylene oxide and embedded in epon-araldite.
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Results |
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Purification and characterization of antibacterial peptides from endometrial scraping samples
The acid-soluble proteins of endometrial scraping samples contained three most cationic molecular species that killed E. coli ML-35p, as demonstrated by the clear zone, lacking bacterial colonies (Fig. 1). The antibacterial protein bands were cut out and further purified by PAGE and RP-HPLC. As shown in Fig. 2, the HPLC profiles of the samples from three different subjects were similar. The antibacterial activities from fraction 31 to fraction 44 were observed. A highly purified peptide had potent antibacterial activity against E. coli ML-35P, as indicated by the agar radial diffusion assay. The peptide was eluted at 40% acetonitrile from a C-18 column and the molecular mass was
6 kDa, confirmed by Tricine–SDS–PAGE with silver staining (Fig. 2). As lysozyme usually elutes at 40 min on RP-HPLC, the lysozyme activity of this antibacterial peptide was detected on a lysoplate. No such activity was observed, even at high concentration (data not shown).
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Lysozyme activity of purified peptide
Under the same RP-HPLC elution conditions as used for the separation of antibacterial peptides from the flora of bladder or tracheal mucus, lysozyme was also eluted at the same time. To find out whether the sample was mixed with leucocytes, a lysozyme activity assay of the antibacterial peptide was performed (Fig. 3). As the concentration of purified peptide (198 µg/ml) is
5000 times higher than the minimum inhibitory concentration (MIC) for lysozyme (0.039 µg/ml), the results clearly indicated that the purified peptide was not lysozyme.
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Antimicrobial properties of the purified peptide
The agar radial diffusion assay indicated that the HPLC fraction 40 from three different samples had potent antibacterial activities toward E. coli ML-35p, E. coli ATCC 25922, and clinical isolate of E. coli ATCC 54080 (Table I), whereas no activity against S. aureus ATCC 25923 and C. albicans ATCC 90028 was detected in this assay system.
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Characterization of the purified peptide
The first 10 AA residues of the N-terminal sequences of HPLC fraction 40 were F L S F P T T K T Y, corresponding to AA residues 32–41 of human hemoglobin
chain, as indicated by NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Mass spectrometric analysis of the purified peptide revealed the same molecular mass (m/z = 6776.8) as hemoglobin 32–93 (32–93 AA fragment of human hemoglobin chain) (Fig. 4). These data suggested that the purified peptide should be a fragment of human hemoglobin chain. OMIGA protein structure software analysis revealed that hemoglobin 32–93 contains at least three -helices (Fig. 5).
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Monitoring estrous cycle of experimental rats
The vaginal smears of rats after removal of both ovaries showed a state of diestrus with many leucocytes and some pavement epithelium cells (Fig. 6A), whereas on the other hand, the smears of rats treated with estradiol benzoate showed the rats in a state of estrus, with cuticular epithelium cells (Fig. 6B). If the smears were not the same as A or B, the rats were not used for this study.
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Histological changes in rat uterus and vagina
To observe the curative effect of hemoglobin, some rats were administered hemoglobin intravaginally and compared with the control groups. Pathological sections of uterus and vagina were observed and the results are shown in Fig. 7. The vagina in the normal group comprised a mucous layer (stratified pavement epithelium and lamina propria), muscular layer and adventitia (Fig. 7A). The blank group showed a thickening of stratified pavement epithelium with other layers the same as the normal group (Fig. 7B). The matrix control group had no smoothing of stratified pavement epithelium, and showed massive congestion and cell infiltration in the vaginal lamina propria (Fig. 7C). Comparing the hemoglobin group with the matrix control group (no treatment after infection), the superficial layer of vaginal stratified squamous epithelium was smoother, inflammatory cell infiltration was significantly relieved in the lamina propria and the congestion pattern was obviously decreased (Fig. 7D).
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Discussion |
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We initially focus on the identification of unknown antimicrobial peptides or proteins that might be secreted by uterine epithelial cells. The target molecule was isolated and further purified by RP-HPLC. The peaks at
40 min (elution time) were displayed on HPLC profiles from three independent samples. We originally thought this fraction was lysozyme, because its retention time is very similar to lysozyme in our elution condition. However, the result of lysozyme activity assay demonstrated no lysozyme activity of this fraction, even when at least 200-fold higher concentration than lysozyme MIC. SDS–PAGE showed that the molecular mass of the purified peptide was 6kDa, far less than that of lysozyme (14 kDa). Therefore, it was apparently not lysozyme and we further characterized this purified peptide.
On the basis of its N-terminal AA sequence and mass spectrometric value, the purified peptide was expected to be the 32–93 AA fragment of human hemoglobin chain. Its antibacterial activity was similar to the 35–56 AA fragment of -hemoglobin (HB 35–56) isolated from menstrual blood (Mak et al., 2004), or the 33–76 AA fragment (HB 33–76) cleaved by cyanogen bromide (CN Br) from human peripheral blood (Parish et al., 2001), as well as the efficacy of these fragments against gram-negative bacteria. The only difference was a lack of activity toward gram-positive bacteria. The MIC of the synthetic peptide HB 35–56 against E. coli was 105 µM (=262 µg/ml), and that of HB 33–76 was 100 µg/ml. Although we were not able to compare the antibacterial effect of our peptide with peptides HB 35–56 and HB 33–76, we found its anti-gram-negative bacteria action was so powerful that its anti-E. coli activity was 100-fold higher than lysozyme in the agar radial diffusion assay (the clear zone of 200 ng of our peptide equals that of 25 µg lysozyme).
In addition to its primordial function as an oxygen carrier, the hemoglobin released from erythrocytes also displayed multiple biological functions that have been documented by several authors since 1970s. These hemoglobin-derived peptides either exhibited adrenocorticotrophic hormone-releasing, growth hormone-releasing, opioid-like activity (Schally et al., 1978; Brantl et al., 1986) or had hemopoietic, immunomodulatory actions (Karelin et al., 1995; Ivanov et al., 1997; Petrov et al., 1997). As for the interaction of hemoglobin with micro-organisms, some researchers believed that the hemoglobin might promote bacterial growth through providing iron or interfering with leukocyte oxygen metabolism under certain conditions (Asghar and Cormane, 1976; Welch et al., 1982; Hand et al., 1984; Olczak et al., 2005). Since the discovery in 1999 of ticks utilizing bovine hemoglobin as a defense against micro-organisms (Fogaca et al., 1999), growing evidence suggests that hemoglobin-derived peptides should be the host defense peptides, and were named as hemocidins (Mak et al., 2000; Liepke et al., 2003). Hemocidins exhibited a broad-spectrum of antimicrobial activity (Parish et al., 2001).
As far as we know, the antibiotic mechanism of endogenous antimicrobial peptides is related to several features. Previous studies have suggested that the cationic charge and amphipathic -helix are crucial for the antimicrobial activity of such molecules. The structure responsible for the antimicrobial activity of hemoglobin may resemble that of the cationic antimicrobial peptides, such as magainins, defensins and LL-37 (Mak et al., 2000). Our peptide (HB 32–93) had a pI of 8 and at least three intact -helices, which would be the molecular structural basis for its antibacterial activity. Moreover, it is believed that the length of the hemoglobin fragments is related to their antibacterial activity (Mak et al., 2000). The length of hemoglobin-derived fragments, whether they were isolated from blood, scleaved by CN Br or chemically synthesized, must be more than 16 AA residues (Parish et al., 2001; Mak et al., 2004) as the very short fragments would completely abolish the antibacterial activity, or even could stimulate bacterial growth. For example, a five AA fragment from bovine β-hemoglobin exhibited a gram-negative bacteria growth-stimulating activity (Zhao et al., 1996). These phenomenon suggest that the longer peptides might help in forming a stable -helical structure for destruction of the bacterial membrane.
In the antimicrobial peptide database (http://aps.unmc.edu/AP/main.php), the peptides were divided into four groups based on diversity of antimicrobial activity: antiviral, antifungal, anticancer and antibacterial peptides. Among these types of antibacterial peptides, bee apidaecin IB and cow BACTENECIN 5, seven have been verified to exhibit antibacterial activity only toward gram-negative bacteria. As early as 1994, Casteels advanced the hypothesis that Apidaecin-type peptides were composed of constant regions which determine their antibacterial activity, such as core region and the histidine constituents like drosocin from Drosophila (any modifications to these would abolish all function) (Casteels et al., 1994). In addition, the antibacterial peptides were divided into three categories, and Drosophila cecropin A was selected into the first family (corresponding to peptides which are preferentially active on gram-negative bacteria) because this molecule belongs to the family of -helical antimicrobial peptides that kill preferentially gram-negative bacteria through membrane permeabilization (Rabel et al., 2004). On the mechanism of antibacterial action, the review of Hoffmann and Reichhart (2002) inspired us so much. Two distinct signaling pathways (Toll and Immune deficiency or Imd) have been identified, which control the expression of these antimicrobial peptides. The Toll pathway was shown to participate in the defense against infections by fungi and gram-positive bacteria, whereas an infection with gram-negative bacteria activates the Imd pathway.
The peptide purified by our laboratory has been characterized as a hemoglobin-derived fragment. The result of agar radial diffusion assay indicated that HPLC fraction 40 from three different samples had potent antibacterial activity against E. coli ML-35p, E. coli ATCC 25922 and clinical isolate of E. coli ATCC 54080, but no effect on gram-negative bacterium. These targeted, narrow spectra may be controlled by constant regions and/or activated in different ways. We will further explore this issue in the follow-up experiments.
Recently, 20 hemoglobin fragments have been isolated directly from female menstruation vaginal blood. Such peptides all displayed bactericidal activity against E. coli in vitro. Increased levels of similar hemoglobin-derived fragments were also detected in uterine secretion (lochia) samples from woman after Cesarean delivery and might help in maintaining human vaginal homeostasis (Mak et al., 2004, 2007). Meanwhile, hemoglobin may be degraded under certain conditions in vivo. Ivanov proposed a concept of ‘tissue specific peptide pool’, which suggests the hemoglobin could be degraded in erythrocytes and then digested specifically into various fragments by different proteolytic enzymes (Ivanov et al., 1997, 1998, 2000). Subsequently, Mak further proposed that both matrix metalloproteinases and leukocyte protease activity are directly responsible for tissue dissolution and forming of menstrual discharge (Mak et al., 2004; Geisler et al., 2004). We found the intact hemoglobin, separated chains , β and their fragments had similar antibacterial effects against E. coli (data not shown). We established rat models of vaginal infection with E. coli to exam whether the hemoglobin showed antibacterial activity in vivo. Histopathological data demonstrated that, in the experimental group, stratified pavement epithelium became smooth, congestion lightened and inflammatory cell infiltration decreased, suggesting hemoglobin and/or its derived peptides were able to alleviate inflammation induced by E. coli.
By experimental measurements and theoretical estimation, the average bleeding volume of adult female menstruation is 30–60 ml containing 2200–9000 mg hemoglobin (Wicherek et al., 2007). In some gynecologic diseases, such as uterine fibroids, the patient often suffers menorrhagia with a menstrual blood loss of >80 ml (Goldstein, 2004; Sulaiman et al., 2004). Under these circumstances, the production of hemoglobin is far greater than other known antibacterial peptides, such as , β-defensins originating from leukocytes and epithelial cells (Lathbury and Salamonsen, 2000; Salamonsen and Lathbury, 2000). So the erythrocytes from endometrium may serve as an antibacterial peptide bank to maintain the homeostasis of female genital tract (Zhang et al., 2000). The antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against micro-organisms (Fogaca et al., 1999). We maintain that if such a large amount of blood was viewed as useless, our bodies would lose efficiency of a natural defense strategy, and our reproductive tract would be much more susceptible when faced with invading micro-organisms. The view from Obstetrics and Gynaecology in cases of uterine pathology, such as myoma (symptom: menorrhagia, lower abdominal pain, lumbago and dysmenorrhea), is that androgen and LHRH are used to control estrogen levels in order to relieve anemia. However, from the aspect of anti-infection, during this pathology, the self-defensive mechanism should be applied to control and prevent infection, for instance, the antibacterial activity of hemoglobin. The infection would be much worse if hemoglobin had no antimicrobial activity. So we suggest utilizing a drug that has a synergistic antibacterial effect to hemoglobin and, at the same time, correcting anemia.
Taken together, this discovery may have significant impact not only in the basic antimicrobial biology of hemoglobin, but also in the understanding of pathologies associated with abnormal bleeding of the uterus and microbial infection, among others.
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This work was supported by Chinese Medical Board of New York (96-681) and National Natural Science Foundation of China (30671963 and 30470763).
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Submitted on December 11, 2007; resubmitted on May 20, 2008; accepted on June 30, 2008.
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