Streszczenie

Cel: Celem pracy było zbadanie i porównanie poziomu TNF-α w surowicy krwi kobiet w wieku me­nopauzalnym, leczonych i nieleczonych za pomocą hormonalnej terapii zastępczej (HTZ).
Projekt pracy: Badanie zaprojektowano w sposób umożliwiający sprawdzenie istnienia korelacji pomiędzy stę­żeniami tej cytokiny a gęstością mineralną kości (BMD).
Materiał/metody: Badania przeprowadzono w grupie 60 kobiet w okresie menopauzy, 30 nieleczonych (grupa kon­trolna) i 30 leczonych HTZ (grupa badawcza). U połowy pacjentek menopauza została wywo­łana przez usunięcie jajników. Od pacjentek pobierano próbki krwi. Badania densytometryczne przeprowadzono na kościach kręgosłupa. Aby ocenić wyniki badania densytometrycznego dla każdej z pacjentek określono wartość wskaźnika T-score.
Wyniki: Indeks T-score w grupie kontrolnej osiągnął wartości poniżej (-2). Wyniki T-score w grupie ba­dawczej były istotnie wyższe niż w grupie kontrolnej. Hormonalna terapia zastępcza stosowana u kobiet z grupy badawczej powodowała zmniejszenie stężenia TNF-α w surowicy, w porówna­niu z grupą kontrolną.
Wnioski: Korzystny wpływ HTZ na tkankę kostną może być wywierany m.in. przez zmniejszenie stęże­nia TNF-α w surowicy. HTZ umożliwia utrzymanie stałej gęstości mineralnej kości, co prowa­dzi do prewencji rozwoju osteoporozy.

Słowa kluczowe:TNF-alfa • osteoporoza • menopauza • Hormonalna Terapia Zastępcza

Summary

Objective: The aim of the study was to investigate and compare levels of TNF-α in serum of menopausal women treated and not treated with hormone replacement therapy (HRT).
Design: The study was designed to verify whether there is a correlation between the concentrations of this cytokine and bone mineral density (BMD).
Material/Methods: The study was carried out on a group of 60 women during menopause – 30 untreated (control group) and 30 treated with HRT (study group). Half of the patients were after natural menopause and the other half were after ovariectomy. Blood samples were collected. Densitometry was con­ducted on the vertebral column. To evaluate the results of densitometric examination the T-score index was calculated.
Results: The T-score index of the control group reached values below -2. T-score results for the study group were significantly higher than in the control group. Hormone replacement therapy used by women from the study group caused a decrease in the TNF content in serum, compared with the control group.
Conclusions: Beneficial effects of HRT on bone tissue may be exerted through decreased concentration of TNF-α in serum. The use of HRT allows constant bone mineral density to be maintained, which leads to prevention of osteoporotic changes.

Key words:TNF-alpha • osteoporosis • menopause • hormone replacement therapy

Introduction

Menopause is defined as termination of the menstrual cyc­le due to lack of ovarian hormonal function (natural me­nopause), or surgical removal of ovaries (surgically indu­ced menopause). Currently one third of women’s lifespan is the postmenopausal period [3]. The loss of sex hormo­nes results in the development of a range of ailments, cal­led “climacteric syndrome”. Climacteric syndrome occurs in all women, but its intensity varies and depends on many factors, including hormonal, socio-economic, psychologi­cal and genetic factors (Fig. 1).

Figure 1. Radiographic image showing bone resorption of corpus of mandible

Female sex hormones have a protective effect on the con­nective tissue. One of the major results of estrogen defi­ciency is an adverse effect on bone tissue. The deficit of estrogen results in disorder of homeostasis of the skeleton and the emergence of pathological lesions. Imbalance be­tween bone formation and the process of bone resorption leads to bone loss, impaired bone microarchitecture and the development of systemic metabolic disease of bone tis­sue – postmenopausal osteoporosis.

The mechanism of this process is complex and multifac­torial. Recent studies indicated the possibility of linkages between metabolic processes occurring in the skeleton and the immune system. Estrogen deficiency leads to increased production of proinflammatory cytokines (IL-1, IL-6, IL-8, IL-15, TNF-α, TGF) [11]. Cytokines are produced mainly by immune cells: lymphocytes, macrophages and fibrobla­sts [10]. Cytokines have a biological effect through the in­fluence on target cells via receptors on the surface of these cells. Cytokines take part in the development of inflamma­tion, stimulate the rise of body temperature, regulate cell morphogenesis and have a cytotoxic effect.

Cytokines can be divided according to their functions and structure. One of the major groups is the superfamily of TNF (tumor necrosis factor), which includes more than 20 protein molecules of similar structure. One of the most im­portant cytokines is TNF-α (tumor necrosis factor alpha, cachectin), which is secreted by activated macrophages and monocytes, osteoblasts, NK cells, and T and B lymphocy­tes. Cachectin has antitumor and immunomodulating pro­perties. It is involved in the physiological immune response and in inflammatory processes. TNF-α also has a signifi­cant impact on bone tissue by stimulating osteoclastoge­nesis [7]. TNF-α increases the metabolism of connective tissue by activating transcription of proteases, which de­grade unmineralized coat protein, facilitating the access of osteoclasts to the mineralized tissue and slowing down the maturation of the extracellular matrix formed by osteobla­sts. Increased levels of TNF-α have been found in such di­seases as rheumatoid arthritis or Crohn’s disease.

There are various standards of therapy used to avoid de­velopment of climacteric syndrome and its consequences, such as the destruction of the skeletal system. One of the most effective methods of treatment is hormone replace­ment therapy (HRT).HRT may be used either as estrogen monotherapy or estrogen-progestagen (combination thera­py). The use of HRT is aimed at preventing disorders as­sociated with hormone deficiency. Supplementary hormo­ne therapy improves the quality of life of wome’s life by preventing long-term effects of menopause, among which some of the most significant are osteoporotic changes. The present study was aimed to investigate the effect of HRT on the osseous system in menopausal patients. The study was designed to demonstrate the possible relation between the state of bone tissue and the level of TNF-α in serum.

Materials and Methods

The study was conducted on a group of 30 postmenopausal women undergoing HRT for at least 6 months (age range 49-59 years, mean age 53.0 years) (study group). The con­trol group consisted of 30 postmenopausal women, at le­ast 12 months after the last menstruation (age range 53-59 years, mean age 55.4 years). Patients in the control group did not receive HRT. Examined patients were treated in the out-patient gynecological clinic of Public Hospital No. 4 in Lublin. All patients gave their consent to the examination and research protocol. Testing procedures received appro­val of the Local Ethics Committee in Lublin, no of deci­sion: KE-0254/140/2005 and KE-0254/47/2010. The study was carried out in accordance with the ethical principles contained in the Declaration of Helsinki.

Patients were divided into four subgroups according to es­tablished guidelines: M – a group of menopausal women; OV – a group of women after surgical removal of ovaries; OV + HRT – a group of women after surgical removal of ovaries, using HRT; M + HRT – a group of menopausal women using HRT. Patients from groups OV and OV + HRT (mean age 54.2 years) underwent surgery at least 3 years before the study was conducted. Surgical procedu­res were performed as treatment of diseases of the repro­ductive system (neoplasms, prolapse of the uterus, endo­metriosis). In the case of patients with malignant tumors no metastases were detected. None of the patients received chemo- or radiotherapy at least for 2 years before the stu­dy. Women from groups M + HRT and OV + HRT were supplemented with estrogen and progesterone in combi­nation (combination therapy). Patients were administered Femoston in tablets (2 mg of estradiol hemihydrate and 10 mg of dydrogesterone). Estrogen was taken on a daily ba­sis and progesterone was added for the last 14 days of each course (a course lasted 28 days).

An individual anamnesis chart was developed in order to obtain exact information about health of patients. The qu­estions in the survey included age, occupation, social con­ditions, date of last menstrual period, number of births, du­ration of HRT, addictions, physical activity, medications and surgeries. Women qualified for the study did not suf­fer from any severe general diseases. Patients had no ad­dictions and have not been taking any medications continu­ously. During the examination of the oral cavity attention was paid to the condition of teeth and oral mucosa, perio­dontal status, the presence of dentures and the time of the­ir use and other possible problems occurring from the oral cavity. Patients who were selected for further tests did not have any aggressive inflammations. Patients with periodon­tal sockets deeper than 5 mm were excluded from the stu­dy. Patients did not need any extractions of teeth and had average or good oral hygiene. After examination unstimu­lated saliva and venous blood were collected from women in the fasting state. Blood and saliva samples were collec­ted in morning hours (7-9 a.m.). Chewing gum was prohi­bited for 2 hours before collection of diagnostic material. Before collection of saliva patients were asked to rinse the mouth with distilled water and relax for 5 minutes. After this time, patients were asked to lean their head forward and keep their mouth open for 5 minutes in order to allow saliva to drain into the testing tube. At the end of the time of collection, patients were asked to spit remaining saliva into the tube. Venous blood was collected from a cubital vain. After centrifugation of the material, obtained serum (blood) was stored until biochemical tests at a temperature of -70°C. The concentration of TNF-α in serum was de­termined by enzyme-linked immunosorbent assay (ELISA) using an ELISA Kit from BD Biosciences Pharmingen. The examination was performed according to procedures specified by the manufacturer.

The study of bone mineral density (BMD) of the vertebral column was performed in the Densitometric Laboratory of the Institute of Agricultural Medicine in Lublin, by me­ans of the DPX-A Luna equipment and absorptiometry of X-ray beams of two energies. Bone density was specified in g/cm². To evaluate the results of densitometric examina­tion the T-score index was calculated. T-score is the ratio of BMD of the examined patient to the average bone den­sity of young people. T-score values characterizing bone quality are defined as follows:
• healthy bone – T-score higher than -1 (bone density hi­gher than 833 mg/cm²),
• osteopenia – T-score between -1 and -2.5 (bone densi­ty between 833 and mg/cm²),
• osteoporosis – T-score less than -2.5 (bone density be­low 648 mg/cm²).

Obtained results were statistically analyzed. The arithme­tic mean (M) and standard deviation (SD) were calculated. The significance of differences between groups is based on confidence intervals (NIR) determined from the analy­sis of variance (ANOVA). The interdependence between selected traits was expressed by Pearson’s correlation co­efficient. The paired t-test was used in order to determine whether results from 2005 differ significantly from those in 2010. Results were considered significant if the p-value was equal to or less than 0.05.

Results

Results of BMD examination conducted on the vertebral column in 2005 are shown in table 1. The data obtained from control and study groups differed slightly. The lowest value of BMD occurred in the OV group (0.969 g/cm²) and the highest in the OV + HRT group (1.06 g/cm²). The results of densitometric examination performed on the same patients after 5 years revealed a significant decrease in vertebral column BMD in groups not receiving HRT. In group M, the BMD was on average 0.95 g/cm², while in group OV the average value was 0.92 g/cm². BMD in the study group had the following values: in group M + HRT 1.11 g/cm² and in group OV + HRT 1.09 g/cm². The differences between study and control groups were stati­stically significant.

Table 1. BMD levels in groups of women

T-score results are presented in table 2. Data obtained in 2005 showed no statistically significant differences betwe­en the groups. The T-score index in 2010 significantly de­creased in the control groups M and OV, compared to le­vels from 2005, and reached values below -2. In groups M + HRT and OV + HRT there was no significant change in the index, compared to results from 2005.

Table 2. Correlation between T-score values in 2005 and 2010

The results for TNF-α level in serum are presented in table 3. TNF-α level in serum of women from group M had an average value of 4.14 pg/ml and in group OV 3.76 pg/ml (the difference was statistically insignificant). Hormone replacement therapy used by women from gro­up M + HRT and group OV + HRT was associated with a decrease of TNF-α content in serum, compared with control groups. The differences in the levels of this cy­tokine between groups M and M + HRT, and OV and OV + HRT were significant, but not high enough to be sta­tistically relevant.

Table 3. Level of TNF-α in blood serum in 2010

Discussion

Hormone replacement therapy is regarded as one of the most effective methods of preventing the emergence of effects of sex hormone deficiency. The results obtained in this study allow us to draw the conclusion that HRT has a beneficial effect on BMD and allows a high level of bone mineral density to be maintained during menopause.

Administration of combined HRT resulted in maintenance of a constant value of the T-score index in the study gro­up, whereas there was a statistically significant decrease in the level of this index within 5 years between densito­metric examinations in patients from the control group. If this process would continue, in future years patients from the control group would develop osteoporosis. These re­sults are consistent with the results obtained by Stanosz and coworkers, who studied the effects of HRT on hormo­ne levels and BMD of women with diagnosed osteopenia [14]. Their results supported the conclusion that appropria­te hormonal supplementation in women with hormone de­ficiency leads to normalization of estrogen levels and in­crease in BMD. Similar results were obtained by Miller and associates, who analyzed the impact of HRT on mar­kers of bone metabolism [8]. Mitwally and colleagues stu­died the effectiveness of combined estrogen-progestagen therapy. Their work showed that HRT has a beneficial ef­fect on bone status and patient comfort [9].

The analysis of mechanisms occurring in bone tissue asso­ciated with bone resorption and osteoporosis have been the subject of numerous studies in recent years. Knowledge of the role of cytokines in cell interactions is still not comple­te. Too high production of these protein substances may be important in the etiology of certain diseases, including dise­ases of the skeleton. Groblewska and colleagues described in their work increased levels of proinflammatory cytoki­nes in such disorders as Paget’s disease, rheumatoid arth­ritis and hyperparathyroidism [4]. Knowledge of concen­trations of various cytokines in a state of homeostasis and certain pathological processes may be used to reveal the­ir biological importance. Analysis of cytokine levels in the human organism may be an additional diagnostic parame­ter. It was found that the diseases causing the loss of bone mass have a common pathogenesis at the molecular level. Researchers are still analyzing the effects of cytokines on the occurrence of pathological processes, poor healing or lack of osseointegration of implants [15]. The subject of many studies is correlation between the decrease in estrogen le­vels and increased production of cytokines such as TNF-α, IL-1, and IL-6 [1,2]. Results obtained in the present study indicate that TNF-α levels in serum of women receiving HRT were lower than in women from the control group.

The obtained data indicate a negative correlation between TNF-α serum concentration and the level of BMD of the vertebral column. Based on the test results it can also be concluded that hormone supplementation may have an oste­oprotective effect through cytokine TNF-α. Lack of hormo­nal supplementation results in increased levels of cachectin and reduced BMD. This statement is consistent with the viewpoint of Kastelan and colleagues [5], who found that patients with low levels of estrogen have elevated levels of tumor necrosis factor. These conclusions are also in ac­cordance with data presented by Warenik-Szymankiewicz. These authors reported that patients with low levels of sex hormones have significantly higher levels of bioactive TNF in serum compared with the contents of this cytokine in se­rum of women from the control group [17]. Studies deter­mining the extent of bone resorption showed that activity of TNF-α is one hundred times higher in the presence of IL-1. It seems that both cytokines synergistically determi­ne bone destruction. It would be worth carrying out futu­re studies to examine a possible link between these cyto­kines in patients with low levels of sex hormones [6,16].

Drugs which lead to neutralization of TNF-α activity (eta­nercept, infliximab, adalimumab) are currently used in such diseases as psoriasis, arthropathies and Crohn’s disease. These medicaments affect the inhibition of elevated acti­vity of cachectin. The results of the present study allow us to advance the hypothesis that these drugs could also be effective in the prevention of osteoporotic changes resul­ting from estrogen deficiency. Studies carried out on ani­mals by Saidenberg-Kermanach have indicated the effecti­veness of systemic administration of osteoprotegerin (OPG) and anti-TNF-alpha [12]. Their results proved the effective­ness of these compounds in osteoprotection in inflammato­ry diseases. The positive impact of compounds neutralizing activity of cachectin on the state of the skeleton was also confirmed by a study conducted by Seriolo and colleagues [13].The researchers analyzed the effect of TNF blockers in 30 patients suffering from rheumatoid arthritis. Six-month therapy with anti-TNF drugs led to a slight increase in the level of bone formation and reduction of bone resorption. Further studies carried out on a larger group of menopau­sal women could allow a clear therapeutic effect of TNF blockers to be achieved in the treatment of osteoporosis.

Conclusions

The results obtained in this study clearly show that the use of hormone replacement therapy in women with estrogen de­ficiency has a beneficial effect on the skeleton. The prophy­lactic effect of sex hormones is multidirectional. Hormone supplementation leads, inter alia, to the reduction of concen­tration of TNF-α in serum, and thus has a protective effect on BMD. The use of HRT allows constant BMD to be mainta­ined, which leads to the prevention of osteoporotic changes. What is more, administration of agents neutralizing cachec­tin seems to be a promising way of preventing the develop­ment of osteoporosis. Further studies should be conducted on the effect of TNF on the skeleton, performed on a bro­ader group of patients, to clearly confirm these observations.

REFERENCES

[1] Cantatore F.P., Loverro G., Ingrosso A.M., Lacanna R., Sassanelli E., Selvaggi L., Carrozzo M.: Effect of oestrogen replacement on bone metabolism and cytokines in surgical menopause. Clin. Rheumatol., 1995; 14: 157-160
[PubMed]  

[2] Chaudhary L.R., Spelsberg T.C., Riggs B.L.: Production of various cytokines by normal human osteoblast-like cells in response to interleukin-1 beta and tumor necrosis factor-alpha: lack of regulation by 17 beta-estradiol. Endocrinology, 1992; 130: 2528-2534
[PubMed]  

[3] Freeman E., Sammel M., Lin H., Gracia C., Pien G., Nelson D., Sheng L.: Symptoms associated with menopausal transition and reproductive hormones in midlife women. Obstet. Gynecol., 2007; 110: 230-240
[PubMed]  

[4] Groblewska M., Mroczko B., Czygier M., Szmitkowski M.: Cytokiny jako markery osteolizy w diagnostyce pacjentów z przerzutami nowotworowymi do kości. Postępy Hig. Med. Dośw., 2008; 62: 668-675
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[5] Kastelan D., Kastelan M., Massari L., Korsic M.: Possible association of psoriasis and reduced bone mineral density due to increased TNF-alpha and IL-6 concentrations. Med. Hypotheses, 2006; 67: 1403-1405
[PubMed]  

[6] Kmieć Z., Sokołowska I.: Rola cytokin z rodziny czynnika martwicy nowotworu w przebiegu reumatoidalnego zapalenia stawów-nowe możliwości terapii. Pol. Merkur. Lekarski, 2007; 22; 300-304
[PubMed]  [Full Text PDF]  

[7] McMahon M., Ueki Y.: Does anti-TNF-alpha have a role in the treatment of osteoporosis? Bull. NYU Hosp. Jt. Dis., 2008; 66: 280-281
[PubMed]  [Full Text PDF]  

[8] Miller B.E., De Souza M.J., Slade K., Luciano A.A.: Sublingual administration of micronized estradiol and progesterone, with and without micronized testosterone: effect on biochemical markers of bone metabolism and bone mineral density. Menopause, 2000; 7: 318-326
[PubMed]  

[9] Mitwally M.F., Gotlieb L., Casper R.F.: Prevention of bone loss and hypoestrogenic symptoms by estrogen and interrupted progestogen add-back in long-term GnRH-agonist down-regulated patients with endometriosis and premenstrual syndrome. Menopause, 2002; 9: 236-241
[PubMed]  

[10] Pacifici R.: Estrogen, cytokines, and pathogenesis of postmenopausal osteoporosis. J. Bone Miner. Res., 1996; 11: 1043-1051
[PubMed]  

[11] Riggs B.L.: Estrogens/progestins and bone. Osteoporos. Int., 2000; 11: 44

[12] Saidenberg-Kermanach N., Corrado A., Lemeiter D., deVernjoul M.C., Boissier M.C., Cohen-Solal M.E.: TNF-alpha antibodies and osteoprotegerin decrease systemic bone loss associated with inflammation through distinct mechanisms in collagen-induced arthritis. Bone, 2004; 35: 1200-1207
[PubMed]  

[13] Seriolo B., Paolino S., Sulli A., Ferretti V., Cutolo M.: Bone metabolism changes during anti-TNF-alpha therapy in patients with active rheumatoid arthritis. Ann. NY Acad. Sci., 2006; 1069: 420-427
[PubMed]  

[14] Stanosz S., Zochowska E., Safranow K., Sieja K., Stanosz M.: Influence of modified transdermal hormone replacement therapy on the concentrations of hormones, growth factors, and bone mineral density in women with osteopenia. Metabolism, 2009; 58: 1-7

[15] Stashenko P., Jandinski J.J., Fujiyoshi P., Rynar J., Socransky S.S.: Tissue levels of bone resorptive cytokines in periodontal disease. J. Periodontol., 1991; 62: 504-509

[16] Taichman R.S., Hauschka P.V.: Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteoclacin and mineralized extracellular matrix in vitro. Inflammation, 1992; 16: 587-601
[PubMed]  

[17] Warenik-Szymankiewicz A.: Hormonal therapy in postmenopausal osteoporosis. Osteoporoza, 2001; 1: 6

The authors have no potential conflicts of interest to declare.

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