THERAPEUTIC PROPERTIES OF SALT MINES FROM ROMANIA

THERAPEUTIC PROPERTIES OF SALT MINES FROM ROMANIA Constantin Munteanu1, Diana Munteanu2, Mihai Hoteteu1 Romanian Association of Balneology, Bucharest, Romania, www.bioclima.ro, office@bioclima.ro 2 National Institute of Rehabilitation, Physical Medicine and Balneoclimatology, Bucharest, Romania; 1 Abstract: Main therapeutic indications of salt mines and caves are represented by respiratory diseases, especially asthma. Asthma is a disease characterized by chronic inflammation of the airways which make them hyperresponsive and change in their architecture, a process called remodeling. To solve the existing problems in allergy, pulmonology and medical recovery field and for use of natural therapeutic factors in patient treatement with different pathologies, international scientific community reviewed the therapeutic properties of caves and salt mines. Our objective was to explore the effects of speleotherapy on cellular morphology and physiology of pulmonary and dermal fibroblasts obtained from tissues of Wistar rats, in normal and Ovalbumin challenged, “asthmatic” conditions. 60 Wistar rats of 75100 g weight were divided in two lots: control and ovalbumin challenged animals. Ten animals of each lot were send to Cacica, Turda and Dej Salt Mine for 14 days and maintained in the salt mine medium, as in speleotherapy treatment. Pulmonary and dermal fibroblasts cultures were prepared from Wistar rat lung and respectively dermal tissue. The complex picture of results was analysed and explained through biological mechanisms comparing to the control cell cultures obtained from healthy, untreated Wistar rats. In this article, we describe the supposed biological mechanisms that explain the protective effects of speleotherapy. Speleotherapy induces changes on the morphology and protein expression of pulmonary and dermal fibroblasts in vitro, and these changes - by comparing with ovalbumin sensitised animals, supports the beneficial effects of speleotherapy. 1. Introduction Speleotherapy, the use of the climate of salt mines and caves, is an accepted but not widely known therapeutic measure in the treatment of chronic obstructive airway diseases. It is established that the microclimate of some caves can beneficially affect respiratory disorders and the salt mine or cave should be considered as an optimal environment for complex respiratory rehabilitation. Speleotherapy – a special kind of climatotherapy, uses the certain conditions of caves and salt mines to cure several diseases, especially respiratory and skin diseases. The cave air is very low on dust, which could cause allergic reactions or 3qirritation; the symptoms of the diseases are reduced or eliminated completely, while the patient is in the cave. But that does not explain how it should have a longer lasting effect. Curing asthma involves spending 2-3 hours a day underground in subterranean caves or salt mines over a 1-2 month period. An old study describes a speleotherapy course, which was 4 hours a day for 6-8 weeks, with 100 COPD (Chronic Obstructive Pulmonary Disease) and asthma patients and reported improvement that lasted 6 months to 7 years (Skulimowski, 1965). Asthma is a disorder characterized by chronic inflammation of the airways, airways hyperresponsiveness, and changes in airway architecture, termed remodeling. The cells responsible for maintenance of lung structure are the parenchymal cells of the lung, including epithelial cells, mesenchymal cells, and endothelial cells. Recent studies have suggested that the function of epithelial cells, smooth muscle cells, and fibroblasts cultured from lungs of individuals with asthma differs from the function of cells similarly cultured from individuals without asthma. These functional differences, particularly as they relate to repair and remodeling, could contribute to airway structural alterations (Sugiura et al., 2007). Therapy with bronchodilators, corticosteroids, leukotriene inhibitors, mastoid cells stabilizers and recent with IgE receptor antagonists have been shown an improvement of asthma symptoms. The new scientific and practical directions in therapy of the most severe allergic deseases - bronchial asthma - use underground medium of salt mines and caves. This therapy method was name speleotharapy from greece „spelaion”- cave, gap and „therapy”- treatment. Today the speleotherapy is regognized as therapy in underground of salt mines and caves with natural theraoeutic factors for many deseases (Iu.Simionca şi al.,2005, 2008). Primary cell cultures can readily be obtained from human and animal skin using the explant method or trypsynisation. Full thickness skin, also called the integument, is a composite of three layers (epidermis, dermis and subcutaneous tissue), none of which constitutes a homogenous entity. Epidermis normally is composed of keratinocytes, which represent the largest population numerically, and lesser numbers of melanocytes, Langerhans’cells, and occasional cells of the lympho-reticular system, which are, however, transient members of the community. Although the bulk of the dermis is noncellular (collagen and ground substance), within this compartment is also a variety of cell types, including fibroblasts, histicytes, mast cells, macrophages, lymphocytes and Schwann cells, endothelial cells of blood vessels and lymphatics, striated muscle cells of erector pili muscles, and smooth muscle of blood vessels. The subcutaneous tissue includes most of the dermal cell types and fat cells as well (Flaxman, 1974). The current study was designed to investigate the influence of salt mine medium from Cacica, Turda and Dej Salt Mines upon the cell morphology and electrophoretic expression of pulmonary and dermal fibroblasts in vitro obtained from Wistar rats tissues, in normal and Ovalbumin - “asthmatic” conditions. Fibroblasts were cultured from lung and dermal parenchyma of control, ovalbumin-sensitized, and speleotherapy treated rats after ovalbuminsensitization. Fibroblasts shape in culture can vary in accordance with the substrate, which on they is growing, and the space they have for movement. Using pulmonary and dermal fibroblasts cultures to verify the therapeutic properties of saline mines medium represents an innovative and scientific new way to establish the medical methodology of preventing, treating and recovery of patients with various skin and pulmonary problems. 2. Geography and geology TURDA SALT MINE is one of the historical monuments of Romania, from Cluj and a touristic attraction at national and international level especially for Bai Sarate Turda, Durgau salted lakes and the ruins of Potaissa roman castrum where was stationed the Vth Macedonica Legion 2000 years ago. Figure 1. Turda Salt Mine The exploitation of salt from Turda in current microdepression of Baile Sarate has a special interest during the roman occupation in Dacia. The first documentary of mine attestation dating from XII century when avid rocks, minerals and fossils collector - Joanne Fridvaldscky says- „is so famous that has no equal in all eastern”. Turda Salt Mine joined to touristic circuit in 1992 (Ov. Mera si al., 2010) and benefit from EU funding under PHARE CES Programme 2005 through „Improving the attractiveness of the tourist potential of the balneray resort Lacurile Sãrate-Zona Durgãu-Valea Sãratã and Turda Salt Mine” project; modernization works of Turda Salt Mine has start in 2008 and have lasted two years. Turda Salt Mine has legally all prerequisites, for therapeutic use: mines with furhished rooms, tailored for both tourists and sick persons, including disabled persons, mines rooms are large space, isolated rooms; no exploition activities; in Terezia Mine there are a salin lake adapted for recereation. Official opening of modernized Turda Salt Mine took place on 22 January 2010. Figure 2. Turda Salt Mine CACICA SALT MINE - it is situated in the locality with the same name, in the N-E part of the Romania, at 42 km W from Suceava Town and the 17 km N from Gura Humorului. The air strongly ozonized, the purity and beauty of nature, make from this place an attractive destination in any season, both for rest, pleasure and the treatment of respiratory disorders. The entrance into the salt mine is made on fir tree stairs that are over 200 years old, mineralized by the salty water that penetrated the wood. The work by chisel gab and sledge hammer of the miners that ones worked here left real works of art, that bear the seal of the talent access stairs cut in the salt massif, vaulted ceilings or huge galleries. The real measure of the craftsmanship of those who dug the salt with the hammer is given by the small church built in salt at a depth of 27 metres and the dance hall located at a depth of 37 metres. The Catholic chapel sanctified in 1800 has been gathering all the inhabitants, for the last two centuries, on the feast of Sf. Varvara protector saint of the miners. Figure 3. Cacica Salt Mine OCNA DEJ SALT MINE is located in Romania, in the iddle of the Transylvanian Basin 3 km from the city of Dej and 60 km from Cluj-Napoca. Importance of salt in the development of human civilization and the exceptional quality of the salt deposit made the salt to be exploited since antiquity in Ocna Dej. The first statement concerning the Ocna Dej salt exploitation dating from Roman times can be observed today in the form of excavation remains clogged. Figure 4. Ocna Dej Salt Mine Today, Ocna Dej salt mine is part of National Salt Company SA and its main activity is extraction, preparation and marketing of gemstones salt. The Ocna Dej salt mine is characterised by: temperature: 12.4– 14.5 0C, pressure: 1,018–1,020 hPa, humidity: 65–71%, the presence of saline aerosols, lighting artificial and own ventilation system. A higher concentration of NaCl is ensured by continuous operation of the mine. These environmental conditions provided by the Ocna Dej salt mine led researchers to undertake studies on evaluating the possibility of using this mine, not only for salt extraction, but also for the development of the radon therapy and speleotherapy in Romania (Calin M.R.. and Calin M.A., 2010) 3. Methods 30 0,04 25 0,03 20 0,02 15 10 0,01 5 0 0 40 2,5 1 2 3 4 5 30 6 1 8 2 3 4 S1 2 7 5 10 1 2 3 0 4 5 6 1 S1 7 8 2 3 4 9 Ligand A Wnt PI PLC PIP2 IP3 γ β α D M A U G N C PI-3K Frizzled + Akt/PKB IP IMPase - IP2 + Ca 2+ APC IMPase - + AXIN + + Li PKC Dsh - PP2A - - GSK-3β GBP Li + P Phosphorylation, Translocation of transcription factors β-catenin Lithium-Responsive Gene Network Regulation of PKC responsive genes SYNAPTIC FUNCTION Long-term Lithium Effects NEUROPLASTICITY Fig.5 Experimental Design S1 9 0,5 1 CMP-PA 7 8 1,5 0 inositol 6 9 20 β-catenin Protein degradation β-catenin TCF/LEF TCF/LEF responsive genes NEUROPROTECTION 5 6 S1 7 8 9 Materials: Phosphate Buffer Solution (PBS: NaCl 0,13M + KCl 2,6mM + Na2HPO4 x12 H2O 8mM + KH2PO4 1,4mM); HAM-F12 culture medium (Sigma); penicillin 100 U/ml, streptomycin 100µg/ml; neomycin 50µg/ml, fetal bovine serum (Sigma). Rat Wistar Model of Allergic Asthma - Wistar rats of 75-100g weights were sensitized to Ovalbumin by i.m. injections. Primary pulmonary fibroblasts culture After anaesthesia with chloroform, rats were killed. The thorax was opened and then the lungs were removed en bloc in a laminar flow hood using sterile technique and put into ice-cold sterile Phosphate Buffer Solution (PBS: NaCl 0,13M + KCl 2,6mM + Na2HPO4 x12 H2O 8mM + KH2PO4 1,4mM). 1mm tissue pieces were suspended in 0.125% trypsin and 0.001% DNase and repeatedly stirred for 6 minutes and centrifuged at 1000g. The pellet was resuspended in HAM-F12 medium with 4500mg/l glucose, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml neomycin and 10% fetal bovine serum (Sugiura et al, 2007; Foster et al, 1990; Nunez et al, 1995). Primary dermal fibroblasts culture After anaesthesia with chloroform, rats were killed. Skin sampels were removed en bloc in a laminar flow hood using sterile technique and put into ice-cold sterile Phosphate Buffer Solution (PBS: NaCl 0,13M + KCl 2,6mM + Na 2 HPO 4 x12 H 2 O 8mM + KH 2 PO 4 1,4mM). 1mm tissue pieces were suspended in 0.125% trypsin and 0.001% DNase and repeatedly stirred for 6 minutes and centrifuged at 1000g. The pellet was resuspended in HAM-F12 medium with 4500mg/l glucose, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml neomycin and 10% fetal bovine serum (Sugiura et al, 2007; Foster et al, 1990; Nunez et al, 1995). Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrast-enhancing optical technique that can be utilized to produce highcontrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles). SDS-PAGE Electrophoresis The proteins electrophoresis from the total homogenate has as the purpose to establish the changes, which are revealed at the proteic level of fibroblasts cultures obtained from rats held on saline mine medium for the speleotherapy. The proteins electrophoresis in gel of polyacrylamide was done in the denaturated conditions in the conformity with the techniques described by Laemmli (1979). The cultures have been washed with PBS, curetted from the culture plate and lyzed in buffer containing 0,5M Tris-HCl, pH 6,8 + 0,05% BPB + 10% glycerol + SDS 10%. Samples of 10µl were loaded into wells in the gel. One lane was reserved for Sigma molecular markers mixture of 205; 116; 97; 66; 55; 45; 36; 29; 24; 20,1; 14,2 and 6,5 Kda Following electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250, that allowed visualization of the separated proteins. After staining, different proteins appeared as distinct bands within the gel (Towbin et al., 1979). Analysis with GeneTools version 4 software from SynGene of each track of the electrophoresis, allowed us to compare the profiles of the total proteins expression. The air composition of underground environment in the Cacica and Ocna Dej salt mines were determined with RAE gas detection tubes and the following gases and volatile compounds were assayed: O2 (%), CO2 (%), CO (ppmv), SO2 (ppmv), O3 (ppmv), Cl2 (ppmv), NH3 (ppmv), NOX (ppmv), HC (ppmv), H2O (mg/L). Total protein concentration was assayed by microplate biuret reaction, the readings being made with a Modulus Multimode Microplate Reader (Turner Biosystems). Serum protein electrophoresis was performed on cellulose acetate using a Genio S (Interlab) electrophoresis automated system. In order to study the electrolyte balance animals were kept for 24 hours in individual metabolic cages without food and with free access to a saline solution. After 24 hours were measured water volume (ml/24 h) and the amount of sodium intake (mEq/24 h), urine volume (ml/24 h) and concentrations of sodium and potassium in urine (mEq/24 h) using a Ciba Corning 480 flame photometer. From these values were calculated the urinary Na/K ratio as an expression of the mineralocorticoid response of adrenals in the experimental conditions. The experimental data obtained show that the air composition of the both studied salines is characterized by an increased level of CO2, absence of toxic compounds and the presence of hydrocarbons. Onto the serum protein fractions the salt mine speleotherapy cause a variation of their values depending on the pathology and used salt mine. Experimental cure of speleotherapy in Ocna Dej and Cacica salt mines normalize hydric and electrolyte balance parameters in most cases (water and sodium intake, diuresis and renal sodium concentration ability) of rats with induced pathologies due to the direct effect of salt microclimate on the wounds and burns, and also due to saline load of the body in the presence of NaCl aerosol. 4. Results Speleoteraputic treatment of Wistar rats resulted in significant differences in morphology and protein expression of dermal and pulmonary fibroblasts cultures. These differences support the protective effects of speleotherapy compared with data obtained from untreated animals sensitized with ovalbumin and with state induced experimental asthma. 4.1. Speleotherapy results on dermal fibroblasts Control skin cells culture of 7 days has a heterogenic aspect with a high pre-confluence level. The cell division is to a high level and the cell morphology shows a typical microscopic view, described in the specific literature. There are two types of cells: epithelial and fibroblastic. Skin cells cultures of 7 days obtained from Ovalbumin sensitized rats presents many morphological changes from the control skin cell culture, being observed an sensible number reducing of dermal fibroblasts in culture, the diminished cellular dividing frequency and an accentuated cellular morphopathology of the cells in culture. After 7 days of culturing, the pre-confluence level is much lower than in the control case. Skin cells cultures of 7 days obtained from Ovalbumin sensitized rats and treated by speleotherapy in Cacica Salt Mine shows an improvement of the morphological parameters of the cells comparative with the cultures obtained from Ovalbumin-challenged rats. By phase contrast microscopy, it is possible to observe a rising of the cells number. Skin cells cultures of 7 days obtained from Ovalbumin sensitized and treated by speleotherapy in Dej Salt Mine shows also an improvement of the morphological parameters of the cells comparative with the cultures obtained from Ovalbumin-challenged rats. It is observed the rising of the cell population density and that of cell viability. Skin cells cultures were homogenized with Laemmli buffer pH 6,8, and the proteins of the obtained homogenate were separated by 10 % SDS polyacrylamide gel electrophoresis that maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure. 4.2. Speleotherapy results on pulmonary fibroblasts Control pulmonary fibroblasts culture of 9 days has a homogenic aspect with a high pre-confluence level. The cell division is to a high level and the cell morphology shows a typical microscopic view, described in the specific literature. Pulmonary fibroblasts cultures of 9 days obtained from Ovalbumin sensitized rats presents many morphological changes from the control pulmonary fibroblasts culture, being observed an sensible number reducing of pulmonary fibroblasts in culture, the diminished cellular dividing frequency and an accentuated cellular morphopathology of the cells in culture. After 9 days of culturing, the pre-confluence level is much lower than in the control case. Pulmonary fibroblasts cultures of 9 days obtained from Ovalbumin sensitized rats and treated by speleotherapy in Cacica Salt Mine shows an improvement of the morphological parameters of the cells comparative with the cultures obtained from Ovalbumin-challenged asthmatic rats. By phase contrast microscopy, it is possible to observe a rising of the cells number. Pulmonary fibroblasts cultures of 9 days obtained from Ovalbumin sensitized and treated by speleotherapy in Dej Salt Mine shows also an improvement of the morphological parameters of the cells comparative with the cultures obtained from Ovalbumin-challenged asthmatic rats. It is observed the rising of the cell population density and that of cell viability. Dermal fibroblasts Healthy rats Ovalbumin challenged rats Speleotherapy treated rats at Cacica Dej Pulmonary fibroblasts Healthy rats Ovalbumin challenged rats Speleotherapy treated rats at Cacica Dej Turda 5. Total amount of proteins (yg) / 10 yl protein amount 200 160 155 130 150 100 50 Series1 0 1 Series1 155 2 3 130 160 sample Discussion The present study evaluated morphological phenotypes related to repair and remodeling in fibroblasts and epithelial cells obtained from control Wistar rats and from Ovalbumin-sensitized and -challenged rats. Compared with control culture, cells cultures from Ovalbumin-sensitized rats and Ovalbumin-sensitized treated in Cacica, Turda and Dej Salt Mines rats demonstrated the positive role of the saline medium for the sensitized rats. The current study focused on skin and pulmonary cells, which are believed to play a major role in the organism – environment interaction. In this context, fibroblasts are believed to play a key role in maintaining and altering tissue structure. The ability of fibroblasts to migrate in response to chemotactic stimuli and to proliferate in response to specific growth factors is believed to control their accumulation at sites undergoing tissue repair. The ability of fibroblasts to produce and remodel extracellular matrix is thought to contribute to tissue structural changes. Remodeling of tissues likely involves fibroblast contractile activity. In summary, the present study supports the concept that phenotypically altered fibroblasts can contribute to lesion repair in dermatological and pulmonary problems. Cells cultured from the skin of chronically OVA-sensitized and -challenged animals demonstrated consistently augmented repair responses for a number of functional assays (Sugiura et al., 2007). Induced pathology (burns, wounds, sensitization) does not cause a significant change in mineralocorticoid function of the adrenal glands regardless of animal age, instead speleotherapy cure lead to an increase in that, possibly due to a stimulation of the renin – angiotensin - aldosterone system in Wistar white rats exposed to salt mines microclimate. 6. Conclusions This study supports the idea that phenotypically altered fibroblasts may contribute to airway remodeling in asthma. Fibroblasts cultured from the lungs of chronic ovalbumin sensitized animals showed consistently increased reparative responses to several functional tests. Phase contrast microscopy analyses of primary skin cells cultures reveals an cellular regeneration after animal exposure to saline medium in Cacica and Dej Salt Mines, comparative with the cells morphology of cultures from Ovalbumin sensitised rats. The morphological observations are confirmed by the electrophoretic analyses, which demonstrate through rising of the expression of many proteins and of total protein amount that the exposure of Ovalbuminsensitised animals to the saline medium from Cacica and Dej Salt Mines is reversing the cells morphopathology of skin cells in cultures; Wistar rats sensitised with Ovalbumin have a low number fibroblasts in skin cells cultures, with a more sensitive morphopatologic level. Phase contrast microscopy analyses of primary fibroblasts cultures reveals an cellular regeneration after animal exposure to saline medium in Cacica and Dej Salt Mines, comparative with the cells morphology of cultures from Ovalbumin rats. The morphological observations are confirmed by the electrophoretic analyses, which demonstrate through rising of the expression of many proteins and of total protein amount that the exposure of Ovalbuminsensitized animals to the saline medium from Cacica and Dej Salt Mines is reversing the cells morphopathology of pulmonary fibroblasts in cultures; Wistar rats sensitized with Ovalbumin have a low number pulmonary fibroblasts output cultures, with a more sensitive morphopatologic level. Acknowledgments This study was finished in 2011 and was granted by The National Authority for Research - CNMP, contract nr. 42120/2008, project: Complex of medicalbiological study of potential therapeutic factors related to salt mines and karst environments for effective use in health and balneo-turism; development and modelling solutions of these factors”, coordinated by Dr. Iuri Simionca. References Calin MR, Calin MA, 2010. 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