Diacetylrhein and rhein:In vivo andin vitro effect on lymphocyte membrane fluidity

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DIACETYLRHEIN AND RHEIN : IN VIVO AND IN VITRO EFFECT ON LYMPHOCYTE MEMBRANE FLUIDITY ELSA BECCERICA, GIANNA FERRETTI*, GIOVANNA CURATOLA* and CLAUDIO CERVINI Rheumatology Unit and the *Institute of Biochemistry, Medical Faculty, University of Ancona, Italy Received in final form 12 October 1989

SUMMARY The effect of diacetylrhein on lymphocyte membrane fluidity in osteoarthritis patients before and after 10 and 30 days of treatment was studied using the 1,6diphenyl-1,3,5-hexatriene fluorescence polarization . Moreover we studied the in vitro effect of rhein, the active metabolite of diacetylrhein, on lymphocyte membrane fluidity of controls using the fluorescence polarization of 1,6-diphenyl1,3,5-hexatriene and its cationic derivative 1-(4-trimethylaminophenyl)-6-phenyl1,3,5-hexatriene. Our results showed that the patients with active osteoarthritis have higher fluorescence polarization values than those in the other osteoarthritis patients and in the controls . Moreover after 10 days of diacetylrhein treatment, we observed a significant decrease of fluorescence polarization values only in lymphocytes of the patients who showed active osteoarthritis before therapy . However after 30 days of treatment, significant changes of fluorescence polarization values were observed also in those patients who did not show painful osteoarthritis before diacetylrhein treatment . We observed that in vitro rhein also induces an increase in lymphocyte membrane fluidity, more evident with 1-(4trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene with respect to 1,6-diphenyl1,3,5-hexatriene. WORDS : diacetylrhein, fluorescence polarization, lymphocyte membrane fluidity, osteoarthritis, them . KEY

INTRODUCTION Osteoarthritis (OA) is a degenerative joint disease characterized by fibrillation, thinning and erosion of articular cartilage, depletion of proteoglycans, abnormal replication of chondrocytes and formation of osteophytes at joint margins . In addition in OA there is evidence of inflammatory events which increase the destruction of articular cartilage and determine gradual development of joint pain, Correspondence to: Prof. Giovanna Curatola, Istituto Biochimica, Facoltà di Medicina e Chirurgia, Università di Ancona, via Ranieri, 60131 Ancona, Italy .

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© 1990 The Italian Pharmacological Society



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stiffness and limitation of motion . The inflammatory process of OA is determined by the intervention of polymorphonuclear leucocytes and mononuclear cells which release lysosomal enzymes and oxygen free radicals [1] . Moreover both OA and rheumatoid arthritis have connections with peroxidative processes [2]. Rhein is an anthraquinone compound and active metabolite of diacetylrhein (DAR: 1,8-diacetoxy-9,10-dioxo-dihydroanthracene-3-carboxylic acid), a new antirheumatic drug which is particularly suitable for the treatment of OA . The antiinflammatory effect of rhein is linked to mechanisms which have not yet been completely clarified . In particular, in contrast to other non-steroidal antiinflammatory drugs, rhein does not inhibit prostaglandin synthesis but increases prostaglandin production both in vitro and in vivo [3-5] . Rhein is known to inhibit the super'oxide (O, - ) production by A 23187 and Nformyl-methionyl-leucyl-phenylalanine (FMLP) stimulated neutrophils in a dose dependent manner [3, 6] . At concentrations comparable to the blood levels reached during DAR treatment, rhein affects the release and the activity of lysosomal enzymes such as ß-glucuronidase, elastase and myeloperoxidase [3, 6, 7] . These effects could be related to a direct action of rhein on plasma and intracellular membranes . In this study we investigated the physicochemical state of the lymphocyte membrane in patients with OA and the effects of in vivo DAR treatment on lymphocyte membrane fluidity . Moreover we studied the in vitro effect of rhein on lymphocyte membrane fluidity of healthy subjects in order to study its pharmacological action at molecular level . Our interest in the use of the lymphocyte membrane as a model in which to study the effect of DAR treatment is based on the following considerations . In a previous report we observed a decrease of membrane fluidity in lymphocytes of rheumatoid arthritis patients [8] . The positive correlations of fluorescence polarization values with disease activity indices, such as erythrocyte sedimentation rate and plasma fibrinogen concentrations, suggested that lymphocyte membrane fluidity could be used as a disease monitoring index . Recently we observed that treatment of rheumatoid arthritis patients with various antirheumatic drugs such as auranofin and hydroxychloroquine induced changes of lymphocyte membrane fluidity ; therefore lymphocyte membrane fluidity appears to be a useful model also for investigating the pharmacological action of antirheumatic drugs at the molecular level [9] .

MATERIALS AND METHODS Patients

Ten patients (eight women, two men ; mean age 58-6±7, range 49-69 years) with OA were studied before and after 10 and 30 days of rhein treatment (100 mg/ day) . On the basis of the clinical examination, two groups of patients with OA were characterized . In the first group (four women, one man ; mean age 57 ± 3 . 7, range 51-60 years) the patients showed active OA with pain and swelling of joints . In the second group, the patients (four women, one man ; mean age 58 . 4 ± 7 . 8, range 49-67 years) did not show any evidence of a painful occurrence of synovitis . No



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patients took non-steroidal anti-inflammatory drugs (NSAID) and/or other drugs for at least 7 days before rhein administration and during the observation period . Twelve healthy subjects (eight women, four men ; mean age 58-2±8-6, range 49-76 years) were used as controls .

Lymphocytes

Lymphocytes were obtained from freshly drawn heparinized blood by FicollIsopaque (Lymphoprep ; Nyegard and Co., A/S, Oslo, Norway) centrifugal sedimentation [10] . The procedure gave a population of cells which was 98% lymphocytes . Possible contamination by monocytes was evaluated by using anaphtholacetate esterase staining [11] . The isolated lymphocytes were washed twice, resuspended in phosphate buffered saline (PBS) to a concentration of 2 X 10' cells/ml and immediately used for fluorescence measurements .

Incubation of lymphocytes with rhein

Rhein (kindly supplied by Istituto Gentili S.p .A ., Pisa, Italy) was dissolved and diluted in NaOH . 2 X 10' lymphocytes obtained from healthy subjects were preincubated with rhein for 10 min at 37°C . The concentrations of rhein used were 1 µg/ml, 5 ,ug/ml, 10 µg/ml . After 10 min of incubation the lymphocytes were washed, resuspended in PBS and immediately used for fluorescence measurements .

Fluorescence labelling of lymphocytes and fluorescence polarization analysis

In this study the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and a cationic derivate of DPH, the 1-(4-trimethylaminophenyl)-6-phenyl1,3,5-hexatriene (TMA-DPH) were used to investigate membrane fluidity [12-14] . In comparison with DPH, TMA-DPH is rapidly incorporated into the plasma membranes of intact cells and it does not penetrate into the cell for 20 min ; therefore, its use has been recommended for specific plasma membrane fluidity measurements in living cells [14] . DPH was dissolved in tetrahydrofuran at a concentration of 2 x 10 - ; M . For cell labelling, the solution of DPH was diluted 1000-fold by mixing into vigorously stirred PBS . The resulting dispersion of 2 x 10 -6' M DPH was clear and practically lacking in fluorescence . One ml of this fluorescent probe stock solution was added to a 1 ml cellular suspension (2 x 10' cells/ml) to give a final probe concentration of 10 - 'm [12, 13] . The incorporation of DPH into lymphocyte membranes was followed by a steep increase in fluorescence intensity . After 45 minutes' incubation at room temperature, the labelled cells were used for fluorescence studies . TMA DPH was dissolved in ethanol at a concentration of 2 x 1 .0 - " M . A 2 µl aliquot of the probe solution was added to 2 ml of cellular suspension (2 X 10`/ml) to give a final concentration of 2 X 10 - ' M . DPH and TMA-DPH fluorescence polarization was measured with a Perkin Elmer spectrofluorimeter MPF 44A . The excitation and emission wavelengths were respectively 365 nm and 430 nm for both probes . The degree of DPH fluorescence polarization (P,) was obtained by the following equation :



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IVV- IVHg

P_ IVV+IVHg Ivv

and Iv, are respectively the emission intensities polarized vertically and horizontally to the direction of the polarized light . The grating correction factor (g) corrects for parallel diffraction anomalies introduced by the monochromator grating (g=IHV/IHH ) and any anisotropy introduced by the glass of the phototube . For each patient two or three determinations of fluorescence polarization measurements were carried out . The variation of these measurements was always less than 2% . Steady-state fluorescence polarization (Pf) is widely used as an index of membrane fluidity [14-16] . For DPH and TMADPH, the fluorescence polarization values depend on probe rotational mobility and on the degree of order of membrane molecules . Increases in P values reflect increases in order, which are associated with decreases in membrane fluidity [15-17] . Statistical analysis

All results are expressed as means ± SD . Data were analysed using the Mann-Whitney U-test and Wilcoxon paired test .

RESULTS In vivo In lymphocyte membranes from untreated OA patients, we observed a mean value of DPH polarization (Pf) of 0-215±0-010 . It seems that no significant difference was found between the OA patient group and the control group ; however, we observed a striking difference in membrane fluidity in OA patients having different types of disease activity (Table I) . In fact patients with active OA exhibited higher values of Pf with respect to the other patients and controls (Fig . 1) . Table I DPH fluorescence polarization (Pf) values in lymphocyte membranes from controls and patients with osteoarthritis (OA) before diacetylrhein treatment Subjects

Pf

Controls

(12)

0 . 215 ± 0 .002

Patients with active OA with painless OA total

(5) (5) (10)

0 . 224 ±0-006* 0 . 207 ± 0 .002*t 0. 215 ±O-010

Values are presented as mean ± SD. The number of examined subjects is in parentheses . *P< 0 . 01, versus controls ; tP< 0 . 01, versus patients with active OA .



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)-240 ∎ ∎

.

0 220

a

----------------------

0 .210

∎ 0 .200 -

Controls

OA patients

Fig . 1 . Fluorescence polarization (Pf) of 1,6-diphenyl-1,3,5-hexatriene in lymphocyte membranes obtained from controls and untreated osteoarthritis patients . The continuous line indicates the control mean fluorescence polarization value and the dashed lines the sip of values for the controls.

The mean values of Pf were respectively 0 . 224 ± 0 . 006 for patients with active OA and 0 . 207 ± 0 . 002 for patients with painless OA (P< 0 . 01) (Table I) . As shown in Fig. 2 and summarized in Table II, DAR treatment induced an increase of lymphocyte membrane fluidity in OA patients . After 10 days of DAR treatment, we observed a significant decrease of fluorescence polarization values only in

0 . 240

.

0 230

El

0220 0 .210

4

A

o

Q 0 0 .200

g ∎

.

0 190

O

.

0 180

o

o\

.

0 170 Before

10 days

30 days

Fig. 2 . Fluorescence polarization (Pf ) values of 1,6-diphenyl-1,3,5-hexatriene in lymphocyte membranes obtained from osteoarthritis patients before, after 10 days and after 30 days of diacetylrhein treatment . Each patient is represented by a specific symbol . The continuous line indicates the control mean fluorescence polarization value .



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Table II DPH fluorescence polarization values in lymphocyte membranes from osteoarthritis (OA) patients before and after diacetylrhein treatment After treatment Before treatment

Patients

10 days

30 days

With active OA before therapy

0 . 224±0 . 006(5)

0 . 209±0 . 003(5)*

0 . 199±0 . 010(5)*

With painless OA before therapy

0 . 207±0 . 002(5)

0 . 201±0 . 012(5)

0 . 180±0 . 007(5)*

Total

0 .215±0 .001(10)

0 .205±0 .010(10)*

0 .189±0 .013(10)**

Values are presented as mean ± SD . The number of examined patients is in parentheses . **P< 0 versus OA patients before treatment . *P< 0 lymphocytes of the patients who showed active OA before therapy (Fig . 2, Table II) . However, after 30 days of DAR treatment, significant changes of fluorescence polarization values were observed also in those patients who did not show painful OA before treatment (Fig . 2, Table II) . It is possible to observe that treatment with DAR for 30 days induces a decrease of Pf values which are actually lower than those of controls .



In vitro The effect of various concentrations of rhein on fluorescence polarization values of DPH and TMA DPH in lymphocyte membranes of controls is shown in Fig . 3 .

0-00 C

o ô

0 .01

N

° 0 . 02

n

0.03 CC O

0 . 04 0 . 05 I I I 5 10 1 RhRin concentration (11q/ml )

Fig. 3 . Effect of rhein on fluorescence polarization of DPH ( •) and TMA DPH (O) in lymphocyte membranes . The results are presented as decreases of fluorescence polarization values with respect to lymphocyte membranes in absence of rhein . One representative experiment of 3 is shown .



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Rhein induces a decrease of fluorescence polarization values . This rhein effect has been observed both with DPH and its derivative TMA DPH . Using DPH, a decrease of Pf values has been observed at the concentration of 1 ,ug/ml ; increasing concentrations of rhein failed to induce further changes of Pf. On the contrary, using the TMA DPH that specifically labels the plasma membrane in whole cells [14], the decrease of Pf is much higher and concentration dependent (Fig . 3) .

DISCUSSION In the present study, we observed that patients with active OA exhibit a decreased lymphocyte membrane fluidity with respect to painless OA patients and healthy controls . Moreover we observed that DAR treatment induces remarkable effects on lymphocyte membrane fluidity of OA patients . At present some hypotheses relating to the molecular mechanism involved in the changes of membrane physical state shown in OA patients before and after DAR treatment can be proposed . In various diseases free radical generation causes various kinds of cellular damage and induces peroxidation of the polyunsaturated fatty acids of membrane phospholipids [18] . Several lines of evidence show perturbation of membrane components and a decrease of membrane lipid fluidity associated with lipid peroxidation [19-22] . In a previous study we observed a decreased lymphocyte membrane fluidity in patients affected by rheumatoid arthritis [8] and we advanced the hypothesis that the increase in membrane rigidity could be related to peroxidation of membrane lipids . Both rheumatoid arthritis and OA involve peroxidative processes [2] . In fact in OA, in common with rheumatoid arthritis, there is evidence of increased superoxide (O 2 - ) production by stimulated polymorphonuclear leucocytes and high levels of free radical oxidation products have been observed in the plasma of OA patients [23] . Increase in lipid peroxidation could induce changes in lymphocyte membrane fluidity of OA patients as occurs in rheumatoid arthritis . As far as DAR is concerned, our results clearly indicate that treatment induces changes of fluidity of lymphocyte membranes . An increase of lymphocyte membrane fluidity in patients treated with various antirheumatic drugs was described in our earlier report [9] . Membrane fluidity is involved in various functions, such as permeability, membrane associated enzyme and hormone receptor functions [15] . It is known that even small modifications of fluidity can affect membrane functions through the control exerted by the lipid matrix on membrane protein activities [15, 24] . During DAR treatment, the average decrease of fluorescence polarization values ranged from 9% to 11% with respect to the values before therapy . Changes of Pf values similar to those found by us have been reported to produce remarkable changes in membrane functions [15] . Dependence on the physicochemical state of the membrane as regards complex membrane functions such as immunological reactions has been clearly demonstrated and changes in membrane composition and fluidity were found to markedly affect lymphocyte activity and responses [25, 26] .



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The changes in fluorescence polarization observed in lymphocytes of OA patients during DAR treatment could be a consequence of pharmacological effect or could be related to a direct in vivo interaction of DAR or its metabolite rhein with plasma and/or intracellular membranes . Our results have shown that in vitro exposure of lymphocytes to pharmacological concentrations of rhein decreases the fluorescence polarization of DPH and TMA DPH incorporated into lymphocytes . DPH is a non-polar molecule which distributes in plasma as well as in intracellular membranes ; on the contrary, TMA DPH labels specifically the plasma membranes of intact cells . It is therefore probable that also under in vivo conditions, rhein will interact with the plasma and intracellular membranes of lymphocytes modulating its physicochemical state . Moreover, studies of Mian et al. [6] have shown that rhein inhibits the production of oxygen free radicals in FMLP stimulated human polymorphonuclear leucocytes . There is evidence that in rat liver microsomal membranes in vitro rhein inhibits CC], induced lipid peroxidation, determined by thiobarbituric acid [3] . Although it has not been established if rhein in vivo inhibits peroxidation of membrane phospholipid polyunsaturated fatty acids, this mechanism could also explain the concomitant increase of membrane fluidity in biological membranes .

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