Anti-inflammatory effect of plant compositions of Tibetan medicine in vitro on early rheumatoid arthritis

doi:10.4236/health.2011.39096

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Vol.3, No.9, 560-565 (2011)

doi:10.4236/health.2011.39096

Health

Anti-inflammatory effect of plant compositions of

Tibetan medicine in vitro on early rheumatoid arthritis

L. Yu. Semenova*, J. M. Salmasi, G. V. Poryadin

Department pathophysiology, The Russian State Medical University, Moscow, Russia;

*Corresponding Author: sememovaly@mail.ru

Received 27 May 2010; revised 9 June 2010; accepted 22 June 2010.

ABSTRACT

Materials and Methods: lymphocytes of 10 pa-

tient s hav i ng early rheumato id ar thriti s (RA ) (the

duration of the illness was 3 - 6 months) w ith a

marked exudational process in joints were ex-

amined. The content of lymphocytes expressing

the CD3, CD4, CD8, CD16, CD56, CD20, CD72,

CD38, CD23, CD25, CD71, HLA-DR, CD95, CD30,

CD54, mIgM, mIgG antigens was determined.

Results: the “Taban-Arshan” extract corrects

the changes of the immune system character-

ized by the evident activation of the B-cell part

of the immune system and normalizes immune

parameters of the lymphocytes taken from the

patients with autoimmune diseases (early

rheumatoid arthritis). The immunocorrective

effect of the “Ta ban-Arsh an” extract i s related to

its abilit y to suppress the lymphocyte increased

activation by normalizing expression of the

main activation antigens (CD23, CD25, CD71,

HLA-DR, CD54).

Keywords: Earl y Rheumatoid Arthritis;

Subpopulation of Lymphocytes; Tibetan Medicine;

Immunoregulators

1. INTRODUCTION

The elucidation of the role of the immune system in

physiological and pathophysiological processes creates

prerequisites for developing disease treatment methods

based on directional regulation of immune reactions.

[1-4].

Current experience in application of immunoregula-

tors in treatment of autoimmune diseases demonstrated

certain success in this direction; however, some immu-

nomodulators used in clinical medicine nowadays have

adverse actions upon the organism. Thereupon research-

ers’ attention is drawn by phytogenous immunotropic

drugs that have been widely studies over the past years.

At present phytogenous drug compositions prepared

according to Tibet medicine prescriptions arouse a seri-

ous interest. It is known that herb extracts and tinctures

have no adverse effects and possess apparent regulating

properties. Effectiveness of Tibetan medicine treatment

methods has been checked by a centuries-old practice

and has raised no doubts till now [5-12].

One of the promising Tibetan medicine plant compo-

sitions is “Taban-Arshan” that is mostly helpful in dis-

orders of the locomotor apparatus. A decoction of this

phyto collection saturates the organism with oxygen,

produces a favorable action on metabolism, as well as

removes edemata, pains in joints and reflex muscle

spasms. “Taban-Arshan” is used in a folk medicine to

treat diseases of joints, osteochondrosis, kidney and

urogenital diseases, female disorders and skin diseases

[12,13]. “Polyphytokhol”, a complex plant drug of the

Tibetan medicine has hepatoprotective and hemostimu-

lating effects and is used in diseases of the hepatobiliary

system [14-16]. Another combined phytodrug―“Bai-

kalsky”―produces anti-inflamatory and expectorative

effects and is used in diseases of the upper respiratory

tract [17].

Three herbal drug compositions of the Tibetan medi-

cine were offered for our studies by the Ulan-Ude Center

of Eastern Medicine. Since collections of herbs of the

traditional Tibetan medicine have been used from old

times to treat inflammatory processes, it was of interest

to define more exactly if their anti-inflammatory effect is

connected with their influence on the immune system.

The aim of the present research work was to study the

expression of surface antigens by lymphocytes of

healthy donors and sick persons under the effect of the

extracts “Taban-Arshan”, “Polyphytokhol” and “Bai-

kalsky” extracts of the Tibetan medicine.

2. MATERIALS AND METHODS

Lymphocytes of 10 patients having early rheumatoid

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arthritis (RA) (the duration of the illness was 3-6 months)

with a marked exudational process in joints were exam-

ined. The RA diagnosis was based on the American

Rheumatologic Association’s criteria. All patients’ di-

agnoses were confirmed by X-ray data. At the time of

the study the patients received no basis therapy. 30 prac-

tically healthy persons of 20 - 30 years old served as con-

trols. To conduct the immunological analysis, the blood

was taken from the ulnar vein and stabilized by Trilon B

solution. The lymphocyte suspension was obtained in the

density gradient of phycol-verographin according to

Boyum’s method. The content of lymphocytes express-

ing the CD3, CD4, CD8, CD16, CD56, CD20, CD72,

CD38, CD23, CD25, CD71, HLA-DR, CD95, CD30,

CD54, mIgM, mIgG antigens was determined by the

method of indirect immunofluorescence using mono-

clonal antibodies of the IKO and LT series (Russia).

To determine the effect of the extracts on the expres-

sion of lymphocyte surface receptors, a sterile separation

and cultivation of cells in the final concentration of 2.5

million/ml in a minimal medium199 with no antibiotic in

the atmosphere of 5% СО2 at a temperature of 37˚С for

16 hours were conducted. The concentration of the

preparations in the incubation medium was 10–7 М.

The results were computer processed according to the

method of variational statistics by the Statistika applica-

tion program package; the validity of differences was

estimated by Student’s criteria.

3. RESULTS AND DIS CUSSION

To evaluate the patients’ immune status, population

and subpopulation composition of T-lymphocytes was

studied as well as quantitative composition of B-lym-

phocytes, content of NK-cells and parameters of the ac-

tivation process in lymphocytes were determined.

The effect of the extracts on the expression of surface

antigens of lymphocytes of 10 healthy donors during the

cultivation process was previously analyzed.

The conducted studies have shown that the cultivation

process of cells in vitro for 16 hours causes a change in

the expression of some subpopulations of lymphocytes

(CD3, CD8, CD16). Perhaps it is due to a loss of recep-

tors or an increase of their expression (Table 1).

The obtained results show that under the influence of

the “Taban-Arshan” extract in vitro there are neither

essential nor valid changes in the subpopulation of the

T-lymphocytes picked out of the healthy donors―total

content of the T-lymphocytes, helper (CD4+) and cyto-

toxic (CD8+) lymphocytes. In this study it was also

shown that the content of the subpopulation of natural

killers (CD16) and natural killers that realize their cyto-

toxic function (CD56) did not differ much from the lev-

els of the healthy persons with the “Taban-Arshan” ex-

tract used.

The obtained results show that the “Polyphytokhol”

extract causes an increase of the expression of the total

quantity of T-lymphocytes (CD3) as well as a decrease

of quantity of the natural killers realizing their cytotoxic

function (CD56). The cell cultivation with the “Baikal-

sky” extract results in a valid increase (p < 0.05) of the

contents the cytotoxic (CD8+) lymphocytes in healthy

donors and a rise of the surface receptor (CD16) expres-

sion (Table 1).

During the studies of the B-cell part of the immune

system, no valid changes in B-lympocytes content

(CD20+-cells) and their subpopulation composition were

registered in the cell culture picked out from the healthy

donors under the influence of the “Taban-Arshan” ex-

tract (Table 2).

Under the influence of the drug compositions “Poly-

phytokhol” and “Baikalsky” a reduction of B-lympho-

cyte (CD20+) expression was observed. Moreover, the

“Baikalsky” extract changed the B-lymphocyte subpopu-

lation composition of a various degree of differentiation

making the CD38, mIgG expression lower (Table 2).

Changes of the cell culture content of various activa-

tion antigens in vitro did not differ significantly from

similar values of the healthy persons with the “Ta-

ban-Arshan” extract used.

The surface phenotype of the B-lymphocytes ex-

pressing marker CD23 in the process of cultivation of

lymphocytes taken from the healthy donors under influ-

ence of the “Baikalsky” extract showed a tendency to

increasing CD23 quantity (Table 3). The expression of

the receptor for transferrin―CD71 (that is an early acti-

vation marker) had a tendency to increase this factor

when coupled with the “Baikalsky” extract. It should be

noted that the quantity of lymphocytes taken from the

healthy donors that expressed “the late” activation anti-

gen HLA-DR in the presence of the “Polyphytokhol”

and “Baikalsky” extracts did not correspond to the simi-

lar factor of the healthy persons during cultivation with-

out the extracts. The quantity of lymphocytes that ex-

pressed the HLA-DR activation antigen was reduced

compared with the reference level (Table 3).

A tendency to increasing the number of lymphocytes

expressing the ICAM-1 (CD54) adhesion marker, which

were taken from the healthy donors was registered with

the use of “Baikalsky” extract. It means that there is no

full stop of the stimulation process of the immune sys-

tem.

The results of the investigation of the “Polyphytok-

hol” and “Baikalsky” extracts effects on the expression

of surface markers of the lymphocytes taken from the

healthy donors testify the absence of any toxic effect of

the examined extracts of the used concentration. The

L. Y. Semenova et al. / Health 3 (2011) 560-5 65

562

Table 1. Change of the expression of surface antigens of subpopulations of T- and NK-lymphocytes taken from the healthy donors

during cultivation under the influence of the “Taban-Arshan”, “Polyphytokhol” and “Baikalsky” extracts (in %).

Cultivation for 16 hours

Lymphocyte

markers

Lymphocytes of

healthy donors before

cultivation n = 10 Lymphocytes of healthy

donors without extracts

n = 10

Lymphocytes of

donors + “Taban-Arshan”

n = 10

Lymphocytes of

donors + “Polyphytok-hol”

n = 10

Lymphocytes of

donors + “Baikalsky”

n = 10

СD3 68.44 ± 2.7 54.93 ± 2.2 57.78 ± 2.1 62.30 ± 2.2* 55.60 ± 3.1

CD4 35.57 ± 1.4 36.97 ± 2.9 37.96 ± 1.7 39.30 ± 1.7 33.00 ± 1.7

CD8 25.85 ± 1.6 28.47 ± 1.2 26.19 ± 0.8 29.75 ± 2.1 32.50 ± 1.7*

CD16 10.82 ± 0.5 7.47 ± 0.3 8.20 ± 1.1 10.80 ± 1.1 12.90 ± 1.1*

CD56 6.95 ± 2.4 7.78 ± 2.0 7.32 ± 2.5 2.40 ± 2.5* 7.00 ± 2.5

p < 0.05 (compared with lymphocytes of the healthy donors with no extracts used).

Table 2. Change of the expression of surface antigens of subpopulations of B-lymphocytes taken from the healthy donors during

cultivation under the influence of the “Taban-Arshan”, “Polyphytokhol” and “Baikalsky” extracts (in %).

Cultivation for 16 hours

Lymphocyte

markers

Lymphocytes of

healthy donors before

cultivation n = 10 Lymphocytes of healthy

donors without extracts

n = 10

Lymphocytes of

donors + “Taban-Arshan”

n = 10

Lymphocytes of

donors + “Polyphytok-hol”

n = 10

Lymphocytes of

donors + “Baikalsky”

n = 10

CD20 10.77 ± 0.5 12.18 ± 0.4 10.20 ± 0.6 7.90 ± 0.6* 7.20 ± 0.63*

CD72 10.92 ± 0.7 9.70 ± 0.6 11.32 ± 2.2 7.10 ± 2.1 9.60 ± 1.21

CD38 7.40 ± 2.1 6.28 ± 1.2 5.78 ± 0.7 7.20 ± 0.7 2.80 ± 0.73*

mIgM 7.25 ± 0.5 6.72 ± 0.9 7.20 ± 1.2 5.10 ± 1.2 6.70 ± 1.23

mIgG 6.53 ± 0.6 6.32 ± 0.8 6.90 ± 1.9 5.10 ± 1.9 3.70 ± 1.94

-chain Ig 6.75 ± 0.9 6.68 ± 0.8 5.95 ± 1.0 6.00 ± 0.8 4.30 ± 1.26

-chain Ig 12.48 ± 2.9 9.38 ± 1.6 8.35 ± 1.9 9.00 ± 1.2 8.20 ± 1.06

p < 0.05 (compared with lymphocytes of the healthy donors with no extracts used).

Table 3. Change of the expression of activation antigens of lymphocytes taken from the healthy donors during cultivation under the

influence of the “Taban-Arshan”, “Polyphytokhol” and “Baikalsky” extracts (in %).

Cultivation for 16 hours

Activation

markers of

lymphocytes

Lymphocytes of

healthy donors before

cultivation n = 10

Lymphocytes of healthy

donors with-out extracts

n = 10

Lymphocytes of

donors + “Taban-Arshan”

n = 10

Lymphocytes of

donors + “Polyphytok-hol”

n = 10

Lymphocytes of

donors + “Baikalsky”

n = 10

СD23 5.73 ± 0.40 4.90 ± 0.01 6.80 ± 2.10 6.05 ± 2.85 8.25 ± 3.75

CD25 5.88 ± 0.47 5.18 ± 0.29 5.40 ± 0.42 5.00 ± 0.42 6.30 ± 0.42

CD71 7.60 ± 1.43 8.20 ± 1.38 7.34 ± 0.71 5.70 ± 0.71 10.60 ± 0.71

HLA-DR 12.15 ± 2.9 10.78 ± 2.27 10.40 ± 2.34 5.60 ± 2.3* 4.00 ± 2.34*

CD95 6.57 ± 1.68 7.64 ± 1.44 7.05 ± 1.06 6.20 ± 1.06 6.40 ± 1.06

CD54 5.72 ± 0.79 4.91 ± 0.65 5.10 ± 0.75 5.50 ± 0.50 7.80 ± 0.20

p < 0.05 (compared with lymphocytes of the healthy donors with no extracts used).

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investigation has also demonstrated immunosuppressive

effects of the “Polyphytokhol” and “Baikalsky” extracts

on the processes of lymphocytes differentiation. The

conducted studies make it possible to conclude that the

“Taban-Arshan” extract of the Tibetan medicine is the

only one of the tested drugs, which has neither activating

nor inhibiting effects on the expression of the surface

receptors of the healthy donors’ lymphocytes.

At the next step of the research effects of the “Ta-

ban-Arshan”,“Polyphytokhol” and “Baikalsky” extracts

on the lymphocytes taken from the patients with early

rheumatoid arthritis were investigated.

Previous studies have shown that a reduction of

T-lymphocytes in the blood due to helper cells and a

decrease of quantity of mature B-lymphocytes primed by

an antigen are typical of the early rheumatoid arthritis.

An increase of lymphocyte expression CD54 receptor of

cell adhesion and CD23, CD25, CD71, HLA-DR, CD95

activation markers was also noticed [18-25].

During the cultivation of the lymphocytes taken from

the patients with early rheumatoid arthritis no changes

were registered related to normalization or improvement

of any disturbed parameters of the surface lymphocyte

markers when the “Polyphytokhol” and “Baikalsky”

extracts were used. The “Polyphytokhol” extract raised

the mIgG, CD71, HLA-DR expression, and the “Bai-

kalsky” extract considerably increased the quantity of

CD8, CD56, which had already been high in the cell

culture of the patients with early rheumatoid arthritis. It

was also noticed that due to the extract effect, the num-

ber of CD4-lymphocytes was decreased though their

level was already low in such patients. One should pay

attention to the fact that the “Polyphytokhol” and “Bai-

kalsky” extracts increase ICAM-1 expression, which

indicates advisability of their use as anti-inflammatory

drugs of folk medicine. However, taking into account

their negative effects on the immune system, it is not

expedient to use them as immunomodulators in case of

autoimmune diseases.

The results of the analysis of the “Taban-Arshan” ex-

tract’s effect on the lymphocytes picked out of the pa-

tients with early RA show that it does not affect the ex-

pression of T-lymphocytes and CD16 and CD56 markers.

If the extract is added to the lymphocytes of patients

with early RA, during the cultivation there appears a

tendency to lowering the expression of the marker of

B-lymphocytes CD20. It indicates a decrease of potential

readiness for B-cells activation. In this connection an

analysis of changes in the content of B-lymphocytes of

various degree of differentiation patients with the early

RA during the cultivation process was performed. The

“Taban-Arshan” extract affects the content of differenti-

ated forms of the B-lymphocytes obtained from the ex-

amined patients. A valid reduction of the number of

plasma cells (CD38) р < 0.05 under influence of the ex-

tract (Figure 1) compared with the similar index for the

healthy one is regular.

A low level of expression of both receptors of CD38

and mIgM+-, mIgG+-lymphocytes confirms the fact that

mature B-cells affected by the extract are neither acti-

vated nor transformed into antibody producing cells.

The examined extract exerts a marked inhibitory ac-

tion on the activated B-lymphocytes that express the

FcRII (CD23+-cells) receptor; in the “Taban-Arshan”

extract presence (Figure 2), this index goes down from

14.49% ± 1.16% to 8.21% ± 0.54%. It means that there

is deceleration of activation processes within the B-cell

part of the immune system.

The effect of the examined phytopreparation results in

a tendency of lowering the expression of the receptor for

interleukin-2 (CD25), the receptor for transferrin (CD71)

and the expression of the late activation antigen

(HLA-DR+-lymphocytes), that reflects processes of dif-

ferentiation of lymphocytes, the expression of which

approached similar values of the healthy donors. The

increase of the lymphocytes expressing the adhesion

ICAM (CD54) in the cell cultures of the examined pa-

tients show that in case of early rheumatoid arthritis the

Figure 1. Expression of the surface antigens of the B-lym-

phocyte subpopulation in the patients with early rheumtoid

arthritis with the “Taban-Arshan” extract used.

L. Y. Semenova et al. / Health 3 (2011) 560-5 65

564

Figure 2. Expression of activation antigens by lymphocytes in

the patients with early rheumatoid arthritis with the “Taban-

Arshan” extract used.

processes of the immune system stimulation are not

switched off. [26-28]. The “Taban-Arshan” extract

causes a valid reduction in the expression of this marker

to the level of the healthy donors (Figure 2 ).

The increase of the expression of membrane CD30

during an autoimmune inflammation is based on a

mechanism of involving the inflammatory cells in an

antigen response reaction. So the patients with early

rheumatoid arthritis have an increase of the CD30 sur-

face receptor from 0.73% ± 0.76% up to 3.68% ± 0.60%

registered for the donors [29,30], however in the pres-

ence of the extract this figure decreases to 2.90% ±

0.25%.

The results of this experimental section prove that, on

the one hand, there is no significant effect of the exam-

ined extract on expression of either T-lymphocyte mark-

ers or NK-cells (CD16) and activated NK-cells (CD56)

in the patients with early rheumatoid arthritis.

On the other hand, it has been noticed that the “Ta-

ban-Arshan” extract exerts a slight inhibitory effect on

the expression of early activation antigens of lympho-

cytes CD25, CD71, as well as of late activation antigen

HLA-DR and surface immunoglobulins M and G.

It has been determined that the “Taban-Arshan” extract

reduces the expression of molecules of cell-cell adhesion

ICAM-1, expression of low-affinity receptor for IgE on

activated B-lymphocytes, the quantity of plasma cells

CD38 and expression of surface receptor CD30.

Thus, the “Taban-Arshan” extract of the Tibetan

medicine produces an immunomodulatory effect: it in-

hibits apparent activation of the B-cell part of the im-

mune system in patients with early rheumatoid arthritis.

The revealed immunomodulatory effect suggests that the

“Taban-Arshan” extract can be used for patients with

other autoimmune diseases, which have similar immune

status disorders.

The conducted comparative studies of the Tibetan

medicine extracts make it possible to single out the “Ta-

ban-Arshan” extract as having the apparent immuno-

modulatory effect. Our studies have proved the “Ta-

ban-Arshan” extract ability to correct an immune re-

sponse in inflammatory diseases: the “Taban-Arshan”

extract corrects the changes of the immune system char-

acterized by evident activation of the B-cell part of the

immune system and normalizes immune parameters of

the lymphocytes taken from the patients with autoim-

mune diseases (early rheumatoid arthritis). The results of

our studies confirm that the immunocorrective effect of

the “Taban-Arshan” extract is connected with its ability

to suppress increased activation of lymphocytes by nor-

malizing expression of the main activation antigens

(CD23, CD25, CD71, HLA-DR, CD54).

Among the three examined preparations, the “Ta-

ban-Arshan” extract proves to be most effective for cor-

rection of immune disturbances in early rheumatoid ar-

thritis. Evidently, the immunomodulatory function of the

extract during the cell cultivation process, which we

have revealed, can produce similar actions on activated

lymphocytes in the human organism.

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