當歸內酯對免疫抑制小鼠免疫功能的重建作用

龍 銳,楊 芳,杜俊蓉＊,王 蓓

1重慶醫科大學附屬第一醫院藥劑科,重慶 400016;2四川大學華西藥學院藥理學與生物制藥系,成都 610041

當歸內酯對免疫抑制小鼠免疫功能的重建作用

龍 銳1,2,楊 芳2,杜俊蓉2＊,王 蓓2

1重慶醫科大學附屬第一醫院藥劑科,重慶 400016;2四川大學華西藥學院藥理學與生物制藥系,成都 610041

探討當歸內酯(ASDL)對免疫抑制小鼠免疫功能的重構作用。通過小鼠腹腔注射環磷酰胺建立免疫抑制動物模型。采用免疫器官重量法和小鼠腹腔巨噬細胞吞噬雞紅細胞實驗檢測了 ASDL對非特異性免疫功能的影響;用血清溶血素分光光度法檢測了對體液免疫功能的作用;用MTT法進行了致分裂原誘導的小鼠脾淋巴細胞的增值反應實驗,再用乳酸脫氫酶法測定了NK和 CTL細胞活性,從而確定ASDL對小鼠細胞免疫功能的影響。結果表明:ASDL能夠對免疫低下小鼠的非特性和特異性免疫功能起到一定的重構作用。但是這種效果并不是劑量依賴性的,20 mg/kg這個劑量的效果明顯好于 5和 80 mg/kg這兩個劑量。上述結果表明 ASDL能夠顯著提高免疫低下小鼠的免疫功能。

當歸;當歸內酯;環磷酰胺;免疫抑制;小鼠

Introduction

During the past several decades,a considerable number of Chinese medical herbs haven been found to be immunomodulatores upon screening them for inhibition of immunosuppressive reaction,especially in cancer patients treated by chemotherapy.Angelicasinensis(Oliv.)Diels(ASD),called Danggui in China,plays multiple roles in phar macological and biological functions as a well-known Chinese traditionalmedicine.The major componentsofASD are volatile oil andwater-soluble substance[1].It was reported that ethanol extract fromAngelica sinensiscould significantly enhance the mouse cell-mediated immune function in vitro[2].However,it is unclearwhich component in ASD volatile oil can increase the immunocompromised host immune function.In addition,the restoring mechanis ms of immune function of ASD are still not fully understood. Lactones were the main components of ASD volatile oil.Lots of studies showAngelica sinensislactones(ASDL)has various bioactivities,such as it’s effects onthe uterus s mooth muscle,cardiovascular system,respiratory system,central nerve system and so on.However,the immunomodulation of ASDL in immunocompromised model has not been reported yet.The aim of this report is to reveal the effectiveness of oral ASDL administration to evaluate the thymus and spleen index,macrophage phagocytosis,serum hemolysin concentration,spleen lymphosyte proliferation,cytotoxic T lymphocyte and nature killer activity in CTX-treated I CR mice.

Materials andM ethods

Preparation of ASDL

Danggui was purchased from the cultivating base of Good Agricultural Practice in Min Xian County,Gansu Province,China.Its identitywas confirmed by comparison with descriptions of characteristics and appropriate monograph in Chinese Phar macopoeia.ASDL was extracted,separated and purified with Supercritical Fluid Extraction technique in our laboratory.The concentration of lactoneswas found to be＞90%.

Exper imental an imals

Studies were carried out in male I CR mice weighing 18.0～22.0 g,and these mice were obtained from the experimental an imal institution(Academy of Medical Sciences,Sichuan,China).The animals were randomly divided into five groups(n=10):nor mal control group,cyclophosphamide(CTX)and 5,20,80 mg/kg ASDL-treated group.ASDL was diluted in 3%Tween-80 and administrated p.o.for 7 days once daily.The normal control group and CTX group mice were administrated volume-matched solvent.Mice were given a single intraperitoneal(i.p.)dose of CTX (80 mg/ kg)except the mice of normal control group.On day 8,all animal were executed.The mice,thymus and spleen were weighed.Thymus and spleen indexwas expressed as the thymus and spleen weight relative as bodyweight.

Macrophage phagocytosis assay

Phagocytosis ofmice was performed using the methods ofLinet alwith some modifications[3].The mice were injected i.p.with 1.0 mL 2% soluble starch 3 days prior to sacrifice.On day 8,themicewere injected i.p. with 0.5 ml 5%cock red blood cells(CRBC).After 30 min,the macrophages were collected,fixed with methanol and stained withW right-Giemsa.The number of CRBC ingested by 100 macrophages was counted in an optical microscope and expressed as phagocytosis index(PI).Besides,the percentages of macrophages that ingested CRBC were deter mined and expressed as phagocytic efficiency index(PEI).

PI=numberof total ingested CRBC/numberofmacrophage ingesting CRBC

PEI(%)=(number of macrophage ingesting CRBC /number of totalmacrophage)×100

Quantitative hemolysis of sheep red blood cells (QHS)assay

QHS assaywas detected using the method described by Xu and Li[4].In brief,0.2 mL of 20%sheep red blood cells(SRBC)prepared in normal saline was injected to animals,i.p.4 days prior to the assay.On day 4 following immunization,mice were bled and serum samples was diluted with normal saline to 1:500.A total of 1.0 mL 5%SRBC and 1.0 mL 10%guinea pig serum were mixed with 1.0 mL diluted serum and incubated for 30 min.Hemolysin concentration was determined by colorimetric method.

M itogen induced splenocyte proliferation

The extirpated spleenswere treated in ger m-free condition.Single spleen cells suspension were prepared in cold phosphate buffered saline (PBS) by forcing spleen fragments through a fine nylon mesh.Washed twice in PBS and in low speed centrifugation,1000 r/ min for 5 min,the cellswere resuspended to a concentration of 1×106cells/ml in RP M I1640 completely cultivated liquid (HyClone,USA).100μL single spleen cells suspension was placed on a 96-wellmicroplate and 100μL concanavalinA(ConA,10 mg/L)or lipopolysaccharides(LPS,100 mg/L)was added to each testwell,and 100μL RPM I1640 cultivated liquid was added to controlwell.After treatment in 5%CO2-air mixture at 37℃for 44 h,10μL MTT(5 g/L)was added to each well and the plate was incubated for another 4 h.The supernatants were discarded.A total of 150μL dimethyl sulfoxide was added to each well.The plate was then shaken until crystalswere dissolved.Theabsorbance A570 was detected on theMicroplate Reader(Bio-Rad,Model 550,USA)

Assays of cytotoxic T lymphocyte(CTL)

The assay for cytotoxic T lymphocyte(CTL)activity was performed as described previouslywith some modification[5].The splenocytes from each mouse were sensitized in vitro with mitomycin C-treated P815 mastocytoma cells at a responder/sensitizer ratio of 50:1 in a 5%CO2-airmixture at 37℃for 5 days.For the preparation ofmitomycin C-treated P815 cells,cells(1×105cells/ml)were incubated in the dark with mitomycin C at a concentration of 0.25μg/mL for 30 min at 37℃, then the cells were washed four times.Following the sensitization phase,cultured spleen cellswere harvested and resuspended in RPM I 1640 complete cultivated liquid,then were counted and diluted to 1×106cells/ mL for determination of CTL activity.P815 cells(1× 105cells/ml)were co-cultured in duplicate with 100 μL splenic effector cells in a 96-well microplate(the cell ration of effector-to-target is 10:1).The plates were incubated for 4 h at 37℃.Lactate dehydrogenase (LDH)activitywasmeasured in 50μL/well of the supernatant by addition of the enzyme substrate and the absorbance was recorded at 490 nm.To deter mine the percentage of killed target cells,the following equation was used:%lysis=(E-S)/(M-S)×100%;where E represents the experimental release of LDH activity from target cells incubated in the presence of lymphocytes;M represents the max imum release of the LDH activity deter mined by lysing the target cells with 1% ofNP-40;S is the spontaneous release of the LDH activity from target cells incubated in the absence of lymphocytes.

Assay of NK cells activity

100μL single spleen cells suspension(1×106cells/ mL)and 100μL target cells YAC-1(1×105cells/ ml)were placed on a 96-wellmicroplate(effect:target =10:1).The cells were incubated in a 5%CO2–air mixture at 37℃ for 4 h.Lactate dehydrogenase (LDH)activitywasmeasured as described above.

Statistical analysis

Resultswere expressed as mean ±standard deviation (S.D.).Statistical significance was performed with SPSS 13.0.Analyses of variance were followed by Fisher’s least significant difference test.Differences were considered to be significantwhenP＜0.05.

Results

Effects of ASDL on nonspecific immune function in im munocomprom ised m ice

Table 1 Effect ASDL on thymus and spleen indexes,macrophage phagocytosis in immunocomprom ised m ice(mean±S. D.,n=10)

As shown in Table 1,5 and 20 mg/kg ASDL caused a significant increase in the thymus,spleen index and macrophage phagocytosis compared with CTX-treated control group.In addition,ASDL 20 mg/kg could regulate nonspecific immunity in immunocompromised mice nearly to that of nor mal control.Treatment with ASDL (80 mg/kg)also resulted in an increase in the thymus,spleen index and macrophage phagocytosis,although the increase did not reach the level of statistical significance.Effects of ASDL on humoral immunity in immunocompromised mice 20 mg/kg ASDL could significantly increase the hemolysin concentration in immunocompromised mice by QHS assay(P＜0.05). Although 5 and 80 mg/kg ASDL also could increasethe antibody concentration in mouse serum,the statistically significant differenceswere not found(Fig.1).

Fig.1 Effects of ASDL on the humoral immune function in immunocomprom ised m ice.(n=10)

Effects of ASDL on cellular immun ity in immunocomprom ised m ice

The effect ofASDL on the induced proliferation of splenic lymphocytes was examined by MTT method. The results in figure 2 demonstrated the effect of different dose ofASDL(5,20,80 mg/kg)on the proliferation of splenic lymphocytes induced by Con A and LPS.Con A-induced T lymphocyte proliferation was significantly increased at allASDL dose(5,20,80 mg/ kg),and the proliferation rate is 35.5%,41.1%and 18.1%,relatively.LPS-induced B lymphocyte proliferation was also significantly increased at allASDL dose, and the proliferation rate is 24.2%,36.3% and 17.1%,relatively.

The enhancement of proliferation of splenic lymphocytes by ASDL suggests that this compound may also affect the generation of cell-mediated cytotoxic responses.To test this,we examined the effect ofASDL on the production of antigen-specific cytotoxic T lymphocytes (CTLs).The cytolytic activity of effector cells against p815 target cellswas deter mined in a Lactate dehydrogenase activity assay.5,20,80 mg/kg ASDL could remarkably increase CTL activity in immunocompromised mice(P＜0.001)(Fig.3A).

Fig.2 Effects of ASDL on the T(A)and B lymphocyte proliferation(B)in immunocomprom ised m ice.(n=10)

To investigate the effect ofASDL on the developmentof non-specific cytotoxic cells,the cytotoxic activity ofNK cellswas determined in LDH release assay using YAC-1 target cells.The effectofASDL on the developmentof NK cell-mediated cytotoxicitywas significant at the doses of 5,20 and 80mg/kg(P＜0.001)(Fig.3B).

Fig.3 Effects of ASDL on the CLT(A)and NK cell activity(B)in immunocomprom ised m ice(n=10)

D iscussion

Host immune fuction is composed of nonspecific,humoral and cellular immunity.Phagocytes act as regulator and effector cells in the immune system and phagocytosis represents the indispensable step of the immunological defense system[6].The humoral defence via antibody response ismediated byB cells and other immune cells involved in antigen processing and immunization.The antigen– antibody complex can counteract toxin and defend the infection induced by pathogen. Cell-mediated immune defense is mediated specifically by T cells including cytotoxic T cells.T cells produce many lymphocyte factors consisting of macrophage mobile factor,lymphotoxin,transfer factor and interferon, which can enhance macrophage phagocytosis and the capacity of killing target cells.NK cells are directly cytotoxic for target cells and play primary role in regulating immune responses aswell[7].

Although CTX is one of the commonly used chemotherapeutic agents in clinic,this agent usually comes with side effects,such as immunosuppressive reaction.CTX has a profound effect on the humoral immune response in exper imental animalmodels,particularly in the early events following its administration[8].The compound may also lower the humoral response affecting IgA production in Peyer’s patches,accompanied by a significant decrease in IL-2 and IL-4 production by st imulated T cells.The proliferative response of lymphocytes to mitogens was still markedly suppressed after 5 weeks following CTX administration[9].CTX was also shown to inhibit the cellular immune response in guinea pigsand mice-.In this study,we found thatASDL could significantly increase macrophage phagocytosis,hemolysin concentration,spleen lymphocyte proliferation,CTL activity in CTX-treated ICR mice,which suggested ASDL could enhance the nonspecific,humoral and cell-mediated immunity in immunocompromised mice.

In this experiment,the immunomodulative activity of ASDL was in a dose-dependentmannerwhen the doses ofASDL range between 5 mg/kg to 20 mg/kg.However,the effect ofASDL 80 mg/kgwas lower than 5 mg/ kg.Hence,we recommend ASDL 20 mg/kg to enhance host immune function in immunocompromised mice. In conclusion,ASDL can reduce the side and toxic effect caused by chemotherapy by inhibiting the immunosuppressive reaction.Hence,in the process of tumor chemotherapy,we propose thatASDL may be find clinical application for immunocompromised patients as an adjuvant therapy a imed at the reconstitution of host immune function.

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Reconstitution of Immune Response byAngelica sinensis(O liv.)D iels lactones in Immunocomprom isedM ice

LONG Rui1,2,YANG Fang2,DU Jun-rong2＊,WANGBei2

1The First Affiliated Hospital,Chongqing M edical University,Chongqing 400016,China;2Departm ent of Phar m acology,and B iophar maceuticsW est China School of phar m acy,Sichuan University,Chengdu 610041,China

This paper studied the effects of lactone extracts fromAngelica sinensis(Oliv.)Diels(ASDL)on immunity functions in immunocompromised mice by differentmethods.The nonspecific immune function wasmeasured by thymus, spleen index and macrophage phagocytosis.Determination of hemolysin concentration in serum byQuantitative hemolysis of sheep red blood cells assay revealed the humoral function.In addition,the proliferative response of lymphocyte to concanavalin A nd lipopolysaccharides,the activity of cytotoxic T lymphocyte and nature killer cells activitywere studied to demonstrate the effects of ASDL on cellular immunogical functions.These effects were not dose-dependent in a linear fashion.A total of 20 mg/kg dose is more effective than 5 and 80mg/kg doses.In summary,we conclude that the oral treatment of immunocompromised mice with ASDL led to partial reconstitution of the nonspecific,humoral and cellular immune response.

Angelica sinensis;Angelica sinensis(Oliv.)Diels lactones;cyclophosphamide;immunocompromise;mice

December 14,2009;Accepted February 10,2010

R282.71;Q949

A

1001-6880(2010)03-0398-05

＊Corresponding author Tel:86-28-85503938;E-mail:lrzj820@yahoo. com.cn