bone marrow is the major hematopoietic organ . bone marrow hematopoietic tissues are highly sensitive to toxins , including radiation and chemotherapeutics , which may induce myelosuppression .
the cytoactivities of primitive hematopoietic cells may , therefore , be depressed , and the number of peripheral blood cells may decrease substantially .
in addition , severe myelosuppression may cause infections , inflammation , and bleeding [ 1 , 2 ] .  
it is generally accepted that maintenance of normal hematopoiesis relies on the stabilization of the hematopoietic microenvironment . as the major component of this microenvironment ,
hematopoietic cytokines play a key role in the regulation of hematopoiesis , controlling the proliferation , differentiation , and maturation of primitive hematopoietic cells [ 3 , 4 ] .
il-6 is a kind of cytokine with a wide range of biological activities , produced mainly by t and b lymphocytes , monocytes , fibroblasts , and so forth [ 5 , 6 ] .
recent studies showed that il-6 involved not only in the progress of the stress reaction , autoimmune , and neoplastic diseases of the body but also in regulation of the proliferation and differentiation of primitive hematopoietic cells , and it is considered as a permissive factor of primitive hematopoiesis .
of all the cytokines , il-6 is the first one found to be associated with the pathogenesis of the disease , due to its close relation with a variety of diseases under overexpression .   in the present study , a mouse model of aplastic anemia
was established by using cyclophosphamide in combination with chloramphenicol and co radiation . after modeling ,
the in vitro proliferation of bmsc was tested by mtt assay , and the il-6 secretion levels of bmsc were analyzed .
this study gives new insights into the hematopoiesis regulating properties of il-6 in mice with aplastic anemia .
the procedures of our experiment are in compliance with the principles of laboratory animal care .
imdm tissue culture media and il-6 elisa kits were purchased from the jingmei company ( chengdu , china ) .
the tunel kit was purchased from the boster company ( wuhan , china ) . the fully automatic blood cell analyzer ( hs-18 )
was made in italy ; the inverted phase contrast microscope by the chong qing optical instrument factory ( china ) ; the co2 incubator ( mcd-15a ) in japan ; and the fully - automatic elisa meter by the thermo company ( usa ) .
healthy balb / c male mice , weighing 1822  g , aged 68 weeks , were provided by the experimental animal center of chengdu university of traditional chinese medicine .
animals were housed in a warm , quiet environment with free access to food and water .
the radiation source ( co ) was provided by sichuan academy of agricultural sciences .  
briefly , the mice were irradiated with 2.0  gy co and then treated with daily intraperitoneal injections of cyclophosphamide at 40  mg / kg / day and chloramphenicol at 50  mg / kg / day for the next three days .   on day 7 after modeling ,
20  l blood was taken from the orbital vein of the mouse and diluted with the manufacturer recommended diluent before analysis with the hs-18 fully automatic blood cell analyzer .
eight mice from each group were sacrificed , and the femurs of each mouse were removed .
the bmc suspension was prepared according to zhang 's method , and the number of bmc was counted . on day 7 ,
two mice from each group were randomly chosen , the femurs and spleens were removed , fixed in 4% neutral formalin , decalcified , and desiccated , and embedded in paraffin , and then semithin sections were cut .
after hematoxylin - eosin staining , the pathomorphological changes of bone marrow and spleen were observed with a light microscope . on day 7 , bmc smears were prepared , and bmc apoptosis was analyzed by tunel , according to the specifications of the tunel kit .
5 microscopic fields of each slide were randomly chosen and observed under microscope , and the apoptosis ratio was calculated .
on the 4th , 7th , and 10th days after modeling , bmc suspensions were prepared as described above then adjusted to a concentration of 1  10  cells / ml .
bmc were cultured in imdm supplemented with 15% fetal calf serum at 37c in 5% co2 and saturated humidity . after 48  h of cultivation , culture supernatants were harvested and stored at 20c for il-6 analysis .
mtt was added to every well , and the culture medium was put back to the incubator .
4  h later , the mtt was removed and the cells were washed by pbs twice . 100  l of isopropyl alcohol was added to split the cells . then , the enzyme - linked immunosorbent assay was used at the 570  nm to detect the a value of every well .
all data were expressed as mean  standard deviation ( x-s ) , and the t - test was used to evaluate the difference between groups , with p < 0.05 considered to be statistically significant .
as measured by the fully automatic blood cell analyzer , the number of peripheral blood cells in the aplastic anemia mouse model was notably decreased ( p < 0.05 ) . besides , the number of bmc was markedly decreased ( p < 0.05 ) .
these data indicated that the aplastic anemia model had been successfully established ( figure 1 ) .  
the morphology of the bone marrow tissue had changed obviously after modeling . in our model ,
the amount of hematopoietic tissue , as well as the number of hematogenous cells in bone marrow , was obviously reduced .
the number of lymphocytes , fat cells , and other nonhematogenous cells , however , increased markedly .
these changes were accompanied by interstitial edema and blood sinus expansion ( figure 2 ) .
the spleen atrophied , the splenic corpuscle shrank or disappeared , and its structure was clearly compromised .
spleen blood sinuses dilated , hematopoietic focus disappeared , and megakaryocytes decreased or were absent ( figure 3 ) .  
the apoptosis rate of bmc from the aplastic anemia group ( 9.75  2.43% ) was significantly higher than that of normal controls ( 3.63  1.06% ; figure 4 ) ( p < 0.05 ) .
nuclei of the apoptotic bmc were stained red with fast red while nuclei of nonapoptotic bmc were stained blue with hematoxylin ( figure 5 ) . 

figure 6 showed that at the three time points ( 4th day , 7th day , and 10th day ) , the level of il-6 in cultured supernatants of bmsc from the anemia group was significantly higher than that of the normal group ( p < 0.05 ) .
bmsc displayed long fusiform shape under invert phase contrast microscope cultured more that 2 days ( figure 7 ) .
the a value of model group at the three time points ( 4th day , 7th day , and 10th day ) was significantly lower than that of normal group ( p < 0.05 ) ( figure 8) .
it has many target cells including macrophages , liver cells , stationary t cells , activated b cells , and plasma cells .
it can play important roles in the acute phase of the immune response , hematopoietic regulation , and other processes .
recent studies also found that il-6 can stimulate the formation of myeloid , erythroid , megakaryocyte , and macrophage cloning , in combination with epo , il-3 , and other hematopoietic cytokines .
as il-6 has important functions in proliferation , differentiation , and maturation of early hsc , it is considered as a permissive factor of primitive hematopoiesis .  
defective bone marrow stroma , or microenvironment , has been proposed as one of several mechanisms to account for bone marrow failure .
this could involve defects in positive- or negative - acting hemopoietic regulator expression by stroma or alteration of normal stroma - stem cell interactions . as the permissive factor of primitive hematopoiesis ,
the role of il-6 in the bone marrow hematopoiesis disorder has been subject of intense attention in recent years .
so far , the precise mechanism of il-6 in hematopoiesis regulation still remains largely unclear . in the present study , a mouse model of aplastic anemia
was established by using cyclophosphamide in combination with chloramphenicol and co radiation . after modeling ,
the peripheral blood cells and bmc were significantly reduced and the apoptosis of bmc was obviously increased . decreased hematogenous cells and increased fat cells in the bone marrow of the mouse model were easily observable with a microscope .
furthermore , the spleen shrank , the splenic sinus was enlarged , and the hemopoietic focus were evidently reduced .
these results demonstrated that the bone marrow hematopoietic function was severely injured , and the mice model was successfully constructed .   to investigate the level of il-6 secreted by bone marrow stromal cells from the mouse model of aplastic anemia , in the present study , in vitro cultivation of bone marrow stromal cells was employed , and the il-6 level was measured with elisa .
the result showed that at the three time points ( 4th day , 7th day , and 10th day ) , the level of il-6 in cultured supernatants of bmsc from the anemia group was significantly higher than that of the normal group , which indicated that the il-6 secretion level may be enhanced to some extent by the induction of aplastic anemia caused by irradiation and chemotherapeutic drugs .
as a cytokine with a wide range of biological activities , il-6 has very complex functions .
it is a key component of the inflammatory mediator network , with an important role in the inflammatory response . as an anti - inflammatory cytokine
however , il-6 is also a proinflammatory factor and , when overexpressed , can lead to a variety of diseases .
significantly elevated il-6 levels were detected in the serum of rheumatoid arthritis , diabetes , acute pancreatitis , hiv , and cancer patients [ 1316 ] .  
herodin et al . reported that in radiation - induced bone marrow depressed baboons , a substantial precocious and transient il-6 blood release was detected within hours after irradiation .
the origin of the precocious transient il-6 peak observed just after irradiation was not clear .
it was supposed that the increase of il-6 may be caused by a more precocious il-1 and/or tnf release . in a clinical experiment ,
fu et al . found that the pretreatment level of il-6 in serum and bone marrow of 37 aplastic anemia patients were slightly higher than that of normal control , but there was no significant difference between the aplastic anemia group and the normal control group .
supposed that the change of il-6 level in aplastic anemia patients may be due to an association with inflammatory response , and il-6 may have an effect of immunoregulation and mediate induction in the destruction pathology of bone marrow cells , but the concrete mechanism still needs to be elucidated . in our study , we noticed that in the anemia group , with the increase of il-6 level , the proliferation of the bmsc was obviously impeded .
this result indicated that high level of il-6 might be harmful to the proliferation of bone marrow stromal cells and might interfere with the stability of the bone marrow hematopoietic microenvironment . as the main component of the bone marrow hematopoietic microenvironment , bmsc adhere to hematopoietic stem cells , support and regulate the internal environment for the settling , differentiation , proliferation , and maturation of the primitive hematopoietic cells , and secrete a variety of cytokines to regulate hematopoiesis ; therefore , defects of the bone marrow stromal cells can cause hematopoietic disorders of the bone marrow [ 19 , 20 ] .
the results of our study suggested that the il-6 secretion level may be enhanced to some extent by the induction of aplastic anemia caused by irradiation and chemotherapeutic drugs and that the abnormal level of il-6 might probably interfere with the stability of the bone marrow hematopoietic microenvironment .
currently , the role of il-6 in the regulation of hematopoiesis process and the specific mechanism remain unclear , so further investigations are needed .
 parasecretion of the hematopoietic cytokines is considered as one of the mechanisms account for bone marrow hematopoiesis disorder . in this study , the level of il-6 secreted by bone marrow stromal cells from a mouse model of aplastic anemia was analyzed . 
 the aplastic anemia mouse model was established with cyclophosphamide in combination with chloramphenicol and 60co radiation . 
 the impairment of bone marrow hematopoiesis induced by irradiation and chemotherapeutic drugs was subsequently characterized by peripheral blood cell count , pathomorphological changes , and apoptosis rate . 
 furthermore , the in vitro proliferation of bone marrow stromal cells ( bmsc ) and the il-6 secretion levels of bmsc were analyzed . in our model of aplastic anemia , 
 the number of peripheral blood cells and bone marrow cells ( bmc ) were notably decreased , and the apoptosis rate of bmc increased . 
 furthermore , the proliferation of bmsc was obviously impeded while the il-6 secretion levels of bmsc significantly increased . 
 the findings of our study suggested that the il-6 secretion level may be enhanced to some extent by the induction of aplastic anemia caused by irradiation and chemotherapeutic drugs and that the abnormal level of il-6 might probably interfere with the stability of the bone marrow hematopoietic microenvironment . 