BACKGROUND
as the primary effector cell, mast cell is actively involved in the pathogenesis of both acute and chronic allergic diseases <cit> .
upon activation, mast cell can release not only its preformed but also newly generated mediators to fulfill its biological functions.
besides histamine, heparin and proteases, mast cells can synthesize and secrete a variety of cytokines <cit>  such as il- <dig>  il- <dig> and il- <dig>  these cytokines have been well documented for their ability to regulate cell behavior including growth, secretion and migration in physiological and pathological conditions.
in recent years, pars have been indentified as receptors for serine proteinases.
pars are a subfamily of seven transmembrane g-protein-coupled receptors.
among them, par- <dig>  par- <dig> and par- <dig> serve as a receptor of thrombin <cit> ; par- <dig>  par- <dig> and par- <dig> are receptors of trypsin and par- <dig> is a receptor of tryptase.
these serine proteinases have been discovered to play a crucial role in allergic inflammation.
tryptase was reported to be able to stimulate microvascular leakage in the skin of guinea pigs <cit> , to induce inflammatory cell accumulation in the peritoneum of mice <cit> , to elicit histamine release from mast cells <dig> and to enhance monocyte chemoattractant protein- <dig>  and il- <dig> production in human endothelial cells <cit> .
trypsin was found to be able to induce histamine release from human tonsil and skin mast cells <cit>  and to stimulate il- <dig> and il- <dig> release from human respiratory epithelial cells <cit> .
rnai is a process in which double-stranded rna  induces the posttranscriptional degradation of homologous transcripts.
it has been observed in variety of organisms including plants, fungi, insects, protozans and mammals <cit> .
rnai can be initiated by exposing cells to dsrna either via transfection or endogenous expression.
dsrnas are processed into 21- to 23-nt double-stranded fragments known as sirnas <cit> .
with unprecedented speed, rnai has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool in mammals.
the aim of this study is to investigate the effect of par- <dig>  par- <dig> and par- <dig> gene silencing on the expression of pars and cytokine production in p <dig> cells by using rnai.
RESULTS
reduction of par- <dig> expression in p <dig> cells by sirna
three target sequences in the coding region of par- <dig> mrna were selected as sirna sequences, namely sirna par- <dig> , sirna par- <dig>  and sirna par- <dig> .
among the  <dig> sirna par-1s, sirna par- <dig>  appeared most effective in inhibition of par- <dig> mrna expression in p <dig> cells .
approximately up to  <dig> % reduction of par- <dig> mrna expression was observed when p <dig> cells were transfected with  <dig> nm of sirna par- <dig>  for  <dig> h. at  <dig> or  <dig> h following transfection, however sirna par- <dig>  showed less degree of inhibition of par- <dig> mrna expression .
flow cytometry analysis revealed that par- <dig> protein expression was eliminated by up to  <dig> % when p <dig> cells were transfected with  <dig> nm of sirna par- <dig>  for  <dig> h .
because sirna par- <dig>  showed greater inhibitory effect on par- <dig> expression at  <dig> h than at  <dig> or  <dig> h following incubation, the  <dig> h incubation period was chosen as the fixed time point for the following western blot and immunofluorescent analysis.
approximately up to  <dig> % and  <dig> % inhibition of par- <dig> expression was observed by western blot  and immunofluorescent analysis , respectively.
sirna par- <dig>  and sirna par- <dig>  also showed significant inhibition of par- <dig> protein expression in p <dig> cells .
reduction of par- <dig> expression in p <dig> cells by sirna
of the three sirna par-2s, sirna par- <dig>  and par- <dig> , but not par- <dig>  downregulated par- <dig> mrna expression by up to  <dig> % and  <dig> % at  <dig> h following transfection .
therefore sirna par- <dig>  was employed as a representative throughout the study.
sirna par- <dig>  also suppressed par- <dig> protein expression during flow cytometry, western blot and immunofluorescence staining analysis by up to  <dig> % ,  <dig> %  and  <dig> % , respectively following  <dig> h incubation period.
at  <dig> and  <dig> h following incubation, sirna par- <dig>  showed significant downregulation of par- <dig> expression on both mrna and protein levels.
reduction of par- <dig> expression in p <dig> cells by sirna
all three sirnas of par- <dig>  namely sirna par- <dig> , sirna par- <dig>  and sirna par- <dig>  were able to downregulate the par- <dig> expression on both mrna and protein levels in p <dig> cells .
however, sirna par- <dig>  showed the most potent effect on p <dig> cells among the  <dig> sirnas.
it was observed that sirna par- <dig>  suppressed approximately up to  <dig> % par- <dig> mrna expression at  <dig> and  <dig> h following transfection .
sirna par- <dig>  inhibited also up to  <dig> % par- <dig> protein expression on flow cytometry analysis at  <dig> h .
similarly, sirna par- <dig>  eliminated par- <dig> expression by  <dig>  and  <dig> % on western blot  and immunofluorescence staining analysis  in p <dig> cells at  <dig> h following transfection.
inhibition of il- <dig> induced changes in par expression in p <dig> cells by sirna
il- <dig> at the concentration of  <dig> ng/ml induced approximately  <dig>  and  <dig>  fold upregulation of expression of par- <dig>  and par- <dig> mrnas  in p <dig> cells, respectively at  <dig> h following incubation.
in contrast,  <dig> ng/ml of il- <dig> downregulated par- <dig> mrna expression in p <dig> cells by approximately up to  <dig> % .
it was observed that il- <dig> induced upregulation of par- <dig> , but downregulation of par- <dig> protein expression .
sirna par- <dig>  at  <dig> nm and sirna par- <dig>  at  <dig> nm significantly reduced il- <dig> induced upregulation of par- <dig>  and par- <dig> expression , respectively when p <dig> cells were transfected with them for  <dig> h. sirna par- <dig>  at  <dig> nm was able to block il- <dig> induced downregulation of par- <dig> expression on both mrna  and protein  levels in p <dig> cells.
inhibition of protease-induced cytokine release by sirna
tryptase at  <dig> μg/ml, trypsin at  <dig> ng/ml, sfllr-nh <dig> at  <dig> μm and sligkv-nh <dig> at  <dig> μm induced  <dig> ,  <dig> ,  <dig>  and  <dig>  fold increase in il- <dig> release from p <dig> cells, respectively at  <dig> h following incubation .
however, thrombin at  <dig> u/ml, rllfs-nh <dig> at  <dig> μm, vkgils-nh <dig> at  <dig> μm, gypgqv-nh <dig> at  <dig> μm and vqgpyg-nh <dig> at  <dig> μm failed to elicit il- <dig> release from p <dig> cells following  <dig> h incubation period .
trypsin induced il- <dig> release was almost completely abolished by  <dig> nm of sirna par- <dig> , and reduced approximately  <dig> % by  <dig> nm of sirna par- <dig> .
similarly, sligkv-nh <dig> induced il- <dig> release was inhibited by  <dig> % and  <dig> % by  <dig> nm of sirna par- <dig>  and sirna par- <dig> , respectively.
it was observed that tryptase- induced il- <dig> release was eliminated by approximately  <dig> % by  <dig> nm of sirna par- <dig> , and sfllr-nh <dig> induced il- <dig> release was diminished by approximately  <dig> % by  <dig> nm of sirna par- <dig>  .
tryptase at  <dig> μg/ml, but not thrombin at  <dig> u/ml, trypsin at  <dig> ng/ml, sfllr-nh <dig> at  <dig> μm, sligkv-nh <dig> at  <dig> μm and gypgqv-nh <dig> at  <dig> μm was able to provoke  <dig>  fold increase in il- <dig> secretion from p <dig> cells at  <dig> h following incubation.
however, sirna par- <dig> , sirna par- <dig>  and sirna par- <dig>  at  <dig> nm did not show any effect on tryptase-induced il- <dig> release from p <dig> cells .
discussion
delivery of synthetic sirna or vector-based sirna expression systems to target cells can reverse the expression and function of the gene under consideration.
in recent years, sirna technique has been widely used as one of the most powerful tools for investigation of protein functions in mammalian cells  <cit> .
although p <dig> cell is a mouse mastocytoma cell line, rather than primary mast cells, it has been shown to express par- <dig>  par- <dig>  par- <dig> and par- <dig> genes and proteins <cit> .
to confirm these findings, we examined the effect of sirnas on par expression in the present study.
our data showed, for the first time, that sirnas of par- <dig>  par- <dig> and par- <dig> are able to reduce par- <dig>  par- <dig> and par- <dig> expression to a great extent, indicating these small rnas disrupt the normal par generation process of p <dig> cells.
as for most dsrnas, sirnas of par- <dig>  par- <dig> and par- <dig> fail to completely block par production in p <dig> cells.
incomplete sirna-induced gene suppression may result from the presence of a fraction of mrna in a protected compartment such as spliceosomes, other nuclear locations or non-transfected cells <cit> .
another study found that the activity of sirna in mammalian cells is related to structural target accessibility <cit> .
par- <dig>  par- <dig> and par- <dig> gene silencing can block il- <dig> induced alteration of expression of par mrnas and proteins in p <dig> cells, confirming that il- <dig> is an effective modulator of par expression in mast cells.
to our knowledge, this is the first work demonstrates the effects of par sirnas on cytokine induced alteration of par expression.
il- <dig> regulates th <dig> cell differentiation, while suppressing the expansion of th <dig> cell clones <cit> .
it has been implicated in the pathogenesis of allergy <cit> .
since mast cell has long been recognized as the primary effector cell of allergy and upregulation of par- <dig> expression was found in the airways of asthma <cit> , we anticipate that il- <dig> is likely to be involved in the pathogenesis of asthma through its ability of regulation of par expression on mast cells.
we have previously showed that tryptase and trypsin can induce il- <dig> release from p <dig> cells via a par- <dig> dependent mechanism <cit> .
using sirna technique in the present study, it is confirmed that trypsin provoked il- <dig> release is mainly through par- <dig> and partially via par- <dig> related mechanisms, whereas tryptase elicited il- <dig> release is dependent on activation of par- <dig>  in contrast, tryptase induced il- <dig> release is independent on the activation of pars as sirnas of par- <dig>  par- <dig> and par- <dig> did not show any influence on the event.
since tryptase is a unique secretory product of mast cells and il- <dig> is a classic th <dig> cytokine which is actively involved in the pathogenesis of allergic reactions, induction of il- <dig> release from mast cells by tryptase may suggest a self-amplification mechanism of allergic reactions.
inhibition of par dependent cytokine release by sirnas of pars has been reported before.
thus, thrombin induced il- <dig> and vegf release from prostate cancer cells <cit>  and il- <dig> production from synovial fibroblasts <cit>  was blocked by sirna of par- <dig>  trypsin induced il- <dig> production from human gastric epithelial cells  was inhibited by sirna of par- <dig> <cit> .
par independent release of il- <dig> from p <dig> cells induced by tryptase was unexpected.
however, a study demonstrated that beta-tryptase regulates il- <dig> expression in airway smooth muscle cells by a par-2-independent mechanism <cit>  may help to explain our above observation.
CONCLUSIONS
in conclusion, sirnas of par- <dig>  par- <dig>  par- <dig> not only block their corresponding par expression, inhibit il- <dig> induced alteration of expression of pars, but also reduce tryptase and trypsin provoked il- <dig> release from p <dig> cells, confirming that mast cell pars are likely to be involved in the pathogenesis of allergic reactions.
however, a further study using primary human mast cells would be better for evaluation the role of mast cell pars in allergic reactions.

 as the primary effector cell, mast cell is actively involved in the pathogenesis of both acute and chronic allergic diseases. upon activation, mast cell can release not only its preformed but also newly generated mediators to fulfill its biological functions. besides histamine, heparin and proteases, mast cells can synthesize and secrete a variety of cytokines such as il- il- and il- these cytokines have been well documented for their ability to regulate cell behavior including growth, secretion and migration in physiological and pathological conditions. in recent years, pars have been indentified as receptors for serine proteinases. pars are a subfamily of seven transmembrane g-protein-coupled receptors. among them, par- par- and par- serve as a receptor of thrombin; par- par- and par- are receptors of trypsin and par- is a receptor of tryptase. these serine proteinases have been discovered to play a crucial role in allergic inflammation. tryptase was reported to be able to stimulate microvascular leakage in the skin of guinea pigs, to induce inflammatory cell accumulation in the peritoneum of mice, to elicit histamine release from mast cells and to enhance monocyte chemoattractant protein- and il- production in human endothelial cells. trypsin was found to be able to induce histamine release from human tonsil and skin mast cells and to stimulate il- and il- release from human respiratory epithelial cells. rnai is a process in which double-stranded rna induces the posttranscriptional degradation of homologous transcripts. it has been observed in variety of organisms including plants, fungi, insects, protozans and mammals. rnai can be initiated by exposing cells to dsrna either via transfection or endogenous expression. dsrnas are processed into 21- to 23-nt double-stranded fragments known as sirnas. with unprecedented speed, rnai has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool in mammals. the aim of this study is to investigate the effect of par- par- and par- gene silencing on the expression of pars and cytokine production in p cells by using rnai.  reduction of par- expression in p cells by sirna three target sequences in the coding region of par- mrna were selected as sirna sequences, namely sirna par-, sirna par- and sirna par-. among the sirna par-1s, sirna par- appeared most effective in inhibition of par- mrna expression in p cells. approximately up to % reduction of par- mrna expression was observed when p cells were transfected with nm of sirna par- for h. at or h following transfection, however sirna par- showed less degree of inhibition of par- mrna expression. flow cytometry analysis revealed that par- protein expression was eliminated by up to % when p cells were transfected with nm of sirna par- for h. because sirna par- showed greater inhibitory effect on par- expression at h than at or h following incubation, the h incubation period was chosen as the fixed time point for the following western blot and immunofluorescent analysis. approximately up to % and % inhibition of par- expression was observed by western blot and immunofluorescent analysis, respectively. sirna par- and sirna par- also showed significant inhibition of par- protein expression in p cells. reduction of par- expression in p cells by sirna of the three sirna par-2s, sirna par- and par-, but not par- downregulated par- mrna expression by up to % and % at h following transfection. therefore sirna par- was employed as a representative throughout the study. sirna par- also suppressed par- protein expression during flow cytometry, western blot and immunofluorescence staining analysis by up to %, % and %, respectively following h incubation period. at and h following incubation, sirna par- showed significant downregulation of par- expression on both mrna and protein levels. reduction of par- expression in p cells by sirna all three sirnas of par- namely sirna par-, sirna par- and sirna par- were able to downregulate the par- expression on both mrna and protein levels in p cells. however, sirna par- showed the most potent effect on p cells among the sirnas. it was observed that sirna par- suppressed approximately up to % par- mrna expression at and h following transfection. sirna par- inhibited also up to % par- protein expression on flow cytometry analysis at h. similarly, sirna par- eliminated par- expression by and % on western blot and immunofluorescence staining analysis in p cells at h following transfection. inhibition of il- induced changes in par expression in p cells by sirna il- at the concentration of ng/ml induced approximately and fold upregulation of expression of par- and par- mrnas in p cells, respectively at h following incubation. in contrast, ng/ml of il- downregulated par- mrna expression in p cells by approximately up to %. it was observed that il- induced upregulation of par-, but downregulation of par- protein expression. sirna par- at nm and sirna par- at nm significantly reduced il- induced upregulation of par- and par- expression, respectively when p cells were transfected with them for h. sirna par- at nm was able to block il- induced downregulation of par- expression on both mrna and protein levels in p cells. inhibition of protease-induced cytokine release by sirna tryptase at μg/ml, trypsin at ng/ml, sfllr-nh at μm and sligkv-nh at μm induced,, and fold increase in il- release from p cells, respectively at h following incubation. however, thrombin at u/ml, rllfs-nh at μm, vkgils-nh at μm, gypgqv-nh at μm and vqgpyg-nh at μm failed to elicit il- release from p cells following h incubation period. trypsin induced il- release was almost completely abolished by nm of sirna par-, and reduced approximately % by nm of sirna par-. similarly, sligkv-nh induced il- release was inhibited by % and % by nm of sirna par- and sirna par-, respectively. it was observed that tryptase- induced il- release was eliminated by approximately % by nm of sirna par-, and sfllr-nh induced il- release was diminished by approximately % by nm of sirna par-. tryptase at μg/ml, but not thrombin at u/ml, trypsin at ng/ml, sfllr-nh at μm, sligkv-nh at μm and gypgqv-nh at μm was able to provoke fold increase in il- secretion from p cells at h following incubation. however, sirna par-, sirna par- and sirna par- at nm did not show any effect on tryptase-induced il- release from p cells. discussion delivery of synthetic sirna or vector-based sirna expression systems to target cells can reverse the expression and function of the gene under consideration. in recent years, sirna technique has been widely used as one of the most powerful tools for investigation of protein functions in mammalian cells. although p cell is a mouse mastocytoma cell line, rather than primary mast cells, it has been shown to express par- par- par- and par- genes and proteins. to confirm these findings, we examined the effect of sirnas on par expression in the present study. our data showed, for the first time, that sirnas of par- par- and par- are able to reduce par- par- and par- expression to a great extent, indicating these small rnas disrupt the normal par generation process of p cells. as for most dsrnas, sirnas of par- par- and par- fail to completely block par production in p cells. incomplete sirna-induced gene suppression may result from the presence of a fraction of mrna in a protected compartment such as spliceosomes, other nuclear locations or non-transfected cells. another study found that the activity of sirna in mammalian cells is related to structural target accessibility. par- par- and par- gene silencing can block il- induced alteration of expression of par mrnas and proteins in p cells, confirming that il- is an effective modulator of par expression in mast cells. to our knowledge, this is the first work demonstrates the effects of par sirnas on cytokine induced alteration of par expression. il- regulates th cell differentiation, while suppressing the expansion of th cell clones. it has been implicated in the pathogenesis of allergy. since mast cell has long been recognized as the primary effector cell of allergy and upregulation of par- expression was found in the airways of asthma, we anticipate that il- is likely to be involved in the pathogenesis of asthma through its ability of regulation of par expression on mast cells. we have previously showed that tryptase and trypsin can induce il- release from p cells via a par- dependent mechanism. using sirna technique in the present study, it is confirmed that trypsin provoked il- release is mainly through par- and partially via par- related mechanisms, whereas tryptase elicited il- release is dependent on activation of par- in contrast, tryptase induced il- release is independent on the activation of pars as sirnas of par- par- and par- did not show any influence on the event. since tryptase is a unique secretory product of mast cells and il- is a classic th cytokine which is actively involved in the pathogenesis of allergic reactions, induction of il- release from mast cells by tryptase may suggest a self-amplification mechanism of allergic reactions. inhibition of par dependent cytokine release by sirnas of pars has been reported before. thus, thrombin induced il- and vegf release from prostate cancer cells and il- production from synovial fibroblasts was blocked by sirna of par- trypsin induced il- production from human gastric epithelial cells was inhibited by sirna of par-. par independent release of il- from p cells induced by tryptase was unexpected. however, a study demonstrated that beta-tryptase regulates il- expression in airway smooth muscle cells by a par-2-independent mechanism may help to explain our above observation.  in conclusion, sirnas of par- par- par- not only block their corresponding par expression, inhibit il- induced alteration of expression of pars, but also reduce tryptase and trypsin provoked il- release from p cells, confirming that mast cell pars are likely to be involved in the pathogenesis of allergic reactions. however, a further study using primary human mast cells would be better for evaluation the role of mast cell pars in allergic reactions. 
mast cell has been identified to be able to express pars and release an array of cytokines upon activation. recently, it was reported that interleukin - could regulate the expression of pars in mast cells, and tryptase could induce il- and il- release from mast cells. in conclusion, sirnas of pars can modulate par expression and par related cytokine production in mast cells, confirming that pars are likely to play a role in allergic reactions. the results showed that sirnas for par- par- and par- significantly downregulated expression of par- par- and par- mrnas and proteins in p cells at and h following transfection. sirna par- and sirna par- significantly reduced il- induced upregulation of par- and par- expression, respectively when p cells were transfected with them for h. sirna par- blocked il- induced downregulation of par- expression on both mrna and protein levels.
 protease-activated receptors are seven transmembrane g-coupled receptors comprising four genes. mast cell has been identified to be able to express pars and release an array of cytokines upon activation. recently, it was reported that interleukin - could regulate the expression of pars in mast cells, and tryptase could induce il- and il- release from mast cells. in order to further investigate the issues, rna interference technique was employed and small interfering rnas of pars were transfected in p cells.  the results showed that sirnas for par- par- and par- significantly downregulated expression of par- par- and par- mrnas and proteins in p cells at and h following transfection. sirna par- and sirna par- significantly reduced il- induced upregulation of par- and par- expression, respectively when p cells were transfected with them for h. sirna par- blocked il- induced downregulation of par- expression on both mrna and protein levels. it was also observed that sirna par- and sirna par- reduced trypsin induced il- release by approximately % and %, and sligkv-nh induced il- release by % and %, respectively. similarly, sirna par- eliminated tryptase-induced il- release by %, and sirna par- diminished sfllr-nh induced il- release by %. however, sirna par-, sirna par- and sirna par- at nm did not show any effect on tryptase-induced il- release from p cells.  in conclusion, sirnas of pars can modulate par expression and par related cytokine production in mast cells, confirming that pars are likely to play a role in allergic reactions. 