narcolepsy is an obstinate neurological disorder characterized by excessive daytime sleepiness , cataplexy , hypnagogic hallucinations , sleep paralysis , and disturbed nocturnal sleep patterns . deficiency of the hypocretin ( hcrt ) system has been confirmed as an underlying cause of most cases of narcolepsy . the orexigenic peptides , hcrt-1 and hcrt-2 ( hcrt , also known as orexin ( ox)-a and ox - b ) , are proteolytically cleaved from a common precursor , prepro - ox , and share 46% amino acid sequence identity . these peptides also play a role in the central regulation of feeding and energy homeostasis . the physiological effects of ox - a and ox - b are mediated by two g - protein coupled receptors , ox receptor 1 ( ox1r ) and ox2r , also known as hcrt receptor 1 ( hcrtr-1 ) and hcrtr-2 . knockout of ox - a , ox - b , ox1r , or ox2r produces a narcoleptic mouse model . furthermore , natural mutations of the ox2r are directly linked to the pathology of narcolepsy . the intracellular signaling pathways mediating the physiologic function of oxs and their receptors have been under intense investigation . recent studies have demonstrated that ox - a induces extracellular signal - regulated kinase ( erk1/2 ) activation via multiple signaling mechanisms in the over - expressing ox1r chinese hamster ovary cells in vitro . furthermore , ox - a - induced erk1/2 activation appears to be protective , whereas p38 mitogen - activated protein kinase ( mapk ) activation induces cell death . the activation of erk1/2 , p38 mapk , and c - jun n - terminal kinase ( jnk ) are important in cell proliferation and apoptosis . nevertheless , the precise roles of the signaling pathways of hcrt in narcolepsy remain unclear . our previous studies showed that spleen deficiency and the stagnated heat in the liver were the most common syndromes in narcolepsy . xingshentongqiao decoction ( xstq ) , a significantly effective traditional chinese medicine preparation for narcolepsy , is intended to correct the basic pathogenesis of the imbalance of yin and yang for narcolepsy , and this preparation uses herbs that have been proven to have obvious effects targeting the signaling pathways of hcrt . this study aimed to explore the molecular mechanism of xstq in targeting the hcrt signaling pathway for narcolepsy . the herbs of xstq were obtained from the pharmacy of traditional medicine of peking university people 's hospital ( beijing , china ) . the human sh - sy5y cell line was obtained from the cell resource center of peking union medical college . cell counting kit-8 ( cck-8 ) was purchased from dojindo laboratories ( kumamoto , japan ) . annexin v - fluorescein isothiocyanate ( fitc ) apoptosis detection kits were obtained from bd biosciences ( san jose , california , usa ) . the superscript iii first - strand synthesis system was purchased from invitrogen corporation ( carlsbad , ca ) . 2.5  sybr green real master mix was purchased from tiangen biotech co ltd ( beijing , china ) . nitrocellulose filter membranes were purchased from whatman ( maidstone , uk ) , and supersignal west pico chemiluminescent substrate was purchased from pierce biotechnology ( rockford , usa ) . antibodies to p - erk1/2 , erk , p - p38 mapk , p38 mapk , p - jnk , jnk and anti - rabbit immunoglobulin g ( igg ) , horseradish peroxidase ( hrp)-linked antibody were purchased from cell signaling technology inc ( danvers , ma , usa ) . xingshentongqiao decoction was made by the water decocting method and then filtered and degermed using 0.22 m filters . sh - sy5y cells were grown in dulbecco 's modified eagle medium : nutrient mixture f-12 ( 1:1 ) medium ( dmem / f12 ) ( gibco , invitrogen , carlsbad , california , usa ) with 10% fetal bovine serum , 100 units / ml penicillin , and 100 g / ml streptomycin ( gibco , invitrogen ) at 37c under 5% co2 and 95% ambient air . dose and time dependent cell viability was monitored using the cck-8 assay , a sensitive nonradioactive colorimetric assay used to determine the number of viable cells in proliferation and cytotoxicity assays . sh - sy5y cells were cultured in 96-well plates containing growth culture medium at a density of 2  10 cells / well . the cells were divided into seven groups : control group and xstq treatment groups ( 100 , 200 , 400 , 600 , 800 , and 1000 g / ml xstq ) . each group was further treated for 0 , 24 , 48 , 72 , or 96 hours separately as described , after which cck-8 solution was added to the cell culture medium to a final concentration of 10 l/100 l , and the cells were incubated for another 3 hours at 37c . absorbance was measured at 450 nm to determine cell viability as a percentage using a microplate reader ( imark ; bio - rad , hercules , ca , usa ) . cells were seeded by placing 1  10/ml of cell culture in 10 cm cell culture dishes and maintained for 24 h at 37c . xstq ( 0 , 10 , 100 or 1000 g / ml ) was directly added to the dishes , which were incubated for an additional 24 hours . the annexin v - fitc apoptosis detection kit was used to measure apoptosis levels according to the manufacturer 's instructions . total ribonucleic acid ( rna ) was isolated from sh - sy5y cells using rneasy mini kits according to the manufacturer 's instructions . the purity and concentration of rna were measured using a gene quant pro device ( nanovue spectrophotometer , ge healthcare , london , uk ) . complementary deoxyribonucleic acid ( cdna ) was synthesized with 2.5 g total rna using the superscript iii first - strand synthesis system according to standard protocol . quantitative real - time polymerase chain reaction ( qrt - pcr ) was performed using a detector ( bio - rad , munich , germany ) . mol / l ) , 4.0 l 2.5  sybr green real master mix , and 0.25 l cdna with a final volume of 10 l . the following primers were used : human ox1r sense : 5-cag cct atg tgg ctg tgt tcg t-3 , antisense : 5-agt tgg tga ctg tcc tca tgt gg-3 ) ; human ox2r sense : 5-gtg ttc gtc gtg gct ctc att g-3 , antisense : 5-cag gtg atg gtc acg agc aca t-3 ) and human -actin sense : 5-ctg gaa cgg tga agg tga ca-3 , antisense : 5-aag gga ctt cct gta aca atg ca-3. the reaction conditions for amplifying dna were 94c for 2 minutes , followed by 45 cycles of 94c for 20 seconds , 62c for 20 seconds , and 72c for 30 seconds . the messenger rna ( mrna ) expression was normalized to the expression level of -actin and was calculated using the following equation : fold change = 2 . after 24 hours serum deprivation , sh - sy5y cells were treated with xstq at indicated concentrations for the times indicated . then they were washed in phosphate buffered saline twice and homogenized in an ice cold lysis buffer containing 1% triton x-100 , 20 mmol / l tris - hcl ( ph 7.5 ) , 150 mmol / l nacl , 1 mmol / l hepes , 10% glycerol , 1 mmol / l naf , 1 mmol / l na3vo4 , and 1% protease inhibitors ( amresco corporation , usa ) . the homogenates were centrifuged at 12,000 an aliquot of the supernatant was removed for protein assay , and the remaining supernatant was added to an equal volume of sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) sample buffer . samples ( 125 g protein / lane ) were subjected to 10% sds - page and transferred electrophoretically onto nitrocellulose membranes using a mini protean 4 cell device ( mini - protean 4 , bio - rad , hercules , ca , usa ) and mini trans - blot electrophoretic transfer cell ( mini - protean 4 , bio - rad ) at 100 v for 2 hours . the membranes were incubated with 5% nonfat dried milk for 2 h at room temperature to block nonspecific binding and then incubated with rabbit anti - p - erk1/2 ( 1:1000 ) , anti - p - p38 mapk ( thr180/tyr182 ) ( 1:500 ) , anti - p - jnk ( thr183/tyr185 ) ( 1:1000 ) , anti - erk1/2 ( 1:1000 ) , anti - p38 mapk ( 1:1000 ) or anti - jnk ( 1:1000 ) antibodies at 4c overnight . the membranes were washed three times for 10 minutes each in tris - buffered saline with tween 20 ( tbst ) ( 20 mmol / l tris - hcl , 137 mmol / l nacl , 0.05% tween-20 ) and then incubated for 1 hour with anti - rabbit igg , hrp - linked antibody ( 1:2000 ) . the membranes were washed three additional times for 10 minutes each in tbst , and the signals were detected using the supersignal west pico chemiluminescent substrate . the bands were captured , and their optical density was analyzed with software image j. each experiment was repeated three times , and the mean value was presented as the experimental result . the statistical analysis was performed using software statistical package for the social sciences ( spss ) version 18.0 ( spss , chicago , il , usa ) . data are expressed as mean  standard deviation ( sd ) . statistical analysis was performed using one - way analysis of variance or the student 's t - test . the herbs of xstq were obtained from the pharmacy of traditional medicine of peking university people 's hospital ( beijing , china ) . the human sh - sy5y cell line was obtained from the cell resource center of peking union medical college . cell counting kit-8 ( cck-8 ) was purchased from dojindo laboratories ( kumamoto , japan ) . annexin v - fluorescein isothiocyanate ( fitc ) apoptosis detection kits were obtained from bd biosciences ( san jose , california , usa ) . the superscript iii first - strand synthesis system was purchased from invitrogen corporation ( carlsbad , ca ) . 2.5  sybr green real master mix was purchased from tiangen biotech co ltd ( beijing , china ) . nitrocellulose filter membranes were purchased from whatman ( maidstone , uk ) , and supersignal west pico chemiluminescent substrate was purchased from pierce biotechnology ( rockford , usa ) . antibodies to p - erk1/2 , erk , p - p38 mapk , p38 mapk , p - jnk , jnk and anti - rabbit immunoglobulin g ( igg ) , horseradish peroxidase ( hrp)-linked antibody were purchased from cell signaling technology inc ( danvers , ma , usa ) . xingshentongqiao decoction was made by the water decocting method and then filtered and degermed using 0.22 m filters . sh - sy5y cells were grown in dulbecco 's modified eagle medium : nutrient mixture f-12 ( 1:1 ) medium ( dmem / f12 ) ( gibco , invitrogen , carlsbad , california , usa ) with 10% fetal bovine serum , 100 units / ml penicillin , and 100 g / ml streptomycin ( gibco , invitrogen ) at 37c under 5% co2 and 95% ambient air . dose and time dependent cell viability was monitored using the cck-8 assay , a sensitive nonradioactive colorimetric assay used to determine the number of viable cells in proliferation and cytotoxicity assays . sh - sy5y cells were cultured in 96-well plates containing growth culture medium at a density of 2  10 cells / well . the cells were divided into seven groups : control group and xstq treatment groups ( 100 , 200 , 400 , 600 , 800 , and 1000 g / ml xstq ) . each group was further treated for 0 , 24 , 48 , 72 , or 96 hours separately as described , after which cck-8 solution was added to the cell culture medium to a final concentration of 10 l/100 l , and the cells were incubated for another 3 hours at 37c . absorbance was measured at 450 nm to determine cell viability as a percentage using a microplate reader ( imark ; bio - rad , hercules , ca , usa ) . cells were seeded by placing 1  10/ml of cell culture in 10 cm cell culture dishes and maintained for 24 h at 37c . xstq ( 0 , 10 , 100 or 1000 g / ml ) was directly added to the dishes , which were incubated for an additional 24 hours . the annexin v - fitc apoptosis detection kit was used to measure apoptosis levels according to the manufacturer 's instructions . total ribonucleic acid ( rna ) was isolated from sh - sy5y cells using rneasy mini kits according to the manufacturer 's instructions . the purity and concentration of rna were measured using a gene quant pro device ( nanovue spectrophotometer , ge healthcare , london , uk ) . complementary deoxyribonucleic acid ( cdna ) was synthesized with 2.5 g total rna using the superscript iii first - strand synthesis system according to standard protocol . quantitative real - time polymerase chain reaction ( qrt - pcr ) was performed using a detector ( bio - rad , munich , germany ) . mol / l ) , 4.0 l 2.5  sybr green real master mix , and 0.25 l cdna with a final volume of 10 l . the following primers were used : human ox1r sense : 5-cag cct atg tgg ctg tgt tcg t-3 , antisense : 5-agt tgg tga ctg tcc tca tgt gg-3 ) ; human ox2r sense : 5-gtg ttc gtc gtg gct ctc att g-3 , antisense : 5-cag gtg atg gtc acg agc aca t-3 ) and human -actin sense : 5-ctg gaa cgg tga agg tga ca-3 , antisense : 5-aag gga ctt cct gta aca atg ca-3. the reaction conditions for amplifying dna were 94c for 2 minutes , followed by 45 cycles of 94c for 20 seconds , 62c for 20 seconds , and 72c for 30 seconds . the messenger rna ( mrna ) expression was normalized to the expression level of -actin and was calculated using the following equation : fold change = 2 . after 24 hours serum deprivation , sh - sy5y cells were treated with xstq at indicated concentrations for the times indicated . then they were washed in phosphate buffered saline twice and homogenized in an ice cold lysis buffer containing 1% triton x-100 , 20 mmol / l tris - hcl ( ph 7.5 ) , 150 mmol / l nacl , 1 mmol / l hepes , 10% glycerol , 1 mmol / l naf , 1 mmol / l na3vo4 , and 1% protease inhibitors ( amresco corporation , usa ) . an aliquot of the supernatant was removed for protein assay , and the remaining supernatant was added to an equal volume of sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) sample buffer . samples ( 125 g protein / lane ) were subjected to 10% sds - page and transferred electrophoretically onto nitrocellulose membranes using a mini protean 4 cell device ( mini - protean 4 , bio - rad , hercules , ca , usa ) and mini trans - blot electrophoretic transfer cell ( mini - protean 4 , bio - rad ) at 100 v for 2 hours . the membranes were incubated with 5% nonfat dried milk for 2 h at room temperature to block nonspecific binding and then incubated with rabbit anti - p - erk1/2 ( 1:1000 ) , anti - p - p38 mapk ( thr180/tyr182 ) ( 1:500 ) , anti - p - jnk ( thr183/tyr185 ) ( 1:1000 ) , anti - erk1/2 ( 1:1000 ) , anti - p38 mapk ( 1:1000 ) or anti - jnk ( 1:1000 ) antibodies at 4c overnight . the membranes were washed three times for 10 minutes each in tris - buffered saline with tween 20 ( tbst ) ( 20 mmol / l tris - hcl , 137 mmol / l nacl , 0.05% tween-20 ) and then incubated for 1 hour with anti - rabbit igg , hrp - linked antibody ( 1:2000 ) . the membranes were washed three additional times for 10 minutes each in tbst , and the signals were detected using the supersignal west pico chemiluminescent substrate . the bands were captured , and their optical density was analyzed with software image j. each experiment was repeated three times , and the mean value was presented as the experimental result . the statistical analysis was performed using software statistical package for the social sciences ( spss ) version 18.0 ( spss , chicago , il , usa ) . statistical analysis was performed using one - way analysis of variance or the student 's t - test . to determine whether xstq affects cell proliferation , we treated sh - sy5y cells with a range of doses and times of xstq . sh - sy5y proliferation was inhibited in a dose - dependent manner after 72 hours and 96 hours treatment with xstq at concentrations of 4001000 g / ml [ table 1 , figure 1 ] . inhibition of sh - sy5y after treatment with xstq at different concentrations or times ( mean  sd , n = 9 ) * * p<0.01 , * p<0.05 versus untreated control . ( a ) sh - sy5y cell proliferation was determined by cell counting kit-8 assay after 0 , 24 , 48 , 72 , or 96 hours incubation without xstq ( control ) or with xstq at a concentration of 100 , 200 , 400 , 600 , 800 , or 1000 g / ml . results are standardized to 1.0 at 0 hour . ( b ) the average proliferation of xstq - treated - sh - sy5y cells is shown after 72 hours . ( c ) average proliferation of xstq - treated - sh - sy5y cells is shown after 96 h. results are standardized to an average of 1.0 in the control . data represent the mean  standard deviation of three independent experiments ( * * p < 0.01 , * p < 0.05 , vs. untreated control ) . to determine whether xstq also affects cell apoptosis , we performed annexin v - fitc staining assays after 24 h treatment of sh - sy5y cells with a range of doses of xstq ( 0 , 10 , 100 and 1000 g / ml ) . the apoptotic rates were 3.65%  0.12% , 3.05%  0.18% , 3.23%  0.22% and 4.72%  0.16% , respectively . statistical analysis suggests that the apoptotic rate of cells treated with 1000 g / ml xstq was higher than that of the control group [ figure 2 ] . ( a ) apoptosis was measured using an annexin v - fluorescein isothiocyanate apoptosis detection kit for sh - sy5y cells incubated with xstq ( 0 , 10 , 100 or 1000 g / ml ) for 24 h. ( b ) the results from panel a were quantified for three independent experiments . the data are expressed as mean  standard deviation ( * * p < 0.01 vs. untreated control ) . deficiency of the hcrt system has been confirmed as an underlying cause of most cases of narcolepsy . to test whether xstq counteracts narcolepsy by modulating components within the hcrt signaling cascade , we assessed levels of expression of the hcrt receptors ox1r and ox2r . mrna was collected from sh - sy5y cells treated with xstq ( 1000 g / ml ) for 0 , 24 , 48 , and 72 hours , and transcript levels were quantified by qrt - pcr . xstq induced an obvious increase in ox1r and ox2r mrna expression , reaching a maximum at 2448 hours [ figure 3 ] . these results suggest that xstq may counteract narcolepsy by enhancing the expression of hcrt signaling receptors . xingshentongqiao decoction ( xstq ) enhances orexin receptor 1 ( ox1r ) and orexin receptor 2 ( ox2r ) expression in sh - sy5y cells . induction of ox1r ( a ) and ox2r ( b ) was determined by quantitative real - time polymerase chain reaction at a range of times of exposure to 1000 g / ml xstq . the data are expressed as mean  standard deviation of ox1r or ox2r messenger ribonucleic acid ( mrna ) expression relative to -actin mrna expression for three independent experiments and are standardized to 1.0 in the control sample ( * * p < 0.01 , * p < 0.05 vs. untreated control ) . to identify hcrt - related signaling pathways that may be affected by xstq , we examined the effects on erk1/2 , p38 mapk and jnk activation . increasing concentrations of xstq were added to sh - sy5y cells , and levels of phosphorylated and total protein were assessed by western blotting . xstq significantly downregulated the phosphorylation of erk1/2 , p38 mapk and jnk at 1000 g / ml concentration . furthermore , xstq downregulated the phosphorylation of erk1/2 and jnk at 100 g / ml and erk1/2 at 10 g / ml [ figure 4 ] . xingshentongqiao decoction ( xstq ) mediates dose - dependent downregulation of the phosphorylation of extracellular signal - regulated kinase ( erk1/2 ) , p38 mitogen - activated protein kinase ( mapk ) and c - jun n - terminal kinase ( jnk ) . ( a ) sh - sy5y cells were incubated with xstq ( 0 , 10 , 100 or 1000 g / ml ) for 2 hours , and levels of phosphorylated erk1/2 , p38 mapk and jnk were determined relative to total erk1/2 , p38 mapk and jnk levels by western blotting analysis . ( b - d ) levels of phosphorylated proteins were normalized to levels of total protein by densitometry . data represent the mean  standard deviation of three independent experiments and are standardized to 1.0 in the control ( untreated ) cells ( * * p < 0.01 vs. untreated control ) . to verify the inhibition of erk1/2 , p38 and jnk activation by xstq , we examined the time dependence of the inhibition at 1000 g / ml xstq . xstq promoted a time - dependent - downregulation of the phosphorylation of all three proteins compared with the control group [ figure 5 ] . these results confirm the effects of xstq in inhibiting these three mapk signaling pathways and provide an additional mechanism to explain the xstq - dependent effects on proliferation and cell death . xingshentongqiao decoction ( xstq ) mediates time - dependent downregulation of the phosphorylation of extracellular signal - regulated kinase ( erk1/2 ) , p38 mitogen - activated protein kinase ( mapk ) and c - jun n - terminal kinase ( jnk ) . ( a ) sh - sy5y cells were incubated with 1000 g / ml xstq for 0 , 5 , 15 , 30 , 60 , 90 , or 120 minutes , and levels of phosphorylated erk1/2 , p38 mapk and jnk were determined relative to total erk1/2 , p38 mapk and jnk by western blotting . ( b - d ) levels of phosphorylated proteins were normalized to levels of total protein by densitometry . data represent the mean  standard deviation of three independent experiments and are standardized to 1.0 at time 0 ( * * p < 0.01 , * p < 0.05 , vs. untreated control ) . to determine whether xstq affects cell proliferation , we treated sh - sy5y cells with a range of doses and times of xstq . sh - sy5y proliferation was inhibited in a dose - dependent manner after 72 hours and 96 hours treatment with xstq at concentrations of 4001000 g / ml [ table 1 , figure 1 ] . inhibition of sh - sy5y after treatment with xstq at different concentrations or times ( mean  sd , n = 9 ) * * p<0.01 , * p<0.05 versus untreated control . ( a ) sh - sy5y cell proliferation was determined by cell counting kit-8 assay after 0 , 24 , 48 , 72 , or 96 hours incubation without xstq ( control ) or with xstq at a concentration of 100 , 200 , 400 , 600 , 800 , or 1000 g / ml . results are standardized to 1.0 at 0 hour . ( b ) the average proliferation of xstq - treated - sh - sy5y cells is shown after 72 hours . ( c ) average proliferation of xstq - treated - sh - sy5y cells is shown after 96 h. results are standardized to an average of 1.0 in the control . data represent the mean  standard deviation of three independent experiments ( * * p < 0.01 , * p < 0.05 , vs. untreated control ) . to determine whether xstq also affects cell apoptosis , we performed annexin v - fitc staining assays after 24 h treatment of sh - sy5y cells with a range of doses of xstq ( 0 , 10 , 100 and 1000 g / ml ) . the apoptotic rates were 3.65%  0.12% , 3.05%  0.18% , 3.23%  0.22% and 4.72%  0.16% , respectively . statistical analysis suggests that the apoptotic rate of cells treated with 1000 g / ml xstq was higher than that of the control group [ figure 2 ] . ( a ) apoptosis was measured using an annexin v - fluorescein isothiocyanate apoptosis detection kit for sh - sy5y cells incubated with xstq ( 0 , 10 , 100 or 1000 g / ml ) for 24 h. ( b ) the results from panel a were quantified for three independent experiments . the data are expressed as mean  standard deviation ( * * p < 0.01 vs. untreated control ) . deficiency of the hcrt system has been confirmed as an underlying cause of most cases of narcolepsy . to test whether xstq counteracts narcolepsy by modulating components within the hcrt signaling cascade , we assessed levels of expression of the hcrt receptors ox1r and ox2r . mrna was collected from sh - sy5y cells treated with xstq ( 1000 g / ml ) for 0 , 24 , 48 , and 72 hours , and transcript levels were quantified by qrt - pcr . xstq induced an obvious increase in ox1r and ox2r mrna expression , reaching a maximum at 2448 hours [ figure 3 ] . these results suggest that xstq may counteract narcolepsy by enhancing the expression of hcrt signaling receptors . xingshentongqiao decoction ( xstq ) enhances orexin receptor 1 ( ox1r ) and orexin receptor 2 ( ox2r ) expression in sh - sy5y cells . induction of ox1r ( a ) and ox2r ( b ) was determined by quantitative real - time polymerase chain reaction at a range of times of exposure to 1000 g / ml xstq . the data are expressed as mean  standard deviation of ox1r or ox2r messenger ribonucleic acid ( mrna ) expression relative to -actin mrna expression for three independent experiments and are standardized to 1.0 in the control sample ( * * p < 0.01 , * p < 0.05 vs. untreated control ) . to identify hcrt - related signaling pathways that may be affected by xstq , we examined the effects on erk1/2 , p38 mapk and jnk activation . increasing concentrations of xstq were added to sh - sy5y cells , and levels of phosphorylated and total protein were assessed by western blotting . xstq significantly downregulated the phosphorylation of erk1/2 , p38 mapk and jnk at 1000 g / ml concentration . furthermore , xstq downregulated the phosphorylation of erk1/2 and jnk at 100 g / ml and erk1/2 at 10 g / ml [ figure 4 ] . xingshentongqiao decoction ( xstq ) mediates dose - dependent downregulation of the phosphorylation of extracellular signal - regulated kinase ( erk1/2 ) , p38 mitogen - activated protein kinase ( mapk ) and c - jun n - terminal kinase ( jnk ) . ( a ) sh - sy5y cells were incubated with xstq ( 0 , 10 , 100 or 1000 g / ml ) for 2 hours , and levels of phosphorylated erk1/2 , p38 mapk and jnk were determined relative to total erk1/2 , p38 mapk and jnk levels by western blotting analysis . ( b - d ) levels of phosphorylated proteins were normalized to levels of total protein by densitometry . data represent the mean  standard deviation of three independent experiments and are standardized to 1.0 in the control ( untreated ) cells ( * * p < 0.01 vs. untreated control ) . to verify the inhibition of erk1/2 , p38 and jnk activation by xstq , we examined the time dependence of the inhibition at 1000 g / ml xstq . xstq promoted a time - dependent - downregulation of the phosphorylation of all three proteins compared with the control group [ figure 5 ] . these results confirm the effects of xstq in inhibiting these three mapk signaling pathways and provide an additional mechanism to explain the xstq - dependent effects on proliferation and cell death . xingshentongqiao decoction ( xstq ) mediates time - dependent downregulation of the phosphorylation of extracellular signal - regulated kinase ( erk1/2 ) , p38 mitogen - activated protein kinase ( mapk ) and c - jun n - terminal kinase ( jnk ) . ( a ) sh - sy5y cells were incubated with 1000 g / ml xstq for 0 , 5 , 15 , 30 , 60 , 90 , or 120 minutes , and levels of phosphorylated erk1/2 , p38 mapk and jnk were determined relative to total erk1/2 , p38 mapk and jnk by western blotting . ( b - d ) levels of phosphorylated proteins were normalized to levels of total protein by densitometry . data represent the mean  standard deviation of three independent experiments and are standardized to 1.0 at time 0 ( * * p < 0.01 , * p < 0.05 , vs. untreated control ) . orexins , also known as hcrts , are neuropeptides produced in neurons of the lateral and dorsomedial hypothalamus and perifornical area , which project widely throughout the brain . blockade of ox1r and ox2r antagonism elicits a dysregulation of rapid eye movement ( rem ) sleep by shifting the balance in favor of rem sleep at the expense of nonrem sleep and may increase the risk of adverse events . moreover , knockout of either ox1r or ox2r produces a narcoleptic rat model . in this study , we found that xstq upregulated ox1r and ox2r expression in sh - sy5y cells in vitro . the deficiency of ox1r or ox2r is one of the critical factors contributing to the pathogenesis of narcolepsy . in addition , hcrt ( ox ) deficiency ( < 40 pg / ml ) is highly associated with clinical narcolepsy and cataplexy ( 89.5% ) , and the hcrt content in the hypothalamus is decreased significantly in autopsies of narcoleptic patients . these observations indicate that upregulation of ox1r and ox2r expression may be one of the critical molecular mechanisms of xstq in protecting against narcolepsy . we also examined the ability of xstq to affect cell proliferation and apoptosis as an additional potential mechanism of protection against narcolepsy . narcolepsy is accompanied by the proliferation of histamine neurons , which can cause classic symptoms of narcolepsy , such as cataplexy and hypnagogic hallucinations . in addition , an increase of cholinergic receptors is one of the most significant features of narcolepsy , which might comprise an adaptive response to the malfunction of the secretion of acetylcholine and dopamine . previous studies show that retinoic acid - differentiated sh - sy5y cells have a daergic - like phenotype , and play a critical role in the secretion of histamine and dopamine . in the current study , incubation of sh - sy5y cells with 4001000 g / ml xstq was shown to significantly inhibit cell proliferation after 72 hours and 96 hours , as determined by cck-8 assay . the effect of xstq on sh - sy5y cell apoptosis at 1000 g / ml was also verified by annexin v - fitc apoptosis detection . our study indicates that xstq can inhibit sh - sy5y cell proliferation and promote sh - sy5y cell apoptosis in vitro . these results are consistent with the possibility that xstq may regulate abnormal proliferation and apoptosis of histamine neurons in narcolepsy . to gain further insight into the molecular mechanisms by which xstq mediates proliferation and apoptosis in sh - sy5y cells , we examined intracellular signaling pathways . treatment of sh - sy5y cells with various concentrations of xstq significantly decreased phosphorylation of erk1/2 , p38 mapk and jnk . phosphorylation of erk1/2 , p38 mapk and jnk are involved in cell proliferation and apoptosis . furthermore , tissue kallikrein promotes sh - sy5y cell proliferation through the erk1/2/mapk signaling pathway , and effects of propofol on proliferation and anti - apoptosis of sh - sy5y cells are mediated via erk1/2/mapk signaling . sphingomyelinase metabolites also control survival and apoptotic death in sh - sy5y cells through p38 mapk signaling pathways , while jnk can mediates either cell death and proliferation of sh - sy5y cells . therefore , the observation that xstq reduces the phosphorylation of these signaling proteins provides a mechanism for this preparation in the suppression of proliferation and activation of sh - sy5y cell death . xingshentongqiao decoction upregulates ox1r and ox2r expression in sh - sy5y cells in vitro , which could play a critical role in the activation of hcrt signaling pathways in narcolepsy . furthermore , xstq inhibits proliferation and induces apoptosis in sh - sy5y cells , which may be mediated through its effects in repressing the erk1/2 , p38 mapk and jnk pathways . this study expands our knowledge of the molecular mechanisms of xstq on the hcrt signaling pathways in narcolepsy and suggests that hcrt signaling may provide a target for the beneficial effects of xstq in the treatment of narcolepsy with syndromes of spleen deficiency and the stagnated heat in the liver .
background : hypocretin ( hcrt ) signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by chinese herbal therapy . 
 our previous study showed that xingshentongqiao decoction ( xstq ) is clinically effective for the treatment of narcolepsy . to determine whether xstq improves narcolepsy by modulating hcrt signaling 
 , we investigated its effects on sh - sy5y cell proliferation , apoptosis , and hcrt receptor 1/2 ( orexin receptor 1 [ ox1r ] and orexin receptor 2 [ ox2r ] ) expression . 
 the signaling pathways involved in these processes were also assessed.methods:the effects of xstq on proliferation and apoptosis in sh - sy5y cells were assessed using cell counting kit-8 and annexin v - fluorescein isothiocyanate assays . ox1r and 
 ox2r expression was assessed by quantitative real - time polymerase chain reaction analysis . 
 western blotting for mitogen - activated protein kinase ( mapk ) pathway activation was performed to further assess the signaling mechanism of xstq.results:xstq reduced the proliferation and induced apoptosis of sh - sy5y cells . 
 this effect was accompanied by the upregulation of ox1r and ox2r expression and the reduced phosphorylation of extracellular signal - regulated kinase ( erk ) 1/2 , p38 mapk and c - jun n - terminal kinase ( jnk).conclusions : xstq inhibits proliferation and induces apoptosis in sh - sy5y cells . 
 xstq also promotes ox1r and ox2r expression . 
 these effects are associated with the repression of the erk1/2 , p38 mapk , and jnk signaling pathways . 
 these results define a molecular mechanism for xstq in regulating hcrt and mapk activation , which may explain its ability to treat narcolepsy .