Study On The Molecular Mechanism Of Xingshen Tongqiao Decoction In The Treatment Of Narcolepsy

Narcolepsy is an obstinate neurological disorder characterized by excessive daytime sleepiness,cataplexy,hypnagonic hallucinations,sleep paralysis,and disturbed nocturnal sleep patterns.The malfunction of the signaling pathways of hypocretin is proved to play a great role in the pathogenesis of narcolepsy,and it is confirmed that the hypocretin system can be influenced by Chinese herbal therapy.Xingshentongiqao decoction(XSTQ),a significantly effective TCM preparation for narcolepsy,is made based on the basic pathogenesis of the imbalance of yin and yang for narcolepsy,some herbs of which were proved to have active effects targeting the signaling pathways of hypocretin.For providing new methods for therapies and mechanisms of drug action on narcolepsy,the molecular biological methods were used to investigate the molecular mechanism of XSTQ for narcolepsy targeting the signaling pathways of hypocretin.Objective:1)To investigate the molecular mechanism of XSTQ targeting the signaling pathways of hypocretin.2)To explore the molecular mechanism of XSTQ for narcolepsy.Methods:1)To prepare the Xingshentongqiao decoction(XSTQ)and observe the effect of XSTQ on SH-SY5Y cell proliferation by CCK-8 assay.2)To observe the effect of XSTQ on SH-SY5Y cell migration by transwell assay.3)To observe the effect of XSTQ on SH-SY5Y cell apoptosis by Annexin V-FITC assay.4)To observe the effect of XSTQ on SH-SY5Y cell gene expression of OX1R and OX2R by quantitative real-time polymerase chain reaction(qRT-PCR).5)To observe the effect of XSTQ on SH-SY5Y cell protein expression of the signaling pathways of hypocretin related protein by Western blotting.6)To construct SH-SY5Y gene overexpress cell model for OX1R gene and observe the effect of OX1R gene overexpression on SH-SY5Y cell proliferation,migration,apoptosis and protein expression of the signaling pathways of hypocretin related protein.7)To observe the effect of XSTQ on SH-SY5Y gene overexpress cell model for OX1R gene on cell proliferation,migration,apoptosis.Results:1)For CCK-8 assay,XSTQ treatment of lmg/ml,800ug/ml,600ug/ml,400ug/ml inhibit SH-SY5Y cell proliferation after 72h,which is stronger after 96h.The XSTQ lmg/ml group has the strongest inhibition roles after 96h.2)For transwell migration experiment,compared with the control group,XSTQ treatment of lmg/ml increases the cell numbers of SH-SY5Y cells(P<0.01).3)For Annexin V-FITC apoptosis experiment,compared with the control group,XSTQ treatment of lmg/ml increases the cell apoptosis rates of SH-SY5Y cells P<0.01).4)XSTQ treatment of lmg/ml increases the mRNA levels of OX1R and OX2R(P<0.01,P<0.05).The XSTQ lmg/ml group has the strongest promoting roles after 48h for the mRNA level of OX1R,and it has the strongest promoting roles after 24h for the mRNA level of OX2R.5)XSTQ treatment of lmg/ml decreases the total protein levels of phospho-ERK1/2,phospho-p38 MAPK and phospho-JNK protein(P<0.01).6)After gene overexpression of OX1R,the SH-SH5Y cell proliferation and migration were inhibited,and the cell apoptosis was promoted.And the total protein levels of phospho-ERK1/2 protein were increased(P<0.01),while that of phospho-p38 MAPK and phospho-JNK were decreased(P<0.01).7)XSTQ treatment inhibits the OX1R gene overexpress SH-SH5Y cell proliferation after 48h,which is stronger after 72h and 96h,and XSTQ 600ug/ml group increases the above model cell migration,while XSTQ lmg/ml group increases the above model cell apoptosis(P<0.01,P<0.05).Conclusion:1)The molecular mechanisms of XSTQ targeting hypocretin related signaling pathways may effectively act via inhibiting SH-SY5Y cell proliferation,promoting SH-SY5Y cell migration and apoptosis,increasing the mRNA levels of OX1R and OX2R and decreasing the total protein levels of phospho-ERK 1/2,phospho-p38 MAPK and phospho-JNK protein.2)The gene overexpression of OX1R can inhibit the SH-SY5Y cell proliferation and migration,promote the SH-SY5Y cell apoptosis,increase the total protein levels of phospho-ERK 1/2 protein,and decrease the total protein levels of phospho-p38 MAPK and phospho-JNK protein.3)XSTQ may inhibit the OX1R gene overexpress SH-SH5Y cell proliferation,promote the OX1R gene overexpress SH-SH5Y cell migration and apoptosis.4)The curative effect of XSTQ on narcolepsy may act through the inhibition on cell proliferation,the promotion on cell migration and apoptosis,the increase on the mRNA levels of OX1R and OX2R via ERK1/2 MAPK signaling pathways.