Whole Transcriptomics Study On The Therapeutic Effect Of Mongolian Medicine Warm Needle On Insomnia Model Rat

Insomnia is the most prevalent sleep disorder,which affects about 30%of the general population worldwide.In China,the combined prevalence of insomnia is 15%,and about 207 million people have sleep problems.It is characterized by difficulty with sleep initiation,maintenance,or early morning awakening,and accompanied by daytime impairment.Accumulating evidence indicated that insomnia was associated with a wide range of negative health consequences,such as mental disorder,cardiovascular dysfunction and obesity.At present,benzodiazepines and non-benzodiazepine sedatives are mainly used in clinical treatment of insomnia.Medications for insomnia patients can cause side effects such as daytime sleepiness and cognitive decline.Current pharmacotherapy treatment options are usually associated with adverse effects and withdrawal phenomena.Traditional therapy of Mongolian Medicine has a long history in the treatment of insomnia.In recent years,relevant research has been carried out,which confirmed that Mongolian medical warm-acupuncture not only has significant curative effects,but also has many advantages,such as efficiency,safety,and no drug dependence of insomnia by clinical trials and animal experiments.However,the regulatory mechanism and molecular pathway of Mongolian medicine warm needle therapy for insomnia have not been fully explained,it restricts the development and application of traditional therapy of Mongolian Medicine under the conditions of modern science and technology to an extent.In this study,It uses high-throughput sequencing technology combined with bioinformatics analysis to explore the pathological mechanism of Mongolian medicine warm needle therapy for insomnia in an all-round and in-depth manner.Objective:It is based on total transcriptomics sequencing of rat hippocampal tissue to explore the relevant targets and pathways of Mongolian medical warm needles for the treatment of insomnia by observing the regulatory effect of Mongolian medicine temperature on PCPA insomnia rats.Method:50 SD rats were randomly selected and 10 of them were used as control group.The remaining 40 SD rats were intraperitoneally injected with a suspension of pchlorophenylalanine(PCPA).Forty AIT model rats were established following intraperitoneal injection of a suspension of PCPA.After the successful establishment of the insomnia rats model,The insomnia rats were randomly divided into four groups:①Model(n=10):no treatment after the model;②SMWA(n=10):on the 3rd day after model establishment,rats were given blunt-tip acupuncture stimulation(the needles touch the skin but do not enter the body)on the acupoints of Dinghui,Heyi,and Xin acupoints once daily for seven consecutive days.③MWA(n=10):rats were treated with MMWA on the acupoints of Dinghui,Heyi,and Xin acupoints once daily for seven consecutive days.The acupoints were stimulated with MMWA for 15 min each time at～40 ℃.④Estazolam(n=10):The rats were injected intraperitoneally with 0.92 mg/kg/D estazolam 3 days after the model was established and treated once a day for 7 days.The body weight and spontaneous activity of rats were measured before and after model establishment and after treatment.The behavioral changes were observed before and after treatment.The changes of 5-HT,ACH,NE and GABA in serum were detected by ELISA after treatment.The hippocampal tissues of 3 rats were randomly selected from Control,Model and MWA for high throughput sequencing analysis,and then the expression patterns of lncRNAs,mRNAs,circRNAs and miRNAs were analyzed.At last,the differentially expressed RNA between Control,Model and MWA were screened,and the GO and KEGG enrichment analysis of the differentially expressed mRNA were carried out to explore the genes and signal pathways related to insomnia.Five rats were randomly selected from the Control,Model and MWA for the verification experiment,the mRNA expression of GABRG3,EGR1,NTRK1 and HRH3 of which was abundant and significantly different in the transcriptome was confirmed by RT-PCR.Results:1.The weight effects of insomnia rats after treatment with warm acupuncture in Mongolian medicine:There was no significant difference in body weight of rats in each group(P>0.05).Compared with the Control,the weight of Model,SMWA,Estazolam and MEA decreased significantly after the model was established(P<0.05).After treatment,compared with Model,the weight of the Estazolam and MWA increased significantly(P<0.05),and the weight of the SMWA had no significant difference(P>0.05).2.The Ethological effects of insomnia rats after treatment with warm acupuncture in Mongolian medicine:In the open-field test,there were no significant differences on the exercise distance,exercise time,and number of arm lifting in each group(P>0.05).Compared with Control,the rats in Model,SMWA,Estazolam and MWA were significantly increased in standing times,exercise distance and exercise time after the model was established(p<0.05).After treatment,compared with Model,exercise distance,exercise time,and number of arm lifting in Estazolam and MWA were significantly reduced(p<0.05),there were no significant differences exercise distance,exercise time,and number of arm lifting in SMWA(p>0.05).3.The effects of neurotransmitters in insomnia rats after treatment with warm acupuncture in Mongolian medicine:Compared with the Control,the levels of GABA and 5-HT in Model and SMWA were significantly lower(p<0.05),NE were increased significantly and Ach in Model was significantly increased(p<0.05).Compared with Model,the content of 5-HT in the serum of Estazolam and MWA was significantly increased(p<0.05),ACH and NE were decreased significantly(p<0.05).The content of GABA in the serum of rats in MWA was significantly increased(p<0.05)while no significant difference in Estazolam(P>0.05).And there was no significant difference in the contents of GABA,5-HT,Ach and NE in SMWA(P>0.05).4.High throughput sequencing analysis of whole transcriptome:①A total of 136 differential expression miRNAs were detected.Compared with Control,Model had 88 differentially expressed miRNAs,69 of which were up-regulated and 19 down-regulated.Compared with Model,MWA had 86 differentially expressed miRNAs,23 of which were up-regulated and 63 down-regulated.②A total of 321 differentially expressed mRNAs were detected.Compared with Control,Model group had 294 differentially expressed mRNAs,of which 155 were up-regulated and 139 were down-regulated.Compared with Model,there were 32 differentially expressed mRNAs in MWA,6 of which were up-regulated and 26 down-regulated.③A total of 6 differentially expressed lncRNA were detected.Compared with Control,Model had 6 different expressions of lncRNA,5 of which were up-regulated and 1 down-regulated.And there was no significant difference in lncRNA expression between the MWA and Model.④A total of 527 differentially expressed circRNA were detected.Compared with Control,there were 240 differentially expressed circRNA in Model,137 of which were up-regulated and 103 down-regulated.Compared with Model,MWA had 304 differentially expressed circRNA,of which 169 were up-regulated and 135 were down-regulated.Based on Go and KEGG enrichment analysis,mRNAs related to circadian rhythm,anxiety and cognitive function regulation were further screened,and 19 key differentially expressed genes(Ryrl,Egr1,Kcnj6,Atf5,Erbb3,Prox1,Ntrkl,Tafa2,Ntf3,Nrg1,Rora,Itga3,Gabrg3,Kcnk4,Gabra3,Pak6,Gng13,Tpbg,Hrh3)were obtained.The analysis of KEGG pathway showed that the genes differentially expressed were mainly enriched to GABAergic synapses(KCNJ6,GABRA3,GNG13,Gabrg3)and Calcium signaling pathway(NTRK1,RYR1,Erbb3),Retrograde endocannabinoids signaling(KCNJ6,GABRA3,GNG13,Gabrg3)and Neuroactive ligand-receptor interaction(GABRA3,GABRG3,HRH3),etc.Based on the results of total Cerna detection,we further screened 19 candidate genes for possible Cerna Formation.In the Cerna formed by mRNAs,miRNAs and lncRNAs,we detected 121 possible Cerna formed by 19 candidate genes,Among the 121 Cerna,the most linked miRNAs which are 22 were rno-miR-330-5p(11),rno-miR-296-5p(8),rno-miR-326-3p(6),rno-miR702-3p(6),RNO-MIR-1224(6)and so on.The lncRNAs with the largest number of linked genes were 6 lncRNAs,including LOC102553329(14),LOC102550621(14),LOC100910802(13)and LOC 102549893(12).5.RT-PCR results:RT-PCR method was used to verify the sequence results.The results showed that the expression trend was consistent with the sequence results,so it confirmed the reliability of transcriptome sequencing results.Conclusion:1.Mongolian medicine warm acupuncture therapy can effectively alleviate the influence of insomnia on body weight and spontaneous activity of rats,and make it return to normal.2.By regulating the contents of neurotransmitters related to insomnia,restoring the normal Dynamic equilibrium and inhibiting the excitability of nervous system,traditional Mongolian medicine warm acupuncture therapy can improve insomnia,improve learning,memory and cognitive function.3.In this study,19 key differentially expressed genes were obtained from the whole transcriptome analysis of the hippocampus of insomnia rats.The differentially expressed genes were mainly enriched in GABAaergic synapses and Calcium signaling pathways,retrograde endocannabinoids signaling and Neuroactive ligand-receptor interaction,etc.Therefore,it is speculated that the warming needle of Mongolian medicine can affect the expression of corresponding genes in the above pathway to achieve the therapeutic effect of insomnia.