| Objective:In this study,a clinical randomized controlled trial was conducted for evaluating the effectiveness and safety of acupuncture for patients with chronic spontaneous urticaria(CSU).At the same time,differentially expressed lncRNA and mRNA genes of acupuncture treatment for CSU were analyzed by Long non-codingRNA sequencing technology to further clarify the molecular biological mechanism of acupuncture.Methods:1.Clinical TrialIn this randomized controlled trial,a total of 80 participants were randomly assigned to receive active acupuncture on a fixed prescription of acupoints or sham acupuncture with superficial acupuncture on non-acupuncture points five sessions per week for 2 weeks.The participants were followed for 2 weeks.The primary outcome was Urticaria Activity Scores(UAS7)after treatment at 2 weeks.Secondary outcomes included the Visual analogue scale(VAS)score of itching intensity,Dermatology Life Quality Index(DLQI),Hamilton Depression Scale(HAMD),Hamilton Anxiety Scale(HAMA),and incidence of adverse events(AEs).UAS7,VAS of itching intensity and DLQI were evaluated at baseline,week 1,week 2,week 3,and week 4,respectively;HAMA and HAMD were evaluated at baseline and week 2.The incidence of adverse events was evaluated at week 1 and week 2.2.Analysis of Differentially Expressed GenesAccording to the result of clinical trial,six CSU patients in acupuncture group and sham acupuncture group were selected,and six healthy participants were included in this part of the study.The venous blood samples were collected from healthy participants and CSU patients before and after treatment.The totalRNAs were extracted and purified,and the gene expression profiles of the samples were sequenced by BGISEQ-500 platform.DEGs were screened out from the gene expression profiles.The function of DEGs were annotate with Gene Ontology(GO)and the functional enrichments of pathways related to DEGs were analyzed with the Kyoto Encyclopedia of Genes and Genomes(KEGG).Analysis of differential lncRNA target genes and interaction analysis of differential lncRNA and target genes.Finally,the selected possible related differential genes were verified by real-time quantitative polymerase chain reaction.Results:1.Clinical TrialUAS7:The decrease from baseline in the mean UAS7 total score at week 2 in the active acupuncture group was 8.63(95%CI,5.49 to 11.78)and 6.21(95%CI,-2.98 to 9.43)in the sham acupuncture group.At weeks 4 the reduction from baseline in the mean UAS7 total score is 11.17(95%CI,7.57 to 14.78)and 9.56(95%CI,5.87 to 13.26),respectively.The two groups had not a significant difference(P=0.286>0.05,P=0.537>0.05)at weeks 2 and 4,respectively.VAS score of itching intensity: The decrease from baseline in the mean VAS total score in the active acupuncture group was 13.78(95%CI,8.99 to 18.57)and 17.34(95%CI,12.12 to 22.57)and the sham acupuncture group was 12.97(95%CI,8.07 to 17.88)and18.64(95%CI,13.29 to 24.00).The two groups had not a significant difference(P=0.816>0.05,P=0.730>0.05)at weeks 2 and 4,respectively.DLQI: The change in the DLQI from baseline was greater in the active acupuncture group than the sham acupuncture group,but had not a significant difference(each P>0.05).Besides,the active acupuncture group had greater improvement in the HAMA and HAMD than the sham acupuncture after treatment at 2 weeks,but between-group difference weren't significant(P=0.623>0.05,P=0.931>0.05).The two group had no significant statistical difference in the incidence of AEs(P=0.415>0.05).As for the credibility of blinding,no statistical difference was found between groups in the proportion of participants who guessed that they received active acupuncture at week 2(P=0.609>0.05).2.Analysis of Differentially Expressed Genes(1)Alignment of key differentially expressed genesAccording to the sequencing results,734 differentially expressed lncRNAs were screened out in CSU patients,of which LOC100294145 was most significantly up-regulated lncRNAs(FC:6.2252139)and ZFP91-CNTF was most significantly down-regulated lncRNAs(FC:-9.013792578).1037 differentially expressed mRNAs were screened out in CSU patients,of which PHYHIP was most significantly up-regulated mRNAs(FC:6.216993448)and TBC1D3 F was most significantly down-regulated mRNAs(FC:-5.59935025).After acupuncture treatment,68 lncRNAs in the CSU patients' expression profile changed,of which LOC107985235 was most significantly up-regulated lncRNAs(FC:1.762494242)and LOC105369690 was most significantly down-regulated lncRNAs(FC:-2.945325006).102 mRNA changed,of which MECOM was most significantly up-regulated mRNAs(FC: 2.814131088)and HBA1 was most significantly down-regulated mRNAs(FC:-2.341879038).After sham acupuncture treatment,63 lncRNAs in the CSU patients' expression profile changed,of which C21orf62-AS1 was most significantly up-regulated lncRNA(FC: 2.062793325)and LOC105369690 was most significantly down-regulated lncRNA.121 mRNAs changed,of which SLC35F1 was most significantly up-regulated(FC:4.15416432)and HBB were most significantly down-regulated(FC:-3.715161998).(2)GO analysislncRNA target genes:1891 lncRNAs target genes were differentially expressed between healthy participants and CSU patients.In terms of cellular components,biological functions,and biological processes of GO analysis,nucleosome,protein binding,RNA polymer pair transcription were the most enrichments.After acupuncture treatment,nucleosomes,protein heterodimer activity,and nucleosome were the most enrichments in the 183 differentially expressed lncRNA target genes.After sham acupuncture treatment,MHC class II protein complexes,peptide-antigen binding,and immune system process were the most enrichments in the 161 differentially expressed lncRNA target genes.mRNA:1037 mRNAs were differentially expressed between healthy participants and CSU patients,ribosome,ribosome structural components,SRP-dependent co-translational protein were the most enrichments.After acupuncture treatment,hemoglobin complex,oxygen carrier activity,platelet degranulation were the most enrichments in the 102 differentially expressed lncRNA target genes.After sham acupuncture treatment,cortical cytoskeleton,oxygen carrier activity,bicarbonate transport were the most enrichments in the 121 differentially expressed lncRNA target genes..(3)KEGG Pathway analysislncRNA target genes: 1891 lncRNA target genes were differentially expressed between healthy participants and CSU patients,participating in 341 signaling pathways and the most enrichments was the systemic lupus erythematosus.After acupuncture treatment,183 differentially expressed lncRNA target genes were involved in 172 signaling pathways and the most enrichments was the systemic lupus erythematosus.By searching the NCBI,HGNC,Gene Card,Ensenmbl databases and consulting the literature,four signaling pathways may be related to urticaria were screened.The result found that the HLA family genes closely related to urticaria in the four signaling pathways were up-regulated and HLA family genes had obvious protein interactions after acupuncture treatment.HLA-C and its associated lncRNA-LOC101928573 were selected for RT-PCR verification.No significant difference appeared in the RT-PCR verification(P>0.05).After sham acupuncture treatment,161 differentially expressed lncRNA target genes were involved in 134 signaling pathways and and the most enrichments was the Ig A's intestinal immune network.Five pathways may be related to urticaria were screened out.The result found that compared with acupuncture treatment,sham acupuncture treatment also caused changes in HLA family genes.At the same time,it also caused upregulation of IL15 RA expression in the JAK-STAT signaling pathway.RT-PCR was performed on IL15 RA and its associated lncRNA-LOC107984200,but there was still no significant difference(P>0.05).mRNA:1037 mRNAs were differentially expressed between healthy participants and CSU patients,participating in 304 signaling pathways and the most enrichments was the ribosome signaling pathway.After acupuncture treatment,102 differentially expressed mRNA were involved in 127 signaling pathways and the most enrichments was the ribosome signaling pathway.Three signaling pathways may be related to urticaria were screened out.MPL was down-regulated in the JAK-STAT signaling pathway after acupuncture treatment.After sham acupuncture treatment,121 differentially expressed mRNA were involved in 135 signaling pathways and the most enrichments was the malaria signaling pathway.Four signaling pathways may be related to urticaria were screened out.Compared with acupuncture,sham acupuncture also caused down-regulation of MPL in the JAK-STAT signaling pathway.RT-PCR verified this result.Conclusion:1.Both acupuncture and sham acupuncture improve the relief of symptoms,the quality of life and regulation of anxiety in CSU patients,and acupuncture had a greater improvement.Because the sample size was small and according to the theory of TCM dermatology,the sham acupuncture with superficial acupuncture on non-acupuncture points is an inactive sham control.2.Transcriptome sequencing was used to reveal the abnormal expression of lncRNA in CSU patients.Acupuncture can regulate the expression of lncRNA and mRNA in CSU patients.3.Acupuncture treatment of CSU may be effective by regulating JAK-STAT signaling pathway and down-regulating the expression of related genes such as MPL,but further experiments are needed to verify it.4.Transcriptome sequencing was used to confirm that the sham acupuncture with superficial acupuncture on non-acupuncture points can cause changes in expression levels of lncRNA and mRNA similar to acupuncture and it is a certain therapeutic effect,not a placebo effect in the treatment of urticaria. |
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