Abstract: BACKGROUND: As a common clinical digestive disorder, irritable bowel syndrome becomes an advantageous disease of acupuncture treatment. However, the therapeutic mechanisms remain unclear. The methodological characteristics of omics coincide with the multi-target and multi-level characteristics of acupuncture, providing the possibility of revealing the principle of acupuncture in the treatment of the disease.
OBJECTIVE: To investigate the pathogenesis of irritable bowel syndrome with diarrhea (IBS-D) and the effect of acupuncture at the combined points (selected based on etiologies and symptoms) on IBS-D based on proteomics. 
METHODS: Twelve 3-month-old male Sprague-Dawley rats were randomly divided into three groups: a control group, an IBS-D model group and an acupuncture group. The IBS-D rat models were prepared using the CAS method. After successful modeling, bilateral Zusanli points, bilateral Neiguan points and Guanyuan points were selected for acupuncture treatment in the acupuncture group, with a frequency of 120 times/minute, 1 minute of acupuncture every 4 minutes, and 15 minutes of needle retention, at an interval of 1 day every 6 days, for 28 days in total. Rats in the normal control group and the model group were not given any intervention. The pressure threshold of rat abdominal retraction reflex was measured to evaluate the visceral hypersensitivity of rats. Proteomics analysis was performed using the liquid chromatography-tandem mass spectrometry -based platform. MaxQuant software, Perseus software and DAVID, KOBAS, VENNY, STRING online tools were used for the bioinformatics analysis of proteomic data. Visualization analysis was done using Cytoscape 3.7.1 software. 
RESULTS AND CONCLUSION: There were 47 differentially expressed proteins between the IBS-D model and control groups. Function analysis of differentially expressed proteins revealed that the pathogenic mechanism of IBS-D was associated with abnormal energy metabolism, the imbalance of colon motor function and increased visceral sensitivity. Important proteins related to IBS-D pathogenesis included Atp5a1, Atp5c1, Idh3b, Atp2a3, Pdhb, Ppp1ca and Mapk3. Sixty-one differentially expressed proteins were identified between the acupuncture group and IBS-D model group. Acupuncture at the combined points reversed the up-regulation of nine differentially expressed proteins and the down-regulation of nine differentially expressed proteins. Bioinformatics analysis revealed that acupuncture at the combined points for IBS-D could function via multi-targets and multi-pathways, reverse the damage of energy metabolism caused by IBS-D, and play a role against oxidative stress and inflammation, thereby relieving pain and regulating the imbalance of intestinal function. Important proteins related to acupuncture effects included Atp5a1, Atp5c1, Pdhb, Sars, Uqcrc2, Prdx2, Prdx4, Ppp1ca, Manf and Tmsb4x3. All these findings preliminarily illustrate the potential molecular mechanisms of IBS-D and the effect of acupuncture at the combined points in the treatment of IBS-D at the protein level, which provide a basis for the clinical application of acupuncture at the combined points.  

Key words: irritable bowel syndrome with diarrhea, IBS-D, the combined use of points based on etiologies and symptoms, acupuncture, proteomics, bioinformatics analysis