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.
ABSTRACT
ObjectiveThrough the correlation analysis between intestinal absorption profile and inhibition of macrophage foaming， the pharmacodynamic components of Zhuriheng dripping pills（ZRH） were explored to provide a basis for establishing its quality standard. MethodIntestinal absorption fluids with 0， 5， 10， 15， 20 times clinical equivalent doses were prepared by a rat everted gut sac（EGS）， and the oxidized low density lipoprotein（ox-LDL）-induced RAW264.7 macrophage foaming model was used to investigate the effect of intestinal absorption fluid with different doses on the accumulation of lipids in RAW264.7 cells by oil red O staining and cholesterol content determination， and to screen for the optimal dose. Ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used to analyze and identify intestinal absorption fractions of ZRH intestinal absorption fluids， and partial least squares-discriminant analysis（PLS-DA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were performed on different doses of ZRH intestinal absorption fluids using SIMCA 13.0 with peak area as the independent variable and the pharmacodynamic indicators as the dependent variables to screen the compounds with variable importance in the projection（VIP） value>1.0 as contributing components， and Pearson correlation analysis was used to determine the spectral effect relationship， determined the compounds and positive correlation with pharmacodynamic were as active ingredients. Molecular docking was used to verify the binding energy of peroxisome proliferator-activated receptor α（PPARα）， PPARγ， PPARβ， human retinoid X receptor α（RXRA） and nuclear transcription factor-κB（NF-κB） with the active ingredients in ZRH intestinal absorption fluids. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was performed to detect the mRNA levels of PPARγ， scavenger receptor A1（SRA1） and adenosine triphosphate-binding cassette transporter A1（ABCA1） in RAW264.7 cells， Westen blot was used to detect the expression level of PPARγ protein in RAW264.7 cells， and enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of interleukin（IL）-1β and NF-κB in RAW264.7 cells. ResultAccording to the results of oil red O staining and cholesterol content determination， the ZRH intestinal absorption fluids could significantly reduce macrophage foaming， and intestinal absorption fluids with 15， 20 times clinical equivalent doses had the best effect， the 15-fold ZRH intestinal absorption fluid was finally determined as the study subject. Spectral effect relationship showed that 52 corresponding peaks in the ZRH-containing intestinal fluid were positively correlated with the efficacy， including organic acids， phenylpropanoids， iridoids， flavonoids， bile acids， coumarins and chromones. Target validation results showed that 86.9%-96.2% of the total components processed good binding activities with the key targets of PPARα， PPARγ， PPARβ， RXRA and NF-κB， and the docking energy values were all less than -6.0 kcal·mol-1（1 cal≈4.19 J）. The results of validation showed that， compared with the normal group， the model group showed a significant increase in the levels of SRA1 and PPARγ mRNA expression， a significant decrease in ABCA1 mRNA expression， a significant increase in the level of PPARγ protein expression， and a significant increase in the levels of IL-1β and NF-κB（P<0.01）， compared with the model group， the 15-fold intestinal absorption fluid group showed a significant decrease in the levels of SRA1 and PPARγ mRNA expression（P<0.05， P<0.01）， ABCA1 mRNA expression level was significantly up-regulated， the levels of IL-1β and NF-κB were significantly reduced（P<0.01）， and PPARγ protein expression level was significantly reduced（P<0.05）. ConclusionThis study identifies 52 components and their metabolites in ZRH intestinal absorption fluid that are positively correlated with the inhibition of macrophage foaming， which may be related to the regulation of the PPARs pathway in cells and the reduction of the levels of inflammatory factors， and can provide a reference for the quality control and clinical application of ZRH.