Our team found that Tuina is effective for treating insomnia in clinical trials. In order to explore the relevant mechanisms deeply, we recruited 27 healthy people and 29 patients with insomnia. The standard deviation of the mean age of the control group participants was 6.3 ± 2.0 months, and the insomnia patients was 6.8 ± 2.1 months.
Afterwards, we performed Tuina therapy for the patients with insomnia, and the effect was also achieved. For patients with insomnia, we took plasma sample before and after the Tuina treatment (BTT, ATT). For the HC group, we took plasma sample without taking any treatment measures, and carried out subsequent experiments using samples from the three groups.
The table lists gender distribution, average age and weight (data not shown). In the iTRAQ analysis, the genders were matched. In the ELISA analysis, the gender distribution and average age between the two groups were similar.
The summery of iTRAQ-based proteomics analysis
In this part of the study, we randomly selected 9 BTT and 9 ATT plasma samples from 29 recruited insomnia patients, and 9 plasma samples from 27 recruited healthy controls. Subsequently, the plasma from three persons in the same group was randomly mixed together at a ratio of 1:1:1, and the mixed 9 standard samples (HC-1, HC-2, HC-3; BTT-1, BTT-2, BTT-3; ATT-1, ATT-2, ATT-3) were identified by mass spectrometry.
Before analyzing the proteins identified from the high throughput assay, we first looked at the quality of the data that obtained from the mass spectrometry. For the same we looked at these features such as the distribution of unique peptide number, peptide length, the distribution of coverage identified, and repeatability using parameters such as coefficient of variation. Unique peptides are defined as the peptides that are found only for one protein. From the presence of this type of peptide, the existence of the corresponding protein can be uniquely determined. It shows the coordinate distribution of the number of unique peptides contained in all the proteins identified in this assay (Fig. 1A). The x-axis is the number of unique peptides that contained in the protein, the left y-axis depicts the number of proteins corresponding to the x-axis and the right y-axis corresponds to the ratio of total protein. For example, there are 325 proteins with 2 as the unique number of peptides, which is 80.65 % of the total number of proteins obtained. To increase the number of unique peptides, this inference could be further made. Next, the distribution of peptide length was analyzed (Fig. 1B). As it could be seen from the figure, the average length of the polypeptide identified in the assay was 14.43, which was within a reasonable range of the peptide length. The figure also showed that the length of peptide was mainly concentrated between 7 and 19 with a length of 11 peptides number showing maximum number of peptides. For an identified protein, the more peptides that support the protein, the higher the confidence of the protein. Therefore, the identification coverage of the protein indirectly reflected the overall accuracy of the identification results (Fig. 1C). The different colored sectors in the pie chart represented the percentage of proteins with different ranges of determined coverage. It was clear from the figure that 73.20% of the identified proteins had equal to or more than 10% of the peptide coverage, and 58.06% had equal to or more than 20% of the peptide coverage. The 520 proteins were classified through Blast2go to assess gene ontology (GO) enrichment. Based on different biological process, these grouped proteins were as follows: metabolic process (8.18%), immune system process (4.24%), cellular component organization or biogenesis (5.72%), response to stimulus (7.66%), biological regulation (8.29%). The common proteins are mainly involved in metabolic, structural, and regulating processes (Fig. 1D).
Exploration of differentially expression proteins (DEPs)
We consider the cut-off value of all iTRAQ ratios as a 1.2-fold change, that is, ratios > 1.2 or < 0.80, and classify proteins as up-regulated or down-regulated, respectively.
In BTT vs HC, 97 DEPs were screened successfully, of which 55 were up-regulated and 42 were down-regulated (Fig. 2) (Table S1). It suggested that in ATT vs HC, there were 46 up-regulated and 46 down-regulated DEPs (Table S2). In ATT vs BTT, 63 DEPs were identified, including 23 up-regulated and 40 down-regulated DEPs (Table S3). In addition, there were 17 common DEPs were identified between all three comparisons (Fig. 2).
Functional analysis of DEPs
To identify the primary functions in which the DEPs (Fig. 3A, C & E) were involved, GO (Gene Ontology) enrichment analysis was carried out. There were 55, 63 and 47 terms were enriched successfully in the BTT vs HC, ATT vs HC and ATT vs BTT comparisons (P < 0.01) (Table S4-6; Fig. 3B, D & F), respectively.
In BTT vs HC, it was found that multiple enriched terms were associated with immunity, including “regulation of humoral immune response” (GO:0002920), “regulation of complement, activation” (GO:0030449), “inflammatory response (GO:0006954)”, “regulation of acute inflammatory response (GO:0002673)”, “acute inflammatory response” (GO:0002526), “regulation of immune effector process” (GO:0002697) and “regulation of inflammatory response (GO:0050727)”, “complement activation, alternative pathway” (GO:0006957),“immune effector process” (GO:0002252), “regulation of immune system process” (GO:0002682), “regulation of complement activation, lectin pathway” (GO:0001868), “negative regulation of complement activation, lectin pathway” (GO:0001869), “activation of plasma proteins involved in acute inflammatory response” (GO:0002541) and “complement activation” (GO:0006956) (Table S4, Fig. 3B). It suggested that insomnia might be associated with disorder of the immune system. Moreover, six terms were involved in stress were found including “response to wounding” (GO:0009611), “response to stress” (GO:0006950), “regulation of response to external stimulus” (GO:0032101)༌“response to external stimulus” (GO:0009605), “response to stimulus” (GO:0050896) and “regulation of response to stress” (GO:0080134), suggesting that insomnia might be invloved with response of stress.
In ATT vs HC, it was found that seven enriched terms were associated with immunity, including "inflammatory response" (GO:0006954), "acute inflammatory response" (GO:0002526), "regulation of acute inflammatory response" (GO:0002673), "regulation of immune system process" (GO:0002682), "regulation of complement activation, lectin pathway" (GO:0001868), "negative regulation of complement activation, lectin pathway" (GO:0001869), and "regulation of humoral immune response" (GO:0002920), and three terms were involved in stress were found, including "regulation of response to external stimulus" (GO: 0032101), "response to stress" (GO: 0006950), "response to external stimulus" (GO: 0009605) (Table S5, Fig. 3D).
In ATT vs BTT, it was found that multiple enriched terms were associated with immunity, including "inflammatory response" (GO:0006954), "regulation of humoral immune response" (GO:0002920), "acute inflammatory response" (GO:0002526), "regulation of complement activation" (GO:0030449), "regulation of acute inflammatory response" (GO:0002673), "regulation of inflammatory response" (GO:0050727), "complement activation, alternative pathway" (GO:0006957), "regulation of immune system process" (GO:0002682), "regulation of immune effector process" (GO:0002697), "adaptive immune response" (GO:0002250), "leukocyte mediated immunity" (GO:0002443),"activation of plasma proteins involved in acute inflammatory response" (GO:0002541), "complement activation" (GO:0006956), "humoral immune response mediated by circulating immunoglobulin" (GO:0002455) and "complement activation, classical pathway" (GO:0006958) (Table S6, Fig. 3F). It strongly suggested that the Tuina treatment might have the capacities to affect the immune system and improve the insomnia by cure the disorder of immune system. Moreover, four terms involved in stress were found including "response to external stimulus" (GO: 0009605), "regulation of response to external stimulus" (GO: 0032101), "regulation of response to stimulus" (GO: 0048583), “positive regulation of response to stimulus” (GO: 0048584), suggesting that the Tuina treatment could also improve the sleep quality by affecting the response to stress.
Validation of protein identification and quantification by ELISA
We performed ELISA to analyze the expression levels of C4, C5 and C5b-9 to confirm the results of iTRAQ-labeled LC-MS/MS analysis. Compared with the HC group, the expression levels of C4, C5 and C5b-9 in the BTT group and ATT group were decreased. Compared with the BTT group, the expression levels of C4, C5 and C5b-9 were increased in the ATT group (Fig. 4). The result of ELISA supported the result of iTRAQ, which proved that our experiment was very reliable.