Proteomic analysis reveals the deregulation of inflammation-related proteins in acupuncture-treated rats with asthma onset

Abstract

Although the beneficial effects of acupuncture in asthma treatment have been well documented, little is known regarding the biological basis of this treatment. Changes in the lung proteome of acupuncture-treated rats with asthma onset were comparatively analyzed using a two-dimensional gel electrophoresis (2DE) and mass-spectrometry- (MS-) based proteomic approach. Acupuncture on specific acupuncture points appeared to improve respiratory function and reduce the total number of leukocytes and eosinophils in bronchoalveolar lavage fluid in OVA-induced asthma onset. Image analysis of 2DE gels revealed 32 differentially expressed acupuncture-specific protein spots in asthma onset; 30 of which were successfully identified as 28 unique proteins using LC-MS/MS. Bioinformatic analyses indicated that these altered proteins are most likely involved in inflammation-related biological functions, and the functional associations of these proteins result in an inflammation signaling pathway. Acupuncture regulates the pathway at different levels by regulating several key nodal proteins, including downregulating of proinflammatory proteins (e.g., S100A8, RAGE, and S100A11) and upregulating of anti-inflammatory proteins (e.g., CC10, ANXA5, and sRAGE). These deregulated inflammation-related proteins may mediate, at least in part, the antiasthmatic effect of acupuncture. Further functional investigation of these acupuncture-specific effector proteins could identify new drug candidates for the prophylaxis and treatment of asthma.

Figure 1

Schematic localization of acupuncture points: GV14 (Dazhui), BL12 (Fengmen), and BL13 (Feishu) stimulated in rats (a), and the corresponding equivalent acupuncture points in the human body (b). (c) Presentation of the experimental procedure; rats were sensitized with OVA at day 0. Acupuncture was repeatedly administered at specific acupuncture points on alternate days for 2 weeks from the first day after sensitization. On day 14, rats were anaesthetized and challenged with OVA by injection into the external jugular vein. The pulmonary resistance (RL), dynamic compliance (Cdyn), and respiratory rate (RR) were recorded before the challenge for 10 min as the baseline values and immediately measured for another 10 min after the OVA challenge. The sample collections were performed immediately after the measurements were taken. Control rats were sensitized and challenged with saline instead of OVA.

Figure 2

Changes in the pulmonary resistance (RL) within 10 min after challenge in NC (n = 15, sensitized and challenged with saline), AS (n = 14, sensitized and challenged with OVA), AA (n = 12, sensitized and challenged with OVA + acupuncture treatment) and NA (n = 15, sensitized and challenged with saline + acupuncture treatment).The changes in the RL are expressed as differential values subtracted from the corresponding baseline values (Figure 1). Each point represents the mean ± SEM. ^# P < 0.05, ^## P < 0.01 when comparing the AS group with the NC group and *P < 0.05, **P < 0.01 when comparing the AA group with the AS group.

Figure 3

The effect of acupuncture on the cellular composition of BALF. BALFs were collected after OVA challenge. Total leukocyte (a) and differential cell counts (b) were performed. Neu, neutrophil; Lym, lymphocyte; Mon, monocyte; Eos, eosinophil; Bas, basophil. Values represent the mean ± SEM. ^# P < 0.05, ^## P < 0.01 when comparing the AS group with the NC group, and **P < 0.01 when comparing the AA group with the AS group.

Figure 4

Protein expression profiles in lung tissues. (a) A representative 2DE gel image of proteins isolated from the AA group. The numerically labeled spots indicate the differentially expressed protein spots among the NC, AS, and AA groups. The numbers correspond to the spot identification numbers listed in Table 1. The molecular weight standards and pH range are shown at the left and bottom of the gels, respectively. (b) Differential expression profiles of five inflammation-related proteins regulated by acupuncture: CC10, S100A8, RAGE* (which is actually sRAGE, Table 1), RhoGDI2, and ANXA5. The cropped images of 2DE gels were symmetrically boxed, and the arrows on the images indicate the relative positions of the protein spots. (c) Quantitative analysis of the five inflammation-related proteins regulated by acupuncture. Each spot volume was quantified from the intensity of the spots using PDQuest software. The bars represent the mean ± SEM of triplicate 2DE gels. ^# P < 0.05, ^## P < 0.01 when comparing the AS group with the NC group. *P < 0.05, **P < 0.01 when comparing the AA group with the AS group.

Figure 5

Bioinformatic analysis of the acupuncture-specific differentially expressed proteins in asthma onset. (a) Functional classification of the identified proteins using the PANTHER classification system. Proteins that were differentially expressed following acupuncture were classified into diverse functional categories according to biological process. (b) The STRING network of known protein-protein interactions among the seven inflammation-related proteins (marked by colored rectangles) regulated by acupuncture. Circle nodes indicate intermediate proteins not detected in the proteomic study (RAC2, RAS-related C3 botulinum substrate 2; GDI2, Rab GDI beta; CDC42, Cell division control protein 42 homolog; ANXA6, Annexin A6; TTF-1, Thyroid transcription factor 1). S100A9 (green circle) is an inflammation-related protein regulated by acupuncture, which was determined in our previous study [16]. The network edges represent the predicted functional associations, and stronger associations are represented by darker lines.

Figure 6

Validation of the expression profiles of five inflammation-related proteins by western blot analysis. (a) A representative western blot visualizing the expression levels of CC10, S100A8, and RhoGDI2. β-actin was used to demonstrate equal loading. (a) Western blot analysis using the anti-rat RAGE extracellular domain monoclonal goat antibody demonstrates that the lung homogenate contains three bands that were approximately 48, 50, and 55 kD in size; the 48-kD band corresponds to sRAGE and the 55 and 50 kD bands correspond to full-length RAGE. (b) The densitometric quantification of individual proteins is expressed as the fold change compared to β-actin. Each bar represents the mean ± SEM of triplicate experiments, and similar results were observed in all experiments. ^# P < 0.05, ^## P < 0.01 when comparing the AS group with the NC group; *P < 0.05, **P < 0.01 when comparing the AA group with the AS group.

Figure 7

A possible inflammatory signaling pathway that results from the functional associations of the identified inflammation-related proteins (marked with an underline), which is regulated by acupuncture in asthma. Acupuncture can downregulate the proteins S100A8, S100A9, RAGE, and RhoGDI2 and upregulate the expression of CC10 and sRAGE. This pathway may explain the anti-inflammatory effect of acupuncture in asthma. Rho inact, inactivated Rho GTPase; Rho act, activated Rho GTPase; RhoGEF, Rho-specific guanine nucleotide exchange factors; GAP, GTPase-activating proteins; NF-κB, nuclear factor-κB; AP1, activator protein-1.