Differential Proteomics Analysis of the Subcutaneous Connective Tissues in Alcian Blue Tracks along Conception Vessel and Adjacent Nonmeridian in Rats

In more than half a century, exploring the biological connotation of the meridians was one of the core components of scientific research studies in traditional Chinese medicine (TCM). Based on the previous works of low hydraulic resistance channel (LHRC) along meridians (LHRCM), the differential proteomics between the Alcian blue track (ABT) on LHRC along the conception vessel (CV) and nonmeridians tissue next to the CV were investigated in this study to explore the material basis and biological function of LHRCM. Proteomics based on LC-MS was introduced into the subcutaneous connective tissues (SCT) of ABT along the CV and the adjacent nonmeridian (1 cm from the CV). A total of 2328 proteins were identified from ABT along the CV and adjacent nonmeridian based on data-dependent acquisition (DDA) mode. In total, 1970 proteins were quantified based on the SWATH (sequential window acquisition of all theoretical fragment ions) label-free model, and the nonstandard and quantitative methods of MSALL were applied to analyze the data. There were 468 proteins differentially expressed. GO analytic results showed that the differential proteins were of varieties in molecular function and biological process. Most of differential proteins were involved in metabolic process, cellular process, response to hormone, and response to wounding. Further analysis showed that the upregulated differential proteins involved in ATP metabolism (ATP5E, GAPDH), redox reactions (Gpx-3), and Ca2+ transmembrane transport (CACNA2D1) were closely related to meridian phenomenon and acupuncture effect. These differential proteins would be potential characteristic proteins of the LHRC along the CV in rats which may be useful to deepen the knowledge on LHRCM.


Introduction
For more than half a century, exploring the biological connotation of meridians has been an attractive topic. With the help of modern methods such as biology, physics, and imaging, scholars worldwide have carried out much research to observe the structural characteristics and material basis of meridians [1,2]. Some scholars have carried out research in the field of specific protein and meridian material bases. Zhou proposed one point of view that there are proteins related to meridians in the cell membrane of the tissues along the meridians. Under certain conditions, these proteins can be sequentially altered, rearranged, and coupled with each other, resulting in complex resonance. e protein molecules along the meridian form an energy band structure with the functions of energy, information transmission, and conversion [3]. Feng considered that fibrin in meridians and acupoints is involved in the generation and transmission of acupuncture effects and that fibrin in the body is an important part of meridian essence [4]. rough a systematic study on the biological characteristics of the sensory nervespecific protein 29-kD protein, Meng et al. [5] proposed that the traditional Chinese medicine (TCM) theory of "no obstruction no pain and vice versa," qi sensation along meridians, acupuncture stimulation, and amplification effect of acupoint injection are all used to transmit stimulation information through the bridging of the 29-kD protein. A common differential protein band in the fat belt is observed along the stomach meridian and conception vessel (CV) but not in the fat belt beside the meridian [6]. In addition, it was found that the expression of connexin 43 was significantly higher in the plantar part of the kidney and the dorsal part of the bladder than in the adjacent nonmeridian control line [7]. ese results suggest that there is a certain type of protein in meridian tissues, which may be one of the material bases of meridians.
At present, the research on the protein specificity along meridian tissues is just beginning. A few reported results are mainly the analysis based on the known results of meridian research and modern life science knowledge. Although it is scientific, the additional experimental evidence is absent. For the specific proteins found in the tissues of meridians, the identification involved in the functions of promoting blood and qi, connections between viscera, and the transmission of acupuncture signals is still unknown. erefore, it is possible to find a new material, energy, and information transmission system in the human body, which will help clarify the essence of meridians and the mechanism of acupuncture and moxibustion.
Zhang proposed that the meridians are the low hydraulic resistance channels (LHRC) distributed in the body's interstitium. e hypothesis was verified by tracing the LHRC along the meridians (LHRCM) [8], observing the microstructure in vivo [9] and blocking the LHRCM [10]. Recently, the LHRC along the CV in rats was shown by injecting Alcian blue (AB) [11]. In this paper, the differential protein expression of the subcutaneous connective tissues between the AB tracks (ABT) on LHRC along the CV and the adjacent nonmeridian area were analyzed for the first time, to explore the material basis and biological function of the meridians. e results will provide new evidence to understand LHRCM and may be helpful to reveal the biological connotation of meridians.

Animals.
In total, 15 healthy adult male rats with an average age of 11 weeks and a weight of 380-420 g were used in this study. All animals were provided by Biotechnology Co., Ltd. (SCXK (Beijing) 2016-0002). ey were treated in accordance with international standards for the care and use of laboratory animals, and the entire experiment was performed in accordance with the protocol approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion at the China Academy of Chinese Medical Science.

e Extraction of Connective Tissue in ABT along CV and
Adjacent Nonmeridian. After anesthesia by 10% urethane, the subcutaneous connective tissues (2 mm wide and 1 mm thick) of the ABT along the CV and adjacent nonmeridian (the control tissue, CT, along the line where the median line of the abdomen is 1 cm horizontally) in the abdominal skin of the rats were cut (Figure 1). e tissues in the same group were divided into 5 parts, approximately 30 mg each, washed with 4°C PBS, and were kept at -80°C.

Extraction and Quantification of Total
Protein. About 0.1 g of sample tissue was cut into pieces and added to cold protein lysate (300 μL/100 g) for homogenization. e mixture was transferred to an EP tube (4°C) and shocked and lysed at 4°C and 120 rpm for 20 min. It was centrifuged at 20000 × g and 4°C for 10 min in a high-speed centrifuge to obtain the clear supernatant, which was the total protein solution of the subcutaneous connective tissues of CV and CT. e total protein concentration in the supernatant was quantified by Bradford assays ( ermo Pierce ™ Rapid Gold BCA Protein Assay Kit, ermo Scientific ™ , USA).

Protein Precipitation.
en, 20% trichloroacetic acid was added to 110 μg of protein, which was extracted from each sample to a final concentration of 10%. After incubation at −80°C for 2 h, the protein was precipitated as much as possible. It was centrifuged at 14000 × g at 4°C for 20 min. Acetone (4°C) was added to the precipitate, and the solution was precipitated at −80°C overnight after ultrasonic suspension and then centrifuged at 14000 × g at 4°C for 20 min. e supernatant was discarded, and the acetone was volatilized at room temperature.

Protein Sample Pretreatment and Peptide Solution
Preparation. Each sample was pretreated, and a peptide solution was prepared according to the following methods. e sample was added to 70 μL of ammonium bicarbonate solution (50 mM) with 0.2% Rapigest SF for ultrasonic dissolving protein. e residual acetone volatilized at 37°C and 300 rpm for 10 min, and then 8 μL of ammonium bicarbonate solution (50 mM) with Tris (2-carboxyethyl) phosphine (100 mM) and 2 μL of ammonium bicarbonate solution (50 mM) were added. e mixture was evenly mixed for reaction at 60°C for 30 min. After cooling to room temperature, 9 μL of ammonium bicarbonate solution  Evidence-Based Complementary and Alternative Medicine (50 mM) with 100 mM iodoacetamide and 1 μL of ammonium bicarbonate solution (50 mM) were added, and the mixture was evenly mixed for a photoprotective reaction for 30 min. en, 5 μL of restricted trypsin (0.4 μg/μL) was added and digested overnight at 37°C and 140 rpm. en, 5 μL of trifluoroacetic acid (10%) was added for termination digestion at 37°C and 140 rpm for 30 min. e supernatant containing the digested peptide was transferred to a 10 kDa ultrafiltration membrane and centrifuged at 14000 r/min for 15 min. e filtrate (1 μg/μL) was preserved at −80°C.

DDA Mass Spectrometry.
e data of mixed samples were acquired based on a data-dependent acquisition (DDA) model by the AB Sciex5600 + TripleTOF platform. e peptide solutions of CV (5 μL) and CT (5 μL) samples were mixed. Mixed sample preconcentration was performed by nanoliquid chromatography and nanoliter-high-performance liquid chromatography elution. A capillary direct injection chromatographic column was used for sample separation and analysis, and the parameters of gradient elution were as follows (Table 1). Phase

Qualitative Identification of Protein.
e FASTA files of rat proteins were downloaded from NCBI and imported into the temp of ProteinPilot. e protein identification method was established to search for results from the identification protein library.

SWATH Analysis.
e sequential window acquisition of all theoretical fragment ions (SWATH) method was established. e mixed peptide data were collected in the range of 350-1250 M/Z. e peptide sample data were collected by nano-LC and SWATH MSALL. ere were 5 biological duplications in the CV group and 4 biological duplications in the CT group. Each sample was collected 3 times.

Data Processing and Biological Information Analysis.
e SWATH data were analyzed by the Peak View SWATH Processing Micro App. e proteins that can be quantified by SWATH were extracted from the identification protein library, and the corresponding peptide and fragment ion information were extracted. e confidence interval was 95%, and the false discovery rate (FDR) was 1.0%. e relative quantitative comparison was made between the CV group and the CT group by Markervie. Differential expression of proteins between the CV group and CT group was selected by t-test, P < 0.05, and CV/CT ≥1.5 or ≤0.067. e total protein and differential protein data were analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and BP link KEGG. e functions of differential proteins and their biological processes were analyzed by consulting the UniProt database. en, according to natural studies of TCM meridians, the relationship between differential proteins and TCM meridians was analyzed, and four or five proteins that are closely related to the function of meridians were identified.

Statistical
Analysis. SPSS 19.0 statistical software was used for statistical analysis. One-way ANOVA was used to compare differences in the same index among the groups, and the LSD test was performed to determine significant differences between two groups. e data are expressed as Evidence-Based Complementary and Alternative Medicine the mean ± SD, with P < 0.05 indicating statistical significance.

Proteomic Identification of ABT along CV and CT.
e subcutaneous connective tissues of ABT along CV and CT in three rats of the same body were chosen as experimental specimen. e pooled and tryptic digested protein samples were analyzed by tandem LC-MS in data-dependent acquisition (DDA) model, and the acquired data were processed by ParagonTM (AB Sciex) ( Figure 2). ProteinPilot identified 169161 secondary spectra with 1% confidence level, 15196 peptide fragments within the global FDR 63.7% and 1% confidence level, and 2328 proteins within the global FDR 97.6% and 1% confidence level ( Figure 3).

Differential Proteomics Analysis of ABTalong CV and CT.
ere is a good linear relationship between the DDA database and SWATH data. In total, 1988 proteins were quantified from 2328 proteins. Among them, 1970 were effectively quantified. e peak areas of all quantified proteins were normalized. CT3 and CV1 were eliminated because there was no significant difference between CT3 and CV1 in principal component analysis (Figure 4(a)). e remaining groups were subjected to PAC and t-test analysis, and 468 proteins were found to be differentially expressed (Figure 4(b)).
GO analysis showed that the differential proteins were distributed in the cell membrane or the structure of organelle intima, such as extracellular membrane-bound organelles, adhesion junction, organelle binding membrane, organelle outer membrane, and organelle inner membrane; in the extracellular matrix; and in the structure of cell membrane or organelle intima, such as extracellular membrane-bound organelles, adhesion junction membrane, organelle outer membrane, organelle inner membrane, endoplasmic reticulum, membrane microstructure domain, and muscle fiber membrane. e differential proteins were also found to be located in the cytoplasm, tricarboxylate cyclase complex, blood cell microparticles, projection neurons, and glial cell projections (Figures 5(a) and 5(b)). e molecular functions of the differential proteins may be isoprotein binding, coenzyme binding, enzyme binding, peptide binding, nucleoside binding, nucleotide binding, cell adhesion molecule binding, protein complex binding, S100 protein binding, vitamin binding, anion binding, oxidoreductase activation, intramolecular oxidoreductase activation, nucleoactivation of carbon oxygen lyase, activation of protein dimers, activation of peptidase regulators, and activation of glutathione oxide enzymes ( Figure 5(c)). Most of the differentially expressed proteins were involved in small molecule metabolism, organic acid, single-organism catabolism, oxidation-reduction, organonitrogen compound, cellular catabolism, cofactor metabolism, organic substance catabolism, inorganic response, sulfur compound metabolism, organic response, cellular lipid metabolism, singleorganism biosynthesis, hormone response, cellular aldehyde metabolism, lipid metabolism, organophosphate metabolism, aging, wounding response, and the tricarboxylic acid cycle ( Figure 5(d)).
Based on previous studies on meridians, ATP metabolism-related proteins are differentially expressed between the ABT along CV tissues and adjacent nonmeridian tissues in rats. Among them, the expression of proteins involved in glycolysis was upregulated. Glyceraldehyde-3-phosphate dehydrogenase (GADPH, P04797) is a key enzyme in glycolysis. It is involved in the first step of catalyzing the biochemical reaction and producing a large amount of ATP. e differential expression of Gxp-3 suggested that the glycolytic metabolism in the ABT along the CV was more active than that in adjacent nonmeridian tissues.
At the same time, ATP synthase, one of the key substances involved in the oxidative phosphorylation process, was differentially expressed between the subcutaneous connective tissues of ABT along the CV and nonmeridian tissue in rats, such as ATP synthase subunit epsilon, mitochondria (P29418), ATP synthase subunit d, mitochondria (P31399), ATP synthase-coupling factor 6, mitochondrial (P21571). ATP synthase subunits are distributed in the inner  membrane of mitochondria. During the process of oxidative phosphorylation, they participate in the biological process of ATPase activation to directly affect ATP synthesis. e differential expression of ATP synthase suggested that the process of oxidative phosphorylation is more active in the subcutaneous connective tissues of ABT along the CV than in that of adjacent nonmeridian tissues and that the efficiency of ATP production was increased.
Proteins involved in the redox reaction process were differentially expressed between the subcutaneous connective tissues of ABT along the CV and nonmeridian tissue in rats. For example, glutathione peroxidase 3 (Gpx-3, P23764) is involved in glutathione metabolism, the hydrogen peroxide catabolism process, and the response to oxidative stress. e differential expression of Gpx-3 suggested that the reactive oxygen species scavenging occurs along the CV. e functional proteins related to Ca 2+ transmembrane transport were differentially expressed between the subcutaneous connective tissues of the ABT along the CV and nonmeridian tissue in rats. ese differentially expressed proteins mainly included the voltage-dependent L-type calcium channel protein family, such as voltage-dependent L-type calcium channel subunit beta-1 (P54283) and voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1, P54290). CACNA2D1 can regulate physical properties to affect the calcium current density and activation/deactivation of calcium channels. It plays an important role in excitation contraction coupling and   Evidence-Based Complementary and Alternative Medicine excitation secretion coupling. Ca 2+ is an important substance for information transmission and sensation transmission along meridians after acupuncture stimulation. e upregulated expression of CACNA2D1 suggested that the transmembrane transport of Ca 2+ occurs along the CV.

KEGG Analysis.
Differential proteins were involved in the following metabolic pathways: metabolism, genetic information processing, environmental information processing, cellular processes, organic systems, and disease formation. Most proteins (207) were involved in metabolic processes, mainly carbon metabolism (22), amino acid biosynthesis (15), metabolic pathways (64), carbohydrate metabolism (61), glucose metabolism (18), glycolysis (13), amino acid metabolism (20), fat metabolism (8), and glutathione metabolism (5). Some of the differentially expressed proteins were involved in genetic information processing, cellular processes, organismal systems, and human diseases ( Figure 6). e GO analysis results indicate that the differential proteins were mainly involved in material and energy metabolism.

Discussion
e results showed that the differentially expressed proteins were mainly distributed in organelles such as vesicles, secretory vesicles, and the structure of the cell membrane or inner membrane of organelles. ey are involved in the transportation, identification, and transfer of transmitters, cytokines, and ions. e molecular functions of the differentially expressed proteins were mainly the binding of proteins, nucleosides, enzymes, mucopolysaccharides, and ions. e biological processes associated with the differential proteins were the material metabolism pathway, response to hormones, and response to mechanical injury. Based on the meridian phenomena and acupuncture effects, we proposed that the expression levels of GAPDH, CACNA2D1, ATP5E, and Gpx-3 were upregulated in the ABT along the SCT of CV compared with nonmeridian tissues in rats. ese proteins are involved in ATP metabolism, redox reactions, and calcium ion transmembrane transport which are related to the function of meridians.    Figure 6: KEGG analysis of differential expressed proteins between the SCT of the ABT along the CV and adjacent nonmeridian tissue.

Evidence-Based Complementary and Alternative Medicine
Meridians in TCM are the channels for running qi and blood. ey can connect between viscera and body surface and between different parts of body surface to form a regulatory system on human body. All material transportation and information transmission must consume energy. erefore, it is inferred that the energy metabolism of meridians tissues should be high. In fact, it was higher infrared radiation [12], ultramicro luminescence, increased oxygen consumption [13], carbon dioxide release [14], and blood perfusion along meridians compared with the adjacent nonmeridian regions [15] which indicated an active energy metabolism along the meridians. In addition, higher nerve ending density, higher K+, Na+, and Ca 2+ concentrations, and aggregated mast cells were observed in the tissues along meridians [16]. It is conducive to an active biological process along the meridians. Of course, a large amount of ATP is involved. ATP is an important high-energy phosphate compound which is the main energy form used by cells directly. ATP synthase is the key substance in the process of catalyzing ATP production in the process of oxidative phosphorylation, which is involved in the activation of ATPase and ATP synthesis [17][18][19][20]. In this study, ATP5E, one of the key substances involved in the process of oxidative phosphorylation, was differentially expressed between ABT along the CV and adjacent nonmeridian tissue. is result indicated that the oxidative phosphorylation process in the subcutaneous connective tissues along the CV was more active and the efficiency of ATP production was increased, providing an evidence for the meridian characteristics of high energy metabolism.
However, when the body is in a state of stress, hypoxia, or strenuous exercise, energy is mainly provided by glycolysis. After acupuncture and moxibustion stimulation, tissues along the meridian path are stressed, and the energy metabolism is mainly provided by glycolysis. e class of cells with extremely active metabolism, such as nerve cells and white blood cells, also need some of the energy provided by glycolysis, even in the absence of hypoxia [21].
ere are abundant nerve endings, neurons, and immune cells in the tissues of acupoints and meridian paths. A large amount of ATP is required for maintaining physiological metabolism of these cells. erefore, glycolysis in tissues along the meridian path is active. GAPDH is the key enzyme during glycolysis and is involved in the 1-step reaction. e quantity and activity of GAPDH directly affect ATP synthesis [22,23]. In this study, GAPDH expression was upregulated in the ABT along the CV, indicating that glycolysis was more active in the SCT along the CV than that in nonmeridian tissues. GAPDH provides energy for metabolic activities and information transmission along meridians which is important for the transmission of energy along meridians.
ATP production in eukaryotic cells mainly occurs in mitochondria with the formation of mitochondrial respiration chains (MRCs) and superoxide anions. Superoxide anions are reduced in mitochondria to produce strong oxidizing reactive oxygen species, H 2 O 2 and hydroxyl radicals (·OH). e chemical properties of these reactive oxygen species are very active which can cause oxidative damage to proteins, DNA, and other macromolecules. Nevertheless, the body can remove active oxygen species in time via endogenous antioxidant enzymes [24]. GPX is an antioxidant enzyme that widely exists in the body and Gpx-3 is one of the main members in GPX family. As the energy metabolism of the tissues along the meridian path is very active, it can be speculated that the production of reactive oxygen species is also increased in these tissues. Guo et al. [25,26] found that the concentration of free radicals is high in the meridian of the abdominal wall of rats. ey suggested that the antioxidant reduction reaction was active in the tissues along the meridians to maintain the steady state of the local internal environment. In this study, we observed that ATP5E expression was upregulated in the ABTalong the CV compared with nonmeridian tissues. It can be inferred that the production of H 2 O 2 and peroxides (R-O-OH) is also increased and a large amount of ATP synthesis occurs thereafter. erefore, a large number of antioxidant enzymes are needed to efficiently remove free radicals. Gpx-3 expression was upregulated in the ABT along the CV, suggesting that the catalytic reduction reaction process may be more active in tissues along the meridian path. Gpx-3 can effectively eliminate the reactive oxygen radicals produced by the high energy metabolism of meridians.
Ca 2+ is an important second messenger in cells. e biological activity of Ca 2+ in acupoints and meridians is closely related to the activities of meridians and the acupuncture effects [27,28]. e results showed that the concentration of Ca 2+ at acupoints was higher than that at non-acupoints, and this trend was more significant after acupuncture [29]. After the Ca 2+ along meridians was complexed to reduce the concentration of Ca 2+ , the recruitment and degranulation of mast cell was inhibited, and then the acupuncture effect on visceral regulation disappeared. Blocking the voltage-gated Ca 2+ channel on the cell membrane of acupoint area or blocking the biological activity of calmodulin in the acupoint area can affect acupuncture effects [30]. Many studies have proven that a high content of Ca 2+ is present in the tissues along meridians and this feature is the key to meridian phenomena and acupuncture effects. e biological function of Ca 2+ is produced by a series of cascade reactions caused by intracellular Ca 2+ transmembrane transport [31,32]. e functional proteins related to Ca 2+ transmembrane transport were differentially expressed between the ABT in CV tissues and nonmeridian tissues in rats. e change in Ca 2+ concentration inside and outside the cell membrane is the key factor in Ca 2+ transmembrane transport, and the important factor affecting the change in Ca 2+ concentration is the biological activity of calcium channel proteins and calmodulin on the cell membrane [33]. As the expression of proteins related to Ca 2+ transmembrane transport, such as CACNA2D1, was upregulated in the ABTalong the CV, it is suggested that the biological process of Ca 2+ transmembrane transport was more active in the tissues of ABT than in adjacent nonmeridian tissues. As Ca 2+ in the tissue along meridians is relatively high, when acupuncture, massage, moxibustion, or other physical stimulations are exerted on the acupoints along meridians, nerve endings and MCs are activated to cause a local short reflection and induce the changes of mechanical pressure and voltage of local tissue which may activate the opening of voltage-gated Ca 2+ channel proteins and increase Ca 2+ influx. Additionally, it can cause a potential change in local tissue and excite adjacent cells, further activating the upregulation of CACNA2D1 expression in the tissues along the meridians to cause Ca 2+ influx, triggering a series of cascade reactions. en, the metabolism of the tissue along meridians would be increased accompanied by the release and transmission of information substances like neurotransmitters, ions, and cytokines. In the tissues along meridians, materials, information, and energy are transmitted in a chemotactic way which can produce meridian phenomena and acupuncture effects. Of course, this conjecture needs to be further verified by follow-up studies.

Evidence-Based Complementary and Alternative Medicine
In the subcutaneous connective tissue of the ABT including the LHRCs along the CV, the functional proteins involved in ATP metabolism, redox reactions, and Ca 2+ transmembrane transport were upregulated. e biological functions of these proteins may have a strong correlation with meridian phenomena and acupuncture effects. is study provided new evidence to verify and explain the theory of LHRCM at the protein level. e research of meridian is in a low tide period at present and the study on the essence of meridians has almost been stagnated in the past ten years. Research on the expression of characteristic proteins in meridian tissues is so limited that useful information is scarce. is is the first time to study the material basis of LHRCM using differential proteomics technology. In our study, a very large differential proteomics dataset was obtained. As the information available about the material basis of meridians was limited at present, there were some limitations in the selection of characteristic proteins in the tissues of ABT along meridians. Future exploration of these differential proteomics data is necessary by paying attention to the new progress in meridian essence research and life sciences.

Conclusion
In summary, the present study demonstrates that the ATP5E, CACNA2D1, GAPDH, and Gpx-3 which were upregulated expression in ABT along meridians were closely related to the meridian phenomena and acupuncture effects.
ose proteins could be regarded as the potential characteristic proteins of LHRC along the CV in rats.