Multiple acupuncture manipulations lower blood pressure in SHRs.
To study the antihypertensive effects of different acupuncture manipulations, RF, RD and EA was performed on SHRs. The systolic blood pressure of all experimental rats was measured regularly (Table I). The results demonstrated that there was no significant difference in the blood pressure measurements between the acupuncture-treated groups (RD, RF and EA) and group M (all, P > 0.05) on day 0 (one day prior to acupuncture manipulation). However, the blood pressure measurements of the M, RD, RF and EA groups were significantly higher compared with group WKY at all time points (all P < 0.05). The blood pressure measurements of the RD, RF and EA groups decreased significantly compared with the M group on days 8, 12 and 14 (all, P < 0.05). All three acupuncture manipulation groups exhibited attenuated blood pressures following 14 days of treatment; however, the blood pressure measurements of the RD, RF and EA groups were reduced by varying degrees. The blood pressure measurements of the RD group were lower compared with the RF and EA groups on day 14; however, the differences were not significant (both, P > 0.05). These results indicated that the acupuncture manipulations had a positive effect in lowering the blood pressure measurements of the SHR groups, with RD possibly being the most effective treatment. The blood pressure measurements of the RD group declined over time and there was a significant difference at day 12 vs. day 4 and day 14 vs. day 4(both, P < 0.05). There was a slight decrease in the blood pressure measurements in the RD and EA groups; however, P > 0.05 in all cases (all the others days vs. day 0). These data indicated that RD, RF and EA lowered the blood pressures of the SHRs by varying degrees. Furthermore, the data revealed an instant and long-term protective effect of RD and an instant effect of RF and EA in SHRs.
Table I Comparison of systolic blood pressure (`x ± S,mmHg)
Group
|
N
|
Day0
|
Day4
|
Day8
|
Day12
|
Day14
|
WKY
|
8
|
109.93 ± 1.82
|
111.07 ± 2.43
|
111.57 ± 2.41
|
111.86 ± 2.74
|
111.50 ± 3.01
|
M
|
8
|
166.07 ± 1.33a
|
165.71 ± 2.67a
|
169.14 ± 2.28a
|
171.43 ± 2.06a
|
173.79 ± 2.15a
|
RF
|
8
|
166.14 ± 2.14a
|
164.29 ± 1.64a
|
164.07 ± 2.70ab
|
163.43 ± 3.01ab
|
163.07 ± 2.84ab
|
RD
|
8
|
166.21 ± 1.71a
|
163.93 ± 2.76a
|
162.07 ± 3.08abc
|
160.79 ± 2.33abc
|
158.71 ± 2.84abc
|
EA
|
8
|
166.00 ± 1.92a
|
164.07 ± 3.58a
|
162.71 ± 2.52ab
|
160.86 ± 2.82abc
|
159.93 ± 3.32abc
|
Note: compared with WKY group, aP<0.05; compared with M group, bPaP<0.05; compared with RF group ,cP<0.05.WKY, Wistar-Kyoto; M, model; RF, twirling reinforcing manipulation;RD, twirling reducing manipulation;EA, electroacupuncture. |
Analyses of the proteomics data.
To investigate the effect of acupuncture treatment on the central nervous system, proteomics analysis on the cerebellum of the SHRs was performed. Cerebellum from all rats were subjected to proteomics analysis through a label-free technique to obtain profiling data. The results identified 4,378 protein groups (Fig. 1A). Following this, four comparisons (M/WKY, RF/M, EA/M and RD/M) were performed to identify altered proteins. A total of 397(M/WKY), 96(RF/M), 133 (EA/M) and 216 (RD/M) proteins were identified as DEPs (P < 0.05; fold change ≥ 1.2). Further comparisons were performed to investigate the differences and overlaps in the DEPs induced by the RD, RF and EA manipulations via Venn analysis (Fig. 1B), indicating the presence of numerous rescue effect-related proteins. GO enrichment analysis of DEPs in the groups RF, EA RD and WKY were compared with the M group. Following this, volcano and GO enrichment analyses of these proteins was conducted. (Fig. 1C, 1D,1E)Extracellular exosome and endoplasmic reticulum membrane were among the top 5 most significant (P < 0.05) terms in all of the GO enrichment analyses, indicating that hypertension and acupuncture manipulations affected the expression of secreted proteins in the cerebellum of the SHRs. In summary, these results may elucidate the effect of acupuncture manipulation on cerebellum in the SHRs.
Analyses of KEGG pathway enrichment and protein interoperability network
To identify biological processes most relevant to biological phenomena, we did the analyses of KEGG pathway enrichment and protein interoperability network. There were 41 KEGG pathways significantly enriched (P < 0.05) in M compared with WKY group(Fig. 2A): cAMP signaling pathway, HIF-1 signaling pathway, mTOR signaling pathway, Ras signaling pathway, Huntington's disease,Alzheimer's disease༌human immunodeficiency virus type 1 infection, nonalcoholic fatty liver disease (NAFLD), Parkinson's disease, herpesvirus infection, Kaposi's sarcoma-associated herpesvirus infection, alcoholism, viral carcinogenesis, chemical carcinogenesis, human t cell leukemia virus 1 infection, tuberculosis, anti-folate resistance, hypertrophic cardiomyopathy (HCM), insulin resistance, oxidative phosphorylation, retinol metabolism, pyrimidine metabolism, folate biosynthesis, purine metabolism, other polysaccharide degradation, nicotinic acid and nicotinamide metabolism, steroid biosynthesis, D-glutamine and D-glutamic acid metabolism, sphingolipid metabolism, thermogenesis, glucagon signaling pathway, hematopoietic cell lineage, estrogen signaling pathway, thyroid hormone signaling pathway, cholesterol metabolism, gastric acid secretion, ribosome, spliceosome, endoplasmic reticulum protein processing, cell iron death, oocyte maturation division. There were respectively 12(RF/M), 11(RD/M), 15(EA/M) pathways with significant differences in protein enrichment(Fig. 2B,2C,2D). The overall trend of PRM verification results and Lable free quantitative results is consistent in this experiment. The KEGG annotation of these differential proteins in adipocyte lipolysis regulation, autophagy, gap junction, phosphoinositide metabolism, spliceosome, lysosome, thermogenesis and RNA transport. A total of 397 significant differential proteins was more than 1.2 or less than 0.83 times in Group WKY compared with M group. Furthermore, a total of 96, 216, 133 differentially expressed proteins with up and down regulation of more than 1.2 fold or less than 0.83 were screened in Group RF, RD, EA compared with M group Of all the differential proteins.(Fig. 3A,3B,3C,3D), 13 target proteins were selected for PRM verification. It can be found from the table that the PRM verification results are consistent with the overall trend of Lable free quantitative results, including Acsl6(P33124), Kifap3(A0A0G2K0P1), Atat1(Q6MG11), Pkg(A9LNM8), Rab23(D3ZRM5), Zfyve1(D4A3T4), Nup205(D4A7R3), Apoa2(P04638), Nrbp1(Q3SWT7), Gga1(Q5FVF3), Eif3(Q5RK09), Clint1(Q6DGF2) and Pi4k2ag(Q99M64).(TableⅡ)
TableⅡ Description of 13 target proteins
Protein Number
|
Gene Name
|
Protein Description
|
P33124
|
Acsl6
|
long chain fatty acid A coenzyme 6
|
A0A0G2K0P1
|
Kifap3
|
Kallikrein 3
|
Q6MG11
|
Atat1
|
α-tubulin N-acetyltransferase1
|
A9LNM8
|
Pkg
|
cGMP-dependent protein kinases
|
D3ZRM5
|
Rab23
|
RAB 23,RAS oncogene family members
|
D4A3T4
|
Zfyve1
|
Zinc-containing finger FYVE 1
|
D4A7R3
|
Nup205
|
Nuclear pore size 205
|
P04638
|
Apoa2
|
A-II of apolipoprotein
|
Q3SWT7
|
Nrbp1
|
Nuclear receptor-binding protein
|
Q5FVF3
|
Gga1
|
Golgi-associated ,γ aptamer ear-containing agent, ARF binding protein 1
|
Q5RK09
|
Eif3g
|
Eukaryotic translation initiation factor 3 subunit G
|
Q6DGF2
|
Clint1
|
Cage protein interaction factor
|
Q99M64
|
Pi4k2a
|
phosphatidylinositol 4-kinase 2-α
|