7-1. The effect for reducing blood pressure by EA/CE
To make sure the SHRs were with hypertension, we measured tail-pressures of the rats after adapted for 1-2 weeks. All the systolic, mean and diastolic blood pressure (BPs, BPm and BPd) of the SHRs were significantly higher than the WKYs (P<0.001) (Table 2A).
At the 3rd day of therapy, all the BPs, BPm and BPd of EAT rats were reduced more than 15 mmHg, and significantly lower than the SHR rats (P<0.001) (Table 2B). The similar effect was shown on CET rats, and all the blood pressures of CET rats was slightly higher than EAT rats but not with significance. There was no difference between SHR and both the two sham group (EAS and CES). Like EAT rats, the blood pressures of CET rats were significantly lower than CES rats except BPs (P>0.05, P<0.05, P<0.05) (Table
2B).
At the 7th day of therapy, the blood pressures were continuatively reduced by both electro-acupuncture and catgut-embedding. The average BPs, BPm, and BPd of CET rats were 133.50, 98.17 and, 80.39 mmHg, overtaking those of EAT rats (139.28, 101.56, and 83.56 mmHg) (Table 2C), but there was no significant difference between these two group.
7-2. Histological analysis
As showed in Fig 8A, Cardiac hypertrophy and disarray caused by hypertension can be reversed by stimulation of Taichong point with both electro-acupuncture and catgut-embedding for a week.
Left ventricular of the SHR rat stained by trichrome showed more blue stain of collagen aggregation than the WKY rat, and so did the EAS and CES rats (Fig 8B). After treatment of Taichong with EA/CE for a week, the stain was almost disappeared in LV sections of the EAT and CET rats.
Tissue sections of the rat hearts were stained by TUNEL assay. The
45
-apoptotic nuclei were identified by the characteristic fragmented nuclei. The nuclear location was confirmed with DAPI stain. As shown in Fig 23A, the TUNEL spots were obviously much more in hypertension rats (SHR, EAS, and CES) than the healthy rat (WKY). After a week of therapy with EA/CE on LR-3, the apoptotic cells were significantly fewer in the hearts of EAT and CET rats. The statistical analysis was shown in Fig 23B.
All the histological phenomenon supported that EA/CE on LR-3 could protect heart from injury by hypertension, like remodeling, fibrosis and apoptosis.
7-3. Western blotting
7-3-1. SOD was increased much more in LV of CET rats than EAT rats.
The SOD 1 protein expression in SHR left ventricle volume was lower than the WKY，After treated with electro-acupuncture has rebounded，CET group even higher than that of WKY and EAT groups and achieve significant differences (Fig 12A and B).
7-3-2. EA/CE reduced the cardiac hypertrophy markers induced by hypertension
Some reports supported [3] that cardiac hypertrophy would cause inflammation. In our experiment, We found more TNFα in LV of the hypertension rats than the normal rats by Western blotting(Fig 7-5A). After one week of therapy, TNFα was decreased in LV of all the therapy groups (including sham-point and LR3), among of them, EAT group showed the best effect. We also found another inflammation factor IL-6 and its down-stream hypertrophy marker STAT 3 increased in LV of SHR, EAS, and CES rats. After EA/CE for a week, both IL-6 and STAT 3 were decreased in LV of EAT and CET rats. The effect of catgut embedding therapy is better than electro-acupuncture, but there was no significant difference between these two groups.
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-7-3-3. EA/CE reduced the cardiac apoptotic markers induced by hypertension
To make sure whether hypertension could induce apoptosis of myocardium, western blot was performed for detecting Bcl-2 and Bax in LV of the rats.
We found that hypertension caused decrease of the anti-apoptotic marker Bcl-2, but this result could be reversed by EA/CE for a week. The pro-apoptotic marker Bax showed a opposite tendency from Bcl-2 (Fig 14).
In another cardiac-apoptotic pathway, IGF-2R dependent apoptotic pathway, we also found that the associated protein PLCβ3 and pPKCα were raised in LV of SHR, EAS and EAT rats. After one week of therapy, The EATgroup to pPKCα has a more significant impact, but the CET group to PLCβ3 has a more significant impact (Fig 15 A and B).
7-3-4. Stimulation on LR3 would elevate protein expression of cardiac survival markers which were reduced by hypertension.
IGF-1R dependent survival pathway is an important mechanism against apoptosis in cardiomyocytes. To clarify whether EA and CE would inhibit apoptosis by activating this IGF1-PI3K-Akt pathway, LV tissue were homogenized and measured by Western blot. All the associated markers like IGF-1Rα, PI3K, and the active-form pPI3K were reduced in LV of the hypertension rats (SHR, EAS and CES) (Fig 7-8A). After one week of treatment, all these proteins were increased. Furthermore, PI3K and pPI3K were much more in CET rats than EAT rats (Fig 7-8B). Akt, the downstream protein of PI3K, and the active-form pAktT308 were also reduced in hypertension rats but restored in the treated rats (Fig 7-9A and B).
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-8 Discussion and conclusion
8-1 To observed the protecting the heart of SHR by EAT and catgut embedding treatment (CET)on Taichong point
Dan Huang and Li-De Zhang investigated for past 5 years on hypertension be treated with acupuncture results, it has shown that acupuncture for treating high blood pressure was significant effect [84].
Fang Zuo and Ting Lou have Studied of 518 articles for catgut embedding, their conclusion is that the catgut embedding will have much wider use in future and need to be improved[85]..
Effect by catgut embedding is a unique method, as in the acupoint catgut will turn to softening, decomposition, liquefaction and absorption process , and then the acu-points will get the physiological, physical and biochemical stimulation[86].
Cui-Ying Xu and Feng-Ling Wang found that injection Cuan xong (ligustrazine )in Taichong Acupoint for treating hypertension was an exact method[87].
There were three major be found in this study. First, from the result of H& E stain on heart tissue biopsy, we found that From heart tissue biopsywith TUNEL dyeing we observed that either the electracupuncture or catgut embedding therapy can suppressed some of apoptosis proteins from the heart tissue , and then detected by Western blot to check their pathways.
Apoptosis has made it clear there are three signal transduction pathways, death receptor pathway, mitochondrial pathway, endoplasmic reticulum signaling pathways[88]. The final way is through the activation of Caspases proteases achieved. A research has shown that removing PKC alpha can improved the hearts ability to contract[89]. Interleukin-6 (IL-6) is secreted by T cells and macrophages to stimulate immune response to foreign pathogen.
IL-6 relevant to IL-6 is relevant to atherosclerosis process[90]. Epidemiological studies have revealed an association between inflammation and cardiovascular disease due to hypertension[15, 16]. In our research has
48
-shown that compared with SHR and EAS group, the IL-6 in EAT group was significant decreased. That means acupuncture did work with Anti-inflammatory function.
The main representative of BCL2 family mediated by variety of pathways to apoptosis[91]. Akt regulates cellular survival and metabolism[92].
There was a research has shown that When hypertension in the third stage with complications of heart failure or cerebral infarction, the growth hormone or insulin growth factor-1 will lower than the normal person[93]. The phosphatidylinositol 3-kinase has a key regulatory function for many cellular processes[94]. Superoxide dismutases (SOD)have three forms in human. SOD1 binds copper and zinc ions. Lack of SOD1 will get a shortened lifespan[95]. Nitric oxide (NO) is an important regulator of blood pressure (BP) which produced by endothelial NO synthase (eNOS)[96]. eNOS can prevent enzyme to oxidative stress. Another proteins such as Bcl-2 family members Bax and Bak play a critical role in apoptosis[97].
The bcl2, IGF1-R, Akt, PI3K, SOD and eNOS protein can avoid cytochrome c be released from the mitochondrion. It means they can prevent caspase 3 be induced. We have found catgut embedding therapy(CET) or electroacupuncture therapy (EAT) can suppress the activation of Caspase 8 and Caspase 3 and also can avoid apoptosis while also can suppress the activation of death receptor-independence , apoptosis can be reduced though this process.
After treatment, the pro-apoptosis protein Bak of performance was decreased and anti-apoptosis apoptosis protein SOD, and IGF1-R, and Akt, and PI3K, and Bcl and eNOS were increased.
By Trichrome dyeing can be found either EAT or CET can reduced blue collagen protein fiber produced from the SHR's myocardial organization ., it was shown that the EAT or CET therapy can prevent the heart from fibrosis. According to Western blot analysis, we have found that the proteins of FAS 、FADD、 Bax、 Cytochrom C、 Gαq 、calcineurin 、PLCβ3、
49
-PKCα can be suppressed and FAS,,FADD,Bax,Cytochrom C, G Alpha q, calcineurin, PLC Beta 3, and PKC Alpha protein of performance were decreased and IL-6, STAT3 and BNP were suppressed . the concentration of IL-6 and TNF alpha also decreased . At the same time we also found that both EAT or CET can increased bcl2, IGF1-R, Akt, PI3K, SOD and eNOS protein expression, that means EAT or CET on Taichong point can prevent apoptosis from hypertension.
8-2 To research the EA T/ CET Taichong Point on protecting the heart
Left ventricular hypertrophy is the major problem in patient who has hypertension[98].
The SHR group's myocardial cell got hypertrophy and disarrayed which phenomenon can be observed by the H & E staining ,that means its heart is hypertrophy and remodeling. The groups of sham-point (EAS, CES) also have remodeling situation. After EA / catgut embedding Taichong Point for a week , the shape and arrangement of myocardial cells have clear recovery , and similar with WKY. Trichrome stain also has the same result, SHR, EAS, CES groups have significant collagen aggregation but EAT and CET group did not. TUNEL assay showed that the hypertension will increase in myocardial apoptosis, and EA or catgut embedding on Taichong Point could inhibit apoptosis, the effect of catgut embedding has a little better than EA group (1.3%: 1.5%), but there were no significant difference between two groups (P> 0.05).
8-3 To determine the EA / catgut embedding in Taichong Point on the effect of protecting heart by Western blotting
We further observed by Western blotting for looking for the molecular pathway to see how the Taichong Point affect on the heart , It can be seen in Fig 7-4 SHR, EAS and the CES groups, their volume of SOD 1 in the left ventricular less than WKY, this is indicating a lower ability to resist
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-oxidative damage, and after a week of EA / embedding therapy, the amount of SOD 1 in EAT AND CET Rats have significantly increased in the left ventricular (Fig 7-4A), while the effect of catgut embedding group was better than EA group and achieve significant (P <0.001, Fig 7-4B).
In the aspect of cardiac hypertrophy, the literature pointed out that the myocardial hypertrophy can company with inflammation and the release of TNFα, IL-6 and other cytokine [29] . In our research was found that the amount of TNFα in SHR's left ventricle was higher than WKY and up to 1.5 times (Fig 7.5B)，TNFα expression was reduced among l the EA / catgut embedding ,even sham-point as well, but EA Taichong Point was the best, but the effect of catgut embedding has slightly better than sham-point but not significant difference. Both EAT and catgut embedding sham-point can effectively reduce TNFα, it may be due to stimulation can induce primary immune system itself, The acupuncture Taichong points can further reduced TNFα performance, and achieve significant difference with the EAS group (P<0.01),( Fig 7-5B).
Another cytokine IL-6 and its downstream hypertrophy marker STAT 3 were increased in LV of SHR, EAS and CES rats. but after a week of treatment, EAT and the CET of the IL-6 and STAT 3 mRNA levels were decreased, catgut embedding group was better than EAT group but no significant, this is indicating the catgut embedding was little better than EAT group.
In the anti-apoptotic aspect , we examined the Bcl-2 family of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax. We found that hypertension would reduce Bcl-2 and increase Bax in left ventricular(LV) of the rats.
In the EAT group of Bcl-2 expression was increased slightly and better than catgut embedding group ,but in the catgut embedding group the Bax was better than EAT group, but there were no significant difference (Fig 7-6).
In another IGF-2R dependent apoptotic pathway, we also found that PLCβ3
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-and pPKCα were increased in LV of hypertension rats. Both of EA -and catgut embedding groups can reduce the performance of these two proteins.
EAT was better for pPKCα , but the catgut embedding was better for PLCβ3.
Hypertension (hypertension) is morbidity and risk factor for atherosclerosis (AS), cerebrovascular disease and also closely related to insulin resistance[99], obesity, obstructive Sleep apnea syndrome [100]and terminal kidney disease
[101]. Myocardial cell apoptosis occurs are hypertension and the foundation of development of Cytology [102]. Apoptosis of myocardial cells are affected by a variety of cell signaling pathways controlling large amounts of myocardial cell apoptosis not only can lead to ventricular remodeling and cardiac arrhythmias, can also affect myocardial energy metabolism and contractility[103].
According to TCM theory, for treating hypertension, we pay more attention on the symptom differentiation such as LR-3 for excessive liver-yang syndrome[104], ST40 for phlegm damp obstruction[105], BL23 and KI-3 for kidney essence deficiency[105], ST36 for qi and blood deficiency[105].
Therapeutic effect of acupuncture treatment for hypertension is safe and non-toxic side effects. But clinical researches exist some problem, as effect standard, less of experimental research. Hypertension is a cardiac chronic medical condition .Our goal in future is keep studying forward to getting the best method for prevent complications from high blood pressure in the field of acupuncture.
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-Table 3-1. Compared the rats in each group after acupuncture treatment of changes in blood pressure
A.
Comparisons with BP of rats of at 0 day
Group No. BPs BPm BPd
WKY 11 114.17 ± 16.93 83.49 ± 7.71 67.78 ± 9.86
SHR 18 169.54 ±
13.61*** 131.62 ± 11.19***112.44 ±
12.30***
ps. Compared with WKY, ***P<0.001.
B.
Comparisons with BP of the rats at 3rd day
Group No. BPs BPm BPd
ps. Compared with WKY, ***P<0.001.
Compared with SHR, ##P<0.01; ###P<0.001.
Compared with EAS, P<0.05; △ △△P<0.01.
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-C.
Comparisons with BP of the rats at 19th day
Group No. BPs BPm BPd
WKY 3 117.17 ± 10.77 81.67 ± 10.92 62.83 ± 12.79 SHR 3 188.75 ± 5.77*** 139.75 ± 8.98** 115.17 ± 11.62**
EAS 3 188.83 ± 9.70*** 141.83 ± 5.31*** 118.25 ± 6.25***
EAT 3 168.92 ±
2.63##△ 133.92 ± 6.00 116.25 ± 7.81 ps. Compared with WKY, **P<0.01; ***P<0.001.
Compared with SHR, #P<0.05; ##P<0.01.
Compared with EAS, P<0.05.△
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong) for 3 weeks and measured the BPs, BPm, and BPd (systolic, mean, and diastolic blood pressure) with their tail. A) Baseline BP of the rats before treatment. B) BP of the rats at the 3rd day in treatment. C) BP of the rats at 19th day in treatment. (Data are expressed as mean ± SD)
54
-Table 7-1. compared each group’s rats blood pressure change situation after one week treatment by electro-acupuncture or catgut embedding therapy . A.
ps. Compared with WKY, ***P<0.001.
B.
Comparisons with BP of the rats at 3th day
Group No. BPs BPm BPd
ps. Compared with WKY, ***P<0.001.
Compared with SHR, ##P<0.01; ###P<0.001.
Compared with EAS, P<0.05; △ △△P<0.01.
Compared with CES, §P<0.05.
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-C.
Comparisons with BP of the rats at 7th day
Group No. BPs BPm BPd
ps. Compared with WKY, **P<0.01; ***P<0.001.
Compared with SHR, ##P<0.01; ###P<0.001.
Compared with EAS, P<0.05.△
Compared with CES, §P<0.05; §§P<0.01.
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong; CES, catgut-embedding on sham-point;
CET, catgut-embedding on Taichong) for 3 weeks and measured the BPs, BPm, and BPd (systolic, mean, and diastolic blood pressure) with their tail.
A) Baseline BP of the rats before treatment. B) BP of the rats at the 3rd day in treatment. C) BP of the rats at 19th day in treatment. (Data were expressed as mean ± SD)
56
-Fig 3-1 A.
B.
57
-C.
BP variation of the EAT rats
BPs BPm BPd
blood pressure (mmHg)
0 50 100 150 200
0 day 3rd day 19th day
*
*
Fig 3-1. Comparing BP of the rats at 3rd and 19th day.
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong.) for 3 weeks. A) The BP of the rats at 3rd day. B) The BP of the rats at 19th day. C) BP variation of the EAT rats in this 3-week treatment. (Compared with WKY, ***P<0.001. Compared with SHR,
#P<0.05; ##P<0.01, ###P<0.001. Compared with EAS, P<0.05, △ P<0.05 in Fig 3
△△ -1A and B. Compared with 0 day, *P<0.05 in Fig 3-1C.)
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-Fig 3-2 A.
B
Fig 3-2. transverse sections of LV cardiomyocytes of the rats.
A) H&E staining (400X). The cardiomyocytes of SHR and EAS rats were larger and more disarrayed than WKY and EAT. B) Masson’s trichrome staining (400X). The cardiomyocytes of SHR and EAS showed more blue stain of collagen aggregation (the arrows) than WKY and EAT.
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-Fig 3-3
60
Fig 3-3. TUNEL assay of LV sections of the rats.
A) More TUNEL spots were detected in LV of SHR and EAS rats than WKY, and the LV section of EAT showed less TUNEL spots than SHR and EAS. B) stick plot of Fig 4A, the apoptosis percentage in cardiomyocytes of EAT heart was significantly lower than SHR and EAS. (***P<0.001, compared with WKY: ##P<0.01, compared with SHR; △△P<0.01, compared with EAS.)
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Fig 3-4. Protein levels of the SOD 1 and eNOS.
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong) for 3 weeks and then the hearts were harvested and lysed. (A) Total protein of cell extracts was separated by 12 % SDS-PAGE, and transferred to PVDF membranes. (B) The results were analyzed by one way analysis of variance (ANOVA).Compared with WKY,
**P<0.01. Compared with SHR, ##P<0.01. Compared with EAS, P<0.001.
△△△
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-Fig 3-5 A.
B.
Fig 3-5. Protein levels of the cardiac hypertrophy markers.
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong) for 3 weeks and then the hearts were harvested and lysed. (A) Total protein of cell extracts was separated by 10-12 % SDS-PAGE, and transferred to PVDF membranes. (B) The results were analyzed by one way analysis of variance (ANOVA).Compared with WKY, *P<0.05, **P<0.01, ***P<0.001. Compared with SHR, #P<0.05,
##P<0.01. Compared with EAS, P<0.05, P<0.01, P<0.001.△ △△ △△△
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-Fig 3-6 A.
B.
Fig 3-6. The protein expression levels of FAD and FADD extracted from heart tissue were quantified by Western blotting analysis.
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong) for 3 weeks and then the hearts were harvested and lysed. (A) Total protein of cell extracts was separated by 12 % SDS-PAGE, and transferred to PVDF membranes. (B) The results were analyzed by one way analysis of variance (ANOVA).Compared with WKY,
**P<0.01, ***P<0.001. Compared with SHR, #P<0.05. Compared with EAS, P<0.05.
△
64
-Fig 3-7 A.
B.
Fig 3-7. The protein expression levels of Bax and Cytochrome c extracted from heart tissue were quantified by Western blotting analysis.
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong) for 3 weeks and then the hearts were harvested and lysed. (A) Total protein of cell extracts was separated by 12 %
The rats of all the groups were treated (WKY, the normal control; SHR, the hypertensive control; EAS, electro-acupuncture on sham-point; EAT, electro-acupuncture on Taichong) for 3 weeks and then the hearts were harvested and lysed. (A) Total protein of cell extracts was separated by 12 %