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Auricular concha eletroacupuncture modulates lipid-lipoprotein metabolism in rats submitted to cold stress
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Auricular concha eletroacupuncture modulates lipid-lipoprotein metabolism in rats submitted to cold stress Yu-tian Yu , Xiao Guo , Jin-ling Zhang , Shao-yuan Li , Chun-zhi Tang , Pei-jing Rong PII: DOI: Reference:
S1003-5257(19)30125-4 https://doi.org/10.1016/j.wjam.2019.12.010 WJAM 139
To appear in:
World Journal of Acupuncture – Moxibustion
Please cite this article as: Yu-tian Yu , Xiao Guo , Jin-ling Zhang , Shao-yuan Li , Chun-zhi Tang , Pei-jing Rong , Auricular concha eletroacupuncture modulates lipid-lipoprotein metabolism in rats submitted to cold stress, World Journal of Acupuncture – Moxibustion (2019), doi: https://doi.org/10.1016/j.wjam.2019.12.010
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Experimental Research
Auricular concha eletroacupuncture modulates lipid-lipoprotein metabolism in rats submitted to cold stress☆ Yu-tian YU（俞裕天）a, b, c, # , Xiao GUO（国笑）a, #, Jin-ling ZHANG（张金铃）a, Shao-yuan LI（李少源）a, Chun-zhi TANG（唐纯志）c, Pei-jing RONG（荣培晶）a, c, * a
Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing
100700, China （中国中医科学院针灸研究所，北京 100700，中国） b
Department of Acupuncture Therapy, Beijing Shijitan Hospital, Capital Medical University,
Beijing 100038, China （首都医科大学附属北京世纪坛医院针灸科，北京 100038，中国） c
Clinical Medical College of Acupuncture, Moxibustion and Rehabilitation, Guangzhou
University of Chinese Medicine, Guangzhou 510006, China （广州中医药大学针灸康复临床医
学院，广州 510006，中国） ☆Supported by Major National R & D Program of China: Z161100002616003; Joint Sino-German-Project: GZ1236; The Fundamental Research Funds for the Central Public Welfare Research Institutes: ZZ16012; Chinese Postdoctoral Science Foundation: 2016M590185; Natural Science Foundation of Beijing: 7111007 # YU and GUO contributed equally to this work. * Corresponding author E-mail address：[email protected] (P.-j.Rong)
Keywords: Auricular acupuncture (AA) Auricular concha eletroacupuncture (ACEA) Auricular margin eletroacupuncture (AMEA) Lipid-lipoprotein metabolism
Abstract Objective: To explore whether auricular concha eletroacupuncture (ACEA) is effective in regulating lipid-lipoprotein metabolism in rats submitted to cold stress. Methods: Thirty-six adult male Sprague-Dawley rats were placed in four groups(9 rats in each group), the rats in three groups of which were submitted to cold stress for fourteen days, the last one of which was a control group. After the cold stress process, in those three groups, the rats of one group were with no treatment (stress only), two were treated with either ACEA or auricular margin eletroacupuncture (AMEA) repeated for fourteen days. On the 14th day, all the rats were sacrificed after all experimental procedure for blood sampling. Blood glucose, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were tested by using the collected serum. Plasma lecithin-cholesterol acyltransferase (LCAT) was measured with ELISA kit. Results: ACEA down-regulated the TG level (P<0.05) and LDL-C level (P<0.05), and up-regulated HDL-C level (P<0.05) and LACT level (P<0.05). AMEA did not regulate the bio-markers. Conclusion: ACEA played an important role in regulating lipid-lipoprotein metabolism in rats submitted to cold stress.
Introduction Chronic stress causes marked physiological alterations in the heart, some of which could be of pathogenetic consequence [1]. Chronic cold stress could induce hyperglycemia [2], hyperlipidemia [3], and hypercholesterolemia [4] in animals. These metabolic disorders can evoke coronary heart disease (CHD) clinically [5]. Son et al. [6] reported different effects of cold and heat on mortality from 1996-2010 in São Paulo, a subtropical city in Brazil, where cold is a greater threat to mortality than heat, especially cardiovascular deaths. Ambient temperature is closely related to fat metabolism. Cold temperatures can probably harmfully change plasma lipid concentrations and lead to abnormal thrombosis and chronic atherogenesis [7].
Increased total cholesterol and low-density lipoprotein（LDL）, popular indicators of lipids profiles, are strongly related to atherosclerosis by inducing manifestation of adhesion molecules, and vascular endothelium dysfunction. The high LDL-tohigh-density lipoprotein (HDL) ratio, induced by increased LDL and decreased HDL, significantly increase cardiovascular disease (CVD) risk [8]. Elevated lipid-lipoprotein levels are associated with arteriosclerosis and CHD [9]. Mildly heightened serum triglycerides and cholesterol are thought to be risk factors of arteriosclerosis and CHD [10]. Studies on the distribution of cholesterol among varying lipoprotein fractions have demonstrated that elevated low-density lipoprotein cholesterol (LDL-C) levels and decreased high-density lipoprotein cholesterol (HDL-C) levels were primarily associated with increased risks of arteriosclerosis and CHD [11]. To elucidate the pathological significance of the lipoproteins, multivariate control mechanisms and factors regulating the lipoprotein system need to be considered, including enzymes involved in the metabolism [1]. Lecithin–cholesterol acyltransferase (LCAT) is one of the enzymes which convert free cholesterol into hydrophobic cholesterol called cholesteryl ester. LCAT is secluded into the core of a lipoprotein particle, forming the novel generated HDL-C ball-shaped and driving the reaction monodirectional since the particles are eliminated from the exterior [12].
Huángdì Nèijīng(《黄帝内经》The Yellow Emperor’s Inner Classic) said “the ears are where meridians gather.” The ear acupoints corresponding to the thoracic and abdominal organs are located in the auricular concha. Thus, stimulating the auricular concha by auricular acupuncture can regulate the qi, blood, and function of the zang-fu organs. Auricular acupuncture (AA) was found effective in treating obesity and dyslipidemia clinically [13-15]. Thus, auricular concha electroacupuncture (ACEA) may have stronger effects. Besides, anatomical evidence has shown that the vagus nerve has a branch of afferent projection at the auricular concha [16] in mammals, which means ACEA might be comparable in efficacy to vagus nerve stimulation (VNS). Although there are other afferent nerve fibers in the auricular
concha, ACEA has still been called transcutaneous auricular vagus nerve stimulation (taVNS). Previously, Samniang et al. [17] found that VNS significantly decreased total cholesterol, triglyceride, LDL in obese-insulin resistant rats. It is worth investigating whether ACEA or taVNS has the same effects. In this study, we intended to find whether ACEA could reverse the dyslipidemia in rats caused by chronic cold stress.
Materials and methods Animals and grouping Thirty-six adult Sprague-Dawley rats, male, 180 g–210 g, purchased from the Laboratory Animal Center of China Academy of Military Medical Sciences [License number: SCXK-(Military)-2016-0024], were assigned to four groups randomly in this study: the control group (n=9), received no treatments, the stress only group (n=9) received the cold stress protocol only. The stress + ACEA group (n=9) and the stress + AMEA group (n=9) of animals were both submitted to cold stress and then received either ACEA or AMEA treatment, 30 min after the cold stress procedure for 14 days. Each rat was caged separately. With a 12 h dark/light cycle, the temperature was set in （23±1）°C and the humidity in （50±5）%. The rats were given water and standard chow ad libitum. Before the study, rats were acclimatized for seven days. The experimental protocols followed the Principles of Laboratory Animal Care (WHO 1985) [18] and the legislation for the care and use of experimental animals in the People’s Republic of China. The Ethics Committee of Institute of Acupuncture and Moxibustion, CACMS approved these protocols. Cold stress procedure In these 14 days, rats of the stress only group, the stress+ACEA group, and the stress+AMEA group were placed in cages with 1 cm deep ice/water mixture [0°C -4 °C separately (each rat in a cage) for 1 h daily]. After it, rats of the stress+ACEA group and the stress+AMEA were returned to their home cages, and rats’ body temperature was allowed to normalize for 30 min before initiating the ACEA or AMEA treatments. ACEA administration For ACEA, under 2% inhaling isoflurane anesthesia, electrodes (magnetic) were
placed over the bilateral auricular concha regions, outside (-) and inside (+) each, and the cathode and anode are placed in cymba conchae and cavum conchae respectively(Fig.1). The current can conduct across the tissue, including the auricular vagus nerve fibers . Conducting liquid was used to improve the conduction. A 20-min ACEA procedure was administered to the rats once daily via electrical stimulators (HANS-100, Gensun technology co. LTD, Nanjing, China). The frequency was set in 20 Hz and the intensity in 1 mA. AMEA administration For AMEA, two electrodes (the cathode and anode, +/-) were placed over the auricular margin of the rats, where no vagus nerve fibers were distributed (Fig.1, Fig. 2). Other procedures were the same as the stress+ACEA group.
Fig.1 Locations for ACEA and AMEA in rats
Fig.2 Treatment with ACEA or AMEA in rats.
Blood sampling The rats were sacrificed at day 14 after all experimental procedures in anesthesia. Their neck venous blood was collected for the following tests. The blood was placed in 1.5 mL tubes coated with heparin sodium and centrifuged at 4°C at 7 000 revolutions per minute for 2 min. The plasma was stored at -80°C till analysis. Serum was collected for biochemical measurement in a 1.5 mL low retention sterile tube,
allowed to remain at 0°C -4°C for 15 min and centrifuged at 4°C at 4 000 revolutions per minute for 4 min. It was also stored at -80°C till analysis. Biochemical analyses Blood glucose, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured by the commercial kits (Prodia Diagnostics) by using the collected serum. The atherogenic index and the cardiac risk factor [19] were calculated by applying the following formulas. Atherogenic index = (TC– HDL-C) / HDL-C Cardiac risk factor = TC / HDL-C ELISA analysis of LCAT The ELISA was performed per the manufacturers’ recommended protocols. Rat Lecithin-Cholesterol Acyltransferase (LCAT) ELISA kit (GPBIOTECH) was used to measure LCAT levels. Briefly, 10 μL of plasma was deliquated in binding buffer (0.05% NaN3, 1 × TBS). The final volume was 200 μL. Then, they were incubated at 4°C in microtiter plates overnight. The next day, with washing buffer (1 × TBS and 0.05% Tween-20), the wells were washed 3 times. And with blocking buffer [0.05% Tween-20 in 1 × TBA and 0.5% bovine serum albumin (BSA)], the wells were blocked for 3 h at room temperature and washed again. Blocking buffer with rabbit anti-human LCAT antibody in 1:2 000 dilution was added into the wells and were incubated at room temperature for 3 h. Then, the plates were washed and incubated with 1:2 000 anti-rabbit IgG conjugated with horseradish peroxidase (Sigma) and developed with 1-stepTM Turbo-TMB-ELISA kit (Pierce) and read at 450 nm after
color developed. Statistical analysis GraphPad Prism 6 was used to analyze the data and presented as Mean±SD. One-way ANOVA was used to determine the differences. P<0.05 was identified as statistical significance among groups.
Results Comparison of GLU, TG, TC, HDL-C, LDL-C in the four groups of
rats
Among rats of the four groups, no statistical difference was found in blood glucose (Fig.3, Table.1). The blood glucose levels of each group were not influenced remarkably.
10
C o n tro l S tr e s s O n ly
G L U ( n m o l/L )
8
S tr e s s + A C E A 6
S tr e s s + A M E A
4
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Fig.3 Blood glucose in the four groups of rats Table.1 Blood glucose in the four groups of rats Group
GLU (nmol/L)
Control
6.241.80
Stress Only
7.101.30
Stress+ACEA
6.301.50
Stress+AMEA
6.701.30
Compared to the Control group, the TG levels of the stress only group were raised significantly (P<0.05). ACEA down-regulated it (P<0.05), but AMEA failed to down-regulated it (Fig.4, Table.2). The TC levels of the four groups had no statistical difference (Fig.5, Table.3). Cold stress evoked the rise of LDL-C levels (P<0.05), and ACEA decreased it significantly (P<0.05), however, AMEA could not (Fig.6,
Table.4). Meanwhile, cold stress also initiated the decline of HDL-C levels (P<0.05), and ACEA reversed the situation (P<0.05), but AMEA could hardly do so (Fig.7, Table.5).
2 .0
C o n tro l
T G ( m m o l/L )
*
S tr e s s O n ly
1 .5
S tr e s s + A C E A S tr e s s + A M E A
1 .0 #
0 .5
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Fig.4 TG levels in the four groups of cold stress rats Table.2 TG levels in the four groups of cold stress rats Group
TG (mmol/L)
Control
0.3460.149
Stress Only
1.0410.549*
Stress+ACEA
0.4840.113#
Stress+AMEA
1.1230.394
Note: *Comparison between the control group and the stress only group, P<0.05; #Comparison among the stress only group and the stress+ACEA / stress+AMEA groups, both P<0.05.
2 .0
C o n tro l
T C ( m m o l/L )
S tr e s s O n ly 1 .5
S tr e s s + A C E A S tr e s s + A M E A
1 .0
0 .5
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A
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s s e S
S
tr
tr
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0 .0
Fig.5 TC levels in the four groups of rats Table.3 TC levels in the four groups of rats Group
TC (mmol/L)
Control
1.3830.125
Stress Only
1.1780.340
Stress+ACEA
1.4400.333
Stress+AMEA
1.0340.142
*
L D L - c ( m m o l/L )
0 .8
C o n tro l S tr e s s O n ly
0 .6
S tr e s s + A C E A S tr e s s + A M E A
0 .4
#
0 .2
A
A
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M
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0 .0
Fig.6 LDL-C levels in the four groups of rats Table.4 LDL-C levels in the four groups of rats Group
LDL-C (mmol/L)
Control
0.3470.066
Stress Only
0.5640.185*
Stress+ACEA
0.2750.063#
Stress+AMEA
0.4470.119
Note: *Comparison between the stress only group and the control group, P<0.05; #Comparison among the stress only group and the stress+ACEA / stress+AMEA groups, both P<0.05.
#
1 .0
H D L -c ( m m o l/L )
C o n tro l S tr e s s O n ly
0 .8
*
S tr e s s + A M E A S tr e s s + A C E A
0 .6
0 .4
0 .2
A
A
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Fig.7 HDL-C levels in the four groups of rats Table.5 HDL-C levels in the four groups of rats Group
HDL-C (mmol/L)
Control
0.8550.062
Stress Only
0.5900.085*
Stress+ACEA
0.7820.159#
Stress+AMEA
0.5610.075
Note: *Comparison between the stress only group and the control group, P<0.05; #Comparison among the stress only group and stress+ACEA / stress+AMEA groups, both P<0.05. Comparison of atherogenic index and cardiac risk factor in the four groups of rats Applying the formula mentioned above, we acquired the atherogenic index and the cardiac risk factor of this study (Fig. 8, Table.6, Fig. 9, Table.7). The cold stress up-regulated both the index and factor compared to the control group (P<0.05), but, either ACEA or AMEA down-regulated them insignificantly. This indicated that the stress might aggravate atherosclerosis and increase the risk of heart attack. However, ACEA might need more time to reverse these situations.
A t h e r o g e n ic I n d e x 2 .0
C o n tro l
*
1 .5
S tr e s s O n ly S tr e s s + A C E A S tr e s s + A M E A
1 .0
0 .5
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Fig.8 Atherogenic Index in the four groups of rats Table.6 Atherogenic Index in the four groups of rats Group
Atherogenic Index
Control
0.6160.089
Stress Only
1.0020.512*
Stress+ACEA
0.8320.137
Stress+AMEA
0.8470.122
Note: *Comparison between the stress only group and the control group, P<0.05.
C a r d ia c R is k F a c t o r 3
C o n tro l
*
S tr e s s O n ly S tr e s s + A C E A
2
S tr e s s + A M E A
1
A
A
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+
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tr
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Fig.9 Cardiac Risk Factor in the four groups of cold stress rats Table.7 Cardiac Risk Factor in the four groups of cold stress rats Group
Cardiac Risk Factor
Control
1.6160.089
Stress Only
2.0020.512*
Stress+ACEA
1.8320.137
Stress+AMEA
1.8470.122
Note: *Comparison between the stress only group and the control group, P<0.05. Comparison of LCAT levels in the four groups of rats LCAT activity level of the stress only group was decreased compared with the control group significantly (P<0.05). Only ACEA reversed the situation (P<0.05) (Fig. 10, Table.8).
60
L C A T (u m o l/L )
C o n tro l S tr e s s O n ly
#
S tr e s s + A C E A
40
S tr e s s + A M E A
* 20
A
A
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M
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A
A
+
+
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tr
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Fig.10 LCAT levels in the four groups of rats Table.8 LCAT levels in the four groups of rats Group
LCAT (umol/L)
Control
44.7774.399
Stress Only
22.9441.862*
Stress+ACEA
41.4162.078#
Stress+AMEA
23.6674.546
Notes： *Comparison between the stress only group and the control group, P<0.05; #Comparison among the stress only group and stress+ACEA / stress+AMEA groups, both P<0.05.
Discussion The evidence of this study showed that cold stress exerted profound effects on lipid and lipoprotein metabolism in rats, and ACEA could reverse some situations. ACEA was developed based on the theories of auricular acupuncture (AA), VNS, and anatomical evidence. Classic VNS reduces body fat, blood triglyceride, and cholesterol levels in rats fed by high-fat diet and in obese-insulin resistant rats [17, 20]. Theoretically speaking, ACEA is supposed to have comparable effect as classic VNS. ACEA down-regulated TG levels in this study. Also, ACEA decreased LDL-C levels and up-regulated both HDL-C levels and LCAT activity. In previous study, cholesterol levels have been discovered to increase or to decrease after cold stress, and also nonsignificant alterations are also reported [21]. Studies on cold stress elicited effects on triglyceride levels are similarly contradictory. In this present study, cold stress-induced increased triglyceride levels. However, significant changes in total cholesterol were not revealed. ACEA decreased the elevated triglyceride levels significantly. LDL-C levels were recommended in guidelines to predict CHD risk [22]. Massive clinical data illustrated that LDL-C was the crucial atherogenic lipoprotein [23-25]. In the present study, cold stress elevated the LDL-C levels, and ACEA decreased it. HDL-C levels were deeply affected by stress. HDL-C showed a remarkable decline after stress. Epidemiological studies are supported by clinical results of correlation between coronary angiography confirmed CHD and low HDL-C levels [26, 27]. Lecithin-cholesterol acyltransferase (LCAT) is a lipoprotein-related enzyme which plays an important role in the esterification of free cholesterol, the maturation of high density-lipoprotein (HDL) particles [28]. In this study, both HDL-C levels and LCAT activity were suppressed by cold stress, and ACEA changed
the situations. Atherogenic index was considered as the risk factor value of atherosclerosis, and cardiac risk factor was considered as the risk factor value of CHD [29-31]. Stress elevated these two values. Down-regulations by ACEA were recorded yet without statistical significance, which might due to time limitations.
Conclusion In this study, we discovered that cold stress may be hazardous in relation to CHD and atherosclerosis, and by regulating lipid-lipoprotein metabolism, ACEA played a crucial role in protecting the heart. Further studies need to be done to reveal the deeper mechanisms.
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Auricular concha eletroacupuncture modulates lipid-lipoprotein metabolism in rats submitted to cold stress Yu-tian Yu , Xiao Guo , Jin-ling Zhang , Shao-yuan Li , Chun-zhi Tang , Pei-jing Rong PII: DOI: Reference:
S1003-5257(19)30125-4 https://doi.org/10.1016/j.wjam.2019.12.010 WJAM 139
To appear in:
World Journal of Acupuncture – Moxibustion
Please cite this article as: Yu-tian Yu , Xiao Guo , Jin-ling Zhang , Shao-yuan Li , Chun-zhi Tang , Pei-jing Rong , Auricular concha eletroacupuncture modulates lipid-lipoprotein metabolism in rats submitted to cold stress, World Journal of Acupuncture – Moxibustion (2019), doi: https://doi.org/10.1016/j.wjam.2019.12.010
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Experimental Research
Auricular concha eletroacupuncture modulates lipid-lipoprotein metabolism in rats submitted to cold stress☆ Yu-tian YU（俞裕天）a, b, c, # , Xiao GUO（国笑）a, #, Jin-ling ZHANG（张金铃）a, Shao-yuan LI（李少源）a, Chun-zhi TANG（唐纯志）c, Pei-jing RONG（荣培晶）a, c, * a
Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing
100700, China （中国中医科学院针灸研究所，北京 100700，中国） b
Department of Acupuncture Therapy, Beijing Shijitan Hospital, Capital Medical University,
Beijing 100038, China （首都医科大学附属北京世纪坛医院针灸科，北京 100038，中国） c
Clinical Medical College of Acupuncture, Moxibustion and Rehabilitation, Guangzhou
University of Chinese Medicine, Guangzhou 510006, China （广州中医药大学针灸康复临床医
学院，广州 510006，中国） ☆Supported by Major National R & D Program of China: Z161100002616003; Joint Sino-German-Project: GZ1236; The Fundamental Research Funds for the Central Public Welfare Research Institutes: ZZ16012; Chinese Postdoctoral Science Foundation: 2016M590185; Natural Science Foundation of Beijing: 7111007 # YU and GUO contributed equally to this work. * Corresponding author E-mail address：[email protected] (P.-j.Rong)
Keywords: Auricular acupuncture (AA) Auricular concha eletroacupuncture (ACEA) Auricular margin eletroacupuncture (AMEA) Lipid-lipoprotein metabolism
Abstract Objective: To explore whether auricular concha eletroacupuncture (ACEA) is effective in regulating lipid-lipoprotein metabolism in rats submitted to cold stress. Methods: Thirty-six adult male Sprague-Dawley rats were placed in four groups(9 rats in each group), the rats in three groups of which were submitted to cold stress for fourteen days, the last one of which was a control group. After the cold stress process, in those three groups, the rats of one group were with no treatment (stress only), two were treated with either ACEA or auricular margin eletroacupuncture (AMEA) repeated for fourteen days. On the 14th day, all the rats were sacrificed after all experimental procedure for blood sampling. Blood glucose, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were tested by using the collected serum. Plasma lecithin-cholesterol acyltransferase (LCAT) was measured with ELISA kit. Results: ACEA down-regulated the TG level (P<0.05) and LDL-C level (P<0.05), and up-regulated HDL-C level (P<0.05) and LACT level (P<0.05). AMEA did not regulate the bio-markers. Conclusion: ACEA played an important role in regulating lipid-lipoprotein metabolism in rats submitted to cold stress.
Introduction Chronic stress causes marked physiological alterations in the heart, some of which could be of pathogenetic consequence [1]. Chronic cold stress could induce hyperglycemia [2], hyperlipidemia [3], and hypercholesterolemia [4] in animals. These metabolic disorders can evoke coronary heart disease (CHD) clinically [5]. Son et al. [6] reported different effects of cold and heat on mortality from 1996-2010 in São Paulo, a subtropical city in Brazil, where cold is a greater threat to mortality than heat, especially cardiovascular deaths. Ambient temperature is closely related to fat metabolism. Cold temperatures can probably harmfully change plasma lipid concentrations and lead to abnormal thrombosis and chronic atherogenesis [7].
Increased total cholesterol and low-density lipoprotein（LDL）, popular indicators of lipids profiles, are strongly related to atherosclerosis by inducing manifestation of adhesion molecules, and vascular endothelium dysfunction. The high LDL-tohigh-density lipoprotein (HDL) ratio, induced by increased LDL and decreased HDL, significantly increase cardiovascular disease (CVD) risk [8]. Elevated lipid-lipoprotein levels are associated with arteriosclerosis and CHD [9]. Mildly heightened serum triglycerides and cholesterol are thought to be risk factors of arteriosclerosis and CHD [10]. Studies on the distribution of cholesterol among varying lipoprotein fractions have demonstrated that elevated low-density lipoprotein cholesterol (LDL-C) levels and decreased high-density lipoprotein cholesterol (HDL-C) levels were primarily associated with increased risks of arteriosclerosis and CHD [11]. To elucidate the pathological significance of the lipoproteins, multivariate control mechanisms and factors regulating the lipoprotein system need to be considered, including enzymes involved in the metabolism [1]. Lecithin–cholesterol acyltransferase (LCAT) is one of the enzymes which convert free cholesterol into hydrophobic cholesterol called cholesteryl ester. LCAT is secluded into the core of a lipoprotein particle, forming the novel generated HDL-C ball-shaped and driving the reaction monodirectional since the particles are eliminated from the exterior [12].
Huángdì Nèijīng(《黄帝内经》The Yellow Emperor’s Inner Classic) said “the ears are where meridians gather.” The ear acupoints corresponding to the thoracic and abdominal organs are located in the auricular concha. Thus, stimulating the auricular concha by auricular acupuncture can regulate the qi, blood, and function of the zang-fu organs. Auricular acupuncture (AA) was found effective in treating obesity and dyslipidemia clinically [13-15]. Thus, auricular concha electroacupuncture (ACEA) may have stronger effects. Besides, anatomical evidence has shown that the vagus nerve has a branch of afferent projection at the auricular concha [16] in mammals, which means ACEA might be comparable in efficacy to vagus nerve stimulation (VNS). Although there are other afferent nerve fibers in the auricular
concha, ACEA has still been called transcutaneous auricular vagus nerve stimulation (taVNS). Previously, Samniang et al. [17] found that VNS significantly decreased total cholesterol, triglyceride, LDL in obese-insulin resistant rats. It is worth investigating whether ACEA or taVNS has the same effects. In this study, we intended to find whether ACEA could reverse the dyslipidemia in rats caused by chronic cold stress.
Materials and methods Animals and grouping Thirty-six adult Sprague-Dawley rats, male, 180 g–210 g, purchased from the Laboratory Animal Center of China Academy of Military Medical Sciences [License number: SCXK-(Military)-2016-0024], were assigned to four groups randomly in this study: the control group (n=9), received no treatments, the stress only group (n=9) received the cold stress protocol only. The stress + ACEA group (n=9) and the stress + AMEA group (n=9) of animals were both submitted to cold stress and then received either ACEA or AMEA treatment, 30 min after the cold stress procedure for 14 days. Each rat was caged separately. With a 12 h dark/light cycle, the temperature was set in （23±1）°C and the humidity in （50±5）%. The rats were given water and standard chow ad libitum. Before the study, rats were acclimatized for seven days. The experimental protocols followed the Principles of Laboratory Animal Care (WHO 1985) [18] and the legislation for the care and use of experimental animals in the People’s Republic of China. The Ethics Committee of Institute of Acupuncture and Moxibustion, CACMS approved these protocols. Cold stress procedure In these 14 days, rats of the stress only group, the stress+ACEA group, and the stress+AMEA group were placed in cages with 1 cm deep ice/water mixture [0°C -4 °C separately (each rat in a cage) for 1 h daily]. After it, rats of the stress+ACEA group and the stress+AMEA were returned to their home cages, and rats’ body temperature was allowed to normalize for 30 min before initiating the ACEA or AMEA treatments. ACEA administration For ACEA, under 2% inhaling isoflurane anesthesia, electrodes (magnetic) were
placed over the bilateral auricular concha regions, outside (-) and inside (+) each, and the cathode and anode are placed in cymba conchae and cavum conchae respectively(Fig.1). The current can conduct across the tissue, including the auricular vagus nerve fibers . Conducting liquid was used to improve the conduction. A 20-min ACEA procedure was administered to the rats once daily via electrical stimulators (HANS-100, Gensun technology co. LTD, Nanjing, China). The frequency was set in 20 Hz and the intensity in 1 mA. AMEA administration For AMEA, two electrodes (the cathode and anode, +/-) were placed over the auricular margin of the rats, where no vagus nerve fibers were distributed (Fig.1, Fig. 2). Other procedures were the same as the stress+ACEA group.
Fig.1 Locations for ACEA and AMEA in rats
Fig.2 Treatment with ACEA or AMEA in rats.
Blood sampling The rats were sacrificed at day 14 after all experimental procedures in anesthesia. Their neck venous blood was collected for the following tests. The blood was placed in 1.5 mL tubes coated with heparin sodium and centrifuged at 4°C at 7 000 revolutions per minute for 2 min. The plasma was stored at -80°C till analysis. Serum was collected for biochemical measurement in a 1.5 mL low retention sterile tube,
allowed to remain at 0°C -4°C for 15 min and centrifuged at 4°C at 4 000 revolutions per minute for 4 min. It was also stored at -80°C till analysis. Biochemical analyses Blood glucose, triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured by the commercial kits (Prodia Diagnostics) by using the collected serum. The atherogenic index and the cardiac risk factor [19] were calculated by applying the following formulas. Atherogenic index = (TC– HDL-C) / HDL-C Cardiac risk factor = TC / HDL-C ELISA analysis of LCAT The ELISA was performed per the manufacturers’ recommended protocols. Rat Lecithin-Cholesterol Acyltransferase (LCAT) ELISA kit (GPBIOTECH) was used to measure LCAT levels. Briefly, 10 μL of plasma was deliquated in binding buffer (0.05% NaN3, 1 × TBS). The final volume was 200 μL. Then, they were incubated at 4°C in microtiter plates overnight. The next day, with washing buffer (1 × TBS and 0.05% Tween-20), the wells were washed 3 times. And with blocking buffer [0.05% Tween-20 in 1 × TBA and 0.5% bovine serum albumin (BSA)], the wells were blocked for 3 h at room temperature and washed again. Blocking buffer with rabbit anti-human LCAT antibody in 1:2 000 dilution was added into the wells and were incubated at room temperature for 3 h. Then, the plates were washed and incubated with 1:2 000 anti-rabbit IgG conjugated with horseradish peroxidase (Sigma) and developed with 1-stepTM Turbo-TMB-ELISA kit (Pierce) and read at 450 nm after
color developed. Statistical analysis GraphPad Prism 6 was used to analyze the data and presented as Mean±SD. One-way ANOVA was used to determine the differences. P<0.05 was identified as statistical significance among groups.
Results Comparison of GLU, TG, TC, HDL-C, LDL-C in the four groups of
rats
Among rats of the four groups, no statistical difference was found in blood glucose (Fig.3, Table.1). The blood glucose levels of each group were not influenced remarkably.
10
C o n tro l S tr e s s O n ly
G L U ( n m o l/L )
8
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S tr e s s + A M E A
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Fig.3 Blood glucose in the four groups of rats Table.1 Blood glucose in the four groups of rats Group
GLU (nmol/L)
Control
6.241.80
Stress Only
7.101.30
Stress+ACEA
6.301.50
Stress+AMEA
6.701.30
Compared to the Control group, the TG levels of the stress only group were raised significantly (P<0.05). ACEA down-regulated it (P<0.05), but AMEA failed to down-regulated it (Fig.4, Table.2). The TC levels of the four groups had no statistical difference (Fig.5, Table.3). Cold stress evoked the rise of LDL-C levels (P<0.05), and ACEA decreased it significantly (P<0.05), however, AMEA could not (Fig.6,
Table.4). Meanwhile, cold stress also initiated the decline of HDL-C levels (P<0.05), and ACEA reversed the situation (P<0.05), but AMEA could hardly do so (Fig.7, Table.5).
2 .0
C o n tro l
T G ( m m o l/L )
*
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S tr e s s + A C E A S tr e s s + A M E A
1 .0 #
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Fig.4 TG levels in the four groups of cold stress rats Table.2 TG levels in the four groups of cold stress rats Group
TG (mmol/L)
Control
0.3460.149
Stress Only
1.0410.549*
Stress+ACEA
0.4840.113#
Stress+AMEA
1.1230.394
Note: *Comparison between the control group and the stress only group, P<0.05; #Comparison among the stress only group and the stress+ACEA / stress+AMEA groups, both P<0.05.
2 .0
C o n tro l
T C ( m m o l/L )
S tr e s s O n ly 1 .5
S tr e s s + A C E A S tr e s s + A M E A
1 .0
0 .5
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A
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s s e S
S
tr
tr
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0 .0
Fig.5 TC levels in the four groups of rats Table.3 TC levels in the four groups of rats Group
TC (mmol/L)
Control
1.3830.125
Stress Only
1.1780.340
Stress+ACEA
1.4400.333
Stress+AMEA
1.0340.142
*
L D L - c ( m m o l/L )
0 .8
C o n tro l S tr e s s O n ly
0 .6
S tr e s s + A C E A S tr e s s + A M E A
0 .4
#
0 .2
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0 .0
Fig.6 LDL-C levels in the four groups of rats Table.4 LDL-C levels in the four groups of rats Group
LDL-C (mmol/L)
Control
0.3470.066
Stress Only
0.5640.185*
Stress+ACEA
0.2750.063#
Stress+AMEA
0.4470.119
Note: *Comparison between the stress only group and the control group, P<0.05; #Comparison among the stress only group and the stress+ACEA / stress+AMEA groups, both P<0.05.
#
1 .0
H D L -c ( m m o l/L )
C o n tro l S tr e s s O n ly
0 .8
*
S tr e s s + A M E A S tr e s s + A C E A
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Fig.7 HDL-C levels in the four groups of rats Table.5 HDL-C levels in the four groups of rats Group
HDL-C (mmol/L)
Control
0.8550.062
Stress Only
0.5900.085*
Stress+ACEA
0.7820.159#
Stress+AMEA
0.5610.075
Note: *Comparison between the stress only group and the control group, P<0.05; #Comparison among the stress only group and stress+ACEA / stress+AMEA groups, both P<0.05. Comparison of atherogenic index and cardiac risk factor in the four groups of rats Applying the formula mentioned above, we acquired the atherogenic index and the cardiac risk factor of this study (Fig. 8, Table.6, Fig. 9, Table.7). The cold stress up-regulated both the index and factor compared to the control group (P<0.05), but, either ACEA or AMEA down-regulated them insignificantly. This indicated that the stress might aggravate atherosclerosis and increase the risk of heart attack. However, ACEA might need more time to reverse these situations.
A t h e r o g e n ic I n d e x 2 .0
C o n tro l
*
1 .5
S tr e s s O n ly S tr e s s + A C E A S tr e s s + A M E A
1 .0
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Fig.8 Atherogenic Index in the four groups of rats Table.6 Atherogenic Index in the four groups of rats Group
Atherogenic Index
Control
0.6160.089
Stress Only
1.0020.512*
Stress+ACEA
0.8320.137
Stress+AMEA
0.8470.122
Note: *Comparison between the stress only group and the control group, P<0.05.
C a r d ia c R is k F a c t o r 3
C o n tro l
*
S tr e s s O n ly S tr e s s + A C E A
2
S tr e s s + A M E A
1
A
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Fig.9 Cardiac Risk Factor in the four groups of cold stress rats Table.7 Cardiac Risk Factor in the four groups of cold stress rats Group
Cardiac Risk Factor
Control
1.6160.089
Stress Only
2.0020.512*
Stress+ACEA
1.8320.137
Stress+AMEA
1.8470.122
Note: *Comparison between the stress only group and the control group, P<0.05. Comparison of LCAT levels in the four groups of rats LCAT activity level of the stress only group was decreased compared with the control group significantly (P<0.05). Only ACEA reversed the situation (P<0.05) (Fig. 10, Table.8).
60
L C A T (u m o l/L )
C o n tro l S tr e s s O n ly
#
S tr e s s + A C E A
40
S tr e s s + A M E A
* 20
A
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tr
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Fig.10 LCAT levels in the four groups of rats Table.8 LCAT levels in the four groups of rats Group
LCAT (umol/L)
Control
44.7774.399
Stress Only
22.9441.862*
Stress+ACEA
41.4162.078#
Stress+AMEA
23.6674.546
Notes： *Comparison between the stress only group and the control group, P<0.05; #Comparison among the stress only group and stress+ACEA / stress+AMEA groups, both P<0.05.
Discussion The evidence of this study showed that cold stress exerted profound effects on lipid and lipoprotein metabolism in rats, and ACEA could reverse some situations. ACEA was developed based on the theories of auricular acupuncture (AA), VNS, and anatomical evidence. Classic VNS reduces body fat, blood triglyceride, and cholesterol levels in rats fed by high-fat diet and in obese-insulin resistant rats [17, 20]. Theoretically speaking, ACEA is supposed to have comparable effect as classic VNS. ACEA down-regulated TG levels in this study. Also, ACEA decreased LDL-C levels and up-regulated both HDL-C levels and LCAT activity. In previous study, cholesterol levels have been discovered to increase or to decrease after cold stress, and also nonsignificant alterations are also reported [21]. Studies on cold stress elicited effects on triglyceride levels are similarly contradictory. In this present study, cold stress-induced increased triglyceride levels. However, significant changes in total cholesterol were not revealed. ACEA decreased the elevated triglyceride levels significantly. LDL-C levels were recommended in guidelines to predict CHD risk [22]. Massive clinical data illustrated that LDL-C was the crucial atherogenic lipoprotein [23-25]. In the present study, cold stress elevated the LDL-C levels, and ACEA decreased it. HDL-C levels were deeply affected by stress. HDL-C showed a remarkable decline after stress. Epidemiological studies are supported by clinical results of correlation between coronary angiography confirmed CHD and low HDL-C levels [26, 27]. Lecithin-cholesterol acyltransferase (LCAT) is a lipoprotein-related enzyme which plays an important role in the esterification of free cholesterol, the maturation of high density-lipoprotein (HDL) particles [28]. In this study, both HDL-C levels and LCAT activity were suppressed by cold stress, and ACEA changed
the situations. Atherogenic index was considered as the risk factor value of atherosclerosis, and cardiac risk factor was considered as the risk factor value of CHD [29-31]. Stress elevated these two values. Down-regulations by ACEA were recorded yet without statistical significance, which might due to time limitations.
Conclusion In this study, we discovered that cold stress may be hazardous in relation to CHD and atherosclerosis, and by regulating lipid-lipoprotein metabolism, ACEA played a crucial role in protecting the heart. Further studies need to be done to reveal the deeper mechanisms.
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