Effect of Electro-acupuncture on Ultrastructure in

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Feng-E HE et al./Journal of Acupuncture and Herbs 1 (2015)70-76

Effect of Electro-acupuncture on Ultrastructure in Diabetic Gastroparesis Model Rats Feng-E HEa, Quan-Quan WANa, Yan PENGb, Ya-Ping LINb＊, Jing SHENb a.

Graduate School, Hunan University of Traditional Chinese Medicine, Changsha, 410208, China

b.

Key Acu-Moxibustion Lab. of Biological Information Analyzing, Institute of Acupuncture, Moxibustion and Massage, Hunan University of Traditional Chinese Medicine, Changsha, 410208, China

Received: April 11, 2015

DOI: 10.1515/tcm-2015-0010

Accepted: April 16, 2015

Abstract Objective: To observe the effects of electro-acupuncture (EA) on ultrastructure of gastric antrum smooth muscle cells (GASMCs) in diabetic gastroparesis (DGP) model rats, and to explore the possible mechanism underlying the therapeutic effects of EA. Methods: Sixty Sprague Dawley rats were randomly divided into 5 groups: blank control (group A), DGP model (group B), EA point (group C), EA non-point (group D), Metoclopramide control (group E). DGP rat model was established by 2% STZ intraperitoneal injection at once, and then being fed with high-sugar-high-fat (HSHF) for 8wks. Phenol red lavage method was employed to measure gastric emptying rate (GER) and intestinal migration rate (IMR). The ultrastructure of GASMCs was observed under the electronic microscope. Results: Compared with the blank control group, the GER and IMR in model group were decreased significantly (P＜0.05 or P＜0.01). Compared with the model group, the GER and IMR in EA point group were increased significantly(P＜0.05 or P＜0.01). Electro-acupuncture on “Zu San Li”(ST36) point, etc. has been observed improving the ultrastructure of GASMCs, as well as increasing the number of interstitial cells of Cajal (ICCs). Conclusion: Electro-acupuncture can improve the GI motility promisingly, based on a potential underlying mechanism that the electro-acupuncture can improve the ultrastructure of gastric smooth muscle, and increase the number of ICCs. Keywords: Diabetic Gastroparesis ; ultrastructure; electro-acupuncture; Cajal interstitial cells; gastric smooth muscle

Diabetic gastroparesis (DGP) is one of the major

imbalance, hyperglycemia and helicobacter pylori

chronic complications of diabetes. As a clinical

infection, etc1. So far there’s few study focusing on

syndrome, it is characterized with decreased gastric

the ultrastructure changes of stomach and effects of

antrum movement and delayed gastric emptying.

electro-acupuncture on it. We therefore designed this

Main symptoms of DGP include postprandial fullness,

study to explore how the gastric ultrastructure changes

upset in upper abdomen, hiccup, anorexia, loss of

of DGP in rats and how the EA has influence on it,

appetite, weight loss, etc. The pathogenesis of DGP is

and further to postulate the possible effective

not very clear at present, but has been related to

mechanism of EA on GI movement disorders.

autonomic neuropathy, ultrastructural changes in

1 Materials and methods

gastrointestinal (GI) smooth muscle cells, GI hormone

1.1 Materials 1.1.1 Experimental animals: 60 healthy adult

*Corresponding author. Professor.

Sprague Dawley (SD) rats, grade SPF, half male and

E-mail address: [email protected]

half female, weighing (231±10)g were bought from

Unauthenticated Download Date | 1/25/16 7:24 PM

Feng-E HE et al./JournalofAcupunctureandHerbs1 (2015)70-76

71

Hunan Silaike Jingda Experiment Animal Co. Ltd.

Tes-tape, as well as recorded the weight, food and

The certificate number of animal quality was

drink, fur colour, urination and defecation, activity

43004700002152. All rats were fed in the exprimental

and mental state. The duration of measurement was 8

animal center, Hunan University of Traditional

wks. The rats having a blood sugar less than

Chinese Medicine, under the temperature 22～25℃,

16.7mmol/L were ruled out from the experiment. A

relative humidity 50%～70%, free feeding and drink

successful modeling was confirmed by: ① blood

of water.

sugar≥16.7mmol/L, urine glucose positive ； ② the

1.1.2 Reagents preparation: Streptozotocin (STZ)

general state and stool character were significantly

were bought from Sigma company, USA. Before use

different from the normal control group; ③the GI

the STZ was dissolved in 0.1mmol/L citric acid-citrate

motilities were significantly different from the normal

sodium buffer (PH value 4.2, temperature 4℃) to

group.

make 2% solution. The citric acid, citrate sodium and

1.2.2Animal grouping and process

NaOH were from Hunan Huihong Reagent Company.

60 SD rats were randomly assigned to give groups: A-

Phenol red was from Tianjin Hengxin Chemical

blank control, B-DGP model, C-EA point (model plus

Reagent Company. TCA was from Tianjin Kemiou

EA on points), D-EA non-point (model plus EA on

Chemical Reagent Company. HSHF feedstuff was

controlled non-points), E-Metoclopramide control

prepared by a mixture of common animal feeds, lard

(model

stearin, cane sugar, milk powder, egg by proportion of

administration), 12 animals in each group. Animals in

58:15:20:5:2. Metoclopramide tablet, produced by

Group A and B were simply bound without any

Shanxi Yunpeng Pharmaceutical Co. Ltd, was

“treatment”. Animals in Group C were treated with

prepared

EA for 20 min after bundle, while in group D were

to

1.7%

suspension

by

adding

double-distilled water.

plus

metoclopramide

intragastric

treated the same process on controlled non-points.

1.1.3 Major equipment: Acupuncture needles:

Animals

in

group

E

were

administered

produced by Suzhou Hualun Medical Articles Co. Ltd.

Metoclopramide suspension by a dosage of 1ml/100g,

The electro-acupuncture apparatus was SDZ-Ⅱ type.

once per day for consecutive 15 days. During the

Transmission electron microscope (TEM) was FEI

experiment all the rats could take foods and water

Tecnai G2 Spirit type. Ultramicrotome was Leica UC7

without any limitation.

type. 722 spectrophotometer was Japanese Shimadzu

1.2.3 Location of acupoints and controlled

UV-1800 type.

non-points

1.2 Methods

The location of acupoints were defined by reference to Experimental Acupuncture edited by Zhong-Ren LI,

1.2.1 Modeling of DGP After an adaptive 7-day feeding and then 12 hrs

based on animal acupoint locating and personification

fasting, the rats were given 2% STZ injection in the

method. The navel was identified on the lower 1/4 and

left lower abdominal cavity (55mg/Kg).

upper 3/4

72 hrs later,

intersection

point

on

the line of

the venous blood of tails was taken and measured by

sternoclavicular joint and pubic symphysis connection.

One Touch blood glucose meter and test strips, by

Zu San Li (ST36): posterolateral side of knee, around

which the diabetic rats were identified when the

5mm under the fibular head; San Yin Jiao (SP6):

instant blood sugar was higher than 16.7mmol/L.

10mm above the inner malleolus top on the hind limbs;

From the same date of identification a HSHF feedstuff

Liang Men (ST21): the intersection point of

was administered to the rats irregularly (odd-number

medioclavicular line and the horizontal line which can

date fed only in the morning and even-number date

be identified by the middle point of xiphosternal

fed only in the afternoon). Once per week, we

synchondrosis and navel connection; "Zu San Li"

measured the blood glucose of rats by One Touch

control point: 5mm of the external side same level to

blood glucose meter and test strips, urine glucose by

Zu San Li; "San Yin Jiao" control point: 5mm of the


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internal side same level to San Yin Jiao; "Liang Men"

0.5ml to remove proteins, centrifuged at 3500 r/min

control point: 5mm of the external side same level to

for 10 min, took the supernate to assay optical density

Liang Men (ST21).

(OD)

value

under

560

nm

ultraviolet

spectrophotometer. The formula for calculating gastric

1.2.4 EA method The rats were bound to rat board, by locating

emptying rate: GER=(1-real OD value/standard OD

unilaterally the above mentioned points (acupoints or

value)*100%.

control non-points) were selected. After removing hair

③IMR: the small intestine segments were put on

around points the same trained operator inserted

ice after decoherence, without drag. Observed the

needles (size 0.25mm*25mm) on the selected points

red-stained end of small intestine, then cut a small

in a depth of 5 to 7mm. Then the negative electrode of

ventage with eye scissors. Dropped a small amount of

EA apparatus was connected to the selected points and

0.5mol/L NaOH solution on the ventage. The position

positive electrode to points 2mm under the selected

where turned to be purple was where the PSP arrived,

points. For each rat a total 3 groups of output lines

then dropped more NaOH around to determine the

were connected. The waves were set at an alternate

real arriving position. Then we measured the distance

mode with 5-second 10HZ sparse wave and 10-second

from pylorus to ileal valve (A) and the distance from

50HZ dense wave in shift. Intensity of wave was

pylorus

regulated until the muscles around points shaking

IMR=a/A×100%.

to

where

was

stained

by

PSP

(a).

slightly. The needles were kept in points for 20 min.

④Observation on the ultrastructure of GASMCs.

The EAs were performed every day by changing sides,

The gastric antrum tissue pieces were fixed in 2.5%

for consecutive 15 days.

glutaric dialdehyde at 4 ℃ for 10 to 12 hrs; then were washed by 0.1mol phosphoric acid PBS for three

1.2.5 Sample collection and process At the end of experiment, the rats were restricted

times, 10 min for each time, then were fixed in 1%

foods for 24 hrs and water for 2hrs. 2 ml of 50mg/dL

perosmic acid for 1 hr, washed by PBS again for three

Phenolsulfonphthalein

were

times, 10 min for each time; then were gradient

administered to each rat, after 20 min we collected the

dehydration by acetone and embedded in ethoxyline

samples under anesthesia by abdominal cavity

resin (Epon) 812, solidification at 37℃ for 24 hrs, 60℃

injection of 10% chloral hydrate according to animal

for 24 hrs. Slices were made by ultramicrotome, with

weight (3ml/kg). From each group two rats were

thickness around 50 to 100nm. The slices were double

randomly selected. After cutting the belly the cardia

stained by uranyl acetate and plumbi nitras, and were

and pylorus were ligated, the whole stomach was

observed and photographed by TEM.

(PSP)

solution

picked off. Slivering the stomach along the curvature

1.2.7 Statistics

gastrica major, washing contents with 0.9% NaCl

All data were analyzed by the SPSS 17.0 statistical

solution. Cutting off a small tissue (size around

package. The data were tested normal distribution first,

1*1*3mm) quickly and putting it in 2.5% glutaric

measurement data were expressed as mean±SD (χ±s).

diadehyde solution at 4℃ for further TEM observation.

One-way ANOVA was employed for comparison data

At the same time a total of 20 ml gastric content was

in multiple groups, wherein LSD method for

collected for measuring the GER; small intestine

homogeneity of variance and Dunnett’s T3 method for

segment was acquired for measuring the IMR.

heterogeneity of variance. Data not in normal

1.2.6 Observational indexes

distribution were expressed by interquartile range and

①General state: food and drink, urination and

tested by rank sum method. P＜0.05 was set as the

defecation, fur colors, activity and mental state.

criterion of significant difference.

② Gastric emptying: to mix the 20ml gastric content with 20ml 0.5 mol/L NaOH, after standing for 1 hr we took 5ml supernate, and added 20% TCA

2 Result 2.1 General state



During the experiment, rats in the blank control group

above showed that the modeling was successful.

had been active, with sensitive reaction, smooth fur colors, and normal eating, urination and defecation.

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2.2 GER and IMR comparison among groups (Table 1)

Rats in model group had an increased eating and

From Table 1: compared with blank control, the

drinking from the third date of modeling, then

GER and IMR of rats in model groups were decreased

continuously had increased drinking, eating, loose

with statistical significance (P＜0.01), indicating a

foul stool; from the fourth week, some of them had

prolonged gastric emptying and retarded intestinal

decreased appetite, down spirit,

retarded reaction

migration movement. Compared with either model

and weight loss; from the sixth week, most of the rats,

group (group B) or EA non-point group (group D), the

apart from increased drinking and urination, could

EA point group (group C) had achieved increased

have apparent decreased eating, significant weight

GER and IMR with statistical significance (P＜0.01

loss, altered stool character (loose or hard) with foul

and P＜0.05, respectively). The difference between

smell, dry yellow fur easy to lose, and retarded

acupoints and non-points suggested that acupoints had

reaction. The indexes for evaluation above-mentioned

specific effects on regulating the GI movement.

general state were from

literature2.

The description

Table 1Comparison of GER and IMR among groups (n=12,χ±s) Group

GER(%)

IMR(%)

Blank control (A)

66.69±4.41

62.53±3.08

DGP model (B)

25.46±2.73▲▲

45.97±2.20▲▲

EA acupoint (C)

46.14±6.28▲▲●●

EA non-point (D)

29.01±3.72▲▲

43.81±6.32▲▲

Metoclopramide (E)

37.13±5.55▲▲

53.13±3.11

Note: compared with blank control with EA non-point group,

★

▲▲

★

63.80±3.16●●

P＜0.01; compared with model group

★★

●●

P＜0.01; compared

★★

P＜0.05， P＜0.01.

2.3 Ultrastructural changes of the GASMCs

the blank control. The cell sizes got smaller, structure

Normal control group: In the intercellular stroma

got unclear, with dissolved cytoplasm. There were a

adjacent to muscle plexus neurons we could see

large

interstitial cells of Cajal (ICCs), in oval shape, with

mitochondrial

many cell processes and stubby microvilli, in the

vacuole-like changes, or even dissolve; the rough

cytoplasm we could also see many ring-shaped

endoplasmic reticulum was expanded, ribosome on

mitochondria with clear crest and well-developed

the surface were off, number of free ribosomes was

rough endoplasmic reticulum, attached on the surface

reduced. Nucleus were atrophic, heterochromatin in

by lots of ribosome particles; the nucleus was large

nucleus were gathered into a mass. Dense body and

and regular, nuclear membrane was smooth and less

dense disc were apparently decreased. The smooth

wrinkled, heterochromatin was distributed evenly,

muscle cells in gastric antrum were in irregular

dense patch cytoplasm and dense conspicuous were

distribution.

apparent. The filaments were clearly visible. The gaps

cells and nerve cells were loose, with messy junctions,

among ICCs and smooth muscle cells and nerve cells

or even not connected to a network. Those signs

were tight (Fig. 1).

suggested that the ultrastructure of smooth muscle

DGP model group: the number of ICCs in gastric antrum was apparently decreased by comparison to

number

of

cytoplasmic

swelling,

vacuolization,

disappeared

cristae,

Gaps among ICCs and smooth muscle

cells in the rat gastric antrum had apparent changes after modeling (Fig. 2A and Fig. 2B).


74


EA point group: compared with the model group,

less than EA point group. The shape and structure of

the ICCs in gastric antrum had apparently increased,

cells were slightly blurred, mitochondria were

but less than blank control. The shape and structure of

vacuolar, partially dissolved, gap junctions among

cells were basically normal, but the mitochondria had

cells still existed. The pathological damage of gap

some slight swelling. The gas junctions exist, though

junctions among cells was less than model group, but

has some damages, but less than that in the model

worse than EA point group. This suggested that EA on

group. This suggested that EA on Zu San Li (ST36)

non-point or Metoclopramide had limited repair

could increase the number of ICCs and fix the

effects for ultrastructure of gastric antrum, possibly

damaged ultrastructure of them (Fig. 3).

due

EA non-point group and Metoclopramide group:

to

the

limited

drug

absorption

acupoint-specific effects (Fig.4 and Fig.5).

the number of ICCs was more than model group, but

Fig.1 (2×1000)

Fig.2A (5×1000)

Fig.3 (2×1000)

Fig.2B (2×1000)

Fig.4(2×1000)

Fig.5(2×1000)


and


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3 Discussion

the nerve system and smooth muscle system to form a

There is no corresponding name for DGP in

complex network to regulate the GI movement. The

traditional Chinese medicine (TCM), but by symptom

previous studies indicated that ICCs serve as the

analyses, TCM holds the concept that the Qi

pace-maker cells for GI tract. It can produce and

stagnation in the middle Jiao can affect the functions

spread slow wave signal. It distributes unevenly, but

of spleen and stomach so a bunch of DGP-related

mostly in the gastric antrum part. A decreased ICCs

symptoms such as vomiting, fullness, belching would

can lead to decreased GI movement and digestion5,6.

occur3, therefore DGP can basically be reflected in the

The results of this study showed that in the DGP

TCM diseases “fullness”, “vomiting”, etc. Based on

model group, the ultrastructure of smooth muscle cells

our experience of acupuncture on treating such TCM

in gastric antrum has had apparent changes, especially

diseases, we established this DGP rat model to

in its nucleus and mitochondria, also the number of

observe the effects of EA on Zu San Li (ST36), Liang

ICCs getting decreased, the gap junctions among ICCs

Men (ST21) and San Yin Jiao (SP6) for the gastric

and other cells also had more or less changes,

antrum ultrastructure, by which we hope to further

suggesting that pathological changes in mitochondria

understand the mechanism of EA on DGP from the

might be an important mechanism involved because it

viewpoint of morphology.

can affect the necessary energy of cells underlying the

Zu San Li (ST36) is the He-sea point of stomach.

GI movement. After EA treatment, the pathological

“He-sea can treat diseases belonging to inner Fu

changes of cells have had improvement on certain

organs”, in the very early ABC of Acupuncture it

degree, as described above. The results showed that

wrote “Zu San Li in charge of Zhong Xiao (Diabetes

EA on certain points could increase the quantity of

Middle Jiao type)”, acupuncture on Zu San Li can

ICCs, repair the ultrastructure of smooth muscle cells,

stimulate the GI movement, so that the stomach can

promote

have a quicker emptying. Liang Men (ST21) is also an

symptoms. This might be one of the mechanisms that

important point on stomach meridian, having the

EA could prevent and treat DGP.

function

Competing interests

of

regulating

Middle

Qi,

improving

gastric

emptying

and

improve

DGP

stagnation and indigestion, and harmonizing stomach

The authors declare that they have no competing

and intestine, serving as a common used point for GI

interests.

diseases. San Yin Jiao (SP6) locates on the

Acknowledgements

intersection point of Zu Tai Yin, Zu Shao Yin and Zu

The authors thank for the fund support from National

Jue Yin, so it can strengthen spleen and promote Qi,

Natural Science Foundation of China（No. 81403487）,

cultivate blood and activate blood flow and regulate

Opening

Qi movement. Our clinical experience calls for further

universities of Hunan Province (No.12k088), and

demonstration of those points on DGP.

Youth project from Hunan Education Administration

So far the mechanism of GI disorder of DGP

foundation

for

creation

projects

in

（No.14B128）.

patients is not clear. Most of researchers believe that the ultrastructural changes of gastric antrum smooth muscles might be the main reason of it. Yi

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2

smooth muscle of gastric antrum is the core unit to regulate GI movement. Interstitial cells of Cajal (ICCs)

3

are a special kind of interstitial cells existing between GI nerve end and smooth muscle, they together with 4

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