Yajima Michiko, et al.. Japanese Acupuncture and Moxibustion; 2023; Vol.17: 1–11

Original Research

Increase of molecular chaperone αB-crystallin expression in the rat
soleus muscle induced by moxa needle stimulation
YAJIMA Michiko, SUMIYA Eiji, KAWAKITA Kenji, YANO Tadashi
Course of Acupuncture and Moxibustion, Graduate School of Acupuncture and Moxibustion,
Meiji University of Integrative Medicine
Abstract[Background] The purpose of this study is to investigate whether the expression of αB-crystallin in slow soleus
muscles is affected by thermal stimulation in moxa needle therapy. αB-crystallin is known to be abundant in slow
muscles and to maintain and improve the function of slow antigravity muscles. Moxa needle used to be performed
with silver needles, which have high thermal conductivity, but is currently performed with stainless steel needles,
which have low thermal conductivity. Therefore, we performed thermal stimulation with both types of moxa needles
and compared their effects.
[Methods] We carried out experiments in two steps: Experiments A and B. Rats were divided into moxa needle
combustion groups using silver (CSV group) and stainless steel (CSS group) needles, non-combustion groups using
silver (NSV group) and stainless steel (NSS group) needles, and a control group with no needle insertion (CON group).
The moxa balls in the CSV and CSS groups were burned three times intermittently. In Experiment A, skin surface anddeep tissue temperatures were measured for 50 minutes. Experiment B was performed in a similar manner without
deep tissue temperature measurements. At 3 hours after stimulation, the rats were euthanized. Their soleus andextensor digitorum longus (EDL) muscles were then removed to examine the expression of αB-crystallin and therelationship with deep temperature obtained from Experiment A.
[Results] In Experiment A, higher skin surface and deep temperatures were observed in the CSV than in the CSS
group. In Experiment B, the skin surface temperatures in the CSV and CSS groups followed similar courses to thosein Experiment A. In addition, αB-crystallin expression in soleus after correction for glyceraldehyde 3- phosphate
dehydrogenase (GAPDH) expression, relative to the CON group (100), was 299.29 in the CSV, 160.62 in the CSS,
121.51 in the NSV, and 67.52 in the NSS group, indicating that αB-crystallin expression was significantly higher in
the CSV than in the CON group (P < 0.01). No significant differences were found in the EDL.
[Conclusion] The results of this study suggest that a deep temperature increase of 4–5 °C for about 3 minutes is needed
to increase αB-crystallin expression. Therefore, in moxa needle stimulation to the posterior surface of the lower leg,the use of silver needles with high thermal conductivity could increase the deep temperature and expression of αBcrystallin in soleus muscles.
Key words: molecular chaperone, heat shock protein, αB-crystallin, moxa needle, soleus

I. Introduction
Molecular chaperones are cellular proteins whosefunction is to ensure that the folding of certain otherpolypeptide chains and their assembly into oligomeric
structures occur correctly, and they do not form part of the
final structure1). Molecular chaperones promote efficient
protein folding and prevent or reverse protein
aggregation2). And chaperones and proteases consist of“protein quality control” network, and repair protein
aggregation2).
As the target of this research, we selected αB-crystallin,a small (22 kDa) molecular chaperone. It is presumed that
αB-crystallin protects proteins from proteolysis byrefolding damaged proteins3). Alpha B-crystallin is one of
the major lens structural proteins in the vertebrate eye3),
but it also exists in a variety of tissues, including the heart,brain, kidney, and skeletal muscle4). Among the skeletal

Corresponding author: Yajima Michiko, Course of Acupuncture and Moxibustion, Graduate School of Acupuncture andMoxibustion, Meiji University of Integrative Medicine, Hiyoshicho, Nantan City, Kyoto 629-0392, JAPAN2

Yajima Michiko, et al.. Japanese Acupuncture and Moxibustion; 2023; Vol.17: 1–11

muscles, αB-crystallin is known to be abundant in slow
muscles5). Under microgravity and during atrophy of slow
antigravity muscles, this chaperone tends to decrease
specifically6–8). Alpha B-crystallin exists to maintain andimprove the function of slow antigravity muscles, and its
increased expression stabilizes cell function9,10). The
expression of αB-crystallin is strongly induced by hightemperatures and is effective in suppressing protein
thermal aggregation2,3).
In this study, to provide heat stimulation, we used moxa
needle. The moxa needle therapy (in Japanese kyutoshin;“needle with moxa on the head”) was spread by Tomooki
Sasagawa at the end of the nineteenth century in Japan 11–
13). In moxa needle therapy, acupuncture needles areinserted into the body, and then a small moxa ball is
attached to the handle of the needle and burned with theaim of obtaining the synergistic effects of acupuncture andmoxibustion.
Currently, stainless steel needles are commonly used, but
in the early days of moxa needle therapy, silver needles
were used, and as silver has high thermal conductivity, it
was possible to provide stimulation by not only radiative,
but also conductive heat transfer. Therefore, moxa needle
therapy was once also called “Conductive Heat Transfer
Needle Therapy (dennetsu hari ryohou)”13). It is presumed
that silver needles allow more heat to reach the deep
tissues than do stainless steel needles, so “Conductive
Heat Transfer Needle Therapy” may have an effect on
increasing molecular chaperones as heat shock proteins inmuscle.
To our knowledge, no studies have been conducted on
the effect of moxa needle therapy on the expression ofmolecular chaperones. Previous studies have reported
increases in molecular chaperones after soaking in hot
water or placing in a hot chamber as thermal stimulationfor extended periods of time14–19). However, if a method
can be devised that is more effective and easier to perform
by not requiring lengthy exposure to high temperatures, it
could become more clinically applicable. It is speculated
that moxa needle with silver needles, which can be
expected to raise the deep tissue temperature, could
provide a stressful of temperature rise in a short period oftime.
Given this background, the purpose of this study is toinvestigate whether thermal stimulation in moxa needle
therapy affects the expression of αB-crystallin, a small
molecular chaperone, in slow soleus muscles and tocompare the effectiveness of with silver and stainless steel
needles in moxa needle for delivering thermal stimulation
to deep tissues.
In order to produce a greater stressor, the burning of themoxa ball was performed three times repetitively, and thestressor was also generated by repeating the rise and fallof the temperature. Repeated burning of moxa several
times has also been a clinical technique11,12).
If moxa needle affects the expression of αB-crystallin, it
could lead to the development of a new method formaintaining slow muscles, and thereby a new concept ofacupuncture and moxibustion for frailty and sarcopenia inolder adults in a super-aging society.
II. Methods
1. Heat stress production and temperature
measurements
We measured the temperature change of the needle bodyof moxa needles with silver and stainless steel needles,and examined the difference in heat conduction between
the two. Based on that, we carried out the experiment inrats in two steps: Experiments A and B.
All rats were housed in cages in groups of two to three
in a room with controlled environmental conditions(ambient temperature 24°C ± 2°C, humidity 50% ± 10%,12-hour light/12-hour dark cycle) and were allowed ad
libitum access to food and water.
In total, 60 male Wistar rats, 9 weeks old (Japan SLC,
Inc., Shizuoka, Japan), were randomly divided into 10
groups, with the following five groups each set for bothExperiments A and B (n = 6/group): moxa needle, i.e.,combustion groups using silver needles (CSV) andstainless steel needles (CSS), non-combustion groups
using silver needles (NSV) and stainless steel needles(NSS), and a control group with no needle insertion(CON).
The temperature in the room where the data wasmeasured was 26 ± 0.5°C and the humidity ranged from
25% to 40%.
This study was approved by the Animal Care and Use
Committee of Meiji University of Integrative Medicine.
(Approval No. 2021006, 2021008)(1) Pre-measurement—needle body temperature
Silver or stainless steel needles with a length of 50 mm
and a diameter of 0.27 mm (Maeda Toyokichi Shouten Co.
Ltd., Tokyo, Japan) are inserted 13 mm perpendicular topolystyrene foam surface, the distance from the moxa ballto the surface was 38 mm, and the needle bodytemperature just above the foam surface were measured
every second for 50 minutes (n = 6).
The moxa needle procedure was that a 0.9 g ± 0.005 g
moxa ball with a diameter of 20 mm was attached to thehandle of the needle, the measurements were started, andthen the moxa ball was ignited 10 minutes later. The
ignition point was the top of the moxa ball. After 7
minutes of ignition, the needle was left inserting and theburned moxa ball was removed and replaced with anotherball. The next ignition was 8 minutes and 30 seconds after
the previous ignition, and this procedure was performed
three times in total. After the third burning, the needle
were not removed and the measurement continued until 50
minutes after the start of the measurement.
(2) Experiment A: temperature measurements
Silver has the highest thermal conductivity among allYajima Michiko, et al.. Japanese Acupuncture and Moxibustion; 2023; Vol.17: 1–11

metals, whereas that for stainless steel is extremely low.
In Experiment A, we researched the degree of difference
in deep tissue temperature changes of the soleus muscle
region when moxa needle was performed using needles
made of either silver or stainless steel, by inserting aneedle probe for deep tissue temperature measurements.
In Experiment A, 30 male Wistar rats, 9 weeks old, themean (SD: standard deviation) weight 304.7 (14.6) g,
were divided into five groups (n = 6/group): CSV, CSS,
NSV, NSS, and CON, and were anesthetized with a triple
anesthetic (medetomidine [0.15 mg/0.15 ml/kg],
midazolam [2 mg/0.4 ml/kg], butorphanol [2.5 mg/0.5
ml/kg]; dose 0.25 ml/100 g).
For the rats in the CSV and CSS, combustion groups ofmoxa needle, the lower legs were shaved, and moxa
needle was performed to the posterior aspect of the rightlower leg (midpoint between the knee joint and the ankle
joint on the posterior midline of the lower leg). And aneedle probe for measuring the temperature of deep tissue(MT-26/4HT, Muromachi Kikai Co. Ltd., Tokyo, Japan)
was inserted 16 mm at a 38° ± 5° angle to the needle froma position 10 mm outside the needle insertion point on theskin surface. And another probe for measuring the skinsurface temperature was attached at 2 mm distal from theneedle insertion point along the posterior midline of thelower leg.
Regarding moxa needle, silver or stainless steel needles(length 50 mm, diameter 0.27 mm) were inserted 13 mm± 1 mm perpendicular to the skin surface. The acupunctureneedle passed through gastrocnemius muscle and reachedsoleus muscle, and the apex of the needle was just past theside of the fibula, and this point was confirmed in apreliminary experiment by making an incision where theneedle would pass, and the penetration depth and angle of
the acupuncture needle and probe needle were determined.
And a 0.9 g ± 0.005 g moxa ball (diameter 20 mm) was
attached to the handle of the needle and left for 10 minutes.
After that, the measurements were started and the moxaball was ignited 10 minutes later. Ignition point andintervals were the same as in the pre-experiment, and threecombustions were performed.
From the beginning, the deep tissue temperature at the
tip of the acupuncture needle and the skin surface
temperature near the needle insertion point were measured
every second for 50 minutes, and then the needle wasremoved.
For the non-combustion groups (NSV and NSS), silver
or stainless steel needles were inserted and a moxa ball
was attached, and the same period of time as in the moxa
needle groups passed without ignition. The same period oftime also passed in the CON group, but without theinsertion of acupuncture needles. The time courses of theskin surface and deep tissue temperatures were comparedand examined by the mean value for each group.
In all groups, after the measurements, the rats wereeuthanized with an overdose of pentobarbital (200 mg/kg,
intraperitoneal [i.p.] injection. Pentobarbital Sodium Salt,

3

NACALAI TESQUE, INC., Kyoto, Japan).
(3) Experiment B: αB-crystallin protein expression
measurements
In Experiment B, 30 male Wistar rats, 9 weeks old, themean (SD) weight 306.6 (7.50) g, were used. In
Experiment B, the usual moxa needle stimulation was
performed without insertion of a thermal probe; all otherprocedures were the same as those in Experiment A. Next,
we compared the intensity of expression of αB-crystallinin the soleus (red slow muscle) and the extensor digitorum
longus (EDL, white fast muscle), and examined therelationship with deep tissue temperature measured inExperiment A.
Since the expression of αB-crystallin was greater 3 hours
after the end of stimulation in the pre-study (data notshown), excision of muscle was performed 3 hours after
the end of stimulation in this study. At 3 hours after theend of measurement, the rats were euthanized with an
overdose of pentobarbital (200 mg/kg, i.p.), and their
entire soleus and EDL, respectively, and eyeballs (positive
control) were removed, weighed, quickly frozen by liquid
nitrogen, and stored at −80°C.
2. Muscle preparation, SDS-PAGE, Western blotting
and chemiluminescence detection
Approximately 0.05 g soleus and 0.06 g EDL (each halfof the extracted) were frozen in liquid nitrogen and thenpulverized. The powder was homogenized in Hepes buffer(25 mM Hepes, pH 7.4, 2 mM EDTA, 250 mM sucrose,
0.1% Triton, one tablet of protease inhibitor/50 ml). The
homogenate was centrifuged at 16,000×g for 20 minutes
at 4°C, samples were prepared from supernatants. Treatedsamples were subjected to sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE), and we useda home-made gel. Protein contents were determined using
the Bradford Protein Assay. Each lane was loaded with aprotein sample from a different rat, 10 μg proteins wereapplied per gel lane. The gel was run for 80 minutes at 130V.
Then transferred to polyvinylidene fluoride (PVDF)
membranes at 0.8 mA/cm2 for 45 minutes by a semi-dry
system. As αB-crystallin is a small molecular chaperone,a transfer membrane with a pore size of 0.2 µm was used.
After blocking with 0.1% skim milk powder for 1 hour atroom temperature, the membranes were incubated with
anti-αB-crystallin antibody (15808-1-AP, 1000-folddilution, Proteintech Group, Inc, Tokyo, Japan) for 1 hourat room temperature to conjugate. After rinsing with abuffer containing 0.1% Tween20, the membranes wereincubated with anti-rabbit IgG-HRP (cat. no. 7074, 5000fold dilution, Cell Signaling Technology, Inc, Tokyo,
Japan) at room temperature for 1 hour to conjugate. The
bands were detected by chemiluminescence (Immobilon
Crescendo Western HRP substrate, Merck KGaA,
Darmstadt, Germany), imaged on X-ray film (BioMax
Light-1film, Carestream Health, Inc., Tokyo, Japan),

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digitized with a scanner (DocuCentre-VII C7788,
FUJIFILM Corporation, Tokyo, Japan), quantified with
the image analysis software Image J, and corrected by theexpression level of glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) as internal control with antiGAPDH (M171-7 Anti-GAPDH mAb-HRP-DirecT,
10000-fold dilution, MEDICAL & BIOLOGICALLABORATORIES CO., LTD., Tokyo, Japan). The levelsof αB-crystallin are shown in arbitrary units (a.u.) divided
by those of GAPDH, and the control group was set as 100.
3. Statistics
All statistical analyses were performed using Excel(Microsoft Office 365) with the add-in software Statcel 4(OMS Publishing Inc., Japan).
The data were expressed as mean (SD: standard
deviation), and differences between groups wereconsidered significant at P < 0.05. The data for each groupwere tested for normality of distribution using theKolmogorov-Smirnov test. The homogeneity of thegroups was confirmed by analyzing the data for eachgroup using Bartlett’s test and one-way analysis ofvariance. The homogeneity of the groups for body weight,
extracted muscle weight, room temperature, and humidity
were thereby confirmed. The statistical significance of thetemperature difference between CSV and CSS wasdetermined using an unpaired t-test. Temperature

difference between in non-combustion groups was
examined by Steel-Dwass test. The protein expression
levels were compared using multiple comparison tests,
Dunnett’s test for soleus muscle and Steel’s test forextensor digitorum longus muscle.
III. Results
1. Temperature changes(1) Pre-measurement—needle body temperature
Figure 1 shows the temperature changes in Premeasurement for needle body temperature. The
temperature of the silver needles at the insertion point
increased with each burning, whereas that of the stainlesssteel needles remained nearly equivalent throughout thethree burns. The maximum temperatures of the silver
needles for the three burnings were 1st: 64.91 (3.47)°C,
2nd: 68.64 (2.51)°C, 3rd: 70.25 (3.38)°C, and those of thestainless steel needles were 1st: 58.60 (2.06)°C, 2nd:58.44 (2.80)°C, 3rd: 58.53 (2.66)°C (Fig. 1, Table 1).
The results of the t-test for the maximum temperatures ofthe silver and stainless steel needles were significant at theP < 0.01 level for each burning, indicating that thetemperature of the needle body of the silver needles
increased significantly more than that of the stainless steel
needles.

Figure 1. Needle body temperatures in the pre-measurement during three moxa ball burnings
The temperature of the silver and stainless steel needle bodies at the insertion point to the polystyrene foam.
Verticals bars are standard deviation. Asterisks indicate statistical significance between silver and stainless steel (ttest).

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Table 1.
Pre-measurement: mean (SD) needle body temperature (°C) at the insertion point to the polystyrene foam (each group n = 6)
At start

Max. at
1st burning

Min. before 2nd
burningMax. at
2nd burning

Min. before 3rdburningMax. at
3rd burning

Total integral
amountCSV

26.57

64.91 (3.47)

29.04 (0.58)

68.64 (2.51)

29.35 (0.48)

70.25 (3.37)

105,443.44

CSS

26.52

58.60 (2.06)

28.05 (0.41)

58.44 (2.80)

28.22 (2.29)

58.53 (2.66)

97,507.39

Experiment A: mean (SD) skin surface and deep tissue temperatures (°C) at each burning (each group n = 6)
Skin surface temperature
Min. before
1st burning

Max. at
1st burning

Min. before
2nd burning

Max. at
2nd burning

Min. before 3rdburningMax. at
3rd burning

Total integral
amountCSV

0.00

8.23 (1.71)

1.97 (0.78)

10.11 (1.80)

2.62 (0.72)

11.25 (1.82)

8494.56

CSS

0.00

7.46 (0.40)

1.58 (0.99)

8.41 (0.57)

1.90 (1.20)

8.36 (0.49)

6622.21

Deep tissue temperature
Min. before
1st burning

Max. at
1st burning

Min. before
2nd burning

Max. at
2nd burning

Min. before 3rdburningMax. at
3rd burning

Total integral
amountCSV

0.00

3.09 (0.99)

1.63 (0.67)

4.02 (1.03)

2.19 (0.66)

4.56 (0.92)

4906.03

CSS

0.00

2.32 (1.13)

1.32 (0.85)

2.94 (1.22)

1.63 (0.85)

3.05 (1.21)

3640.69

Experiment B: mean (SD) skin surface temperature (°C) at each burning (each group n = 6)
Min. before
1st burning

Max. at
1st burning

Min. before
2nd burning

Max. at
2nd burning

Min. before 3rdburningMax. at
3rd burning

Total integral
amountCSV

0.00

8.16 (1.37)

1.83 (0.64)

10.31 (1.39)

2.32 (0.84)

11.37 (1.31)

8497.15

CSS

0.00

7.18 (0.68)

1.70 (0.52)

8.14 (0.43)

1.96 (0.45)

8.17 (0.86)

6624.14(2) Experiment A: temperature measurements
As a first step of experiments in rats, we measured theskin surface and deep tissue temperatures simultaneously.
Figure 2 shows the time courses of the skin surface anddeep tissue temperatures, with the lowest temperature ofeach group before the first ignition set as the baseline.
The mean maximum skin surface temperatures for thethree burnings in CSV were 1st: 8.23 (1.71)°C, 2nd: 10.11(1.80)°C, and 3rd: 11.25 (1.82)°C. And those in CSS were
1st: 7.46 (0.40)°C, 2nd: 8.41 (0.57)°C, and 3rd: 8.36
(0.49)°C (Fig. 2, Table 1). The results of the t-test for the
maximum skin surface temperature in the CSV and CSS
groups at each burning were significant at the P < 0.05
level for the first burning, and at the P < 0.01 level for thesecond and third burnings, indicating that the skin surface
temperature increased significantly more in CSV than in
CSS.
The mean maximum deep tissue temperatures for thethree burnings in CSV were 1st: 3.09 (0.99)°C, 2nd: 4.02
(1.03)°C, and 3rd: 4.56 (0.92)°C, and those in CSS were
1st: 2.32 (1.13)°C, 2nd: 2.94 (1.22)°C, and 3rd: 3.05
(1.21)°C. In the CSV group, both the skin surface anddeep tissue temperatures increased stepwise at eachburning, and the deep tissue temperature increased anddecreased about 2 minutes later than did the skin surface
temperature (Fig. 2).

The maximum temperatures at each burning in the CSV
group were significantly higher than those in the CSS
group (Fig. 2, Table 1). The results of the t-test weresignificant at the P < 0.05 level for the first and second
burnings, and at the P < 0.01 level for the third burning,
indicating that the deep tissue temperature increased
significantly more in CSV than in CSS.
Figure 3 shows the temperature components inExperiment A. In the CSS group, at the third burning, thedeep tissue temperature increased by about 3°C frombaseline, and the duration over 3°C was for 65 seconds. In
the CSV group, the deep tissue temperature increased byabout 4.5°C from baseline at third burning, and theduration over 4.5°C was for 61 seconds (Fig. 3).
Throughout the three burnings, temperature increases ofover 4°C, over 3.5°C, and over 3°C lasted for 187 seconds,
417 seconds, and 651 seconds, respectively (Fig. 3). The
sums of the skin surface temperatures from baseline to theend of the measurements were 8494.56 in CSV and
6622.21 in CSS. For the deep tissue temperature, the same
calculation gave 4906.03 in CSV and 3640.69 in CSS(Table 1). In the NSV, NSS, and CON groups, the skinsurface and deep tissue temperatures decreased with time
(Fig. 5).

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Figure 2. Skin surface and deep tissue temperatures in the combustion groups in Experiment A
CSV: combustion with silver needles, CSS: combustion with stainless steel needles.
Verticals bars are standard deviation. Asterisks indicate statistical significance between CSV and CSS (t-test).

Figure 3. Comparison of high temperature integrals of combustion groups
1: Integral second of each rise in deep tissue temperature
2: Integral amount of each rise in deep tissue temperature
Comparison of high temperature durations and temperature rise integrals in the deep temperatures of the
combustion groups in Experiment A. The unit for 1 and 2 is the integrated second and integrated temperature(°C·second), respectively. The unit of °C·second is adding up the temperature in seconds that was in each of theapplicable temperature ranges.
CSV: combustion with silver needles, CSS: combustion with stainless steel needles.
The bars in the 1 and 2 CSVs are the same length, and the graph scales are delimited in the same way.

Yajima Michiko, et al.. Japanese Acupuncture and Moxibustion; 2023; Vol.17: 1–11

Figure 4. Skin surface temperatures of the combustion groups in Experiment B
CSV: combustion with silver needles, CSS: combustion with stainless steel needles.
Verticals bars are standard deviation. Asterisks indicate statistical significance between CSV and CSS (t-test).

Figure 5. Skin surface and deep temperatures of the non-combustion and control groups in Experiments A
and B
NSV: non-combustion with silver needles, NSS: non-combustion with stainless steel needles, CON: control-no
needle7

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(3) Experiment B: αB-crystallin protein expression
measurements
As a second step, without inserting a probe to measure
the deep tissue temperature, we performed usual moxa
needle and measured the skin surface temperature.
Figure 4 shows the time courses of the skin surface
temperatures, with the lowest temperature of each group
before the first ignition set as the baseline. Skin surface
temperatures increased more in CSV than in CSS.
The mean maximum skin surface temperatures in the
CSV group for the three burnings were 1st: 8.16 (1.37)°C,
2nd: 10.31 (1.39)°C, and 3rd: 11.37 (1.31)°C, and those in
the CSS group were 1st: 7.18 (0.68)°C, 2nd: 8.14 (0.43)°C,
and 3rd: 8.17 (0.86)°C.
The maximum temperatures were significantly higher in
CSV than in CSS (Fig. 4, Table 1). The results of the t-test
for the maximum skin surface temperatures in CSV andCSS were significant at the P < 0.01 level for the second
and third burnings, indicating that the skin surface

temperature increased significantly in CSV than in CSS.
The sums of the skin surface temperatures from baselineto the end of the measurements were 8497.15 in CSV and
6624.14 in CSS (Table 1). In the NSV, NSS, and CON
groups, the skin surface temperatures decreased with time
(Fig. 5).

Figure 6. Comparison of αB-crystallin
expression in soleus muscle in the control andintervention groups
The levels of αB-crystallin are shown in arbitrary
units (a.u.) divided by those of GAPDH, the internal
control, and the control group was set as 100.
Statistical significance was examined using
Dunnett’s multiple comparison test.
A representative blot is shown in the upper panel, andthe bottom panel shows the relative quantitation.
CSV: combustion with silver needles
CSS: combustion with stainless steel needles
NSV: non-combustion with silver needles
NSS: non-combustion with stainless steel needles
EYE p.c.: positive control
CON: control-no needle

Figure 7. Comparison of αB-crystallin expression
in the extensor digitorum longus muscle in thecontrol and intervention groups
The levels of αB-crystallin are shown in arbitrary
units (a.u.) divided by those of GAPDH, the internal
control, as the ratio of soleus control group set as 100.
Statistical significance was examined using Steel’s
multiple comparison test.
A representative blot is shown in the upper panel, andthe bottom panel shows the relative quantitation.
CSV: combustion with silver needles
CSS: combustion with stainless steel needles
NSV: non-combustion with silver needles
NSS: non-combustion with stainless steel needles
CON: control-no needle(4) Temperature decreasing in non-combustion groups
The final decreasing temperature data for NSV, NSS, andCON in Experiments A and B were not normally
distributed, so the Steel-Dwass test was used to examine
whether there were any statistically significant differencesamong the nine groups; the results showed no significant
differences.
2. Expression of alpha B-crystallin
Figure 6 shows the representative blots of αB-crystallinin the soleus of each group and GAPDH as the internal
control, and the bar chart of the mean values for eachYajima Michiko, et al.. Japanese Acupuncture and Moxibustion; 2023; Vol.17: 1–11

group. Regarding αB-crystallin expression in the soleus of
the stimulated side after correction for GAPDH
expression, when the CON group was set at 100 (75.23),
CSV was 299.29 (115.57), CSS was 160.62 (85.39), NSV
was 121.51 (84.11), NSS was 67.52 (51.19), and eyeballs
as the positive control was 283.25. Statistical significancewas examined using Dunnett’s multiple comparison test.
The results revealed a significant difference between the
CON and CSV groups, with the CSV group showing
significantly higher expression of αB-crystallin than the
CON group (P < 0.01).
Figure 7 shows the representative blots of αB-crystallinin the EDL of each group and GAPDH as the internal
control, and the bar chart of the mean values of each group.
Regarding αB-crystallin expression in the EDL of thestimulated side after correction for GAPDH expression,
when CON of soleus was set at 100, CSV was 30.73(20.03), CSS was 14.33 (9.62), NSV was 35.12 (31.21),
NSS was 31.05 (26.42), and CON was 32.16 (12.90). As
the data did not follow a normal distribution, Steel’s test
was used for multiple comparisons, and the results showedno significant differences among the intervention andcontrol groups.
IV. Discussion
1. Relationship between the temperature increase
required for chaperone activity and temperaturechanges with different needle materials
The fact that the courses of the skin surface temperaturechange in Experiments A and B were almost similarsuggests that the course of the deep tissue temperature inExperiment B was almost similar to the course ofExperiment A. Therefore, in Experiment B, it must be thedeep tissue temperature of the moxa needle combustion
group increased approximately 4.5°C with silver needles
and approximately 3°C with stainless steel needles. It is
speculated that this difference in temperature increasecaused a difference in the effects on the expression ofmolecular chaperones.
Many previous studies have examined the temperature
setting for thermal stimulation of the skin surface, but not
those for deep tissue 14–18). We consider that heat shockprotein expression in skeletal muscles should be examinedin relation to deep tissue temperature changes.
From the results of this study, it can be inferred that a
deep tissue temperature increase from 4°C to 5°C forabout 3 minutes is required to increase αB-crystallin
expression. In CSS group, the duration of temperature
differences of over 3°C was short, so it is assumed thatsuch a brief and weak stimulation would have little or noeffect on the increase in heat shock proteins.
On the other hand, in the CSV group, the deep tissue
temperature increased by about 4.5°C at the third burning,
and the duration over 3°C was 10 times longer than that in
the CSS group (Fig.3). As it is fairly certain that theradiant heat from the combustion of moxa balls was

9almost similar for both CSV and CSS, the fact that bothskin surface and deep tissue temperatures weresignificantly higher in CSV than in CSS can be attributed
to differences in the heat conduction of the needles.
During a local temperature increase, heat diffusion byblood flow is considered to occur, and unless the heat
supply exceeds that rate, the temperature will not rise
above a certain level. Therefore, it is considered that in
CSV with a large heat supply, the temperature increased
stepwise with each burning, and in CSS with not a large
heat supply, the temperature increased similarly to thesecond and third burnings.
A comparison of the duration of the deep tissue
temperature rise and the integral amount of temperature
revealed that the difference between the CSV and CSS
groups was greater for the integral amount of temperaturerise than for the duration (Fig. 3), which indicated that thedegree of increase during the period of the temperaturerise was smaller in CSS than in CSV, and thus, the rate ofdeep tissue temperature change was faster in CSV than in
CSS.
Our results revealed that moxa needle with silver needles
could rapidly raise the local deep tissue temperature. The
rate of temperature change affects the sensory warm andcold thresholds, with slower temperature changes
thresholds increase steadily20). As the rate of temperaturechange affects sensory perception, it may also affectchaperone expression. Sciatic denervation also affects
chaperone expression in the lower extremities18,21),
suggesting involvement of the central nervous system.
Moreover, the rate of temperature change ascending thesensory pathways may also be a stressor. However, we
recognize that this supposition requires an examination of
the rate of temperature change between CSV and CSS.
2. Characteristics of moxa needle thermal stimulation
Thermal stimulation using moxa needle, unlike
stimulation such as prolonged immersion in hot water orother thermotherapies, is localized, with a short durationof high temperature and a small stimulation area, so there
is no risk of heat stroke.
It has not been clear how the specific thermal stimulationof moxa needles acts as a stressor and leads to theexpression of heat shock proteins. The time course in CSV
that moxa needle with silver needles showed, not only
increases in the skin surface and deep tissue temperature
but also a considerable decrease in temperature after theinternal combustion of the moxa ball is over.
Although the difference was not statistically significant,in the NSV, NSS, and CON groups, both the surface anddeep tissue temperatures showed a slightly downward
trend in the NSV with silver needles compared with NSS
and CON (Fig. 5). The reason for this may be that somebody heat was lost due to conduction by the silver needle.
In the same way, the temperature decrease after
combustion in CSV was likely due to the high thermal
conductivity of silver, which allows heat to escape

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Yajima Michiko, et al.. Japanese Acupuncture and Moxibustion; 2023; Vol.17: 1–11

through the needles. When this increase and decrease wererepeated, it was presumed that the greater the fluctuation,
the greater the stressor, and the greater the increase of αBcrystallin expression.
Moxa needle stimulation does not apply the same amountof heat to all surfaces, unlike stimulation such as
immersion in warm water, and the temperature increase
varies with the depth of the muscle, so comparisonscannot be made under the same conditions. Therefore, theEDL values in this study are reference data. It is known
that the expression of αB-crystallin is low under thermal
stimulation in EDL, a fast-twitch muscle17,18). In thepresent study, αB-crystallin expression was lower in the
EDL than in the soleus muscle, and no stimulus-inducedchanges were observed. It is thought that the entire rat
lower leg was elevated by the combustion of a moxa ballapproximately 2 cm in diameter. However, moxa needle
was applied to the posterior surface of the lower leg, even
if the needle tip is right next to the fibula and in thevicinity of EDL, the temperature rise in the anterior
muscles of the lower leg was considered to be smaller thanthat in the posterior ones.
3. Effects and usefulness of moxa needle with silver
needles in soleus
The results of this study clarified that moxa needle withsilver needles compared to the stainless steel needles
influence the generation of molecular chaperones by
increasing the temperature not only on the skin surface,but also in the deep tissue. It is assumed that the effects ofmoxa needle with silver needles differ from those ofstainless steel needles, which have thermal stimulation by
mainly radiant heat in addition to mechanical stimulationby the insertion of the needle.
It has been reported that heat stress increases Hsp72
expression in soleus, plantaris, and EDL muscles, among
which, muscle mass gain occurs only in the soleus, which
is a slow antigravity muscle19); this suggests that it is not
Hsp72 that is responsible for the muscle mass gain. Alpha
B-crystallin is specific to slow-twitch muscle fibers andincreases by thermal stimulation, as shown in previous
studies22) and also suggested in the present study, and thus
may be functional for the increase in the mass of soleus
muscles which contains many slow-twitch muscle
fibers23).
Heat therapy is a very promising area of physiology
research, as it has the potential to contribute to approaches
addressing disuse atrophy in aging adults or those under
microgravity circumstances. The idea of using metal heat
conduction to raise the deep tissue temperature may
provide an opportunity to explore factors contributing to
the development of not only moxa needle but also further
new methods. It is suggested that a new position may beestablished as an effect of traditional moxa needle, and a
new aspect may be added to the purpose of moxa needle
therapy. The present results showed an increase in αBcrystallin, which is required for the maintenance of slow-

twitch muscle fibers, and so we will continue our researchin order to contribute to the prevention of muscle atrophy
and the strengthening of standing posture retention.
V. Conclusions
The results of this study suggest that a deep tissue
temperature increase from 4°C to 5°C for about 3 minutes
is required to increase αB-crystallin expression. Therefore,
in moxa needle stimulation to the posterior surface of thelower leg, the use of silver needles with high thermal
conductivity could increase the deep tissue temperatureand expression of αB-crystallin in soleus muscles.
Acknowledgments
We are grateful to Dr. Yoshihisa Naruse (Tokoha
University) and Mr. Yoshiyuki Okamoto (Meiji School ofOriental Medicine) for their excellent technical support ofthis study, and also grateful to Dr. Manami Itoi and Dr.
Satomi Ebara (Meiji University of Integrative Medicine)for their encouragement and continuous support.
Conflict of interest
There is no conflict of interest in this study.
The pre-study for this paper was presented at the annual
congress of the Japan Society of Acupuncture andMoxibustion in Tokyo (71th, 2022). The contents of thispaper have not previously been published in any language
and are not under consideration by another journal.
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