Xiong Luo-jie (熊羅節(jié)), Tian Yue-feng (田岳鳳), Fang Hui (方慧), Mao Kai-rong (毛凱榮), Zhi Bo-yuan (支博遠(yuǎn)),Sun Ni-na (孫妮娜), Li Wei (李瑋)

1 Hunan University of Chinese Medicine, Changsha 410208, China

2 Shanxi University of Chinese Medicine, Jinzhong 030619, China

Abstract

Keywords: Moxibustion Therapy; Direct Moxibustion; Indirect Moxibustion; Medicinal Cake-partitioned Moxibustion;Moxibustion Quantity; Acupuncture Points; Skin Temperature

The warming effect of moxibustion has become a hot topic in related basic research because of its important role in treatment. Many studies have reported the warming effect of indirect moxibustion, one of the treatment methods of moxibustion[1-2]. Most studies focus on the influence of moxibustion amount on temperature or the correlation between moxibustion temperature and the conduction of sensation along the meridian of different acupoints; there are also observational studies on the quantitative analysis of moxibustion treatment; however, in terms of quantitative standardization of indirect moxibustion,especially insufficient research on performance of different acupoints under the same moxibustion quantitative condition still remains. To provide relevant references for clinical developing quantitative indicators of moxibustion, the commonly used direct moxibustion and herbal cake-partitioned moxibustion in clinical practice were applied in this study, and the temperature differences between surface and inside of skin with different cone numbers on different acupoints were observed and analyzed.

1 Material and Methods

1.1 Experimental animals and groups

Half male and half female big-ear white rabbits(n=42), body weight (2.27±0.24) kg, were provided by Beijing Changyang Xishan Farm [Animal Certificate No.SCXK (Jing) 2016-0007]. Animals were adaptively fed for one week before experiment with a breeding temperature of 20-25 ℃ and a relative humidity of 50%-70%. According to the random number table method, the 42 rabbits were divided into 6 groups, a 1-cone direct moxibustion group, a 2-cone direct moxibustion group, a 3-cone direct moxibustion group,a 1-cone herbal cake-partitioned moxibustion group, a 2-cone herbal cake-partitioned moxibustion group, and a 3-cone herbal cake-partitioned moxibustion group,with 7 rabbits in each group. The laboratory temperature was maintained at 22-24 ℃ and the relative humidity was maintained at 45%-65% using airconditioning. The doors and windows were tightly closed and covered with opaque curtains without obvious indoor air flow.

1.2 Drugs and equipment

Moxa sticks and moxa wool without drugs [State Food and Drug Administration Approval No. Z32020253,Jiangsu Kangmei Pharmaceutical Co., Ltd., China]; JE502 electronic balance (Shanghai Puchun Measuring Instrument Co., Ltd., China); AI-3506M temperature recorder (Xiamen Yudian Automation Technology Co.,Ltd., China); WRNT-01 K-type thermoelectricity coupled temperature sensor (Xinghua Suma Electric Instrument Co., Ltd., China); PM-9000A+ type animal version multiparameter monitor (Hunan Ruibo Technology Co., Ltd.,China).

1.3 Intervention methods

Intervention was carried out on the second day after adaptive feeding. Shenque (CV 8) and bilateral Shenshu(BL 23) and Zusanli (ST 36) were positioned referring to the commonly used animal acupoint positioning method provided by theExperimental Acupuncture Science[3]and anthropomorphic comparison method.

Zusanli (ST 36) was located at the 1/5 break point on the outside of the calf, approximately 1.2 cm below the capitulum fibulae and 1.0 cm behind the tibial crest;Shenshu (BL 23) was located 1.5 cm away from the middle between spinous processes of the second and third lumbar vertebrae; Shenque (CV 8) was at the center of the navel, on the linea alba abdominus.

1.3.1 Direct moxibustion group

Non-suppurative moxibustion was used for direct moxibustion.

Skin around the acupoints was shaved. Scissors were used to prepare skin with 1 mm of residual root remained, because depilatory cream may affect the surface tissue structure of skin. Rabbits were attached to the rabbit platform in a prone position. After acupoint temperature changes at bilateral Shenshu(BL 23) were observed, rabbits were further attached in a supine position to observe acupoint temperature changes at Shenque (CV 8) and Zusanli (ST 36).Meanwhile, the dynamic monitor was connected to observe the real-time status of rabbits during treatment.Our previously prepared mold was used to make coneshaped moxa cones with base diameter of 1.2 cm,height of 1.5 cm, and mass of about (0.22±0.03) g[4].Moxibustion was applied at above mentioned acupoints,with 1 cone, 2 cones or 3 cones by groups, once every other day for a total of 5 times.

1.3.2 Herbal cake-partitioned moxibustion group

According to the dosage ratio of Liu Wei Di Huang pills, the drugs were ground into powder and passed through a 90-mesh sieve. Before intervention, the powder was mixed with excipients at a ratio of 1:1:1.2 among powder, excipient and water, and then made into cylindrical herbal cakes with base diameter of 1.6 cm, height of 0.4 cm and mass of (0.90±0.05) g; and each herbal cake was pierced for 5 small holes with a 1 mm diameter toothpick. Rabbit hair around acupoints was shaved to prepare the skin; rabbits were then attached to the rabbit platform and connected to dynamic monitor for real-time observation during treatment. The herbal cake was first placed at Shenshu(BL 23) after the rabbit was successfully fixed in the prone position. Rabbits were then attached to the rabbit platform in the supine position and with the herbal-cake placed at Shenque (CV 8) and Zusanli (ST 36)for observation when moxibustion in the prone position was completed. The moxa cones with the same specifications as in the direct moxibustion were put on the herbal cake and lighted with incense thread. The moxa cone was replaced when burnt till two-fifths to ensure that the highest surface temperature did not exceed 50 ℃. Moxibustion with 1 cone, 2 or 3 cones was applied to each acupoint in each group respectively,once every other day for 5 times.

1.4 Temperature measurement method

Placement method of subcutaneous thermoelectricity couple: According to the method of Li Y,et al[5], a K-type ultra-thin thermoelectricity couple was loaded in the green needle (0.8 mm in diameter and 38 mm in length) of a disposable syringe and penetrated under the skin at 1 cm away from the acupoint to be tested. The detecting head reached directly under the moxa cone or herbal cake.

After the thermoelectricity coupled temperature sensor was accurately placed under the acupoint(confirmed with fingers), the skin surface probe was placed at the acupoint. Due to the use of ultra-fine thermoelectricity couples and preventing limb movement caused position change of sensor during binding, at least 1 cm or more of the wire was left under the skin. The thermoelectricity coupled sensor was left in the designated subcutaneous area after the needle was withdrawn from the skin. After the two thermoelectricity couples on the inside and the outside were placed accurately, the temperature recorder was connected to record the temperature. The subcutaneous temperature measurement point can effectively measure the temperature at which the heat of moxibustion penetrates between the carnosus and the deep fascia (the carnosus of a rabbit is equivalent to the deep layer of human superficial fascia)[6].

Method to place the thermoelectricity couple on the skin surface: The ultra-fine thermoelectricity couple was fixed at the acupoint with medical tape, the moxa cone was placed directly above the thermocouple sensor,and the temperature with moxa cone center as the temperature measurement point was measured. The herbal cake was placed directly above the thermoelectricity sensor for herbal cake-partitioned moxibustion, and the herbal cake center was used as the temperature measurement point.

1.5 Temperature and time record

The temperature recorder was set to automatically record once every 5 s. The ignition time of each cone,the time point of skin temperature rising (when the skin temperature was 0.5 ℃ higher than the normal body temperature), the time point of maximum surface temperature, the time point of maximum subcutaneous temperature, the lowest temperature time during cone changing period and the ending temperature were manually recorded. The surface and inside temperature values of skin around different acupoints with different moxibustion methods and different cone numbers were recorded. The difference between surface and inside temperature was determined by subtraction of the highest temperature.

1.6 Statistical methods

SPSS 23.0 software was applied for statistical analysis.Normality test of relevant data was first carried out. The normally distributed measurement data were presented as mean ± standard deviation (±s), and the data not conforming to normal distribution were normalized before further analysis. Repeated measurements were performed for different acupoints[Shenque (CV 8), Shenshu (BL 23) and Zusanli (ST 36)],different moxibustion methods (direct moxibustion,herbal cake-partitioned moxibustion) and different time points (before moxibustion, day 1, day 3, day 5, day 7,and day 9 of moxibustion). After the homogeneity of variance test, a multi-factor analysis of variance (ANOVA)for repeated measurement data was performed to analyze the effects of different grouping factors, time factors, and interactions. One-way ANOVA was used for pairwise comparison of results satisfying the sphere test;the multi-factor ANOVA was used for pairwise comparison if the sphere test was not satisfied. The least significant differencet(LSD-t) test was used for post-hoc analysis. The independent samplet-test was used for comparison between two groups. The test standard wasα=0.05, and the difference was statistically significant withP<0.05.

2 Results

2.1 Comparing the best temperatures for cone changing

The maximum temperature of the skin surface at different acupoints was measured, showing no significant differences among Shenque (CV 8), Shenshu(BL 23) and Zusanli (ST 36), (allP>0.05). Further comparison of cone changing temperature at different acupoints showed no statistically significant differences among different acupoints (allP>0.05), (Table 1).

It can be seen in this experiment that there are no statistically significant differences in the best cone changing temperature between different acupoints. The best cone changing temperature should be about 46 ℃to ensure that the highest skin surface temperature at the acupoints for herbal cake-partitioned moxibustion does not exceed 50 ℃ (Table 1).

2.2 Comparisons of acupoints, cone numbers and moxibustion methods

The skin surface temperature differences among different acupoints [Shenque (CV 8), Shenshu (BL 23)and Zusanli (ST 36)], different cone numbers (1 cone, 2 cones and 3 cones) and different moxibustion methods(direct moxibustion and herbal cake-partitioned moxibustion) at different time points (before moxibustion, day 1, day 3, day 5, day 7, and day 9 of moxibustion) were completely recorded (Table 2).significant (F=5.923,P=0.004). Please see Table 3.

Table 1. Comparing the cone changing temperature and the highest skin surface temperature in herbal cake-partitioned moxibustion at different acupoints ( ±s, ℃)

Table 1. Comparing the cone changing temperature and the highest skin surface temperature in herbal cake-partitioned moxibustion at different acupoints ( ±s, ℃)

Acupoints n Cone changing temperature Highest skin surface temperature Shenque (CV 8) 21 46.41±0.44 Shenshu (BL 23) 21 46.41±0.47 Zusanli (ST 36) 21 46.31±0.61 F-value 1.854 P-value 0.158 49.22±0.51 49.19±0.49 49.18±0.55 0.091 0.913

Table 2. Skin surface and inside temperature differences in two moxibustion methods with different moxa cones at different acupoints ( ±s, ℃)

Table 2. Skin surface and inside temperature differences in two moxibustion methods with different moxa cones at different acupoints ( ±s, ℃)

Time Acupoint Moxibustion method Cone number Before moxibustion Day 1 Day 3 Day 5 Day 7 Day 9 Shenque(CV 8)Shenshu(BL 23)Zusanli(ST 36)Direct moxibustion Herbal cakepartitioned moxibustion Direct moxibustion Herbal cakepartitioned moxibustion Direct moxibustion Herbal cakepartitioned moxibustion 1 cone 2.52±1.05 4.79±2.61 6.46±1.80 4.64±1.40 5.06±1.81 6.71±1.12 2 cones 1.69±0.25 7.10±1.15 7.43±2.21 7.43±2.41 6.64±1.22 6.46±1.22 3 cones 2.28±0.64 6.60±2.63 6.97±1.29 6.03±1.09 6.06±1.19 5.56±1.78 1 cone 2.10±0.44 3.29±1.49 4.69±1.50 4.01±2.61 3.31±1.35 4.46±1.62 2 cones 1.55±0.81 6.10±2.54 5.30±1.27 5.14±1.24 5.50±4.45 4.54±1.44 3 cones 2.23±0.55 3.80±0.93 3.17±0.88 4.09±0.67 3.57±0.51 4.67±1.50 1 cone 1.74±0.45 8.14±1.15 7.51±3.09 8.27±3.01 7.63±2.79 7.70±3.89 2 cones 1.60±0.49 8.80±3.40 9.01±2.07 7.21±2.46 6.99±2.13 8.04±3.09 3 cones 1.78±0.15 7.93±1.79 7.41±1.32 7.64±1.77 9.51±1.19 9.83±2.19 1 cone 1.73±0.45 5.57±2.30 4.47±2.97 6.06±2.20 5.43±2.77 5.49±2.97 2 cones 1.45±0.48 6.57±2.86 6.74±2.03 6.40±2.97 5.73±1.00 5.30±0.93 3 cones 1.76±0.44 5.13±1.62 5.57±0.76 4.77±0.81 6.27±1.25 6.43±1.77 1 cone 1.73±0.66 3.99±1.17 4.50±2.00 5.11±2.28 4.61±0.66 7.19±2.20 2 cones 0.76±0.26 6.16±1.73 5.79±2.46 5.79±1.51 4.83±2.13 4.73±2.17 3 cones 1.25±0.44 5.71±1.29 4.93±1.47 4.64±1.39 4.71±1.60 4.90±1.58 1 cone 1.49±0.22 4.20±1.98 3.99±1.95 4.20±0.98 3.53±1.96 4.03±2.79 2 cones 0.86±0.29 4.97±1.39 4.04±0.95 3.67±1.79 3.71±1.33 4.96±1.60 3 cones 1.14±0.20 4.29±0.83 3.60±1.16 3.00±1.51 2.99±0.72 3.30±1.28

2.2.2 The interaction of acupoints, moxibustion methods and time

Before comparison, Mauchly’s spherical hypothesis test (P=0.181) showed that the interaction of acupoint ×moxibustion method × time had no statistically significant effect on the skin temperature difference(F=0.467,P=0.764). Interaction of two factors showed that the interaction of moxibustion method × acupoint was not statistically significant (F=2.059,P=0.170); the interaction of time × moxibustion method was statistically significant (F=4.962,P=0.002), (Table 3).

Table 3. Multi-factor analysis of variance for within subject effect of repeated measure data

2.2.1 The interaction of acupoints, cone numbers and time

Before comparison, the Mauchly’s spherical hypothesis test (P=0.410) showed that the interaction of acupoint × cone number × time had no statistically significant effect on skin temperature difference(F=1.691,P=0.192). Further analysis showed that interaction of time × cone number had no statistically significant effect on skin temperature difference(F=2.352,P=0.099); the interaction of cone number ×acupoint was statistically significant (F=3.000,P=0.002);the interaction of time × acupoint was statistically

2.3 Comparing the same acupoint with different cone numbers and different moxibustion methods

2.3.1 Comparing results of different cone numbers under the same acupoint

One-way ANOVA for different cone numbers under the same acupoint at different time points showed that the differences at Shenque (CV 8) on day 1 and day 5 of moxibustion were statistically significant (allP<0.05).Comparison of different cone numbers of Shenshu(BL 23) showed statistical differences on day 3, day 7 and day 9 of moxibustion (allP<0.05). There were statistical differences at Zusanli (ST 36) on day 1 and day 9 of moxibustion (allP<0.05), (Table 4).

2.3.2 Comparing results of different moxibustion methods at the same acupoint

Independent samplet-test analysis of different moxibustion methods at the same acupoint at different time points showed that temperature differences at Shenque (CV 8) were statistically different at the 5 time points after moxibustion except for before treatment(allP<0.05). The comparison results of different moxibustion methods at Shenshu (BL 23) showed statistical differences on day 1, day 3, day 7 and day 9 of moxibustion (allP<0.05). There were statistical differences at Zusanli (ST 36) only on day 5 and day 7 of moxibustion (bothP<0.05), (Table 5).

2.4 Comparison of different acupoints with different cone numbers and different moxibustion methods

2.4.1 Comparison of different acupoints with the same cone number

One-way ANOVA was performed for the results of different acupoints with the same cone number, which showed that the temperature differences between the surface and inside of skin at different acupoints with the same cone number were statistically different (allP<0.05); pairwise comparison showed statistical significance between Shenshu (BL 23) and Zusanli (ST 36)when 1 cone was used (allP<0.05); between Shenque(CV 8) and Zusanli (ST 36), between Shenshu (BL 23)and Zusanli (ST 36) when 2 cones were used (allP<0.05);pairwise comparison of acupoints with 3 cones revealed statistically significant differences (allP<0.05), (Table 6).2.4.2 Comparing different acupoints with same moxibustion method

Results of one-way ANOVA for different acupoints under same moxibustion method showed statistical differences between different acupoints after direct moxibustion or herbal cake-partitioned moxibustion (allP<0.05), (Table 7).

Table 4. Comparison of temperature difference between the surface and inside of skin with different cone numbers at the same acupoint ( ±s, ℃)

Table 4. Comparison of temperature difference between the surface and inside of skin with different cone numbers at the same acupoint ( ±s, ℃)

Note: Compared with 1-cone group at the same acupoint, 1) P<0.05; compared with 2-cone group at the same acupoint, 2) P<0.05

Acupoint Cone number n Before moxibustion Day 1 Day T 3i m e Day 5 Day 7 Day 9 1 cone 14 2.31±0.27 4.04±0.41 5.57±0.45 4.33±0.54 4.19±0.48 5.59±0.40 S(C hVe n 8 q)u e2 3 cc oo nn ee ss 11 44 12..62 25± ±00..12 71 65..62 00± ±00..55 89 12))65..30 67± ±00..52 32 65..20 96± ±00..52 40 12))64..08 71± ±00..82 68 55..51 01± ±00..34 72 1 cone 14 1.74±0.11 6.86±0.55 5.99±0.63 7.16±0.65 6.53±0.78 6.59±0.40 S(B hLe n 2 s3h)u 23 cc oo nn ee ss 11 44 11..57 27± ±00..11 51 76..65 93± ±00..74 1 8 6 7..4898±±0 0..26 1 0 2)66..82 11± ±00..72 3 7 7 6..8396±±00..35711)68..61 73± ±00..62 2 81)1 cone 14 1.61±0.13 4.09±0.53 4.24±0.35 4.66±0.45 4.07±0.37 5.61±0.38 Z(S uTs a 3 n6l i)2 3 cc oo nn ee ss 11 44 11..21 19± ±00..10 09 55..50 60± ±00..52 06 11))44..92 16± ±00..63 48 43..78 32± ±00..63 08 43..28 75± ±00..33 64 44..81 40± ±00..54 16 11))2)

Table 5. Comparing skin temperature difference between different moxibustion methods at the same acupoint ( ±s, ℃)

Table 5. Comparing skin temperature difference between different moxibustion methods at the same acupoint ( ±s, ℃)

Note: DM=Direct moxibustion; HPM=Herbal cake-partitioned moxibustion; BM=Before moxibustion; compared with direct moxibustion at the same acupoint, 1) P<0.05, 2) P<0.01

Acupoint Moxibustion method n BM Day 1 Day 3 Time Day 5 Day 7 Day 9 ShenqueDM 21 2.16±0.19 6.16±0.57 6.95±0.50 6.03±0.45 5.92±0.28 6.24±0.23(CV 8)HPM 21 1.96±0.09 4.40±0.461) 4.39±0.292) 4.41±0.371) 4.13±0.451) 4.56±0.172)ShenshuDM 21 1.71±0.08 8.29±0.23 7.98±0.44 7.71±0.50 8.04±0.40 8.52±0.66(BL 23)HPM 21 1.65±0.10 5.76±0.511) 5.60±0.502) 5.74±0.42 5.81±0.451) 5.74±0.402)ZusanliDM 21 1.25±0.09 5.29±0.32 5.07±0.38 5.18±0.59 4.72±0.29 5.61±0.56(ST 36)HPM 21 1.16±0.04 4.49±0.29 3.88±0.37 3.62±0.131) 3.41±0.211) 4.10±0.45

Table 6. Comparing temperature difference between the surface and inside of skin at different acupoints with the same cone number ( ±s, ℃)

Table 6. Comparing temperature difference between the surface and inside of skin at different acupoints with the same cone number ( ±s, ℃)

Note: With the same cone number, compared with Shenque (CV 8), 1) P<0.01, compared with Shenshu (BL 23), 2) P<0.01

Cone number n Shenque (CV 8) S A he cnusphoui n(t BL 23) Zusanli (ST 36)F-value P-value 1 cone 14 4.34±0.12 5.81±0.311) 4.05±0.192) 8.253 0.026 2 cones 14 5.41±0.17 6.15±0.36 4.19±0.061)2) 112.889 0.000 3 cones 14 4.59±0.22 6.17±0.151) 3.71±0.211)2) 38.706 0.001

Table 7. Comparing skin temperature difference at different acupoints with the same moxibustion method ( ±s, ℃)

Table 7. Comparing skin temperature difference at different acupoints with the same moxibustion method ( ±s, ℃)

Note: With the same moxibustion method, compared with Shenque (CV 8), 1) P<0.05, 2) P<0.01; compared with Shenshu (BL 23),3) P<0.05

Moxibustion method n Shenque (CV 8) S A he cnusphoui n(t B L 23) Zusanli (ST 36)F-value P-value Direct moxibustion 21 5.579±0.185 7.042±0.2791) 4.518±0.1232)3) 202.577 0.000 Herbal cake-partitioned moxibustion 21 3.974±0.139 5.048±0.2291) 3.442±0.1601)3) 16.112 0.007

3 Discussion

Warming effect is the main way of moxibustion to exert its therapeutic effect. Some scholars suggested that the size, quantity, and weight should be used as important references in the indicator recommendations for standardization of moxibustion amount[7]. At present, most basic research experiments on moxibustion use the absolute maximum temperature of moxibustion as the observation indicator for further research[8-9]. Our research group identified that acupoints have influences on moxibustion amount in the long-term clinical practice of moxibustion. However,comparative studies on the difference between skin surface and inside temperature of different acupoints under certain surface temperature still remain insufficient.

Studies have reported that the tolerance to thermal pain of human body is 44-48 ℃. The noxious temperature threshold is higher than 44 ℃, and the correlation effect between 48 ℃ and 52 ℃ is relatively small[10]. Settle JAD[11]reported that the skin was quickly damaged when the human skin temperature exceeded 51 ℃. Based on the preliminary experiment of actual heat-resistance observation in rabbits, we revealed that when temperature exceeded 50 ℃, rabbits in the herbal cake-partitioned moxibustion group were mostly restless and intolerant to high temperature. Therefore,this study limited the maximum skin surface temperature to 50 ℃.

Studies have shown that moxibustion temperature not only exerts effects on the surface skin of the acupoints, but also has a certain effect on the deep tissues under the acupoints[8]. During moxibustion treatment, the inside temperature of acupoints was gradually increased with the increase of moxibustion time[12], but the influence of moxibustion heat was also gradually decreased as the tissue depth increased.

Results of this study on temperature difference between the surface and inside of skin at different acupoints showed that under the condition of maximum skin surface temperature at 50 ℃, total heat resistance of herbal cake was limited, so the herbal cake first absorbed the heat produced by burning moxa during moxibustion treatment. When the heat reached the absorption limit of herbal cake, the heat continued to be transferred into skin and then gradually into subcutaneous tissues. By controlling the cone changing temperature in herbal cake-partitioned moxibustion,the highest temperature of skin surface can be accurately controlled under a certain herbal cake amount. This result can provide reliable data support for subsequent comparison of different factors.Subsequent repeated measurement multi-factor comparison results showed no obvious interaction between acupoints and cone numbers, acupoints and moxibustion methods at different times. Mori H,et al[13]identified that repeated moxibustion did not increase the skin surface temperature, which further confirmed the multiple comparison results of this study.

At the same acupoint, comparison of different cone numbers was statistically significantly different. The differences at Shenque (CV 8) on day 1 and day 5 were statistically significant. Temperature differences between the surface and inside of skin with different cone numbers were increased with treatment numbers,which were mostly focusing around 5.5 ℃, but Shenshu(BL 23) in the 3-cone group showed an increasing trend of temperature difference between the surface and inside of skin with increased treatment time;temperature differences with different cone numbers at Zusanli (ST 36) were only statistically significant on day 1 and day 9. These results suggested that at the same acupoint, different cone numbers showed different trends. Similarly, at the same acupoint, the difference between different moxibustion methods was also statistically significant. This showed that at the same acupoint, different moxibustion methods had different effects on temperature difference between the surface and inside of skin. We found that the skin temperature difference of Shenshu (BL 23) was greater than that of Shenque (CV 8) and Zusanli (ST 36) by comparing different acupoints with different cone numbers;comparison of different acupoints with different moxibustion methods also showed similar results.

Based on the results of the same acupoint with different cone numbers and different moxibustion methods, we identified that under the same maximum skin temperature at different acupoints, the subcutaneous temperature varied significantly with cone numbers and moxibustion methods. Moreover,the skin temperature difference between the surface and inside of Shenshu (BL 23) was higher than that of Shenque (CV 8), and the lowest was at Zusanli (ST 36).

Studies have shown that moxibustion at acupoints located at the chest and abdomen (belonging to yin)requires less physical heat than acupoints located at the lumbar and back (belonging to yang)[14]. This may be the reason why meridian difference affects temperature difference between the surface and inside of acupoints.In addition, results showed that skin temperature difference at the same acupoint in the direct moxibustion group was more significant than that in the herbal cake-partitioned moxibustion group at the same acupoint. This is because that skin is a shielding layer between the inside and outside temperature of the body, direct moxibustion has a short burning time and the continuous thermal stimulation is lower than that of herbal cake-partitioned moxibustion, resulting in less influence on deep tissues and stronger skin shielding effect. Therefore, the temperature difference between the surface and inside skin at acupoints was greater in direct moxibustion than in herbal cake-partitioned moxibustion. This also showed that the skin penetration ability of herbal cakes was higher than that of direct moxibustion.

In summary, by controlling the maximum temperature of moxibustion treatment, we found that temperature difference between the surface and inside of skin around different acupoints under the same condition was different, and the time required to reach the highest temperature for different acupoints was also significantly different. Experimental studies have shown that the temperature effect of moxibustion treatment is closely related to transient receptor potential (TRP) receptors; the activation temperature of different receptors is different. Transient receptor potential vanilloid (TRPV) 1 plays a role when temperature is above 43 ℃[15], and the activation temperature of TRPV2 exceeds 53 ℃[16]. These findings suggested that different activation temperatures caused different feedbacks from the body. Results of this study showed that the same activation temperature caused different activation status due to different acupoints.This may be the reason why same moxibustion treatment results in different final curative effects with different acupoints. Previous studies on acupoints have mostly focused on comparing the difference in efficacy of different acupoints[17], but there is little insight into the structural characteristics of the acupoints themselves. Therefore, in the development of quantification standardization for herbal cakepartitioned moxibustion, not only factors of moxa and herbal cake, but also physical and chemical differences of acupoints need to be considered.

In this study, healthy animals were used as observation objects. However, the experimental observations on temperature difference between the surface and inside of skin at different acupoints under different pathological conditions were still insufficient.Meanwhile, as insufficient cone numbers were studied in this work, the change laws of the skin and the overall function with larger cone numbers, such as 6 or 9 cones,need to be supplemented[18]. Studies have shown that the curative effect of moxibustion treatment does not continue to increase as cone numbers increase, that is,there is a limit effect for moxibustion amount during treatment[19-21]. Therefore, comparison of multiple cone numbers and different moxibustion methods under pathological conditions may be performed in the future to further explore the adaptability of quantitative standards for moxibustion under different conditions.

Conflict of Interest

The authors declare that there is no potential conflict of interest in this article.

Acknowledgments

This work was supported by General Project of National Natural Science Foundation of China (國家自然科學(xué)基金面上項(xiàng)目, No. 81674062).

Statement of Human and Animal Rights

The treatment of animals conformed to the ethical criteria in this experiment.Received: 8 July 2020/Accepted: 10 March 2021