Liao Zong-li (廖宗力), Zhu Chong-zheng (朱重政), Tan Jing (譚靜), Luo Feng-jiao (羅鳳姣), Sun Lu (孫璐), Huang Wen-tao (黃文韜), Chen Yan-ping (陳艷萍), Yang Ren-da (陽仁達), Chang Xiao-rong (常小榮)

1 Hunan University of Chinese Medicine, Changsha 410208, China

2 Xiniujiao Community Health Service Station, Jingxi Street, Baiyun District in Guangzhou City, Guangdong 510515, China

3 Huizhou Hospital of Traditional Chinese Medicine, Guangdong 516000, China

4 Department of Stomach Surgery, Sun Yat-sen University Cancer Center, Guangdong 510060, China

5 The Second Affiliated Hospital of Hunan University of Chinese Medicine, Changsha 410005, China

Abstract Objective: To observe the effects of laurocapram and borneol as transdermal penetration enhancers applied to herbal cake-partitioned moxibustion on liver lipids, hormone-sensitive lipase (HSL) and hydroxymethylglutaryl CoA (HMG-CoA) reductase in hyperlipidemia rabbits.

Keywords: Moxibustion Therapy; Herbal Cake-partitioned Moxibustion; Hyperlipidemias; Laurocapram; Isoborneol; Sterol Esterase; Hydroxymethylglutaryl CoA Reductases; Rabbits

Hyperlipidemia (HLP) is an abnormal lipid metabolism characterized by elevated levels of triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) or reduced high-density lipoprotein cholesterol (HDL-C) in blood and tissues. Traditional Chinese medicine (TCM) has been reported to be an effective and simple way to lower lipid levels[1].Da Huang(Radix et Rhizoma Rhei),Ze Xie(Rhizoma Alismatis),Shan Zha(Fructus Crataegi),Dan Shen(Radix Salviae Miltiorrhizae), andYu Jin(Radix Curcumae) have been proved significantly effective in lowering lipids by many experimental and clinical researches[2-5].

Transdermal penetration enhancers can be divided into two categories: chemical transdermal penetration enhancers and natural transdermal penetration enhancers. Chemical transdermal penetration enhancers include many types of chemical synthetic substances, of which the most widely used are alcohols, amides, surfactants and terpenes[6]. Some organizations in the United States approved the marketing of transdermal drug delivery systems that use ethanol, propylene glycol and others as transdermal penetration enhancers[7]. Natural transdermal penetration enhancers mainly derived from Chinese materia medica or their extracts. Compared with chemical transdermal penetration enhancers, natural transdermal enhancers have the advantages of less irritation, faster onset, and fewer adverse reactions and better effects.

In this study, laurocapram (a chemical transdermal penetration enhancer) and borneol (a natural transdermal penetration enhancer) were used as a drug delivery method during herbal cake-partitioned moxibustion for HLP model rabbits. The active drug components in serum were determined and analyzed using high performance liquid chromatography (HPLC); the levels of fat metabolism-related factors in liver tissues and serum were measured by enzyme-linked immunosorbent assay (ELISA). The lipid-lowering effects of different transdermal penetration enhancers in herbal cake-partitioned moxibustion and their effects on hormone-sensitive lipase (HSL) and hydro- xymethylglutaryl CoA (HMG-CoA) reductase will be investigated.

1 Experimental Materials

1.1 Experimental animals

Forty clean grade New-Zealand purebred rabbits, 3-month old, regardless of male or female, were provided by the Animal Experimental Center of Hunan University of Chinese Medicine [animal certificate number: SYXK (Xiang) 2013-0005]. Each rabbit was housed in an individual cage at the animal laboratory. The ambient temperature was kept at 20-25 ℃ and the humidity was maintained at 50%-70%. The cages, cage standers, feeders and water tumblers were sterilized before use. Before the formal experiment, 7 d of adaptive ordinary feeding was required.

1.2 Experimental drugs and reagents

Dan Shen(Radix Salviae Miltiorrhizae),Da Huang(Radix et Rhizoma Rhei),Shan Zha(Fructus Crataegi),Yu Jin(Radix Curcumae) andZe Xie(Rhizoma Alismatis) were provided by the Pharmacy of the First Affiliated Hospital of Hunan University of Chinese Medicine. These ingredients were ground into a fine powder and filtered through a 200 mesh sieve, and mixed at equal proportions.

1.3 Experimental reagents and instruments

Laurocapram (Cat. No.: 59227-89-3, Xinxiang Gaojin Pharmaceutical Co., Ltd., China), precisely measured 5 mL of the liposolubility laurocapram solution dissolved in 100 mL absolute ethanol solution, well mixed for later use; borneol (Cat. No.: 207-352-6, Shandong Baiweitang Herbal Pieces Co., Ltd., China), precisely weighed 2 g of borneol dissolved in 50 mL of absolute ethanol solution, and then added with 35 mL of distilled H2O to dilute; ulatan (Cat. No.: 51-79-6, Shandong Qilu Xinghua Pharmaceutical Co., Ltd., China); normal saline (Cat. No.: 65230, Guizhou Tiandi Pharmaceutical Co., Ltd., China); absolute ethanol (Cat. No.: E7023, SIGMA, Germany); Shenjiu 300 moxa cone (Oriental Type 1, Suzhou Oriental Moxa Factory, China); propylthiouracil (Batch No.: XW00515251) and cholesterol (Batch No.: 69008214), (Sinopharm Pharmaceutical Co., Ltd., China); egg yolk powder (Cat. No.: 57583-35-8, Bozhou Haichuan Egg Products Co., Ltd., China); standard: 23-acetazepine B (Batch No.: Z18J7L17980), emodin (Batch No.: C18F8Q29652), apigenin (Batch No.: Y27A6C1), tanshinone ⅡA (Batch No.: 20J8C40264) and curcumin (Batch No.: R25M6S1) (Shanghai Yuanye Biotechnology Co., Ltd., China); acetonitrile (Cat. No.: AH015-4, Guangzhou Fanhong Trading Co., Ltd., China); methanol (Cat. No.: 40064292, Sinopharm Pharmaceutical Co., Ltd., China). Methanol was chromatographically pure, water was ultrapure water, and other reagents were analytically pure.

SL02 centrifuge (Shanghai Wantong Experimental Instrument Technology Co., Ltd., China); AE100 electronic balance (METTER, Switzerland); Waters 1525 high performance liquid chromatography and Waters 2487 detector (Hangzhou Yalaibo Trading Co., Ltd., China).

1.4 Establishment of animal models

Forty New-Zealand rabbits were numbered by weights, and eight of them were randomly selected as the blank group using the random number table method and fed normally without any intervention.

High-fat diet: Per 100 g feed contained 15 g egg yolk powder, 0.5 g of cholesterol, and 5 g of lard, plus 10 mg/(kg·bw) propylthiouracil and basic fodder. The above ingredients were well mixed proportionally (the lard was pre-heated to be dissolved), then processed into granular high-fat feed. The remaining 32 rabbits were modeled with high-fat diets[8]by feeding 100 g of the high-fat diets daily at 8:00 a.m. The ordinary feed was added after the high-fat feed, and the total intake of food per rabbit per day was controlled between 100 g and 130 g. All rabbits were free to drinking water, for a total of 12 weeks.

Standards of successful modeling: Ten rabbits were randomly selected (3 in the blank group and 7 in the model group). The TC and TG in the serum were significantly increased in rabbits of the model group compared with the blank group (P＜0.05).

After the successful modeling, the remaining 32 rabbits were then randomly divided into 4 groups by random number table method, 8 rabbits in each group. Plus the blank group, there were 5 groups in total.

1.5 Intervention methods

1.5.1 Acupoint selection and positioning

Acupoints: Group A consisted of Juque (CV 14), bilateral Tianshu (ST 25) and Fenglong (ST 40); group B consisted of bilateral Pishu (BL 20), Ganshu (BL 18) and Xinshu (BL 15).

Acupoint locations: Acupoint location was determined according to theExperimental Acupuncture Science[9]and anthropomorphic comparison method. The line from the lower edge of the xiphisternal synchondrosis to the upper edge of the pubic symphysis was divided into 13 equal parts; Juque (CV 14) was 2 cun inferior to the sterno-costal notch. Tianshu (ST 25) was 3 cm lateral to the umbilicus. Fenglong (ST 40) was at the midpoint of the shin and the trailing edge of the fibula. Xinshu (BL 15) was 1.5 cun lateral to the lower border of the spinous process of the fifth thoracic vertebra. Ganshu (BL 18) was 1.5 cun lateral to the lower border of the spinous process of the ninth thoracic vertebra. Pishu (BL 20) was 1.5 cun lateral to the lower border of the spinous process of the eleventh thoracic vertebra.

1.5.2 Intervention and grouping

Blank group: Rabbits were fed normally without any intervention.

Model group: Rabbits were not treated.

Non-transdermal penetration enhancer group: Pure water was used as the solvent to make herbal cakes for moxibustion (without transdermal penetration enhancers).

Laurocapram group: Laurocapram solution was used as the solvent to make herbal cakes for moxibustion.

Borneol group: Borneol solution was used as the solvent to make herbal cakes for moxibustion.

During the intervention, rabbits were fed with about 60 g of normal feed daily at 8 a.m., with free access to drinking water.

1.5.3 Moxibustion methods

The rabbit was fixed on a rabbit stand. After the acupoints were positioned, hair in 2 cm around the acupoints was shaved and depilated with 8% sodium sulfide solution until the skin was exposed. And then the skin was rinsed with saline to prevent the skin from being damaged by the depilatory solution.

Prior to moxibustion, the Chinese medicine powder was made into a paste with different solvents (80 mL solvent per 100 g of powder) according to different groups, and pressed into herbal cakes of 3 mm thick, 1 cm in diameter by a self-made mould. Oriental Type 1 Shenjiu 300 moxa cone was selected. The burning time of each moxa was about 7.5 min at room temperature. At first the herbal cake was placed on the acupoint, and then the moxa was placed on the herbal cake after removal of the base to ensure the herbal cake was close to the acupoint skin, at last the moxa cone was ignited for moxibustion. Moxibustion was alternately performed at a group of acupoints each day with 4 moxa cones for each acupoint (about 30 min), once a day, for 4 consecutive weeks.

1.6 Sample collection and processing

1.6.1 Serum drug concentration

Solution preparation: Emodin, tanshinone ⅡA, curcumin, 23-acetylazenan and apigenin were weighed in appropriate amounts, dissolved in methanol to make up to 5 mL, and filtered through a 0.22 μm microporous filter for later use.

Cardiac blood collection procedures: Fixed the rabbit in a supine position on the rabbit table, and the fur on the left chest was shaved and disinfected with fortified iodine solution. The insertion point was 2.0-2.5 cm (3-4 intercostal) on the left thoracic-axillary level, where the heart beats. The needle should be inserted quickly and punctured at 45° to the horizontal plane toward the inner sternum. The blood was collected when the needle (9 gauge needle) penetrated about 3.5 cm deep. The serum was then separated after the blood was centrifuged at 12 000 r/min for 20 min. Referring to related literature[10], HPLC analysis was performed on each drug component in the serum of each group of animals.

1.6.2 ELISA detection

After 4 weeks of intervention, 3 mL of blood was collected and the rabbit serum was isolated for HSL and HMG-CoA reductase test by ELISA. Blood sample of one rabbit in the borneol group was not successfully collected because it died of diarrhea before the end of the experiment.

1.6.3 Enzyme assay

The rabbits were sacrificed after cardiac blood collection, the abdominal cavity was dissected, 100 mg of liver tissue was taken, and 1 mL of PBS was added for grinding. After grinding, centrifugation was performed at 4 000 r/min for 10 min, and the supernatant was taken for testing or storage. TC and TG were detected by enzyme assay. HDL-C and LDL-C were measured by direct one-step method. Apolipoprotein-A1 (Apo-A1) and apolipoprotein-B (Apo-B) were determined by transmission turbidimetry.

1.7 Statistical methods

All the experimental data were statistically processed by SPSS 18.0 Windows software. The measurement data were first tested for normality, and those conforming to the normal distribution were expressed as mean ± standard deviation (±s). For comparison between groups, the least significant difference (LSD) method was used if the variance was homogeneity; the Tamhane method was used if the variance was uneven. The rank-sum test was used when normal distribution was not satisfied.P＜0.05 was considered statistically significant.

2 Results

2.1 Drug penetration test results

HPLC analysis results showed that the drug penetration of the model group was 0; compared with the model group, the drug metabolism components were detected in the non-transdermal penetration enhancer, the laurocapram, and the borneol groups, and the between-group differences were statistically significant (P＜0.01). Compared with the non- transdermal penetration enhancer group, the drug penetrations in the laurocapram and the borneol groups were increased, and the between-group differences were statistically significant (allP＜0.05). Compared with the laurocapram group, the penetrations of apigenin, curcumin, emodin and 23-acetylazestirol in the borneol group were increased, and the between-group differences were statistically significant (allP＜0.05). There were no significant between-group differences comparing the content of tanshinone ⅡA (P＞0.05), (Table 1).

2.2 Serum HSL and HMG-CoA reductase test results

Compared with the blank group, the levels of HSL and HMG-CoA reductase in the model group increased significantly (bothP＜0.05); compared with the model group, the level of HSL increased in the non- transdermal penetration enhancer, the laurocapram, and the borneol groups, while the HMG-CoA reductase level was significantly decreased, and the between- group differences were statistically significant (allP＜0.05); compared with the non-transdermal penetration enhancer group, the level of HSL was significantly increased, and the level of HMG-CoA reductase was significantly decreased in the laurocapram and the borneol groups, and the between- group differences were statistically significant (allP＜0.05); compared with the laurocapram group, the level of HSL in the borneol group was significantly increased (P＜0.05), while there was no significant difference between groups in HMG-CoA reductase (P＞0.05), (Table 2).

Table 1. Comparison of the main components of drugs in serum (±s, g/mL)

Table 1. Comparison of the main components of drugs in serum (±s, g/mL)

Note: Compared with the model group, 1) P＜0.01; compared with the non-transdermal penetration enhancer group, 2) P＜0.05; compared with the laurocapram group, 3) P＜0.05

Tanshinone IIA 23-acetylazestirol Model 8 0 0 0 0 0 Non-transdermal penetration enhancer 8 0.033±0.0181) 0.057±0.0111) 0.078±0.0141) 0.062±0.0121) 0.033±0.011) Laurocapram 8 0.057±0.0131)2) 0.097±0.0151)2) 0.099±0.0151)2) 0.175±0.0211)2) 0.089±0.0121)2)Borneol 7 0.141±0.0221)2)3) 0.142±0.0231)2)3) 0.112±0.0251)2)3) 0.182±0.0331)2) 0.103±0.0131)2)3)

Table 2. Comparison of the serum HSL and HMG-CoA reductase (±s)

Table 2. Comparison of the serum HSL and HMG-CoA reductase (±s)

Note: NTPEG=Non-transdermal penetration enhancer group; compared with the blank group, 1) P＜0.05; compared with the model group, 2) P＜0.05; compared with the non-transdermal penetration enhancer group, 3) P＜0.05; compared with the laurocapram group, 4) P＜0.05

Group n HSL (U/L) HMG-CoA reductase (ng/mL)Blank 8 1217.4±153.35 62.19±3.48 Model 8 1356.4±81.571) 77.52±4.171) NTPEG 8 1478.5±96.542) 63.37±4.282) Laurocapram 8 1713.3±136.242)3) 56.11±1.152)3)Borneol 7 1877.3±63.532)3)4) 53.29±4.532)3)

2.3 Liver TC, TG, LDL-C and Apo-B test results

Compared with the blank group, the levels of LDL-C, TG, TC and Apo-B in the rabbits of the model group were significantly increased (P＜0.05); compared with the model group, the levels of LDL-C, TG, TC and Apo-B in the non-transdermal penetration enhancer, the laurocapram and the borneol groups were significantly decreased (P＜0.05); compared with the non- transdermal penetration enhancer group, the levels of TG and TC in the laurocapram group were significantly decreased (P＜0.05), and the levels of LDL-C, TG, TC and Apo-B in the borneol group were significantly decreased (P＜0.05); there were significant differences in the levels of LDL-C, TG, and Apo-B between the laurocapram group and the borneol group (P＜0.05), (Table 3).

Table 3. Comparison of liver TC, TG, LDL-C and Apo-B levels (±s, mmol/L)

Table 3. Comparison of liver TC, TG, LDL-C and Apo-B levels (±s, mmol/L)

Note: Compared with the blank group, 1) P＜0.05; compared with the model group, 2) P＜0.05; compared with the non-transdermal penetration enhancer group, 3) P＜0.05; compared with the laurocapram group, 4) P＜0.05

Group n LDL-C TG Blank 8 1.08±0.04 2.81±0.54 Model 8 2.05±0.031) 4.06±0.251) Non-transdermal penetration enhancer 8 1.45±0.022) 3.45±0.062) Laurocapram 8 1.47±0.032) 3.27±0.052)3) Borneol 8 1.27±0.012)3)4) 2.93±0.172)3)4) TC Apo-B 0.46±0.18 0.65±0.01 0.78±0.161) 1.04±0.021) 0.65±0.122) 0.80±0.022) 0.56±0.052)3) 0.77±0.052) 0.55±0.032)3) 0.64±0.032)3)4)

2.4 Liver HDL-C and Apo-A1 test results

Compared with the blank group, the levels of HDL-C and Apo-A1 in the rabbit liver of the model group decreased significantly, and the differences between groups were statistically significant (allP＜0.05). Compared with the model group, the levels of HDL-C and Apo-A1 in rabbit livers were significantly increased in the non-transdermal penetration enhancer, the laurocapram, and the borneol groups, and the between-group differences were statistically significant (allP＜0.05). Compared with the non-transdermal penetration enhancer group, the level of Apo-A1 in the laurocapram group, HDL-C and Apo-A1 in rabbit liver of the borneol group increased, and the between-group differences were statistically significant (allP＜0.05). There were no significant differences in the HDL-C and Apo-A1 levels between the borneol and the laurocapram groups (bothP＞0.05), (Table 4).

Table 4. Comparison of liver HDL-C and Apo-A1 levels (±s, mmol/L)

Table 4. Comparison of liver HDL-C and Apo-A1 levels (±s, mmol/L)

Note: NTPEG=Non-transdermal penetration enhancer group; compared with the blank group, 1) P＜0.05; compared with the model group, 2) P＜0.05; compared with the non-transdermal penetration enhancer group, 3) P＜0.05

Group n HDL-C Apo-A1 Blank 8 0.65±0.02 0.85±0.01 Model 8 0.44±0.031) 0.53±0.031) NTPEG 8 0.58±0.122) 0.62±0.032) Laurocapram 8 0.63±0.062) 0.73±0.132)3) Borneol 8 0.66±0.032)3) 0.74±0.052)3)

3 Discussion

Hyperlipidemia is a complex disorder of abnormal blood lipid metabolism. The name of this condition does not exist in ancient Chinese medicine literature, but it is similar to the ‘oiliness’ and ‘fat’ recorded in ancient literature. It is equivalent to ‘phlegm’, ‘stroke’ and ‘vertigo’ in Chinese medicine. Contributing factors include improper diet, overeating fat and greasy food, lack of exercise, and emotional disorders. These factors may disturb the normal transportation and transformation of qi, blood and body fluids, resulting in phlegm, stasis, or dampness. It is primarily caused by deficiency of the spleen and kidney and closely associated with the liver, gallbladder, heart and lung[11].

As an effective Chinese medicine therapy, herbal cake-partitioned moxibustion combines the triple functions of meridian acupoints, moxibustion, and herbal cake and acts to unblock meridians, regulate functions of the Zang-fu organs and lower blood lipid[12].

In this study,Da Huang(Radix et Rhizoma Rhei),Dan Shen(Radix Salviae Miltiorrhizae),Shan Zha(Fructus Crataegi),Yu Jin(Radix Curcumae), andZe Xie(Rhizoma Alismatis) were mixed at proportions to make herbal cakes for moxibustion. Emodin is the main component ofDa Huang(Radix et Rhizoma Rhei), having a protective effect on liver and kidney[13]. According to Chinese medicine, the effect ofDa Huang(Radix et Rhizoma Rhei) is manifested as the effect of purging turbidity and removing blood stasis. Emodin has a significant lipid-lowering effect on quail hyperlipidemia, which has been experimentally verified[14]. Tanshinone ⅡA is one of the main ingredients ofDan Shen(Radix Salviae Miltiorrhizae). Studies have shown that tanshinone ⅡA antagonizes homocysteine, induces apoptosis of vascular endothelial cells, reduces endoplasmic reticulum stress, and protects vascular endothelial cells[15]. It is reported that tanshinone ⅡA can reduce the levels of TC and LDL-C in rats, thereby effectively lowering blood lipid[16]. Han M,et al[17]found thatZe Xie(Rhizoma Alismatis) can help with diluting the infiltration of moisture, dissolving phlegm, and removing turbidity. Studies found that curcumin is one of the main ingredients inYu Jin(Radix Curcumae) medicinal residue[18-19]. Wu YJ,et al[20]performed an experiment in which hyperlipidemia rats were given ethanol extract ofYu Jin(Radix Curcumae) and found that it had a significant lipid-lowering effect. The main components ofShan Zha(Fructus Crataegi) are flavonoids, flavans and their polymers, organic acids, etc. It acts to lower blood lipids, protect the liver, lower blood pressure, help digestion and strengthen the cardiac function[21]. Studies have confirmed that the extraction ofShan Zha(Fructus Crataegi) has a significant effect in reducing blood lipids in high-fat model rats and rabbits[22]. In this experiment, the drug components were detected in the serum of rabbits. Moreover, the liver lipid-related factor metabolism of each intervention group changed significantly after the intervention, indicating that the effect of the moxibustion with herbal cake is related to the interaction of active ingredients in herbal cakes.

Penetration enhancer refers to all substances that can increase the transdermal speed or amount of the drugs without causing serious irritation and damage to the skin[23]. Its principle of action includes promoting hydration directly on the stratum corneum; forming ion pairs that are easily transdermal with drugs; acting as a drug co-solvent, improving the thermal activity and increasing the diffusion of drugs in the stratum corneum.

Laurocapram is one of the earliest chemical transdermal penetration enhancers. It has a good transdermal effect, the dosage used is relatively small, it is almost non-toxic and the skin irritation is also minimal. It is mainly used clinically in transdermal preparations of Chinese medicine. Laurocapram can be dissolved in both water and alcohol, and has a certain penetration- promoting effect on hydrophilic and lipophilic drugs[24]. Wang XY,et al[25]found that the penetration promotion effect of laurocapram on Chinese medicine was relatively strong among transdermal enhancers. Borneol belongs to the class of aroma-opening Chinese medicine, which has the nature of spicy channeling, presenting aromatic-opening, anti-inflammatory and analgesic effects, and helps the effective ingredients of the drug penetrate into skin. It is a well-known natural penetration enhancer and has been widely used in many fields[26]. This study indicated that herbal cake-partitioned moxibustion can significantly improve the metabolism of lipid factors in hyperlipidemia rabbits, which is consistent with the results of previous studies[27-28]. Observation of adding laurocapram or borneol in the herbal cakes as penetration enhancers confirmed that the two can effectively promote the transdermal penetration of medicinal ingredients in herbal cakes. However, there was a certain difference in the effects of the laurocapram group and the borneol group on liver lipids and related factors in hyperlipidemia rabbits. For example, the down- regulating effects of borneol on liver LDL-C, TG and Apo-B levels were more significant than those of laurocapram, which deserves further study.

HSL is a key enzyme involved in intracellular triglyceride degradation[29]. It is secreted by adipocytes, but it also hydrolyzes triglycerides. The results of this experiment indicated that compared with the blank group, the HSL level of rabbits in the model group was significantly increased, which may be related to high-fat diets. High-fat diets can cause lipid metabolism disorders in animals, which can lead to obesity and increase HSL secretion from fat cells. However, the results of this study also indicated that the serum HSL level of the high-fat model rabbits increased after the use of herbal cake-partitioned moxibustion, thereby increasing the hydrolysis of triglycerides. HMG-CoA reductase is a rate-limiting enzyme for cholesterol synthesis[30]. By inhibiting the expression of HMG-CoA reductase, it can effectively inhibit the synthesis of endogenous cholesterol and reduce the cholesterol in the body. The results of this study also indicate that herbal cake-partitioned moxibustion can help reducing HMG-CoA content in high-fat model rabbits. It can be inferred that the mechanism of regulating liver lipid metabolism in rabbits with hyperlipidemia may be related to the regulation of HSL and HMG-CoA reductase, which are key enzymes of lipid metabolism. In addition, the application of laurocapram and borneol as penetration enhancers in herbal cakes for moxibustion can significantly increase the penetration of the active ingredients in the herbal cakes, suggesting that the metabolism of these two enzymes may be related to the transdermal penetration of the active ingredients in herbal cakes. Therefore, more research and attention need to focus on the study of relevant mechanisms.

In summary, the application of laurocapram and borneol as penetration enhancers in herbal cake- partitioned moxibustion can promote the transdermal penetration of the effective lipid-lowering ingredients of the drugs in the cake. It has a significant effect on the metabolism of HSL and HMG-CoA reductase and the regulation of liver lipids. The results showed that the borneol group had a slightly better effect than the laurocapram group in some aspects. The use of penetration enhancers deserves more research and extensive clinical application.

Conflict of Interest The authors declare that there is no potential conflict of interest in this article. Acknowledgments This work was sponsored by Hunan Province College Innovation Open-end Fund Project (湖南省高校創(chuàng)新平臺開放基金項目, No. 16K068); 2017 Scientific and Research Innovative Project for Postgraduate Students of Hunan Province (2017 年湖南省研究生科研創(chuàng)新項目, No. CX2017B431); Scientific and Research Innovative Project for Undergraduate Students of Hunan Province (湖南省大學生創(chuàng)新實驗項目, No. 2017-132). Statement of Human and Animal Rights The treatment of animals conformed to the ethical criteria.

Received: 28 December 2019/Accepted: 18 January 2020