Using Herbal-Cake-Separated Moxibustion for the Treatment of Rats with Chronic Renal Failure

Summary

The moxibustion experimental platform can fix the rats without restriction, and the rats can undergo multi-acupoint moxibustion in the awake state, ensuring the welfare of the rats, protecting the safety of the operators, ensuring therapeutic efficacy, and improving the efficiency of the experiment.

Abstract

In the process of moxibustion in clinical practice, subjects need to be in a stable mood and comfortable posture to avoid problems such as moxa ash falling, scalding skin, and poor curative effect. Such problems also exist in the rat moxibustion experiment. To simulate clinical practice, it is necessary to introduce an experimental instrument in animal experiments, that is, a moxibustion device with fixed rats and moxibustion treatment synchronization, which can make experimental rats receive moxibustion treatment quietly and comfortably under non-anesthesia.

Our research group designed a rat moxibustion experimental platform. The device was framed by a wooden board with a supporting base plate, multiple fixed components, and partitioned components. The device can achieve the operation mode of moxibustion in rats without binding, avoiding anesthesia and scalding and simultaneously exposing multiple acupoints on the back. This operation can avoid physical and mental injury to rats and operators, which improves the research efficiency and further promotes the development and research of moxibustion animal experiments.

The device has a simple structure, is easy to operate and popularize, is comprehensively and innovatively designed, reusable, and is suitable for rat experiments mainly based on moxibustion. This article mainly introduces the structure of the experimental platform device for rat moxibustion, the basic procedure of herbal-cake-separated-moxibustion in experimental rats using the device and describes the establishment of a rat model of chronic renal failure (CRF) and representative experimental results.

Introduction

Moxibustion appeared with the application of fire. Ancient doctors accidentally found that some parts of the fire-burning body could relieve the symptoms of discomfort. After long-term practice, "Artemisiae Argyi Folium" was gradually selected as the main moxibustion material¹. Moxibustion is one of the important components of traditional Chinese medicine, and when juxtaposed with traditional Chinese medicine decoction and acupoint acupuncture, these three major traditional Chinese medicine therapies are widely used in many diseases.

Chinese doctors often used moxibustion to treat diseases as early as the period of Huangdi Neijing (Yellow Emperor's Inner Classic). Artemisiae Argyi Folium is a type of Chinese herbal medicine with the function of warming meridians to stop bleeding, dispelling colds, and relieving pain. It is commonly used in the external treatment of traditional Chinese medicine and can also be used in the treatment of traditional Chinese medicine decoction. Moxibustion is a kind of external therapy² wherein moxa or other drugs are placed on acupoints on the body surface to burn and use the mild heat of moxibustion fire and the effect of drugs to treat diseases³.

As one of the common methods of treatment, moxibustion has been widely used in clinical practice. With the continuous expansion in moxibustion therapy, a growing number of studies have investigated the mechanism of moxibustion and carried out many animal experiments^4,5.

The moxibustion method can be classified as direct moxibustion and indirect moxibustion. The method of placing moxibustion on the body's skin is called direct moxibustion. Indirect moxibustion separates the surface of the moxa cone and the acupoints with drugs or other materials. Moxibustion can treat various diseases, such as irritable bowel syndrome⁶, chronic fatigue syndrome⁷, and knee osteoarthritis⁸.

This paper mainly discusses the mechanism of herbal-cake-separated-moxibustion. Herbal-cake-separated-moxibustion belongs to the indirect moxibustion method, in which a moxa cone is placed on the medicinal cake to apply moxibustion. Current studies have shown that the infrared and warming effects produced by moxa cone burning, the chemical composition of moxa leaf tar, and acupoint stimulation can achieve disease prevention and treatment⁹ by increasing the penetration of drugs into acupoints. Therefore, herbal-cake-separated-moxibustion mainly produces a "comprehensive effect" through moxibustion, drugs, and acupoints¹⁰.

Previous studies show that moxibustion is an effective method for treating CRF^{11,12,13,14,15}, but the underlying mechanism is still undefined. We have been working on clinical and animal research on CRF. At present, we are conducting a moxibustion experimental study on CRF rats based on herbal-cake-separated-moxibustion-mediated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway of endoplasmic reticulum stress. Through this animal experiment, we aim to preliminarily explain the potential mechanism of moxibustion in treating chronic kidney disease. In this experiment, the treatment of herbal-cake-separated moxibustion of rats is a key operation.

In the previous experiment¹⁶, the lid of the rearing cage was used to bind and fix the rats. To protect the operator, the rats need to be anesthetized before binding. However, Sprague-Dawley (SD) rats are easy to fear and startle. The binding operation can easily cause restlessness in the rats. Problems such as poor posture fixation of rats, deviation of acupoint positioning, increased experiment time, and operator injury often occurred.

In addition, if the binding is too tight, it can easily cause breathing difficulties in rats, which is not conducive to animal welfare. If the binding is too loose, the rats are prone to struggle and move their bodies, causing moxibustion cones to fall and burn their skin, which affects the experimental results. Based on the literature, animal experiments still involve binding^16,17,18, and most experimental devices can only be fixed, hindering the implementation of other techniques such as moxibustion¹⁹. Therefore, achieving the integration of fixation and moxibustion in the experimental device while ensuring animal welfare and experimental specifications and efficiency has become one of the focus areas for investigators.

Protocol

The plan follows the Animal Care Guidelines of the Yunnan University of Chinese Medicine and has been approved by the Animal Ethics Committee of Yunnan University of Chinese Medicine. The experimental animal welfare and ethics review number is R-062021G098.

1. Moxibustion experimental platform device (Figure 1)

NOTE: We designed and constructed the rat moxibustion experimental platform device based on the distance between the hind and front limbs of rats and their behavioral and physiological characteristics.

1. The size of the moxibustion device
   1. Fabricate the device using anti-beaten board material and a wooden board; the overall size is 52 cm L x 23.5 cm W x 64 cm H, and the bottom area of the device is 1222 cm².
      NOTE: The moxibustion operation is carried out on the platform at this height, and the rats can be kept calm.
2. The main frame of the moxibustion device
   1. Make the base of the whole device with anti-beaten board material: The base plate is 52 cm L x 23.5 cm W with a white surface. Ensure that the bottom accounts for a large proportion of the device's weight to stabilize the whole device.
   2. Make the elevated platform of the device with solid wood, 60 cm high.
      NOTE: This is the main frame of the whole instrument, which stabilizes the whole device and supports the placement of the rats in a standing position.
   3. Put five placement slots above the elevated platform, with a size of 10 cm L x 6 cm W x 10 cm H. Use interval boards (10 cm L x 1.3 cm W x 10 cm H) to separate the placement slots, corresponding to the distance between the front and rear limbs of the rat in standing position.
3. The components of the moxibustion device
   1. Fix two stainless steel strips on top of the placement slots. Each fixed strip is a right angle equal edge steel bar 40 cm L x 1.4 cm W (both sides have the same width), with 15 holes. Three holes at the fixed strip correspond to each slot, used to fix the plum handle screw and the moving hose. The distance between adjacent holes is 7 cm.
   2. Place the adjustable plum blossom handle screw at both ends of the heat insulation network made of 304 stainless steel and connect to the fixed heat insulation network and the fixed strip.
   3. Connect the upper end of each placement slot with a liftable heat insulation network surrounded by a galvanized plate frame. Connect this frame to the plum blossom handle screw at both ends. Place 304 stainless steel circular mesh material in the middle of the heat insulation network to prevent the ash from falling and scalding the rats and to fix the rats to facilitate moxibustion.
   4. Affix the bottom end of the stainless steel movable hose (34 cm long) with a stainless steel fixed strip and the upper end with the pipe clamp connection plate.
      NOTE: The movable hose is convenient for adjusting the position of the moxa stick during the suspension moxibustion operation.
   5. Use the tube clamp made of aluminum alloy to connect the stainless steel movable hose and the moxa clip.
   6. Connect the stainless steel moxa clip to the pipe clamp connection plate, and fix 8-10 moxa strips simultaneously for moxibustion.
      NOTE: The movable hose, tube clamp connecting plate, and moxa clip can be used for suspension moxibustion operation in rats.

2. Establish a rat model of chronic renal failure

1. Acclimate 24 specific pathogen-free (SPF) SD male rats (aged 3-4 weeks, weighing 180-200 g) for 1 week. Keep the feeding temperature at 18-24 °C; relative humidity at 40-70%; and a 12 h light and 12 h dark cycle. House six animals in every cage and provide free access to water and common feed.
2. Establish the rat model of CRF. According to the random number table and following this protocol, select six rats as the control group and the remaining 18 as the model group. Treat the control group (n = 6) with distilled water (10 mL∙kg^-1∙day^-1) and normal feed and the model group (n = 18) with adenine suspension (150 mg kg^-1∙day^-1) and high-protein feed for 21 days.
3. Observe and record the body weight, mental status, and diet daily. After the end of 21 days, ensure that all the animals are fasted for 12 h before orbital blood sampling.
4. Collect the orbit blood samples.
   1. Anesthetize the rats briefly with isoflurane. Grab the rat's back with the non-dominant hand, hold the rat's head and face symmetrically between the thumb and forefinger, fully expose one side of the rat's canthus, and hold this position.
   2. With the dominant hand, hold 3 cm long melting point capillaries between the thumb and forefinger and slowly twist the capillary, entering the rat's inner canthus venous plexus at 45° until the blood slowly flows out of the tube.
   3. Collect 150-200 µL of blood samples in 0.5 mL blood collection anticoagulant tubes, gently pull out the capillaries, and press the orbit with cotton.
5. Shake the anticoagulant tube quickly and thoroughly. Measure plasma serum creatinine (Scr) and blood urea nitrogen (BUN) using an automatic biochemical analyzer.
   NOTE: The increase in Scr and BUN indicates the successful establishment of the model of CRF.
6. Divide the rats into four groups: A: six control rats, no treatment; 18 model rats divided into groups B: (model group: no medication or moxibustion), C: (losartan potassium group: administered 2.33 mg∙kg^-1∙day^-1 by gavage needle 12 G), and D: (herbal-cake-separated-moxibustion group: herbal-cake-separated-moxibustion applied to bilateral Shenshu [B23] and bilateral Pishu [B20]).

3. Treatment phase

NOTE: Groups A and B are not given any treatment, while groups C and D will be treated once a day for 24 days, with two days off after 12 days of treatment.

1. Preparations before treatment
   1. Prepare 300 g of dried Radix astragali, 300 g of dried Radix Codonopsis, 200 g of dried Radix Rehmanniae Praeparata, 100 g of roasted Shorthorned Epimedium Herb and 100 g of Cornus officinalis. Mix these powders.
   2. Put 500 g of Artemisiae Argyi Folium into the traditional Chinese medicine crusher (speed of 34,000 rotations/min) for 5 min, transfer the ground powder onto a plate, and then screen with a 600 mesh sieve. After two screening rounds, eliminate the coarse moxa and transfer the fine moxa to a bag for use (Figure 2).
   3. Prepare the moxa cone by filling the fine moxa into the mold to make a small moxa cone of 0.5 cm diameter and 0.5 cm height. Alternatively, malaxate the moxa between the thumbs and index fingers of both hands to form moxa cones (Figure 3A).
   4. Prepare the herbal cake mold by cutting a 5 mL syringe into four 0.5 cm thick circular plastic rings and gluing the four rings on the same horizontal plane (on the corners of a square plane with a side length of 26 mm, Figure 3D).
   5. Make medicine cakes by mixing the powder and water in a 1:1 ratio and filling the mixture into the herbal cake mold (Figure 3E). Leave the medicine cake in the mold and store it in a dry place.
2. Marking the acupoints
   1. Locate the acupoints as follows: Shenshu (B23), both sides of the second lumbar vertebra, one acupoint on each side; Pishu (B20), the intercostal of the 11th thoracic vertebra on both sides, one acupoint on each side (Figure 4).
      NOTE: For the location of the acupoints, refer to the Manual of Acupuncture for Experimental Animals²⁰.
   2. Wear a thick rubber glove on the non-dominant hand to gently cover the rat's head, locate the acupoints on the rat's back with the dominant hand, and then mark the acupoints with a color marker.
3. Placing the rats
   1. Wear thick rubber gloves. Use the thumb and the other four fingers of the dominant hand to slightly pinch the skin on the back and the neck of the rat. Use the thumb and index finger of the non-dominant hand to pinch the rat's tail gently.
   2. Lift the rat, align the head of the rat into the placement slot of the special moxibustion platform, and move the rat slowly forward. Ensure that the front paw of the rat is close to the edge of the placement slot and that the rat climbs into the slot.
      NOTE: Disinfect the entire moxibustion platform before starting. Before catching the rat, touch the back of the rat gently or talk to it to calm it. Adjust the height of the insulation network from a low position (when not in use) to the highest, which is convenient for rats to enter.
4. Rat fixation
   1. Place the thumb and index fingers of both hands at the screw of the plum blossom screw at both ends of the heat insulation network. Unscrew the plum blossom screw to lower the heat insulation net to the appropriate position, close to the back of the rat, but not causing compression, and then tighten the screws.
5. Placing the moxa cone and medicated cake.
   1. Place the prepared mold containing the medicine cake on the heat insulation net corresponding to the acupoints of both Pishu (B20) and Shenshu (B23) according to the marks of acupoints on the rats. There are four acupoints, each of which corresponds to a medicine cake.
   2. Place the moxa cone on the medicated cake smoothly and steadily.
6. Moxibustion
   1. Light the moxa cone with a lighter and start moxibustion till the moxa cone burns out and the residual heat dissipates naturally (the wet medicated cake dries up).
   2. Replace the burnt-out moxa cone with a new moxa cone and medicated cake on the same acupoint and continue moxibustion till the moxa cone burns out and the medicated cake dries. Repeat this for a total of 5x at each acupoint.
      NOTE: Clean and disinfect the liftable heat insulation network before replacing it with a new moxa cone and medicated cake.
   3. Extinguish the moxa cones and the medicated cakes in a water-containing waste tank after burning five moxa cones at each acupoint.
      NOTE: Each acupoint receives five cones of moxibustion; each moxa cone can be replaced after burning out.
7. Returning the rat to the cage
   1. Put the empty rearing cage in front of the rat, close to the lower end of the placement slot of the moxibustion platform, and keep still. Pat lightly the rat's buttock so that the rat can move from the placement slot to the feeding cage. After the rat enters, put down the cage. Use the dominant hand to grasp the fur on the back of the rat, hold the rat firmly, and place it on the ground for further observation.
   2. Observe whether the rats have shortness of breath, whether the mood is stable, and whether the skin surface is scalded. If there is no abnormality, put it back in the cage. If it is scalded, immediately apply the scald ointment on the skin surface and place it in a single cage.

4. Sampling phase

1. After 24 days of treatment, prepare the rats for 12 hours of fasting before sampling.
2. Pour isoflurane into the gas anesthesia machine and anesthetize the rats with 2-3% isoflurane. After 2-3 min, ensure that reflexes are absent (pinch the tail with surgical tweezers to check that they do not have any reflex) and continued existence of a heartbeat and switch to maintenance anesthesia with 1-1.5% isoflurane.
3. Collect 5 mL of blood from the abdominal aorta by inserting the needle into the quasi-abdominal aorta. Let the blood stand for 2 h, centrifuge at 2490 × g for 10 min at 4 °C, and transfer the serum supernatant to a 1.5 mL microcentrifuge tube for further analysis.
4. Deeply anesthetize the rats for a long period of time and take blood from the abdominal aorta under maintenance anesthesia. Further, euthanize the rats by cervical dislocation.
5. Harvest tissues from both kidneys by stripping the bilateral renal fascia and fat with surgical scissors and tweezers and rinsing with 0.9% normal saline. Weigh the left and right kidneys and record the weight and morphology.
6. Preserve the renal tissue by cutting the left kidney into six parts and putting them into a 50 mL centrifuge tube containing 4% paraformaldehyde for soaking, fixation, and preservation. Cut the right kidney into six pieces using a knife in the longitudinal direction and two knives in the transverse direction. Place these pieces into six 2 mL Cryotubes and immediately put them in liquid nitrogen; transfer them to a -80 °C freezer for storage.

5. Detection phase

1. Use the right kidney tissue of appropriate size for protein extraction, gel electrophoresis, membrane transfer, blocking, primary antibody incubation, secondary antibody, chemiluminescence, development, fixation, and film scanning.
2. Use a gel image data analysis system (e.g., Image-Pro Plus 6.0) to analyze the band's gray value and calculate each protein's content.
3. Subject the left kidney tissues to dehydration, clearing, embedding in paraffin, sectioning (thickness of 4 µm), staining with hematoxylin-eosin and Masson's trichrome stain, and observe the pathological changes²¹ in the renal tissue under a light microscope at 200x magnification.

Results

Kidney morphology
As can be seen from Figure 5, compared with the other groups, the kidneys' size, color, and texture in group D (herbal-cake-separated-moxibustion group) were closer to those of the normal group. The kidney morphology of the model and losartan potassium groups showed different degrees white fibrosis.

Kidney weight
Figure 6 shows that group A's left kidney weig...

Discussion

Clinically,  acupuncture and moxibustion treatment requires patients to be calm and relaxed, avoiding excessive anger and hunger and emphasizing 'peace.' However, the current practice of  acupuncture and moxibustion experiments is still based on the premise of binding animals, which is not only contrary to the original intention of the experiment but also does not conform to clinical practice. Binding animals, making animals frightened and angry, will rapidly increase adrenaline and other hormones in ex...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 mL heparin lithium biochemical tube	Shijiazhuang Kangweishi Medical Equipment Co., LTD	220518	Blood collection
0.9% normal saline	Kunming Nanjiang Pharmaceutical Co., LTD	/	Rinse
1.5 mL Eppendorf tube	Beijing Lanjieke Biotechnology Co., LTD	12100024	Subpackage
2 mL cryopreservation tube	Baroque Biotechnology Co., LTD	88-0151	Packaged kidney
4 °C refrigerator	Haier Group Co. LTD	BCD-236WDGL	Store
5 mL syringe	Sichuan New century Medical polymer products Co., LTD	20211115	Gavage
50 mL centrifuge tube	Beijing Langeke Biotechnology Co., LTD	CT-002-50A	Packaged kidney
12 G gavage needle	Beijing Zhongke Hengtian Technology Co., Ltd.	12#55mm	Gavage
96-well plates	Beijing Lanjieke Biotechnology Co., LTD	11510	Determination of protein concentration
-80 °C freezer	Thermo Fisher Scientific	991	Store
Adenine	Beijing Boaotuo Technology Co., LTD	A0607	Molding drug
BCA developer	Biyuntian Biological Engineering Co., LTD	P0010	Determination of protein concentration
BCL-2 protein antibody reagent	Abcam Shanghai Trading Co., LTD	ab196495	Western Blot
Caspase-3 protein antibody reagent	Abcam Shanghai Trading Co., LTD	ab184787	Western Blot
Chemiluminescence imaging system	Shanghai Qinxiang Scientific Instrument Co., LTD	100240073	Protein banding imaging
Chinese medicine powder	The Second Affiliated Hospital of Yunnan University of Chinese Medicine	/	Cure
Direct-load Color Prestained Marker	Beijing Kangrunchengye Biotechnology Co., LTD (GenStar)	M221	Western Blot
Disposable blood collection needle	Liuyang Sanli Medical science and technology development Co., LTD	YZB/ Country 0411-2010	Blood sampling
Disposable human venous blood sample collection container	Liuyang Sanli Medical science and technology development Co., LTD	20210901	Blood sampling
DK-30 Automatic snow ice maker	Henan Brothers instrument equipment Co., LTD	SHDX0023	Ice-making
ECL luminescent substrate kit	Beijing Lanjieke Biotechnology Co., LTD	BL520B	Convert latent images in exposed film into visible images
electronic scales	Shanghai Ran Hao Electronics Co., LTD	JCS-31002C	The body weight and kidney weight of rats were measured
Freezing microtome	Thermo Fisher Scientific	HM525 NX	Microtome
fully automatic biochemical analyser	Tianjin Micro nano core Technology Co., LTD	PV2112001Z02410305	Renal function test
Gas anesthesia system	Zhongshi Technology Co., Ltd	ZS-MV-1	Anesthesia
Glycine	Beijing Soleibao Technology Co., Ltd.	Cat#G8200	Running buffer, Western transfer buffer
HE staining reagent	Beijing Soleibao Technology Co., Ltd.	G1120	Staining
High-protein feed	Jiangsu Synergetics Biological Engineering Co., LTD	XT19008	Feeding
Intelligent laboratory ultra-pure water machine	Chongqing huachuag water treatment engineering co.,LTD	15L	Filtration
Internal reference β-actin	Jiangsu Qinke Biological Research Co., LTD	AF7018	Western Blot
Isoflurane	Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd	20220701	Anesthesia
liquid nitrogen	Kunming Botai dry ice sales Co., LTD	/	Renal tissue freezing
Losartan potassium tablets	Merck Sharp & Dohme (China) Investment Co., LTD	J20180054	Cure
Low temperature high speed tissue grinder	Wuhan Servicebio technology CO.,LTD	SKZ3F20200191	Tissue grinding
Maason staining kit	Beijing Soleibao Technology Co., Ltd.	G1340	Staining
Methanol	Guangdong Guanghua Sci-Tech Co., Ltd	20220519	Western transfer buffer
Mini-PROTEAN Tetra	Bole Life Medical Products (Shanghai) Co., Ltd	552BR 233193	Electrophoresis
Moxa	The Second Affiliated Hospital of Yunnan University of Chinese Medicine	/	Cure
Multiskan Spectrum Microplate Spectrophotometer	TECAN	Spark	The absorbance and concentration of tissue protein were detected
operating scissors	Zhangjiagang city Jinfeng deer knife cutting factory	201118	Cut the skin
Paraformaldehyde	Beijing Lanjieke Biotechnology Co., LTD	BL539A	Fixed tissue
PERK protein antibody reagent	Shanghai Pumai Biotechnology Co., LTD (CST China Agent)	3192	Western Blot
Positive fluorescence microscope	Carl Zeiss Suzhou Co.,Ltd.	5446000456	Capture picture
Prefabricated glue	Derun Cheng (Beijing) Biological Co., LTD (ACE China Agent)	B230418	Western Blot
ProteinFind Goat Anti-Rabbit LgG(H+L), HRP Conjugate	Shanghai Dinran electronic Technology Co., LTD (TRAN)	HS101	Western Blot
pulverizer	Hefei Rongzhida small household appliances Co., LTD	RS-FS1401	Crushed Chinese medicine
PVDF membrane	MerckMillipore Corporation	IPVH00010	Western Blot
Refrigerated centrifuge	Hettich Precision Technology (Zhuhai) Co., LTD	MIKRO 220R	Centrifuge
RIPA lysate	Beijing Solaibao Technology Co., LTD (Solarbio)	R0010	Lytic tissue
Sampling capillary	Jiangsu Kangjianhua Medical Supplies Co., LTD	20160157	Blood from inner canthus
Scald ointment (Zilian ointment)	Yunnan Provincial Hospital of Traditional Chinese Medicine	/	Used for the treatment of scalding from moxibustion
Skim milk powder	Guangzhou Saiguo Biotechnology Co., LTD (BIOFROXX)	1172GR500	Western blocking buffer
surgical blade	Shanghai Medical Equipment Co., LTD	JOBO10	Incision kidney tissue
surgical knife handle	Shanghai Medical Equipment Co., LTD	J11010	Mounting blade
TS-200 Orbital shaker	Haimen Qilin Bell Manufacturing Co., Ltd.	TS-8S	Gel fixation
Tween 80	MedChemExpress	HY-Y1819	TBST
tweezers	Shanghai Medical Equipment Co., LTD	JD1050	Pick up the moxa
Veterinary drugs*Micro core renal function test disc	Tianjin Micro nano core Technology Co., LTD	47031-35.5-0145-0227-62-220750	Renal function test