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 experiments4,5.
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 CRF11,12,13,14,15, 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 binding16,17,18, 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
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Associated Professor Huang and Miss Cui for their strict guidance and support for this paper. The study was supported by: 1. National Natural Science Foundation of China (NSFC), 82160938, "Exploration of mechanism research on renal interstitial fibrosis with cake-separated moxibustion from the interactive effect of ERK and Smad signal pathways mediated by TGF-β1"; 2. The Health Commission of Yunnan Province "2020 High-level TCM Reserve Talents"Incubation Project (Yunwei TCM Development [2021] No.1); 3. Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Chinese Medicine (No.2019FF002-022, No.202101AZ70001-286, No.202301AZ070001-030); 4. Yunnan Province Innovation Team Preventing and Treating cerebropathy with Acupuncture and Tuina (2023).
Materials
| 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 |
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