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, &#34;Exploration of mechanism research on renal interstitial fibrosis with cake-separated moxibustion from the interactive effect of&#160; ERK and Smad signal pathways mediated by TGF-&#946;1&#34;; 2. The Health Commission of Yunnan Province &#34;2020 High-level TCM Reserve Talents&#34;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.&#160;Yunnan Province Innovation Team Preventing and Treating cerebropathy with Acupuncture and Tuina (2023).

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...

A Moxibustion Platform for Herbal Cake-Based Treatment of Rats with Renal Failure

<ol>
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The authors declare that there are no conflicts of interest. The rat moxibustion experimental platform device&#39;s patent&#160;number is ZL202320880924.X.

Clinically, &#160;acupuncture and moxibustion treatment requires patients to be calm and relaxed, avoiding excessive anger and hunger and emphasizing &#39;peace.&#39; However, the current practice of &#160;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...

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, &#34;Artemisiae Argyi Folium&#34; 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&#39;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&#39;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 &#34;comprehensive effect&#34; 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&#160;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.

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			<td>0.9% normal saline</td>
			<td>Kunming Nanjiang Pharmaceutical Co., LTD</td>
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			<td>Rinse</td>
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			<td>1.5 mL Eppendorf tube</td>
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			<td>2 mL cryopreservation tube</td>
			<td>Baroque Biotechnology Co., LTD</td>
			<td>88-0151</td>
			<td>Packaged kidney&#160;</td>
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			<td>4 &#176;C refrigerator</td>
			<td>Haier Group Co. LTD</td>
			<td>BCD-236WDGL</td>
			<td>Store</td>
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			<td>5 mL syringe</td>
			<td>Sichuan New century Medical polymer products Co., LTD</td>
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			<td>Gavage&#160;</td>
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			<td>&#160;50 mL centrifuge tube</td>
			<td>Beijing Langeke Biotechnology Co., LTD</td>
			<td>CT-002-50A</td>
			<td>Packaged kidney&#160;</td>
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			<td>12 G gavage needle</td>
			<td>Beijing Zhongke Hengtian Technology Co., Ltd.</td>
			<td>12#55mm</td>
			<td>Gavage&#160;</td>
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			<td>96-well plates</td>
			<td>Beijing Lanjieke Biotechnology Co., LTD</td>
			<td>11510</td>
			<td>Determination of protein concentration</td>
		</tr>
		<tr>
			<td>&#160;-80 &#176;C freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>991</td>
			<td>Store</td>
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			<td>Adenine</td>
			<td>Beijing Boaotuo Technology Co., LTD</td>
			<td>A0607</td>
			<td>Molding drug</td>
		</tr>
		<tr>
			<td>BCA developer</td>
			<td>Biyuntian Biological Engineering Co., LTD</td>
			<td>P0010</td>
			<td>Determination of protein concentration</td>
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		<tr>
			<td>BCL-2 protein antibody reagent</td>
			<td>Abcam Shanghai Trading Co., LTD</td>
			<td>ab196495</td>
			<td>Western Blot</td>
		</tr>
		<tr>
			<td>Caspase-3 protein antibody reagent</td>
			<td>Abcam Shanghai Trading Co., LTD</td>
			<td>ab184787</td>
			<td>Western Blot</td>
		</tr>
		<tr>
			<td>Chemiluminescence imaging system</td>
			<td>Shanghai Qinxiang Scientific Instrument Co., LTD</td>
			<td>100240073</td>
			<td>Protein banding imaging</td>
		</tr>
		<tr>
			<td>Chinese medicine powder</td>
			<td>The Second Affiliated Hospital of Yunnan University of Chinese Medicine</td>
			<td>/</td>
			<td>Cure</td>
		</tr>
		<tr>
			<td>Direct-load Color Prestained Marker</td>
			<td>Beijing Kangrunchengye Biotechnology Co., LTD (GenStar)</td>
			<td>M221</td>
			<td>Western Blot</td>
		</tr>
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			<td>Disposable blood collection needle</td>
			<td>Liuyang Sanli Medical science and technology development Co., LTD</td>
			<td>YZB/ Country 0411-2010</td>
			<td>Blood sampling</td>
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			<td>Disposable human venous blood sample collection container</td>
			<td>Liuyang Sanli Medical science and technology development Co., LTD</td>
			<td>20210901</td>
			<td>Blood sampling</td>
		</tr>
		<tr>
			<td>DK-30 Automatic snow ice maker</td>
			<td>Henan Brothers instrument equipment Co., LTD</td>
			<td>SHDX0023</td>
			<td>Ice-making</td>
		</tr>
		<tr>
			<td>ECL luminescent substrate kit</td>
			<td>Beijing Lanjieke Biotechnology Co., LTD</td>
			<td>BL520B</td>
			<td>Convert latent images in exposed film into visible images</td>
		</tr>
		<tr>
			<td>electronic scales</td>
			<td>Shanghai Ran Hao Electronics Co., LTD</td>
			<td>JCS-31002C</td>
			<td>The body weight and kidney weight of rats were measured</td>
		</tr>
		<tr>
			<td>Freezing microtome&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>HM525 NX</td>
			<td>Microtome</td>
		</tr>
		<tr>
			<td>fully automatic biochemical analyser</td>
			<td>Tianjin Micro nano core Technology Co., LTD</td>
			<td>PV2112001Z02410305</td>
			<td>Renal function test</td>
		</tr>
		<tr>
			<td>Gas anesthesia system</td>
			<td>Zhongshi Technology Co., Ltd</td>
			<td>ZS-MV-1</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Beijing Soleibao Technology Co., Ltd.</td>
			<td>Cat#G8200</td>
			<td>Running buffer, Western transfer buffer</td>
		</tr>
		<tr>
			<td>HE staining reagent</td>
			<td>Beijing Soleibao Technology Co., Ltd.</td>
			<td>G1120&#160;</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>High-protein feed</td>
			<td>Jiangsu Synergetics Biological Engineering Co., LTD</td>
			<td>XT19008</td>
			<td>Feeding</td>
		</tr>
		<tr>
			<td>Intelligent laboratory ultra-pure water machine</td>
			<td>Chongqing huachuag water treatment engineering co.,LTD</td>
			<td>15L</td>
			<td>Filtration</td>
		</tr>
		<tr>
			<td>Internal reference &#946;-actin</td>
			<td>Jiangsu Qinke Biological Research Co., LTD</td>
			<td>AF7018</td>
			<td>Western Blot</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd</td>
			<td>20220701</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Kunming Botai dry ice sales Co., LTD</td>
			<td>/</td>
			<td>Renal tissue freezing</td>
		</tr>
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			<td>Losartan potassium tablets</td>
			<td>Merck Sharp &#38; Dohme (China) Investment Co., LTD</td>
			<td>J20180054</td>
			<td>Cure</td>
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			<td>Low temperature high speed tissue grinder</td>
			<td>Wuhan Servicebio technology CO.,LTD</td>
			<td>SKZ3F20200191</td>
			<td>Tissue grinding</td>
		</tr>
		<tr>
			<td>Maason staining kit</td>
			<td>Beijing Soleibao Technology Co., Ltd.</td>
			<td>G1340</td>
			<td>Staining</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Guangdong Guanghua Sci-Tech Co., Ltd</td>
			<td>20220519</td>
			<td>Western transfer buffer</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra</td>
			<td>Bole Life Medical Products (Shanghai) Co., Ltd</td>
			<td>552BR 233193</td>
			<td>Electrophoresis</td>
		</tr>
		<tr>
			<td>Moxa</td>
			<td>The Second Affiliated Hospital of Yunnan University of Chinese Medicine</td>
			<td>/</td>
			<td>Cure</td>
		</tr>
		<tr>
			<td>Multiskan Spectrum Microplate Spectrophotometer</td>
			<td>TECAN</td>
			<td>Spark</td>
			<td>The absorbance and concentration of tissue protein were detected</td>
		</tr>
		<tr>
			<td>operating scissors</td>
			<td>Zhangjiagang city Jinfeng deer knife cutting factory</td>
			<td>201118</td>
			<td>Cut the skin</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Beijing Lanjieke Biotechnology Co., LTD</td>
			<td>BL539A</td>
			<td>Fixed tissue</td>
		</tr>
		<tr>
			<td>PERK protein antibody reagent</td>
			<td>Shanghai Pumai Biotechnology Co., LTD (CST China Agent)</td>
			<td>3192</td>
			<td>Western Blot</td>
		</tr>
		<tr>
			<td>Positive fluorescence microscope</td>
			<td>Carl Zeiss Suzhou Co.,Ltd.</td>
			<td>5446000456</td>
			<td>Capture picture</td>
		</tr>
		<tr>
			<td>Prefabricated glue</td>
			<td>Derun Cheng (Beijing) Biological Co., LTD (ACE China Agent)</td>
			<td>B230418</td>
			<td>Western Blot</td>
		</tr>
		<tr>
			<td>ProteinFind Goat Anti-Rabbit LgG(H+L), HRP Conjugate</td>
			<td>Shanghai Dinran electronic Technology Co., LTD (TRAN)</td>
			<td>HS101</td>
			<td>Western Blot</td>
		</tr>
		<tr>
			<td>pulverizer</td>
			<td>Hefei Rongzhida small household appliances Co., LTD</td>
			<td>RS-FS1401</td>
			<td>Crushed Chinese medicine</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>MerckMillipore Corporation&#160;</td>
			<td>IPVH00010&#160;</td>
			<td>Western Blot</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Hettich Precision Technology (Zhuhai) Co., LTD</td>
			<td>MIKRO 220R</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>RIPA lysate</td>
			<td>Beijing Solaibao Technology Co., LTD (Solarbio)</td>
			<td>R0010</td>
			<td>Lytic tissue</td>
		</tr>
		<tr>
			<td>Sampling capillary</td>
			<td>Jiangsu Kangjianhua Medical Supplies Co., LTD</td>
			<td>20160157</td>
			<td>Blood from inner canthus</td>
		</tr>
		<tr>
			<td>Scald ointment (Zilian ointment)</td>
			<td>Yunnan Provincial Hospital of Traditional Chinese Medicine</td>
			<td>/</td>
			<td>Used for the treatment of scalding from moxibustion</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Guangzhou Saiguo Biotechnology Co., LTD (BIOFROXX)</td>
			<td>1172GR500</td>
			<td>Western blocking buffer</td>
		</tr>
		<tr>
			<td>surgical blade</td>
			<td>Shanghai Medical Equipment Co., LTD</td>
			<td>JOBO10</td>
			<td>Incision kidney tissue</td>
		</tr>
		<tr>
			<td>surgical knife handle</td>
			<td>Shanghai Medical Equipment Co., LTD</td>
			<td>J11010</td>
			<td>Mounting blade</td>
		</tr>
		<tr>
			<td>TS-200 Orbital shaker</td>
			<td>Haimen Qilin Bell Manufacturing Co., Ltd.</td>
			<td>TS-8S</td>
			<td>Gel fixation</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>MedChemExpress</td>
			<td>HY-Y1819</td>
			<td>&#160;TBST&#160;</td>
		</tr>
		<tr>
			<td>tweezers</td>
			<td>Shanghai Medical Equipment Co., LTD</td>
			<td>JD1050&#160;</td>
			<td>Pick up the moxa</td>
		</tr>
		<tr>
			<td>Veterinary drugs*Micro core renal function test disc</td>
			<td>Tianjin Micro nano core Technology Co., LTD</td>
			<td>47031-35.5-0145-0227-62-220750</td>
			<td>Renal function test</td>
		</tr>
	</tbody>
</table>

using herbal-cake-separated moxibustion for the treatment of rats with chronic renal failure

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.

Author Spotlight: Understanding the Effect of Herbal-Cake-Separated Moxibustion in Rats with Renal Faliure

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

We studied the effect of herbal-cake-separated moxibustion on chronic renal fails rats. This experiment tries to answer the mechanism of herbal-cake-separated moxibustion. The advantage of this protocol is that the rats are moxibusting unbundled and non-anesthesic conditions, making animal experiments close to clinical practice.The application of moxibustion platform not only protects the animal welfare of rats, but also reduce the moxibustion operation process in the save the time and energy of operators, and promotes the development of animal moxibustion experiment to a certain extent. In the future, our laboratory will further expand the scope of use of the platform, explore the acupuncture effect of the experimental rats. After using the rats and under the same conditions, carry out a comparative study on the effects of moxibustion with the device and other moxibustion methods in rats.

Kidney morphology 
As can be seen from Figure 5, compared with the other groups, the kidneys&#39; 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&#39;s left...

Watch this Scientific Journal Video about Using Herbal-Cake-Separated Moxibustion for the Treatment of Rats with Chronic Renal Failure at JoVE.com

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.

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 ...

using-herbal-cake-separated-moxibustion-for-treatment-rats-with

Research

JoVE Journal

Medicine

Construction of an Experimental Platform Device for Moxibustion

To begin fabricating the device use anti-beaten board material and a wooden board. For constructing the mainframe make the base of the device with anti-beaten board with a white surface measuring 52 by 23.5 centimeters. Construct the elevated platform with solid wood 60 centimeters high.Put five placement slots above the elevated platform. Separate the placement slots with interval boards corresponding to the distance between the front and rear limbs of the rat in a standing position. Next, fix two stainless steel strips on top of the placement slots, each with 15 holes for fixing components.Three holes of the fixed strip correspond to each slot, which will be used to fix the plum handle screw and the moving hose. The distance between adjacent holes is seven centimeters. Attach the upper end of each placement slot with a liftable heat insulation network surrounded by a galvanized plate frame.Position the adjustable plum blossom handle screw at both ends of the heat insulation network made of 304 steel. Insert the 304 stainless steel circular mesh material in the middle of the heat insulation network. Affix the bottom end of the stainless steel movable hose with a stainless steel fixed strip and the upper end with the pipe clamp connection plate.Connect the stainless steel moxa clip to the pipe clamp connection plate and secure eight to 10 moxa strips simultaneously for moxibustion.

Treatment of Rats with Chronic Renal Failure Using a Moxibustion Platform Device

To begin, place the rat on the experimental table. Gently cover the rat's head using the rubber glove on the non-dominant hand. Then use the dominant hand to identify the acupoints on the back of the rat.Identify the acupoint shenshu or B23 located on both sides of the second lumbar vertebra. and pishu or B20, located on the intercostal of the 11th thoracic vertebra on both sides. Mark the acupoints with a colored marker.Next, use the thumb and fingers of the dominant hand to slightly pinch the skin on the back and the neck of the rat. Also, use the thumb and index finger of the non-dominant hand to gently pinch the rat's tail. Lift the rat in this position and move the rat forward slowly.Align the head of the rat into the placement slot of the Moxibustion platform. 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. Next, place the thumb and index fingers of both hands on the plum blossom screw at both ends of the heat insulation network.Unscrew the plum blossom screw and lower the heat insulation net to the appropriate position close to the back of the rat. Then tighten the screws to fix the rat. Place the mold containing the medicine cake on the heat insulation net at the locations corresponding to the pishu and shenshu acupoints.Then, place the moxa cone on the medicated cake in a smooth and steady manner. Then light the moxa cone with a lighter and start Moxibustion till the moxa cone burns out and the residual heat dissipates naturally. Confirm completion by checking if the wet medicated cake has dried up.Replace the burnt out moxa cone and medicated cake and continue Moxibustion till the moxa cone burns out and the medicated cake dries. Once the procedure is complete, extinguish the moxa cones and the medicated cakes in a waste tank containing water. Place an empty rearing cage in front of the rat, close to the lower end of the placement slot of the Moxibustion platform, keeping the cage 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 experimental table. 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 the rat back in the cage.After the herbal cake separated moxibustion procedure, the kidneys of treated rats were similar in size, color, and texture to the control rats, while the kidneys of rats with chronic renal failure showed white fibrosis. Further, BCL-2 was upregulated, while PERK and Caspase-3 were downregulated in treated rats, compared to those with chronic renal failure, indicating a reduction in apoptosis. Also, H&E staining revealed that the infiltration of inflammatory cells in the treated group was reduced compared to rats with chronic renal failure.Similarly, Masson's staining showed reduced deposition of collagen fibers, indicating that this procedure could be anti-fibrotic, and hence useful for treating renal failure.

510 Views.  Yunnan University of Chinese Medicine.  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.

A Moxibustion Platform for Herbal Cake-Based Treatment of Rats with Renal Failure

Summary