Yuqing ZHOU, Guangyuan XIE, Jun LIU, Min HUANG

The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530001, China

Abstract [Objectives] To study the therapeutic effect and mechanism of Qianjin Lotion on eczema in mice. [Methods] A mouse eczema model was established by the 2,4-dinitrochlorobenzene method. The indicators of mouse ear swelling degree, the number of dermal inflammatory cells, the number of body licking reactions, the IL-2 and TNF-α content in serum were detected to explore the therapeutic effect and mechanism of Qianjin Lotion on eczema in mice. [Results] Qianjin Lotion can effectively eliminate skin inflammation, reduce the degree of auricle swelling, reduce the number of dermal inflammation cells, reduce the number of body licking reactions, and reduce the IL-2 and TNF-α content in serum. [Conclusions] Qianjin Lotion can alleviate the symptoms of eczema in mice, reduce inflammatory factors. Combined with oral administration of Qianjin granules, it can treat eczema and achieve good effects.

Key words Qianjin Lotion, Eczema, Mouse

1 Introduction

Eczema, also called "spreading dampness pattern" and "damp toxin" in traditional Chinese medicine, is a common allergic skin disease that is easy to relapse. It often occurs on the head and face, the flexed sides of the limbs, and the perineum. It is spreading or symmetrically distributed. As described inGoldenMirrorofMedicine(Yizong Jinjian), eczema is like scabies in initial occurrence, itching all the time, and spreading without stop, yellow liquid when scratching, dampness spreading, generated from win wind of heart fire and spleen dampness. The names vary according to the different parts of the disease. For example, those occurring in the lower leg are called shank sore (ecthyma), those occurring in the fossa of the elbow or rouge are called "Four Bend Wind", and those occurring in the scrotum are called "Scrotum Wind". Wind, dampness and heat are the main pathogenic factors.

Qianjin Prescription is a clinically proven prescription of the First Affiliated Hospital of Guangxi University of Chinese Medicine. It is composed ofChrysanthemumindicumL.,Blumealaciniata(Roxb.) DC.,Inulacappa(Buch.-Ham.) DC,Hedyotisdiffusa, andGlycyrrhizauralensisFisch. Its oral preparation-Qianjin Granules is a hospital preparation approved by Guangxi Food and Drug Administration, and it has good effect in the clinical treatment of eczema. Whether Qianjin Prescription can be used externally to treat eczema is still under research. Therefore, using the 2,4-dinitrochlorobenzene method, we established the mouse eczema model. Then, we detected indicators of mouse ear swelling degree, the number of dermal inflammatory cells, the number of body licking reactions, the IL-2 and TNF-α content in serum, to explore the therapeutic effect and mechanism of Qianjin Lotion on eczema in mice.

2 Materials and methods

2.1 Experimental animalsFifty Kunming male mice, SPF grade, weight (20±5) g, provided by the Laboratory Animal Center of Guangxi Medical University, license number: SCXK (Gui) 2019-0001.

2.2 Medicines and reagentsC.indicum,B.laciniata,I.cappa,H.diffusa, andG.uralensiswere purchased from Guangxi Xianzhu Chinese Medicine Co., Ltd. Pulverized the above five medicinal materials according to the prescription ratio, soaked 5 times the crude drug in water for one hour, decocted, took the filtrate and added 3 times the crude drug, boiled for 30 min, combined the two filtrates, and sterilized to prepare Qianjin Lotion with 200 mg crude drug in 1 mL.

2.3 Preparation of mouse eczema modelOne day before the experiment, removed the abdominal hair with an area of about 2 cm×2 cm. On the next day, applied 30 μL of 7% DNCB solution (4∶1 acetone and olive oil as a matrix) to the mouse’s abdominal shearing area to sensitize. On the second day, used the same method to intensify the sensitization on the mouse abdominal shearing area. On the 3rdday to 6thday, normally fed the mice. On the 7thday, after 2 h after drug administration, injected subcutaneously 4-AP1 mg/kg into the depilated area on the back of each mouse. Then, immediately observed the number of body licking reactions within 10 min. Criteria for successful modeling: low-concentration DNCB solution repeatedly stimulates the abdominal skin of mice to show various degrees of flushing, papules, blisters, erosion, exudation, crusting, desquamation and other manifestations.

2.4 Experimental grouping, drug administration, and material collectionThe mice were divided into 5 groups according to the random principle, each with 10 mice, namely: model group, Qianjin Lotion low-dose group (1 time/d), Qianjin Lotion medium-dose group (2 times/d), Qianjin Lotion high-dose group (3 times/d), and multi-effect skin external lotion group (3 times/d). On the 8thday of the experiment, 2 h after the administration, applied the right ear shells of 4 groups of mice except the model group with 15 μL of 1% DNCB solution (4∶1 acetone to olive oil solution) to stimulate, and applied the left ear with 15 μL of the substrate as a control. Evenly applied equal volume of saline for the model group.

2.5 Observation indicators

2.5.1Skin appearance. When different degrees of flushing, papules, blisters, erosion, exudation, crusting, and desquamation appear in the skin of the shearing area of the abdomen, it means successful modeling.

2.5.2Degree of auricle swelling. Anesthetized the mice 2 h after the last administration (the 9thday), cut off both ears, and used a skin punch to cut round ears on the symmetrical parts of the two ears, measured the thickness of the ears, weighed the weight of the ears, and calculated the weight difference between two ears, the difference is the degree of swelling, and then calculated the swelling inhibition rate.

Inhibition rate=(Swelling degree of blank group-Swelling degree of administration group)/Swelling degree of blank group×100%

2.5.3Number of body licking reactions. On the 7thday of the experiment, subcutaneously injected 4-AP1mg/kg into the depilated area on the back of each mouse 2 h after the administration, and immediately observed the number of body licking reactions within 10 min. The body licking reaction is the behavior repeated turning of the head and licking of the back on both sides of the mouse. The mouse body licking until a short pause is considered one time of body licking reaction. Calculation requirements: body licking reaction must be licking the drug injection site of the depilatory area on the back for more than 5 times. Only turning the head or licking other parts of the body will not be counted. Requirement for a short pause: the mice have behavior of walking, stretching their heads, or standing upright.

2.5.4Content of IL-2 and TNF-α in serum. After the last stimulation and administration, intraperitoneally injected the mice with 10% chloral hydrate with general anesthesia, and removed the eyeballs to get blood. After the blood was spontaneously coagulated, centrifuged at 3 000 g for 10 min, and collected and stored the serum at -20 ℃. Measured the IL-2 and TNF-α content in serum in accordance with the ELISA kit instructions.

3 Results

3.1 Effects on the skin appearance of mice with eczema

After stimulation with 0.1% DNCB, the shearing area of the abdomen was pink and there was no abnormal condition. After the second stimulation, some mice showed a little red swelling and erythema. After the last stimulation, various degrees of flushing, papules, blisters, erosion, exudation, crusting, and desquamation appeared on the skin of the shearing area of the abdomen. These indicate that the modeling was successful.

3.2 Effects on the degree of auricle swellingCompared with the model group, the auricle swelling of the mice in the Qianjin Lotion treatment group and the multi-effect skin external lotion group alleviated, and there were statistical differences. The results are shown in Table 1.

Table 1 Effects of Qianjin Lotion on the degree of auricle swelling in mice with chronic dermatitis and eczema n=10)

3.3 Effects on the number of body licking reactions in mice with eczemaCompared with the model group, the mice in the Qianjin Lotion treatment group and the multi-effect skin external lotion group had significantly fewer body licking reactions, and there were statistical differences, as shown in Table 2.

Table 2 Effects of Qianjin Lotion on the body licking reactions in mice with chronic dermatitis and eczema n=10)

3.4 Effects on the IL-2 and TNF-α content in the serum of mice with eczemaCompared with the model group, the IL-2 and TNF-α content in the serum of mice in the treatment group was reduced, and there were statistical differences, as shown in Table 3.

Table 3 Effects of Qianjin Lotion on the IL-2 and TNF-α content in the serum of mice with chronic dermatitis and eczema n=10)

4 Discussion

In this experiment, we established the mouse eczema model using 2,4-dinitrochlorobenzene method. We washed the shearing area of mouse abdomen with Qianjin Lotion. The results proved that it can alleviate the symptoms of eczema in mice and reduce inflammatory factors. If combined with Qianjin granules, the treatment of eczema may be able to receive better effects. At present, most western medicines treat eczema with oral antihistamines and antibiotics, or externally use glucocorticoid preparations, immunomodulators,etc.Long-term use may cause symptoms such as skin atrophy, pigmentation and skin telangiectasia, and patients may become dependent on drugs. If the patient stops using the drugs, the rebound phenomenon will aggravate their disease conditions. By comparison, the external treatment of traditional Chinese medicine has good advantages and characteristics in the treatment of eczema, and the adverse reactions are small[1-2]. The external treatment of traditional Chinese medicine is easy to operate, Chinese herbal medicines are cheaper than western medicines and have high medicinal value, and are easy to store. After being processed into different external dosage forms, they are suitable for the treatment of various eczema and thus worthy of clinical promotion. In summary, the external treatment of traditional Chinese medicine can effectively treat children with various syndromes of eczema, absorb heat and damp toxins in the body, prevent local infections, and reduce the infection rate[3-6].