Materials and reagents
The following reagents and materials were used: burn moisturizing scald (BMS; Shantou Meibao Pharmaceutical Co., Ltd.); ELISA Kit (Neobioscience Technology Company); Collagen I/HRP-labeled goat anti-rabbit antibody (Servicebio); Microscope (XSP-C204); Cognex camera; Toe volume measuring instrument (PV-200; TAIMENG Co., Ltd.); Autoclave (LDZX-50KBS; Shanghai Shenan Medical Instrument Factory); Full-wavelength microplate reader 1510 (Thermo Fisher Scientific). Chinese herbal medicine pieces of Rheum palmatum L., Angelica sinensis (Oliv.) Diels, Radlx Codonopsis pilosula (Franch.) Nannf., Asari Radix et Rhizoma, Borneolum, and Hg2Cl2 powder et al were bought from Hubei Tianji Pieces Factory, they were identified by Dr Dingrong Wan’s laboratory, School of Pharmaceutical Science, South-Central University for Nationalities, China. Voucher samples (No. SC-20191022-20191027) were deposited at the Herbarium of Medical Plants located in the College of Pharmacy, South-Central University for Nationalities, China.
Kunming (KM) mice (20 ± 2 g) and Sprague Dawley (SD) rats (200 ± 20 g) were purchased from the Experimental Animal Center (Certificate of experimental animals: SYXK 2016-0089), Institute of Health and Epidemic Prevention (Wuhan, China) and housed in the standard specific pathogen-free (SPF) environment. All animals were allowed free access to food and water.
HPLC analysis of components of BO
A total of 10 mg of BO was extracted with 10 mL methanol by ultrasonic for 20 min, then the resulting extract was filtered and transferred into 10 mL volumetric flask and kept at a constant volume of 10 mL with methanol. By this procedure a concentration of crude medicine equivalent to 1 mg/mL was obtained. For HPLC analysis, the mobile phase was acetomitrile (Aladdin, Shanghai, China): water (20: 80) mixture and the preparative column was Super Co. Inc. Water S Spheripor ODS (particle size: 5 µm, diameter: 4.5 mm, length: 265 nm). Flow rate was set at 1.0 mL/min, and injected 20 µL/ time; the detection was performed at 254 nm. The temperature of the column oven was set at 35℃
Skin irritation experiment in rats
On the day before the experiment, 30 SD rats were shaved with pet electric scissors, and the shaving area was approximately 4 × 2 cm2. The shaved part was then flushed with saline. Rats with no skin wounds were selected. Twenty-four hours later, the rats were randomly divided into two groups: the control group with canola oil treatment (Con) and the test group with BO treatment. The application times were 10:00, 14:00, and 19:00, and the skin was applied to 0.5 g of the drug each time for 3 days. One hour after the last treatment, the treated skin was cleaned with cotton and water. At 0 h, 12 h, 24 h, 48 h, and 72 h, the allergic reactive changes were recorded.
Physical and chemical indicators test
Ten male and female SD rats were randomly selected. After anesthetization with ether anesthesia, blood samples were collected from the abdominal aorta, serum (3000 rpm, 10 min) was taken and sent to the Hubei Provincial People's Hospital for liver and kidney function tests according to the basic standards. Samples from the experimental animals were collected and tested with the same procedure.
The test of rat's toe swelling
Thirty male and female rats (200 ± 20 g) were randomly divided into three groups: the negative Con group and the BO and BMS treatment groups. The animals were treated with 0.5 g drugs on the toes 3 times per day for 5 days. On the sixth day, the right hind foot ankle of each rat was marked, and the volume before and after inflammation was measured by toe volume measuring instrument, and the average of three measurements was recorded. The right foot was injected with 0.1 ml carrageenan (1%), and the foot volume was measured at 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 h after inflammation began. The foot was still treated with the drug every time after the volume was measured. The swelling volume (ΔV) = The predose (V2) – The postdose (V1)
The test of the xylene-induced mouse ear swelling
Thirty male and female mice (20 ± 2 g) were randomly divided into three groups: the negative Con group and the BO and BMS treatment groups. The animals’ ears were treated with 0.5 g of the drugs 3 times per day for 5 days. On the sixth day, 100 µl of xylene was dripped on both sides of the right ear with a pipette. After the model was established successfully, the animal was immediately sacrificed, and both ears were cut off along the root of the ear. The sample was immediately weighed after being punched. The degree of ear swelling: Δm = The right ear mass - The left ear mass
The hot-plate test of mice
Twenty-seven KM mice were screened out of 40. The index of the test was the time that the mouse licked its toe with its tongue at 54 ± 1 °C, which was the mean of 3 times (the interval was repeated every 30 min) to obtain the mouse’s pain threshold for 5ཞ40 s. The mice were randomly divided into the following groups: the negative Con group and the BO and BMS treatment groups, which were coated with 0.5 g drug each time, 3 times a day for 7 days. The test method of the mouse’s pain threshold was same as that 7 days prior. Pain threshold change = After treatment - Before treatment
The test of knife wound
Thirty mice were randomly divided into two groups. The skin of the wound was approximately cut 1.5 cm in size after anesthetized by ether anesthesia. The Con group and the BO treatment group were coated with 0.5 g drug 3 times a day for 16 days.
The test of scalds in rats
Thirty-five SD rats were anesthetized, and their backs were shaved. The mice were burned with a soldering iron (1.5 cm2) for 10 s. After half an hour, all animals were awakened and randomly divided into the negative Con group, the BO treatment group and the positive BMS group, which were coated with 0.5 g drug each time and 3 times a day for 28 days. After the model was established on the second, seventh, fourteenth, and twenty-eighth days, the skin of wound took off and infiltrated by a paraformaldehyde fixative, and the animals’ blood was centrifuged at 3000 rpm for 10 min. The serum was preserved at -80 °C.
The test of H&E staining
The pretreated sample tissues from rats were embedded in paraffin wax. The embedded wax blocks were cut into slices with a thickness of 4 µm. The sections were routinely dewaxed with xylene and washed with ethanol at every step. The reagent incubation order was as follows: the samples were incubated with xylene twice for 10 min each time, followed by incubation with anhydrous ethanol twice for 10 min each time, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 75% ethanol for 5 min and distilled water for 5 min. These sections were soaked for 2–5 min in hematoxylin and rinsed with tap water. Then, the cells were differentiated in 1% hydrochloric acid ethanol for 30 s. After differentiation, the cells were submerged in tap water. Next, their differentiation was performed in 0.6% ammonia water for 30 s. The sections were rinsed with tap water 1 h after differentiation and submerged for 30 s. The samples were incubated with eosin for 2 min and rinsed with tap water. Then, the samples were dehydrated to transparency and sealed: the handled samples were placed into 95% ethanol, anhydrous ethanol, and dimethylbenzene, and every step was repeated twice for 5 min each; then, the samples were sealed with neutral resin. Finally, the pathological sections were placed under a positive fluorescence microscope for observation, photographing, describing and comparing with normal tissues. The observations included the epidermis, dermis, blood vessels, cells and organelles. Another two evaluators used the histological changes in burn wound healing scoring system to perform blind histological evaluations.
The test of immunohistochemistry
The prepared paraffin tissue sections were placed in a wet box filled with citric acid antigen repair buffer (pH 6.0). The box was placed in a microwave to boil for 8 min, allowed to cool for 8 min, kept warm for 8 min and then boiled for 7 min. The sections were washed with PBS three times after naturally cooling. The sections were incubated with 3% hydrogen peroxide protected from light for 25 min and then rinsed with PBS three times for 5 min each. Then, 3% BSA was added, and the solution was kept at room temperature for 30 min. The blocking solution was discarded, and the primary antibody diluted in PBS was added to the box, followed by an incubation at 4 °C overnight. Next, the sections were washed with PBS, and the appropriate secondary antibody was mixed with horseradish peroxide and incubated at room temperature with the sections for 50 min. Then, the sections were cleaned with PBS and excess moisture was removed. The DAB staining solution was prepared in a staining box and added dropwise onto the tissues, and the reaction was monitored by microscopy. The nuclei were counterstained with hematoxylin. Finally, the cover slips were dehydrated with ethanol, anhydrous ethanol and xylene twice for each liquid, and then the tissue sections were sealed. The sections were placed under a positive fluorescence microscope for observation, photographing, describing and comparing with normal tissues. Then we used Image J to semi-quantitative and analysis the results.
The contents of TNF-α, VEGF, and TGF-β1 were measured in the sera of SD rats from the burn test by ELISA following the kit instructions.
The samples were prepared as follows: the skin tissue was removed from the burn wounds in rats, cut into small fragments, placed into a mortar, ground into powder in liquid nitrogen (200µL lysis solution per 20 mg tissue), and centrifuged (12,000 rpm/min, 5 min), to obtain the samples (the supernatant). The proteins from tissue lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto PVDF membranes. Then, the membranes were blocked with a Western blocking solution and incubated using primary antibodies, followed by an incubation with the appropriate secondary antibodies. Finally, the protein levels were normalized against that of GAPDH.
The data were presented as the mean ± SD. Statistical analysis was performed via Student's t test and one-way ANOVA (SPSS Program, version 11.5; SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a significant difference between the groups.