1.1 Animals and groups.
A total of 40 healthy normal SD rats, 20 male and 20 female, were selected for the study. The mean body weight was (190 ± 10) g. The rats were provided by the Anhui Experimental Animal Center, animal license number: SCXK (Wan) 20190003. The animals were fed adaptively in the animal room of the key laboratory at the Anhui Provincial Department of Education for three days. The temperature was (27 ± 1)°C and the humidity was (55 ± 5) %. The bedding, feed, and drinking water were replaced every day. The experimental process strictly followed the "guiding opinions on being kind to Experimental Animals" issued by the Ministry of Science and Technology of the People's Republic of China (2006). The rats were randomly divided into normal control group, low PM2.5 concentration moxa smoke environment group (low-smoke group), medium PM2.5 concentration moxa smoke environment group (middle-smoke group), and high PM2.5 concentration moxa smoke environment group (high-smoke group), with 10 rats in each group. According to a previous study, the PM2.5 concentration of moxa tobacco in the low-smoke, middle-smoke, and high-smoke groups was controlled at 0.11 ± 0.05 mg/m3, 0.23 ± 0.05 mg/m3, and 0.53 ± 0.05 mg/m3, respectively.ELISA kits for IL-1β, IL-6, and TNF-α (Wuhan Genmei Technology Co., Ltd., batch number: GR2020–05), three years 3:1 Chen moxa (18 mm × 200 mm Shanghai Taicheng Science and Technology Development Co., Ltd.), three cuboid plexiglass moxibustion boxes with a volume of 100cm × 80cm × 125cm, enzyme labeling instrument (Lei she, model: RT-6000), optical microscope (Japanese OLYMPUS, model: BX53), centrifuge (Anhui Jiawen, model: JW3021HR), slicing machine (Thermo Fisher Scientific), Whirlpool mixer (its Limbell instrument manufacturer, model: GL-88B), and in situ apoptosis kit (TUNEL-AP Roche company, batch number: 110919200312) were used for the experiment.
1.2 Main reagents and instruments
1.3 Model replication
According to the previous study of our group , three special cuboid plexiglass moxibustion boxes (except the bottom edge, each side has four symmetrical holes with a diameter of 10 mm, see Fig. 1), according to the setting of PM2.5 concentration in previous experiments, the moxa smoke environment (high, medium and low moxa smoke concentration) was simulated by opening and closing the holes. Box 1 as a high concentration moxibustion box left 2 holes on the left and right side of the mg/m3, box, and the other holes closed to adjust the moxa smoke concentration in the box, while box 2 as a medium concentration moxibustion box left 2 holes on the left, right and top of the mg/m3, box, and the other holes were closed to adjust the moxa smoke concentration in the box. Box 3 was used as a low concentration moxibustion box. The concentration of mg/m3, box was 0.11 ± 0.05.There were 4 holes on the left, right and top of the box, and the rest of the holes were closed. Close the doors and windows of the laboratory, open the indoor ventilation, set the air conditioning temperature at 25 ℃, light 3 common moxa bars with a diameter of 1.8cm, insert them into the moxibustion box, put the moxibustion box in the middle of the three specific moxibustion boxes, and close the door of the box. The moxa smoke concentration in the moxibustion box was detected by the environmental particulate matter sampler. When the moxa smoke PM2.5 concentration reached the corresponding concentration, 10 rats in the low smoke group, middle smoke group and high smoke group were put into the moxibustion box. The rats were observed every 0.5 hours. In order to prevent the "stacking" phenomenon, the rats were forced to move in the cage by beating the moxibustion box with a stick. After the burning of moxa sticks (1.5 h),), the new moxa sticks were lit and inserted into the moxibustion box. The time of each fumigation and moxibustion was set at 4 hours. after the experiment, the rats were taken out, put back to the animal room, filled with water, and the window was opened for ventilation. the experiment lasted 3 months, twice a day. The rats in the normal control group were fed without intervention every day during the experiment.
1.4 Observation indicators and detection methods.
Because of the death of rats during the process of modeling, eight rats in each group were randomly selected for detection.
The levels of IL-1β, IL-6, and TNF-α in the rat serum were detected by ELISA. Briefly, the blood from the abdominal aorta of the rats was placed in an anticoagulant tube at 4°C and centrifuged at 3000rpm for 15 min, and the supernatant was separated. The samples were processed strictly according to the instructions of the corresponding kits for IL-1β, IL-6, and TNF-α, and the absorbance (OD value) was determined using the enzyme-labeling instrument at a wavelength of 450nm. The levels of the above indices in serum samples of each group were calculated from the standard curve and then analyzed statistically.
Then, histopathological examination of the nasal mucosa and olfactory bulb was performed. Briefly, after the rats were killed, the nasal mucosa and olfactory bulb of each group were fixed with polyformaldehyde for 48 h, rinsed with distilled water for 10 min, dehydrated in ethanol gradient (50%, 75%, 85%, and 95%) for 1.5 h, dipped in xylene for 30 min each time, embedded in paraffin, and sectioned to a thickness of 4 µm. The sections were then subjected to hematoxylin-eosin (HE) staining. Briefly, the sections were baked in a constant temperature oven at 65°C for 30 min and dewaxed by soaking in xylene I and xylene II for 15 min each. The dewaxed sections were then hydrated by soaking in 100, 95, 85, and 75% ethanol for 5 min each and rinsing for 10 min with tap water each time. The slices were then placed in distilled water and an aqueous solution of hematoxylin for 5 min, followed by color separation in ammonia for 2 min. The slides were then rinsed in running water for 15 min and dehydrated for 10 min each in 70% and 90% ethanol. Then, the slices were stained with ethanol eosin dye 1 for 2 min and dehydrated using absolute ethanol, made transparent by placing them in transparent xylene twice for 3 min each, sealed using neutral gum seal, and put in an oven at 65°C for 15 min. Finally, the routine pathological changes of the nasal mucosa and olfactory bulb were observed and photographed under a light microscope.
The apoptotic cells in the nasal mucosa and olfactory bulb of each group were detected by TUNEL staining according to the following procedure: 1) Paraffin sections were dewaxed in water thrice for 10 min each using the standard technique and then washed using PBS. 2) The sections were then digested in trypsin at 37°C for 30 min and washed thrice in PBS for 10 min each. (3) The labeling solution was then added, incubated at 37°C for 1 h, and washed using PBS thrice for 10 min each. 4) The sections were then incubated at 37°C for 30 min and washed with PBS and AP thrice for 10 min each. 5) The sections were then subjected to NBT/BCIP coloration. (6) Finally, the sections were dehydrated in an alcohol gradient, made transparent in xylene, sealed, and observed under a light microscope. For the negative control, PBS was used instead of labeling solution in step 3.
The olfactory behavior of the rats was analyzed using the buried food pellet test (BFPT) .
(1) Training stage: According to the surface 0.5cm, the rats were put into the restricted rats to find food, and the rats in each group were given adaptive training for three days.
(2) Examination stage: after three days of adaptive training, behavioral tests were conducted on each group. To avoid the influence of circadian rhythm, the experiment began at 6:00–8:00 pm (fasting for 24 h before the experiment). The feeding time (T value), activity distance, and average speed of rats in each group were recorded. A feeding time of more than 300s indicated impaired olfactory sensitivity of the rats.
1.5 Statistical analysis
Statistical analysis was carried out using the statistical software SPSS version 22.0 and GraphPad Prism version 8. The data were expressed by mean ± standard deviation (‾x ± s). If the measurement data were in line with normal distribution and met the homogeneity of variance, the single-factor analysis of variance (one-way ANOVA) was used for the analysis, selecting the least significant difference (least significant difference, LSD) method according to the variance results. For data that were not normally distributed, the non-parametric (rank-sum) test was used. The difference was statistically significant at p-values of < 0.05.