Hong Cai, Jihong Wu

Abstract

Objective: To observe the effects of moxa smoke condensate on the cell proliferation, ROS and phagocytic function of the rat’s alveolar macrophages.

Methods: In this cell proliferation experiment, NR8383 cells cultured in vitro were exposed to different concentrations of moxa smoke condensate for 12、24、36 and 48 hours, then the cell proliferation was detected by CCK-8. In ROS determination and phagocytic function experiments, the concentration design of moxa smoke condensate was the same as before. NR8383 cells were exposed for 24 hours, and ROS was measured using DCFH-DA reagent kit. The phagocytic function of cells was measured using a fluorescence microsphere phagocytosis experiment.

Results: After NR8383 cells were exposed to different concentrations of moxa smoke condensate for 12h, though the cell viability of each experimental group was higher than the control group, there were no statistically significant differences between each experimental group and the control group. After NR8383 cells were exposed to the different concentrations of moxa smoke condensate for 24h and 36h with the concentrations of 0.004 g/L、0.008 g/L、0.012 g/L、0.016 g/L, the cell viability of each experimental group was higher than the control group, and there were statistically significant differences between the control group and each experimental group（P<0.05). When the concentration was 0.020 g/L, although the cell viability of each experimental group was lower than the control group, there were no statistically significant differences between the control group and each experimental group（P>0.05). After NR8383 cells were exposed to different concentrations of moxa smoke condensate for 48 hours, there were statistically significant differences between each experimental group, with the exception of the 0.008 g/L one and the control group of the cell viability（P<0.05). After 24h of exposure to different concentrations of moxa smoke condensate on NR8383 cells, the ROS of NR8383 cells decreased with each increase of moxa smoke condensate concentration. The differences between the experimental groups and the blank control group were statistically significant (P<0.001). As a result, phagocytic function increases with the increase of the concentration of moxa smoke condensate. With the exception of the experimental group with a concentration of 0.004 mg/ml, the differences between the other groups and the blank control group were statistically significant (P<0.05, corrected chi square value).

Conclusion: Moxa smoke condensate has an effect, based on concentration and time, on the proliferation of NR8383 cells. Within a certain concentration range, moxa smoke condensate can reduce the ROS of NR8383 and enhance phagocytic function. This effect may be one of the important factors that demonstrates that moxa smoke can improve the body’s autoimmunity, prevent respiratory diseases, and repair body damage.

Keywords: moxa smoke condensate， alveolar macrophages， cell proliferation， ROS， Phagocytic function

Moxibustion has the functions of expelling wind and relieving the exterior, warming meridians and unblocking collaterals, circulating qi and ascending yang, dispelling cold and relieving pain, strengthening the kidney and spleen, restoring yang, dissipating blood stasis, clearing heat, detoxifying the body, and so forth. However, increasing concerns of health safety, likening moxa smoke to cigarette smoke, have been discussed broadly. Some scholars suggested that long-term inhalation of moxa smoke will lead to various chronic lung diseases in the human body.^［1］ The question of whether moxa smoke condensate has a regulatory effect on the body’s autoimmunity and reparation of body damage provided an experimental basis for the safety evaluation of moxibustion. This study mainly investigated the effects of moxa smoke condensate on cell proliferation of rat’s alveolar macrophages, NR8383 cells, ROS, and phagocytic function.

Materials and methods

1. Materials

1.1 Alveolar macrophage cell line

The NR8383 cells were selected from the laboratory of radiation toxicology and medical protection, Chinese People’s Liberation Army Academy of Military Sciences. The cells were obtained from normal rats during the lung lavage.

1.2 Main equipment and reagents

Three years Chen moxa (Ambrosia artemisiifolia) (Nanyang Han Medical Moxa L. L. C.); Flow Cytomety (BD, USA); CO2 cell incubator (Thermo, United States); Portable PM2.5 sampler; L3030-1ML (Sigma Corporation, United States); Microbiometers (Bio-RAD Inc., United States); Model 2-6 high speed centrifugation (Sigma, USA); Model 6930 cryogenic high speed centrifugation (Japan Kubota Co., Ltd.); DCFH-DA (Biyuntian Institute of Biotechnology, China); Ham’s F-12K culture medium (Invitrogen Corporation, United States); Fetal bovine serum FBS (ExCell, Inc., USA); A Cell Counting Kit-8 (CCK-8) (Tokyo Research Institute, Japan) etc.

2. Methodology

PIn this study, the researchers conducted sufficient pre-experiments, and the relevant data of the pre-experiments showed a corresponding proliferative effect of the Moxa Smoke Condensate on NR8383 cells with a concentration of 0.02 mg / ml or below, and the median lethal concentration (IC50) of 24 h exposure is 0.039 mg / ml. 95% confidence interval 0.0293-0.047.

2.1 Preparation of moxa smoke condensate

①The Cambridge filter was dried and balanced in a glass vessel with silica gel. The weight of the Cambridge filter was weighed 24 hours later. ②A convenient PM2.5 sampler was used to collect moxa, the size-selective inlets was PM10, and the filter needed to be replaced every 8 minutes; ③ After 24 h of silica gel drying and balancing, the Cambridge filter of moxa smoke was weighed; ④ The smokeless part of the Cambridge filter was removed, leaving the smoky part of the Cambridge filter. Then it was put into the flask, with the organic solvent DMSO (Dimethyl sulfoxide) added. The Cambridge filter was dissolved in DMSO for 1h in the flask, then stirred and smashed in the flask with a glass rod. ⑤ The suspension from DMSO was taken to obtain the moxa smoke condensate, which had a 40g / L concentration ; ⑥ The moxa smoke condensate was filtered and sterilized by using a sterile filter membrane of 0.22 μm, then packed in the corresponding EP tube, respectively, and stored in EP tubes in the refrigerator at -80 ° C.

2.2 Cell Culture

PThe cryopreserved rat alveolar macrophages NR8383 cells were resuscitated in F12K medium containing 15% FBS (fetal bovine serum), supplemented with 1.5 g / L NaHCO, 2 mmol L-glutamine, 100 U / mL streptomycin and 100 U / mLpenicillin. NR8383 cells were cultured in a 37C cell incubator containing 5% CO2.

2.3 Cell Proliferation Assay

The probability of survivability of NR8383 cells was detected by CCK-8 method.^［2］. Probability of survivability = (OD of test-OD of null) / (OD of blank-OD of null).

Rat alveolar macrophage NR8383 cells were inoculated at 5×103 / well with a volume of 100 μ L in 4 different 96-well plates (plate 1), as well as in the same volume of F12K culture medium without NR8383 cells in four different 96-well plates (plate 2). The concentrations of the moxa smoke condensate at 0, 0.004 mg/ml、0.008 mg/ml 、 0.012 mg/ml 、0.016 mg/ml、0.02 mg/ml were applied to treat each well, including those with and without NR8383 cells. The incubation time of the moxa smoke condensate was 12hrs, 24hrs, 36hrs and 48hrs, respectively. A Reactant of 10 μL of CCK-8 was added to each well of the plates. After incubating for 3h, the absorbance (OD value) of 96-well plates was read by a microplate reader (450nm).

The probability of NR8383 cell survivability was calculated as follows: (1). A = OD value of Plate 1-OD value of Plate 1 null; (2). B = OD value of blank of plate 1-OD value of null of plate 1; (3). C = OD value of the experiment of the plate 2-OD value of null adjustment of the plate 2; (4). D = OD value of blank of plate 2-OD value of null of plate 2; Probability of survivability = (A-C) / (B-D).

2.4 The reactive oxygen species assay

The macrophage NR8383 cells were inoculated in 6 well plates with 1 × 105 cells / well. NR8383 cells were cultured with a media volume of 2 ml each well in a CO2 incubator. When the NR8383 cell growth reached the density of 70% confluency under a microscopy, the cells were treated with the moxa smoke condensate at the concentrations of 0 mg / ml, 0.008 mg / ml, 0.016 mg / ml, 0.024 mg / ml, 0.032 mg / ml and 0.04 mg / ml, respectively. After 24 hours of exposure, the cell suspension from the 6-well plate was loaded into different centrifuges, and the supernatant was removed by centrifugation at 1500 rpm for 5 min. Then, 2ml diluted DCFH-DA was added into each centrifuge tube, and the cells were resuspended and incubated in an incubator at 37 °C for 20min. The cells were centrifuged again, then washed with 500μl PBS, resuspended again, and the content of reactive oxygen species (ROS) by flow cytometry was determined.

2.5 Cell phagocytosis test

The phagocytic function of rat alveolar macrophage NR8383 cells was determined by flow cytometric quantitation with fluorescent microspheres. The NR8383 cells were inoculated in 6 well plates at 1×105 cells / well. The culture volume was 2 ml per well. mg/ml、0.004 mg/ml、0.008 mg/ml、0.012 mg/ml、0.016 mg/ml、0.02 mg/ml） After 24 h of exposure, the cell suspension in the 6-well plate was loaded into different centrifuge tubes, centrifuged at 1500 rpm for 5 min, and then 1.5 ml diluted microspheres (L-3030) were added into each centrifuge tube. The cells were resuspended and incubated in the incubator at 37 °C for 2 h, then centrifuged again. 500 μl of PBS was added into each centrifuge tube. After washing and resuspending the cells, the phagocytosis rate of the alveolar macrophages NR8383 cells was detected by flow cytometry.

2.6 Statistical analysis

The experimental data were analyzed by SPSS16.0 software. Analysis of variance (ANOVA) was performed between each group and the control group (0 mg / ml). P < 0.05 demonstrated a significant difference and P < 0.01 demonstrated an extremely significant difference. The data of phagocytosis rate detected by flow cytometry were analyzed by a chi-square test, namely the chi-square segmentation method.

Results

1. The effect of moxa smoke condensate on the probability of survivability of NR8383 cells

The probability of survivability of NR8383 cells with moxa smoke condensate treatment was higher than that of NR8383 cells without treatment, but there was no significant difference (P > 0.05). After 24h and 36h, the NR8383 cells treated with moxa smoke condensate at the concentrations of 0.004mg / ml, 0.008mg / ml, 0.012mg / ml, 0.016mg / ml, had a significantly higher probability of survival than that of the control group (P < 0.05). When the concentration was 0.02 mg / ml, the probability of survival of the treated group was lower than that of the control group (P > 0.05). After 48h exposure, the probability of survival of each group (except 0.008mg / ml exposure group) was significantly different from that of the control group (P < 0.05).

Table 1 The cell viability after NR8383 cells were exposed to different concentrations of

moxa smoke condensate for 12h、24h、36h、48h（%； ±s；n=8）

Note: *P<0.05 **P<0.01 compared with control group

NR8383 cells were exposed to different concentrations of moxa smoke condensate for 12, 24, 36 and 48hrs. The viability increased with the increase of the concentration of moxa smoke condensate. It can be seen that the concentration of moxa smoke condensate has an effect on proliferation of NR8383 cells. With each duration, the viability of NR8383 cells first increased, then decreased with the increase of the concentration. Shown in Figure 1.

Fig.1 The relationship between the survivability and moxa smoke condensates after exposures of 12h, 24h, 36h, and 48h.

2.Effects of Different Concentration of Moxa Smoke Condensate on Reactive Oxygen Species (ROS) in NR8383 Cells

The NR8383 cells were treated with different concentrations of moxa smoke condensate for 24h. The higher the concentration of moxa smoke condensate, the lower the ROS of NR8383 cells. When the concentration of moxa smoke condensate reached 0.04mg / ml, the fluorescence intensity of ROS of NR8383 cells decreased by half. There were significant differences between the experimental groups and the control group (P < 0.001). The results are shown in table 2.

Table 2 Reactive oxygen Species intensity (± s) of NR8383 cells after exposed to the different concentration of moxa smoke condensate for 24 hours

3.Effects of Different Concentrations of Moxa Smoke Condensate on the Phagocytosis of NR8383 Cells

After being exposed to moxa smoke condensate for 24 hours, the phagocytic function of NR8383 cells increased with the concentration of moxa smoke condensate. When the concentration of moxa smoke condensate was 0.02 mg / ml, the phagocytic function of NR8383 cells increased to more than 7 times compared to the normal level. Except for the experimental group with the concentration of 0.004 mg / ml, the difference between the other experimental groups and the control group was significant (P < 0.005). Specific results are shown in Table 3:

Table 3 The phagocytic rate of NR8383 cells after exposed to the different concentrations

of moxa smoke condensate (%; x±s）

Note: Compared with the control group (0 mg / ml), * P < 0.005 (Modified chi-square)

Discussion

Rat’s alveolar NR8383 cells are the normal alveolar macrophages from bronchoalveolar lavage. They have the function of phagocytosis and elimination of foreign bodies. The cell is an important part of the immune system. Macrophages are known as “scavengers,” and as specialized bacteria-eating cells in the body. They have the function of recognizing, phagocytizing and processing foreign bodies.^［3］ Alveolar macrophages are also considered to be the main initiating cells of local airway inflammation because they are the frontline cells and have the most opportunities to contact with foreign antigens and pathogenic microorganisms.^［4］ In inflammatory lesions, the proliferation of macrophages plays a specific role in the immune response of the body, and the cell proliferation response is the premise and basis of the differentiation of immune cells into a variety of functional phenotypes.^［5］ When moxa smoke enters the human body, it first acts on the human respiratory system. The advantages and disadvantages of moxa smoke on the human respiratory system have been studied. Several studies have shown that moxibustion can activate the body’s immune system, thereby improving the body’s immune capacity^［6-7］. But there are also related studies. ^［8］When the particle size in the air is less than or equal to 10 μ m, it can enter the interior of the human body, and when the particle size is less than or equal to 2 μ m, it can be stored and remain in the human lung. More than 95% of the substances roduced in moxa smoke are less than or equal to 2 μm, which means that there are many substances in moxa smoke that can deposit and stay in the human lungs, which may have adverse effects on the human body. Therefore, the safety evaluation of moxa smoke will play a crucial role in guiding clinical application.

The results showed that there were certain concentration effects and time effects on the proliferation of rat alveolar macrophage NR8383 cells. The viability of NR8383 cells increased first and then decreased with the increase of the concentration of moxa condensate. In a certain range of experimental concentrations, the higher the concentration of moxa smoke condensate, the lower the ROS in NR8383 cells, the stronger the phagocytic ability. Therefore, researchers believe that the proliferative effect and phagocytic effect of moxa may be one of the important reasons why moxa can be used for anti-cold, anti-inflammatory and anti-infection. The decrease of ROS in NR8383 cells also indicated that moxa could protect the normal structure and function of NR8383 cells to some extent. This effect may also be an important factor in the prevention of respiratory system diseases and the improvement of the immune system. Therefore, when moxa acts on human alveolar macrophage cells, is the cell performance the same as when it acts on rat alveolar macrophage NR8383 cells? What is the optimal dose and duration of treatment for effective protection of the human respiratory system? Can the moxa smoke of big density and remedial time produce toxic side effect to human body? These problems need to be further studied and explored.

Author information:

Hong Cai, Doctor of Yubei District Peoples Hospital of Chongqing

Jihong Wu, Professor, Master tutor of Beijing University of Chinese Medicine