Effects of moxa smoke condensate on the cell proliferation, Reactive Oxygen Species (ROS), and phagocytic function of the rat’s alveolar macrophages the NR8383 cells

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

艾煙冷凝物對大鼠肺泡巨噬細胞NR8383細胞增殖、

ROS和吞噬功能的影響

蔡虹 鄔繼紅

摘要

目的：觀察不同濃度的艾煙冷凝物對NR8383細胞的增殖、活性氧（ROS）和吞噬功能的影響。

方法：把不同濃度的艾煙冷凝物對NR8383分别進行細胞染毒,12h、24h、36h、48h後，采用CCK-8法對細胞的增殖能力進行檢測；艾煙冷凝物的染毒濃度設計同前，均對NR8383進行細胞染毒24h，分别采用DCFH-DA試劑盒測定ROS、熒光微球吞噬實驗測定細胞的吞噬功能。

結果：實驗所設計的不同濃度的艾煙冷凝物對NR8383染毒12h後，各實驗組與空白對照組結果差异無統計學意義（P>0.05）。分别進行染毒24h及36h後，各實驗組(染毒濃度爲0.004 mg/ml、0.008 mg/ml、0.012 mg/ml、0.016 mg/ml)的細胞存活率均高於空白對照組，差异有統計學意義（P<0.05），當實驗組濃度爲0.020 mg/ml時，NR8383的細胞存活率雖低於空白組，但差异無統計學意義（P>0.05）。分别進行染毒48h後，除0.008 mg/ml的實驗組以外，其餘各實驗組的細胞存活率與空白對照組差异均具有統計學意義（P<0.05）。上述不同濃度的艾煙冷凝物對NR8383分别染毒24h後，艾煙冷凝物的濃度越高,NR8383細胞的ROS越低,各實驗組與空白對照組差异具有統計學意義（P<0.001）。艾煙冷凝物的濃度越高,吞噬功能越强,除濃度爲0.004 mg/ml的實驗組以外，其他各組與空白對照組的差异均具有統計學意義（P<0.005，校正卡方值）。

結論：艾煙冷凝物對NR8383的細胞增殖能力具有一定的濃度及時間效應。且在一定濃度範圍内，它可以降低NR8383的ROS、增强NR8383的吞噬功能。

關鍵詞: 艾煙冷凝物，大鼠肺泡巨噬細胞，細胞增殖，細胞活性氧，吞噬功能

灸法因其具有祛風解表、温經通絡、益氣昇陽、散寒止痛、温腎健脾、消散瘀結、拔毒泄熱、回陽固脱等功效，故常被作爲防病保健的重要手段之一,深受大家喜愛。然而，隨着人們對醫療環境的安全要求日益增加，艾煙是否跟香菸一樣有害,成了大家不容忽視的問題。有學者曾經提出，長期吸入艾煙會導致人體出現各種肺部的慢性疾病^［1］。本實驗通過艾煙冷凝物，即艾煙煙氣生成物，對大鼠肺泡巨噬細胞NR8383的細胞增殖能力、ROS和吞噬功能的研究，以探討其對人體自身免疫力和修復機體損傷等方面是否具有一定調節的作用，進而爲艾煙的安全性評價提供一定的實驗依據。

材料和方法

1.材料

1.1肺泡巨噬細胞株

本實驗所選取的NR8383細胞來自我國軍事醫學科學院放射毒理與醫學防護實驗室，它是正常大鼠在肺灌洗時的肺泡巨噬細胞。

1.2 主要器材與試劑

三年陳艾條（南陽漢醫艾絨有限責任公司）；流式細胞儀（BD公司,美國）；CO2細胞培養箱（Thermo 公司,美國）；便携式PM2.5採樣器； L3030-1ML（Sigma公司,美國）；酶標儀（Bio-RAD公司,美國）；2－6型高速離心機（Sigma公司,美國）；6930型低温高速離心機（日本久保田株式會社）；DCFH-DA（中國碧雲天生物技術研究所）； Hams F-12K培養液（Invitrogen公司,美國）；胎牛血清FBS（ExCell公司,美國）；A Cell Counting Kit-8 (CCK-8)（日本Tokyo研究所）等等。

2. 方法

本實驗的研究者進行了充分的預實驗，預實驗的相關研究數據表明：艾煙冷凝物作用於NR8383 細胞後，在染毒濃度爲0.02mg/ml以前可能出現了相應的增殖效應，且染毒24h 的半數致死濃度（即 IC50）爲 0.039mg/ml；95%置信區間爲0.0293-0.047。

2.1 艾煙冷凝物的制備

①將劍橋濾片置於裝有硅膠的玻璃器皿中進行乾燥、平衡，24h後稱取經乾燥、平衡後劍橋濾片的重量；②采用便携式的PM2.5採樣器采集艾煙，其切割頭爲PM10，每間隔8分鐘需要更换一次濾片；③經24h硅膠乾燥、平衡後，稱取帶有艾煙的劍橋濾片的重量；④去掉劍橋濾片上無煙的部分，保留劍橋濾片上有煙的部分，並將其放入三角瓶中，經計算後加入相應劑量的有機溶劑DMSO(Dimethyl sulfoxide，二甲基亞碸)，溶解時間爲1h，溶解期間需用玻璃棒充分搗碎瓶中劍橋濾片；⑤吸出溶於有機溶劑DMSO的艾煙懸液，得到本實驗所需的艾煙冷凝物，其濃度爲40g/L；⑥使用0.22μm的無菌濾膜對上述所得濃度的艾煙冷凝物進行過濾除菌，分别裝在相應的EP管内，並將其存放在-80℃的冰箱中以備用。

2.2 細胞培養

將凍存的大鼠肺泡巨噬細胞NR8383進行復蘇，NR8383細胞培養環境爲：含15%FBS(Fatal bovine serum，胎牛血清)的F12K培養液中添加1.5g/L NaHCO₃、2mmol L-谷氨酰胺、100U/mL的鏈霉素和100U/mL的青霉素。將肺泡巨噬細胞NR8383細胞放在37℃含5%CO2的細胞培養箱中培養。

2.3 細胞增殖檢測試驗

本實驗采用CCK-8法來檢測NR8383的細胞存活率^［2］。細胞存活率=（實驗孔的OD值-調零孔的OD值)/（空白孔的OD值-調零孔的OD值）

將對數生長期的大鼠肺泡巨噬細胞NR8383以5×103 /孔分别接種在4個不同的96孔板中（板1），每個孔的體積均爲100μL。再以相同體積的不含NR8383細胞的F12K培養液分别接種在另外4個不同的96孔板中（板2），以不同濃度(0、0.004 mg/ml、0.008 mg/ml 、 0.012 mg/ml 、0.016 mg/ml、0.02 mg/ml）的艾煙冷凝物對NR8383細胞進行染毒，没有NR8383細胞的96孔板也以相同的方法進行染毒，兩者的染毒時間均分别爲12、24、36、48h，待染毒時間結束後，向每個板中的每個孔均加入CCK-8試劑10μL，培養箱中孵育3h後，再以450 nm波長的酶標儀分别讀取96孔板的吸光度（OD值）。

NR8383細胞存活率計算步驟如下：①A=板1實驗孔的OD值-板1調零孔的OD值；②B=板1空白孔的OD值-板1調零孔的OD值；③C=板2實驗孔的OD值-板2調零孔的OD值；④D=板2空白孔的OD值-板2調零孔的OD值；⑤細胞存活率=(A-C)/(B-D)。

2.4 細胞活性氧測定

將對數生長期的大鼠肺泡巨噬細胞NR8383分别接種於以1×105 cells/孔的6孔板中，每個孔均爲2ml的培養體積，將其置於CO2細胞培養箱中培養，待NR8383細胞長至顯微鏡下視野面積的70%密度時，即可艾煙冷凝物進行染毒。染毒的濃度分别如下：0、0.008 mg/ml 、0.016 mg/ml 、0.024 mg/ml 、0.032 mg/ml、0.04 mg/ml，染毒24h後，將6孔板中的細胞懸液裝入不同的離心管中，〖JP2〗1500rpm離心5min後去掉上清，每個離心管中均加入2ml已經稀釋好的DCFH-DA，重懸細胞，將其放在37℃細胞培養箱内孵育20min後，再次離心，加入500μl的PBS對細胞進行清洗，重懸細胞，最後用流式細胞儀檢測NR8383細胞的活性氧含量。

2.5 細胞吞噬試驗

本實驗采用熒光微球吞噬實驗對大鼠肺泡巨噬細胞NR8383的吞噬功能進行測定。將對數生長期的大鼠肺泡巨噬細胞NR8383細胞以1×105 cells/孔接種在6孔板中，培養體積爲每孔2ml，將不同濃度(0 mg/ml、0.004 mg/ml、0.008 mg/ml、0.012 mg/ml、0.016 mg/ml、0.02 mg/ml）的艾煙冷凝物分别進行細胞染毒，染毒24h後，將6孔板中的細胞懸液分别裝入不同的離心管中，1500rpm離心5min後棄上清，加入1.5ml稀釋好的微球（L-3030）於每個離心管中，重懸細胞，在37℃的細胞培養箱内孵育2h後再次離心，向每個離心管中加入500μl的PBS，清洗並重懸細胞後，以流式細胞儀檢測大鼠肺泡巨噬細胞NR8383的細胞吞噬率。

2.6 統計學分析

用spss16.0統計軟件對上述實驗數據資料進行統計學分析。計量資料用均數(〖XC1.eps,JZ;P〗)±標準差(s)表示，各實驗組與空白對照組（0 mg/ml）進行方差分析，在此基礎上進行多組均數兩兩比較（LSD-t檢驗）。P<0.05爲顯著性差异，P<0.01爲極顯著性差异。流式細胞儀檢測的細胞吞噬率數據采用卡方檢驗，即卡方分割法。

結果

1.不同時間及不同濃度的艾煙冷凝物對NR8383細胞存活率的影響

艾煙冷凝物對大鼠肺泡巨噬細胞NR8383分别進行染毒12h後，各實驗組的細胞存活率均高於空白對照組，但其差异無統計學意義（P>0.05）。細胞染毒24、36h後，各實驗組（染毒濃度爲0.004 mg/ml、0.008 mg/ml、0.012 mg/ml、0.016 mg/ml）的細胞存活率均高於空白組，且與空白組差异有統計學意義（P<0.05）。當實驗組的染毒濃度爲0.02 mg/ml時，其存活率雖然低於空白組，但與空白組的差异無統計學意義（P>0.05）。染毒48h後，各實驗組（染毒濃度爲0.008 mg/ml的實驗組除外）的細胞存活率與空白組的差异有統計學意義（P<0.05）。〖JP2〗當艾煙冷凝物的染毒濃度達到0.02mg/ml時，除染毒時間相對較短的12h外，其餘各組均出現了細胞抑制現象。結果見表1。

表1 不同浓度艾烟冷凝物对细胞进行12h、24h、36h、48h染毒后的细胞存活率（%；±s；n=8）

注:与空白对照组比较*P<0.05 **P<0.01。

實驗所設計的不同濃度的艾煙冷凝物對大鼠肺泡巨噬細胞NR8383細胞分别染毒12、24、36、48h後，其存活率伴隨着艾煙冷凝物濃度的昇高而呈現出了先增後降的趨勢，由此可以看出，艾煙冷凝物對NR8383細胞的增殖作用具有一定的濃度效應。且伴隨着作用時間的不斷增加，NR8383細胞的存活率也隨其濃度的增高而先增後降，由此可以看出，艾煙冷凝物對NR8383細胞的增殖作用亦具有一定的時間效應。如圖1所示。

图1 不同染毒12h、24h、36h、48h后细胞存活率与艾烟冷凝物浓度的关系

2.不同濃度的艾煙冷凝物對NR8383細胞活性氧的影響

采用不同濃度的艾煙冷凝物對大鼠肺泡巨噬細胞NR8383分别染毒24h後，艾煙冷凝物濃度越高，NR8383的細胞活性氧越低，當艾煙冷凝物的染毒濃度達到0.04mg/ml之時，大鼠肺泡巨噬細胞NR8383的細胞活性氧熒光强度降低了一半左右。各實驗組與空白組差异均有統計學意義（P<0.001）。結果見表2。

表2 不同浓度艾烟冷凝物对细胞进行24h染毒后的细胞细胞活性氧荧光强度（±s）

3.不同濃度的艾煙冷凝物對NR8383細胞吞噬功能的影響艾煙冷凝物對大鼠肺泡巨噬細胞NR8383細胞分别染毒24h後，NR8383細胞的吞噬功能隨着艾煙冷凝物濃度的昇高而呈現出增强的趨勢，當艾煙冷凝物濃度爲0.02mg/ml時，NR8383的細胞吞噬功能提高到了正常水平的7倍以上。除染毒濃度爲0.004 mg/ml的實驗組以外，其他各實驗組與空白對照組差异均具有統計學意義（P<0.005）。具體結果如表3所示：

表3 不同浓度艾烟冷凝物对细胞进行24h染毒后的细胞吞噬率（%；±s）

注:与空白对照组(0 mg/ml)比较,*P<0.005(校正卡方值)

討論

大鼠肺泡巨噬細胞NR8383爲正常大鼠在支氣管肺泡灌洗時的肺泡巨噬細胞，它具有吞噬及消除异物的功能，是機體免疫系統中的重要組成部分。巨噬細胞有“清道夫”之稱，其作爲體内專門的食菌細胞，具有識别、吞噬及加工外來异物的作用^［3］。肺泡巨噬細胞也因其可以最早並具有最多的機會去接觸外界的抗原類物質和病原微生物，因此它被認爲是機體氣道局部炎症反應的主要始動細胞^［4］。在炎症病竈中，巨噬細胞的增殖在機體免疫應答中發揮着它特定的作用，而細胞的增殖反應則是機體免疫細胞分化成多種功能表型的前提和基礎^［5］。艾煙進入人體後，首先將會作用於人體的呼吸系統。而艾煙對人體呼吸系統産生的利弊也因實驗研究方向的不同而有所差别。有多個研究表明，艾灸可以激活人體的免疫系統，從而提高人體的免疫能力^［6-7］。但亦有相關研究提出^［8］，當空氣中的顆粒物大小小於等於10μm時就可以進入人體的内部，顆粒物大小小於等於2μm時就可以沉積並停留在人體的肺部。艾煙生成物中，有95%以上的物質都小於等於2μm，這便意味着艾煙中會有多種物質可以在人體的肺部沉積並停留，這種效應可能會對人體産生不良的影響。因此，艾煙的安全性評價將對臨床的指導應用起着至關重要的作用。

本實驗研究結果顯示：艾煙冷凝物對大鼠肺泡巨噬細胞NR8383細胞的增殖存在着一定的濃度效應和時間效應。在實驗所設計的染毒時間上，NR8383細胞均隨着艾煙冷凝物濃度的昇高呈現出了先增加後降低的趨勢，且隨着染毒時間的不斷延長，NR8383細胞的存活率亦隨着艾煙冷凝物濃度的增高而先增加後降低。在一定的實驗濃度範圍内，艾煙冷凝物的染毒濃度越高，NR8383細胞内的ROS越低，其吞噬能力也越强。於是，本實驗研究者認爲，艾煙的這種增殖效應及吞噬效應可能是艾煙能被用於滅菌、抗感冒、抗炎以及抗感染等方面的重要原因之一。NR8383細胞内ROS的降低，也表明艾煙冷凝物對該類細胞的正常結構和功能起到一定程度的保護作用。這種作用也可能是艾煙能够預防呼吸系統疾病和提高機體免疫力的重要因素。那麽，艾煙作用於人體肺泡巨噬細胞時細胞表現是否與其作用於大鼠肺泡巨噬細胞NR8383時的表現一樣呢？怎樣的艾煙濃度及治療時間才能使其對人體呼吸系統的有效保護作用發揮到最佳水平？多大濃度的艾煙及治療時間會對人體産生毒副作用？這些問題都有待我們進一步研究和探索。

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作者簡介:

蔡虹,重慶市渝北區人民醫院醫生

鄔繼紅,教授，北京中醫藥大學碩士生導師