J Complement Integr Med. 2015; 12(1): 43–51

Quan Zhang, Hui Wang, Ming Liang Cheng*, Mingchang Jin, Qing Zhi Meng, Liang Duan and Yun Chen

The Miaoyao Fanggan Sachets regulate humoral immunity and cellular immunity in mice Abstract Background: Although some studies in the southeast part of Guizhou Province have suggested that Miaoyao Fanggan Sachets (MFS) prevent influenza, little is known about its influence on immune systems. Influenza virus mainly infects immune-compromised individuals. The effects of MFS have mainly been recognized in clinical practice. However, there have been relatively few studies on its biological mechanism. Here we investigated whether MFS was able to affect the mucosal immunization and the activation of alveolar macrophages (AM), CD4þ and CD8þ T-cells in vivo. Methods: Eighty Kunming male mice were treated with MFS continuously or intermittently with Yu-Ping-Feng powder (YPF-P) (positive control group) or with normal saline (NS) (control group) for 4 weeks, respectively. Mice treated with MFS were further divided into the continuous inhalation group (12 h daily/4 weeks) and the discontinuous inhalation group (1 h, three times a day for 4 weeks). Mice in both groups were placed under 0.5 m3 masks which had four ventilation holes (10  15 cm) containing 40 g MFS. Positive control mice were orally treated with YPF-P 0.2 mg/10 g/day once a day for 4 weeks. Control mice were orally treated with equal volumes of NS once a day for 4 weeks. MFS was replaced every 6 days. Administration of YPF-P was used as a positive control since it has been used as an established Traditional Chinese Medicine (TCM) treatment before. After 4 weeks, mice in all experimental groups were sacrificed. IgA and IgG1 in lung and blood serum were *Corresponding author: Ming Liang Cheng, Department of Infectious Diseases, Guiyang Medical College and Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, P.R. China, E-mail: [email protected] Quan Zhang, Hui Wang, Department of Infectious Diseases, Guiyang Medical College and Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, P.R. China Mingchang Jin, Department of Traditional Ethnic Medicines, Qiandongnan Rehabilitation Hospital for Nationalites, Kaili, Guizhou Province, P.R. China Qing Zhi Meng, Liang Duan, Yun Chen, Department of Infectious Diseases, Guiyang Medical College and Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, P.R. China

detected by Western blot and enzyme-linked immuno sorbent assay (ELISA). The expression of alveolar macrophages (AM) in mice was analyzed by immunochemistry test based on CD68þ staining. Blood samples were collected in which CD4þ and CD8þ T-cells were analyzed by flow cytometry. Results: Mice continuously and intermittently inhaling MFS showed a moderate increase in IgA and IgG1 protein levels compared with mice in the control groups. There was also a slightly significant increase in the number of AM in the continuous inhalation group compared with mice in the control groups (p < 0.05). Furthermore, compared with controls, there was also a slightly significant increase in the number and percentage of CD4þ and CD8þ T-cells in both the continuous inhalation group and the discontinuous inhalation group (p < 0.05). Conclusions: MFS was able to up-regulate the protein levels of sIgA and IgG1. Meanwhile, MFS could activate AM, CD4þ and CD8þ T-cells in mice. Our data have, for the first time, demonstrated that the protection against influenza by MFS is partly through activation of the innate and adaptive cell-mediated immune responses, indicating MFS as a potential new immune-modulatory agent for respiratory tract infectious disease. Keywords: alveolar macrophage, CD4þ T-cells, CD8þ T-cells, IgG1, influenza, Miaoyao Fanggan Sachet, Yu-Ping-Feng powder, sIgA DOI 10.1515/jcim-2013-0009 Received February 27, 2013; accepted September 17, 2014; previously published online November 11, 2014

Introduction Influenza tremendously affects the entire population, has an enormous impact upon the economic burden and causes a significant number of human deaths [1]. It is essentially important to prevent influenza. Vaccines are considered one of the most important medical interventions for reducing influenza-related morbidity. However, the efficacy of currently available vaccines is often unsatisfying due to virus variations which allow the

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

virus to escape the immune surveillance, side effects and prohibitive cost in some areas. In addition, many individuals are reluctant to get injections [2–4]. Traditional Chinese Medicine (TCM) has been a good supplement for underutilized vaccines. In China, the use of aromatic TCM herbs is a common practice by putting them into small pockets and wearing around the neck to prevent influenza, which is referred to as Xiang-Pei-Liao-Fa (Sachet-therapy) [5, 6]. Xiang-Pei-Liao-Fa is painless and free of side effects, which may encourage a large number of individuals to participate in the prevention program. The Xiang-PeiLiao-Fa was well tolerated with very few adverse effects and extremely low morbidity during the administration period in the pandemic outbreaks [7–10]. Wearing MFS round the neck is one way of the Xiang-Pei-Liao-Fa in Chinese traditional treatments. The major components of MFS, wo ga le, bang wo wu and jia wo jiao mi, are aromatic TCM herbs mainly grown in the Guizhou Province, southwest of China. The unique natural geographical climate contributes to the unique efficacy of TCM herbs in this area. TCM preparations follow different paradigms, that is, use mixtures of compounds belonging to several families and each family regulates one or more targets. The components used for the direct treatment are named “King” components, while other compounds used for relieving the symptom are referred to as “Minister”, “Assistant” or “Messenger” [2]. In the case of MFS, wo ga le (Valeriana officinalis var. latifolia) is the “King”, and other herbs may be “Ministers”, “Assistants” or “Messengers”. MFS have long been used as a folk medicine for the prevention of seasonal influenza in the southeast part of Guizhou Province [11]. However, there have been relatively few studies on the biological actions of these natural aromatic herbs (wo ga le, bang wo wu and jia wo jiao mi). During the global outbreak of influenza, no influenza infection was reported in Guizhou Province. It was therefore speculated that the habit of wearing MFS might be, at least to some extent, helpful. All the practices suggest that MFS therapy be capable of preventing influenza viral infections. To our knowledge, innate immunity is the first line of defense to prevent pathogen entry including influenza virus, and adaptive immunity plays a crucial role in the clearance or containment of viral infections. Influenza virus mainly infects immune-compromised individuals [12]. So we hypothesize that the protection of MFS against influenza might, at least partly, be associated with regulating the immunity functions. Thus, the present study was designed to validate this hypothesis.

Materials and methods Animals Kunming male mice (18–25 g) were purchased from the Experimental Animal Center of the Third Military Medical University (Approval number SCXK, Chong Qing, China, 20070004) and maintained in our animal facility at the Guiyang Medical College (Guiyang, China). All the experimental procedures were conducted following the Guidelines of the Institutional Animal Care and Use Committee, and the research protocol was approved by Guiyang Medical College that enforces compliance with the Chinese Laboratory Animal Use and Care Guidelines.

Pharmaceutics and reagents MFS were composed of wo ga le, bang wo wu and jia wo jiao mi. MFS was purchased from the Ethnopharmacological Research Institution (Lot number 20091209, Qian-Dong-Nan, Guizhou, China), and YPF-P was obtained from Guang Dong Medi-World Pharmaceutical Co., Ltd (Lot number 100706, Guang Dong, China). PageRuler Prestained Protein Ladder was obtained from Fermentas (Lot number 00057114, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Bradford method quantification kits were obtained from the Nanjing Kaiji Biological Engineering Research Institute (Lot number KGP250, Beijing, China). BCA Protein Assay Kits were obtained from Thermo Scientific (Lot number 121779, Rockford, IL, USA). Immobilon Western Chemiluminescent Alkaline Phosphatase Substrate and polyvinylidene fluoride membranes were obtained from Millipore (Lot numbers 1008201 and KICA0037FK, respectively, Billerica, MA, USA). Goat Anti-Mouse IgA (Lot number H0908-SA00), Goat Anti-Mouse IgG1 (Lot number A1210-WB00) and Rabbit Anti- Goat IgG-AP (Lot number 13506-Q919F) were purchased from SouthernBiotech (Southern Biotechnology Associates, Inc., Birmingham, AL, USA). Goat Anti-Rabbit IgG-AP was purchased from Santa Cruz Biotechnology, Inc. (Cat number sc-2034, CA, USA), and β-actin (13E5) Rabbit mAb was purchased from Cell Signaling Technology, Inc. (Cat number 4970S, Beverly, MA, USA). Mouse sIgA ELISA Kit (Cat number F1072) and Mouse IgG1 ELISA Kit (Cat number F10742) were purchased from Westang Biotechnology (Westang, Shang Hai, China). FACS Lysing Solution was obtained from BD Biosciences (Lot number 349202, Franklin Lakes, NJ, USA). PE Rat Anti-Mouse CD8a (Lot number 561095), Percp Anti-Mouse CD3e (Lot number 553067) and FITC Anti-Mouse CD4e (Lot number 557307) were obtained from BD Biosciences. SP Kit was obtained from Zymed Laboratories, Inc. (Lot number SP-0023, San Francisco, CA, USA). DAB (Diaminobenzidine) Kit was obtained from Beijing Biosynthesis Biotechnology Co., Ltd (Lot number C-0010, Beijing, China).

MFS regimes Mice were kept under a controlled environmental condition (temperature: 20  0.5 °C, relative humidity: 40–60 %) with food and water available ad libitum. Mice in the continual inhalation group were kept in a separate room from mice not receiving treatment or mice treated with YPF-P or NS. Mice were placed in plastic cages (5 mice/cage) and allowed to acclimate before the beginning of the experiments. Experiments were performed on 80 mice. Forty Kunming male mice were randomly divided into four groups. The continuous inhalation

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

group (12 h daily/4 weeks) and discontinuous MFS inhalation group (1 h, three times a day for 4 weeks) were placed under 0.5 m3 masks which had four ventilation holes containing 40 g MFS (10  15 cm); mice in the positive control were orally treated with YPF-P 0.2 mg/10 g/day once a day for 4 weeks [13]; mice in the negative control group were treated with equal volumes of NS orally once a day for 4 weeks. MFS was replaced every 6 days. Administration of YPF-P was used as a positive control since it has been used as an established TCM treatment before [14, 15]. After 4 weeks, mice in all the experimental groups were sacrificed. Their lungs and blood plasma were collected to assess IgA and IgG levels. Lungs were stored at –80 °C until examined. The other 40 Kunming mice were randomly divided into four groups; those were continuous inhalation group, discontinuous MFS inhalation group, negative control group and positive control group. Inhalation of MFS, YPF-P or NS was carried out orally as described above. Blood samples were collected after 4 weeks after treatment and analyzed for CD4þ , CD8þ T-cells and CD4þ /CD8þ by flow cytometry. Lung samples were collected and analyzed for AM (CD68þ ) cells by immunochemistry SP method. In the previous experiments, 4-week treatment yielded the similar results as 6-week treatment with respect to TLR2 and TLR4 expressions, therefore, 4-week treatment was used in the present study. MFS typically contains 10 g of material which is placed around the neck to prevent respiratory tract infections. In a pilot study, 40 g was administered as a means of detecting potential toxic effects.

Measurements of IgA and IgG in serum by ELISA Concentrations of IgA and IgG were determined by enzyme-linked immuno sorbent assay (ELISA). Microtiter plates (96-well Nunc microtiter plates, Thermo Fisher Scientific, Inc.) were coated with either anti-mouse IgG (4 µg/mL) or anti-mouse IgA (10 µg/mL) and incubated overnight at 4 °C. After that, plates were washed four times and blocked for 1 h. Diluted samples were added to the plates and incubated for 1 h followed by washing four times and adding the secondary antibody. The secondary antibody was incubated for 1 h in the dark, and the plates were then washed four times and read on a microplate reader, Thermo Scientific Microplate Reader (Thermo Fisher Scientific, Inc.), at a wavelength of 450 nm  10 nm [16, 17].

Western blot analysis

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membranes to X-ray film. β-actin was used as an internal reference [18]. The results are presented as ratios of IgA/actin or IgG1/actin.

Measurement of AM by immunohistochemistry SP method The tissue samples of lungs from mice were preserved in 100 % glutaraldehyde-polyoxymethylene solution, dehydrated and embedded in paraffin according to the routine methods. AM were detected by immunochemistry SP method [19]. The negative control group was carried out with the same steps as described above, but the rabbit anti-mouse CD68þ was replaced by PBS. AM (CD68þ ) expression level in lung tissue: < 9 % is negative, 10–15 % is weak positive (þ ), 15–30 % is positive (þ ), and  31 % is strong positive (þ þ ).

Measurements of CD4þ , CD8þ T-cells and CD4þ / CD8þ by flow cytometry Five microliters of the Percp-CD3e, PE-CD8a, FITC-CD4e reagents were mixed with 50 µL of anticoagulated whole blood, respectively, and 1 mL hemolysin was added to the bottom of the tube using reverse pipetting and incubated for 15 min in the dark at room temperature. Cells were then washed with PBS (phosphate-buffered saline) and diluted in PBS to a final concentration of 1  105 cells/ mL. CD4þ and CD8þ T-cell expressions in mouse peripheral blood cells were then detected by flow cytometry using a BD FACScanto™ flow cytometer (BD Biosciences) [2].

Statistical analysis Group sizes for animal studies were determined from power calculations. Data were imported into SPSS 11.5 software. Quantitative data were expressed as the mean  SD (standard deviation) and subjected to one-way analysis of variance. Ordinal data were analyzed by Ridit analysis. For all statistical tests, a p-Value of less than 0.05 (p < 0.05) was considered significant.

Results

Total protein homogenate was added into PMSF, extracted and quantified using the Bradford method kits as described by the manufacturer. Forty micrograms of total extracted IgA and IgG was boiled in an equal volume of 2  SDS electrophoresis sample buffer for 5 min before being subjected to 10 % and 5 % SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), respectively. After electrophoresis, proteins were transferred onto polyvinylidene fluoride membranes. Membranes were then blocked with 5 % fat-free dry milk-TBST (Tris-buffered saline with 0.05 % tween 20) buffer for 1 h at room temperature. Membranes were washed three times for 5 min in TBST and then incubated with either a 1:2,500 dilution of anti-IgA or a 1:2,500 dilution of anti-IgG overnight. The next day, membranes were washed and then incubated with a 1:5,000 dilution of the respective AP-conjugated secondary antibodies for 1 h. Transferred proteins were visualized with an electrochemiluminescence detection kit using a BioRad Gel Doc XR (BioRad, Hercules, CA, USA) and a short exposure of the

MFS increased IgA secretion sIgA has multiple roles in mucosal defense [20]. It promotes the entrapment of antigens or micro-organisms in the mucus and prevents direct contact of pathogens with the mucosal surface, which is known as “immune exclusion”. Previous studies suggested that respiratory IgA levels in bronchoalveolar lavage fluid (BALF) were significantly increased following MFS treatment and YPF-P [21]. But our present experiment indicated that the sIgA levels in the serum of mice from the continuous MFS inhalation group, YPF-P (positive control) group or discontinuous inhalation group were not increased compared with the

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

MFS increased IgG secretion

Table 1 The level of sIgA and IgG1 in mouse serum (ng/mL,   SD) by ELISA.

MFS continuous inhalation MFS discontinuous inhalation YPF-P treatment Control

sIgA, ng/mL

IgG1, ng/mL

347.08  31.44 338.90  37.93 313.54  104.23 336.37  78.62

241.62  20.97a 217.30  30.09 287.72  31.90† 198.60  35.03

p < 0.05 vs control mice.

a

negative control mice (p > 0.05) (Table 1). However, the IgA levels in the lungs of mice treated with continuous MFS inhalation, discontinuous MFS inhalation or positive control were significantly increased when compared with the negative control mice (p < 0.05) (Table 2, Figure 1). Continuous MFS inhalation mice showed a slight increase in IgA levels in the lungs when compared with the discontinuous MFS inhalation mice. No significant differences existed between continuous MFS inhalation group and positive control group (p > 0.05) (Tables 1 and 2, Figure 1).

Both respiratory system IgG and serum IgG play an important role in preventing influenza [20] Previous studies suggested that respiratory IgG levels in BALF were significantly increased following MFS treatment and YPF-P [21]. Our present experiment indicated that mice from the continuous MFS inhalation group, YPF-P (positive control) group and discontinuous MFS inhalation group were stimulated to produce more IgG than the negative control mice in the lung tissues (p < 0.05; Table 2, Figure 1). Mice in the continuous MFS inhalation group and positive control group showed an increase in serum IgG levels compared with the negative control mice (p < 0.05; Table 1). The IgG levels of serum and lungs from mice in the continuous MFS inhalation group were increased when compared with those of mice in the discontinuous MFS inhalation group. However, no significant differences existed between the continuous MFS inhalation group and positive control group (p > 0.05; Tables 1 and 2, Figure 1).

MFS activated AM (CD68þ ) Table 2

Protein expression of IgA and IgG1 in mouse lung (x  SD).

MFS continuous inhalation MFS intermittent inhalation YPF-P treatment Control

Ratio of IgA/β-action

Ratio of IgG1/β-action

4.05  1.52a 3.90  0.29a 4.25  0.68a 2.42  0.86

3.89  1.95a 2.69  0.89a 2.88  0.83a 1.52  0.38

p < 0.05 vs control mice.

a

AM resides at the air–tissue interface in the lung and is a part of the first lines of defense which interacts with the inhaled micro-organisms and particles [22]. They play a critical role in homeostasis, host defense and tissue remodeling [23]. Whereas, they are also readily infected by influenza [24]. The increases in AM (CD68þ ) expression levels in the lung tissues from mice in the positive control group, continuous inhalation MFS group, discontinuous MFS inhalation group and negative control group were 23.17 %, 27.5 %, 10.64 % and 12.64 %, respectively. Our experiment indicated that mice with the continuous MFS treatment (12 h/day) or YPF-P treatment for 4 weeks had a slightly but significant increase in the CD68þ expression levels (Table 3, Figure 2B, C; p < 0.05) when compared with those of mice from the discontinuous inhalation group and negative control group. Table 3 Effects of MFS on alveolar macrophages (CD68þ ) in lung tissue of mice. Rank of alveolar macrophages (CD68þ )

Figure 1 IgA and IgG protein expressions were assessed by Western blot analysis. Lane 1, blank control mice; Lane 2, continuous inhalation MFS mice; Lane 3, YPF-P positive control mice; Lane 4, discontinuous inhalation MFS. β-actin was used as an internal reference.

MFS continuous inhalation MFS intermittent inhalation YPF-P treatment Control

—

þ

þþþ

þþþ

0 4 0 3

0 1 5 0

5 0 1 4

0 2 0 0

Mean rank

27.50a,b 10.64 23.17a,b 12.64

p < 0.05 vs the MFS discontinuous inhalation group; bp < 0.05 vs control mice.

a

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

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role in the clearance or containment of viral infections. After MFS treatment, blood was collected and analyzed for the presence of CD4þ and CD8þ T-cell markers As shown in Figure 3, both the number and the percentage of CD4þ T-cell in peripheral blood were prominently increased by treatment with continuous MFS inhalation or YPF-P compared with discontinuous MFS inhalation or the negative control (Table 4, Figure 3B, C) (p < 0.05). In addition, the percentage and number of CD8þ T-cell in peripheral blood showed the similar pattern with those of CD4þ T-cells. However, the ratio of CD4þ / CD8þ did not change significantly (p > 0.05; Table 4).

Discussion

Figure 2 Macrophages (CD68þ) expression in lung tissue of mice. Under the fluorescence microscope, five high powered fields of vision were taken where cells were concentrated. The chromaticity-positive cells were expressed with brown or yellow nucleus. Mice in the continuous MFS treatment or mice receiving YPF-P had a slight increase in CD68þ expression (Table 3, Figure 2B, C) (p < 0.05) compared with that of mice in the discontinuous inhalation group and control group. Lung stained with anti CD68þ macrophage antibody in (A) control mice; (B) continuous inhalation MFS mice; (C) YPF-P mice; (D) discontinuous inhalation MFS mice and (E) negative control.

Effects of MFS on CD4þ , CD8þ T-cells and CD4þ /CD8þ in the peripheral blood Although CD4þ and CD8þ T-cells in mucosal tissues cannot prevent pathogen entry, they might have a crucial Table 4

In this study, we investigated the effects of MFS on the immune function, and the pilot experiments showed its major active compound, isorhamnetin [25]. Isorhamnetin is a natural flavone which is a potent inhibitor of virus replication in addition to its anti-inflammatory, antimicrobial, anti-viral, immune-modulatory, cytoprotective, anti-histamine and anti-stress properties [26–29]. Inhalation of MFS is safe, simple and noninvasive. More importantly, it induces systemic and mucosal immune responses. Mucosal sIgA and IgG, non-inflammatory antibodies, represent the first immunological barrier to pathogens when infecting the epithelial surface with a wide cross-protection against variant viruses, while systemic IgA and IgG in blood protect only against homologous strains of virus [30, 31]. Thus, the secretion amount of anti-influenza IgA and IgG produced on the nasopharyngeal and respiratory tract mucosal surface during infection correlates with the early relief of respiratory symptoms. Therefore, protective mucosal immune responses are most effectively induced by mucosal immunization through nasal routes. Our data here indicated that continuous MFS inhalation could provide an important protection by inducing the mucosal immune responses. Both continuous inhalation and discontinuous inhalation of MFS can enhance the sIgA and IgG in BALF

Effects of MFS on CD4þ , CD8þ T-cells and CD4þ /CD8þ in peripheral blood of mice.

CD4þ (cell/µL) CD8þ (cell/µL) CD4þ (%) CD8þ (%) CD4þ /CD8þ

MFS continuous inhalation

MFS intermittent inhalation

YPF-P treatment

Control

2,068.2  855.2a,b 1,505.9  754.48a,b 46.19  6.03a,b 28.82  2.95a,b 1.48  0.3

1,798.8  726.36 1,068.1  422.84 40.51  3.17 22.87  6.30 1.74  0.48

2,057.7  476.84a,b 1,260.0  281.12 48.31  5.47a,b 26.80  3.44 1.67  0.35

1,067.3  274.59 853.9  235.28 40.32  4.79 24.36  4.07 1.27  0.22

p < 0.05 vs the MFS discontinuous inhalation group; bp < 0.05 vs control mice.

a

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

Figure 3 Analysis of CD4þ and CD8þ T-Cell. Peripheral blood from mice was subjected to three-color analysis to determine the expression levels of CD4þ T-cell (CD3þ CD4þ ) and CD8þ T-cell (CD3þ CD8þ ). Peripheral blood cells were simultaneously stained with PerCP-conjugated anti-mouse CD3, FITC-conjugated antimouse CD4 and PE-conjugated anti-mouse CD8 antibodies. (A) Cells from the blank control, (B) cells from mice in the continuous MFS inhalation group, (C) cells from mice in the positive control group and (D) cells from mice in the discontinuous group. The dot plots were derived from the gated events and the light scatter characteristics of viable cells. Red and green in CD4-FITC and CD8-PE indicated the proportion and number of CD4þ (CD3þ CD4þ ) and CD8þ (CD3þ CD8þ ) cells, respectively. Data represented the analysis of 10,000 total events and were expressed as the mean  SD (Table 4).

[17] and lung tissues; however, sIgA and IgG are increased more significantly in the continuous MFS inhalation mice compared with the discontinuous inhalation MFS mice. Our previous observation showed that continuous inhalation of 10 g MFS produced a comparable effect with that of 40 g MFS [32], which might be in a time-dependent manner rather than a dose-dependent manner demonstrated by our study. Mice with continuous MFS inhalation treatment showed an increase in the

serum IgG levels compared with the negative control mice. But the serum IgA level of mice with continuous MFS inhalation treatment did not increase compared with the negative control mice. However, no significant effects of discontinuous inhalation of MFS were observed on the levels of AM, CD4þ and CD8þ T-cells. In addition, our previous study showed that discontinuous inhalation MFS was also unable to enhance TLR2/4 and NKp46 levels. These results suggest that respiratory tract mucous membrane is very sensitive to aromatic stimuli. The present study provides a new insight into the effect of MFS on the functions of mucosal tissues and interplay of innate and adaptive immune responses which results in immune protection at mucosal surfaces. Further works are required to fully elucidate its underlying mechanisms. AM produce a robust innate immune response to influenza. The alveoli is a major site of the AM [33]. Consistent with other studies of avian or human influenza infections in humans and animals, influenza viral antigens have been detected in AM from humans and many animal species [34–40]. In addition, AM are critical for controlling viral replication in vivo [37–39]. Recently, several groups have explored a cell-specific pattern in producing IFN in response to viral infection. It is well known that TLRs are the main PRRs responsible for IFN production against RNA viruses including influenza. TLRs recognize viral nucleic acid in the endosomal compartment [41, 42]. Previous study found that TLR levels in respiratory tract showed a significant increase following MFS treatment. The present study demonstrated that MFS could up-regulate AM expression, which was consistent with previous reports, demonstrating that aromatic TCM herb derivatives were responsible for triggering the innate cell-mediated immune responses [43, 44], whereas isorhamnetin may be essential to the protection of MFS associated with the significant enhancement of AM expression levels resulting in a moderate increase in innate immune responses following exposure to influenza virus. Furthermore, continuous inhalation MFS can enhance the amount of CD4þ and CD8þ cells in the peripheral blood, indicating a beneficial effect of MFS on the cellular immunity. The ratio of CD4þ /CD8þ cells is as normal as the negative control mice, suggesting that MFS can maintain the immunity balance and T lymphocytes are sensitive to aromatic stimuli. Further experiments are required to fully investigate the underlying mechanisms. In addition, whether aromatic stimuli receptors are expressed in the T lymphocytes will also be explored. Recently, various influenza vaccines have been developed but may not be protective. Traditional influenza

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

vaccines for IM injection are expensive to produce. Previous mucosal influenza vaccine candidates include intranasal attenuated influenza, virosomes or DNA vaccine. Despite excellent efficacy in humans, intranasal virosomes have been withdrawn from the market because of an association with the development of Bell’s Palsy [45]. The attenuated cold adapted influenza virus vaccine recently approved by FDA costs more than the parenteral inactivated influenza vaccine and is contradicted in humans with immunodeficiencies [46]. In contrast, MFS is safer, cheaper to produce, easier to administer and given by the nasal natural route for preventing influenza. There are several advantages to prevent influenza via this strategy: (i) It is safe since there has been few reports of serious adverse effects associated with MFS and toxicity is also assessed in a pilot study following prolonged exposure time window (12 months) and high dose (40 g) of MFS in mice, which did not reveal overt toxicity resulting from continuous Sachet inhalation [47]. The Miao Ethnomedicine dose selection is based on long-term practical experience. It is known that 10 g of Miao Ethnomedicine is typically put into pockets (Sachets) worn around the neck to prevent respiratory tract infections. According to the literature, MFS is a generally safe and effective medicinal herb-preparation, however, it should not be used in the individuals with pregnancy or a history of allergic rhinitis [11, 48–50]. (ii) Sachets can be used to prevent influenza before pandemics. (iii) It is easier to convince people to use it with good cost-effectiveness. (iv) This approach is noninvasive, making it easily acceptable. (v) No special storage is required (i.e. it does not need to be refrigerated), and thus it is easy to transport. (vi) Neither its production nor disposal generates environmental pollutants. Data presented in this report suggested that isorhamnetin was essential for the protective properties of MFS associated with the significant enhancement of lgA, IgG and AM expression levels as well as the CD4þ and CD8þ T-cells resulting in a moderate increase in innate immune and adaptive immunity responses following the exposure to influenza virus. Previous studies suggested that respiratory TLR2/4, sIgA and IgG1 levels were significantly increased following MFS treatment, and the present findings suggested that the MFS also provided beneficial effects on the innate immune functions in mice. Furthermore, Hu and his colleagues [11] found in 500 flu-susceptible children that MFS could reduce the frequency and severity of flu attacks. Our findings strongly suggested that MFS therapy be able to induce immune responses and therefore with the potential to prevent influenza virus infections.

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Conclusions Taken together, more knowledge of the therapeutic use of aromatic TCM herbs is acquired through folklore, and only a few activities of these natural extracts are actually supported by scientific studies. Aromatic TCM herbs are an important source for drug discovery and investigations on biological actions of plant medicinal extracts, as well as the understanding of the underlying mechanisms, might drive the development of novel drugs. In this study, aromatic MFS was able to increase the levels of sIgA and IgG1. Meanwhile, MFS could activate AM, CD4þ and CD8þ T-cells in mice. Our data demonstrated that the protection of MFS against influenza is partly through activation of the innate and adaptive cellmediated immune responses, suggesting a potential new immuno-modulating agent for respiratory tract infectious diseases. Our results demonstrated that MFS was able to activate the innate and adaptive mediated immune response and provided convincing evidences for a novel application of this plant extract, except for its known anti-virus properties.

Acknowledgments: We are grateful to the staff at the Department of Infectious Diseases at the Guiyang Medical College for their assistances. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. QZ conceived of the study, designed the experiments, carried out all data analyses, collected and interpreted data and revised the manuscript critically. HW drafted the manuscript and participated in the in vivo study. MLC involved in conceiving of the study and designed the experiments. Other authors carried out the in vivo study. Research funding: This study was supported by GuiYang Science and Technology Department grant 2012 projects of social development and people’s livelihood, contract numbers [2012103], and also supported by the international cooperative project of Gui Zhou Science and Technology Department, contract number [2013GZ7024]. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report or in the decision to submit the report for publication.

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

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Zhang et al.: The Miaoyao Fanggan Sachets regulate immunity in mice

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