MFS is invented and produced in southeast region of Guizhou Province, China, where endemic diseases and epidemics constantly emerge due to its remote location, humid climate and underdeveloped economy. Miao medicine inherited from the traditional Chinese medicine has provided valuable experience for local residents' life and health, disease prevention and control with its unique culture. The design of this experiment coincided with the rapid global spread of the 2009 H1N1 influenza epidemics, and the infected cases showed a trend of aggregation and increased along with the seasonal changes in China. At the beginning of the epidemics, researchers have applied the preventive deodorant sachets in more than 100 local children. Animal experiments have proven that the sachet plays a role in lowering the incidence of influenza, relieving the symptoms of patients, shortening the clinical course, and improving the immunity of respiratory mucosa. On this basis, this study was designed to explore the mechanism underlying the prevention of respiratory tract infection by MFS from the perspective of molecular immunology and taking advantage of the safe and cheap Miao medicine resources in Guizhou Province, China.
Inherent immune response is the first line of defense against the invasion of pathogens . Inherent immune recognition and regulation are mainly realized by a series of embryo-coded pattern recognition receptor (PRR) to recognize those pathogens associated molecular pattern (PAMP) expressed on the pathogenic microorganisms. Toll-like receptors (TLRs) are important pattern recognition receptor families in natural and acquired immunity, and act as pivotal priming proteins for mammals to transmit extracellular antigen signal information into cells . TLR can initiate a series of signal transduction pathways, activate the effect factors in the pathway, amplify the cascade responses of downstream signal pathway, cause the aggregation of granulocytes, macrophages and natural killer cells, increase vascular permeability, stimulate the release of downstream inflammatory mediators, cause slight inflammatory reactions in the body, and finally activate the immune regulation function of the body to achieve the effect of controlling and eliminating pathogens. Currently, 9 TLRs have been reported. TLR1, TLR2, TLR4, TLR5 and TLR6 are located on the cell membrane, which mainly recognize the bacteria, whereas TLR3, TLR7, TLR8 and TLR9 are distributed within cells to primarily recognize viral nucleic acids. TLRS family plays an important role in the discovery and elimination of bacteria and viruses .
Previous researches  have demonstrated that TLRs initiates the downstream inflammatory pathway mainly through two patterns. The first is the myeloid differentiation factor 88 (MyD88) pattern, which is a key adaptor protein in TLRs-mediated intracellular signaling pathway. Under the synergistic effect of myeloid differentiation protein-2 (MD-2), the activated Toll receptor unites with MyD88, and then interleukin-1 receptor-associated kinase (IRAK) is recruited through the domain of MyD88. Recruitment-binding IRAK phosphorylation binds with tumor necrosis factor receptor associated factor 6 (TRAF6), which can activate transforming growth factor β activated kinase 1 (TGF-βactivated kinase 1,TAK1), thereby regulating the NFκB signaling pathway, the activation, expression and immune activation of downstream genes, etc. MyD88 is the first adaptor protein in the inflammatory signaling pathway. Once it receives the cell membrane signal, it can be immediately recruited and activated, and transmit the signal to the downstream pathway. Some scholars have compared MyD88 as the "bottleneck" of the entire signaling pathway. The expression level of MyD88 will significantly affect the signal transmission. The second pattern is the MyD88-independent signaling pathway. After TLR is activated, TRIF-related adaptormol-eeule (TRAM) binds with TIR-domain-containing adaptor inducing interferon B (TRIF), which can induce the expression of interferon and bind with TRAF6 to activate the downstream signaling pathway and secrete cytokines.
TRAF6, as an important conduction protein of inflammatory signals, is able to mediate signal transduction of MyD88-dependent and -independent signaling pathways and activate the nuclear factor kappa B (NFκB) and mitogen-activated protein kinase (MAPK) signaling pathways . Self-activation and degradation can be regulated according to the intensity of inflammatory signals in mediated signal transduction. TRAF6 can activate TAK1 and inhibitor of NFκB (IKK) by binding with IRAK. The degradation of TRAF6 can undergo its own ubiquitination degradation through the monomer polymerization, thereby affecting the regulation of NFκB signaling pathway .
TAK1 is a member of the MAPK family and is functionally located in the inhibitory protein kinase of MAPK and IκB. As an important kinase in the upstream cellular signal transduction in inflammatory response, TAK1 can be activated by the stimulation signals by UV, proinflammatory factors and hormones . Previous studies  have demonstrated that the activated TAK1 can phosphorylate and activate NF-κB induced kinase (NIK), thereby enabling NF-κB to enter the nucleus to participate in gene transcription, play an important role in cell growth, differentiation, apoptosis and regulate the expression of multiple genes, such as inflammatory cytokines, cell adhesion molecules and growth factors.
NFκB is an important transcription factor in the downstream inflammatory pathway, which regulates the transcription of various inflammatory mediators and cytokine genes. The activation of its transduction pathway is the molecular biological mechanism for amplification and persistence of inflammatory response, and ultimately affects the transcription and expression of different pro-inflammatory cytokines and body defense proteins . Under normal circumstances, NFκB binds to the inhibitory protein IκB in the cytoplasm in the form of heterodimer and does not show activity. When stimulated, TLR/MyD88 undergoes a cascade of amplification reactions in the MyD 88-dependent signaling pathway, activates the downstream transcription factor NFκB-induced kinase, provokes the IκB phosphorylation and induces the separation between NFκB and IκB. The activated NFκB can be transferred into nucleus to bind to corresponding target gene sites, which can regulate the over-expression of inflammatory mediators and cytokine genes, leading to the release of a large quantity of inflammatory mediators, infiltration of inflammatory cells and exertion of early immune response . Meantime, the activation of NFκB is explicitly regulated by both positive and negative feedbacks, mainly the negative feedback, which can suppress the activity of NFκB through two intra- and extra-cellular mechanisms, thereby maintaining the balance of cytokine network and preventing excessive inflammatory reaction in the host body .
In terms of the anti-infection immunity, pathogens bind with the IL-1 homologous domain (Toll/IL-1 receptor, TIR). Through the interaction between MyD88 and TIR adaptor protein (Toll/IL-1 receptor domain-containing adaptor protein, TIRAP), it can activate the interleukin-1 (IL-1) receptor associated kinase (IRAK), Traf6, TAK1 in sequence, activate MAP kinase (MAP3K or MKKK)) and NF-κB, stimulate the expression of downstream inflammatory cytokines, such as IFNα/β, IL-1b/6/8/12, thereby regulating the host immunity and exerting anti-infection effect .
Our research team has conducted drug toxicity experiment in the previous study to compare and verify the effect of sachets of different concentrations and durations upon improving the respiratory tract immunity in mouse models. Experimental results have demonstrated that continuous inhalation of 10 g sachets for 12 months fails to increase the mortality rate of mice. The expression levels of TLR2 and SIgA in the mice which continuously inhaled the sachet odor are significantly up-regulated compared with those in the mice that inhaled sachets intermittently. Nevertheless, the results of mice inhaled sachets continuously for 4 weeks are similar to those of mice inhaled for 6 weeks . Based on these preliminary experimental results, our research team optimized and modified the previous experimental conditions by establishing a hypoimmunity model under cold stimulation. In addition, the immunity-enhancing effect between MFS and pidotimod was also statistically compared. Pathological examination revealed that the hypoimmunity model was successfully established. Under normal and cold stimulation conditions, both MFS and pidotimod could up-regulate the expression levels of MyD88, TRAF6, NF-κB (P65) proteins and mRNA in the lung tissues, prompting that MFS probably play an effective role in preventing respiratory tract infection by regulating the TLR-MyD88-NFκB signaling pathway and regulating the inherent immune function of respiratory tract.
Therefore, the previous method for model establishment was adopted and modified in this experiment. The mice were kept into PVC cages with a quantitative air flow rate to ensure the accuracy of drug concentration. Prior to the experiment, the MyD88 knockout mice were bred by Animal Institute of Nanjing University, and the successfully-bred mouse models subject to gene sequencing to ensure the success of model establishment, aiming to verify whether MyD88 is a key factor for the immunity-enhancing effect of MFS and whether MFS can prevent and treat respiratory tract infection via the TLR-MyD88-NFκ b signaling pathway mediated by MyD88.
After 30-d MFS intervention, the mice in each group were in good mental state, had free access to diet and water, sensitive in response, black and bright in hair color, and obtained stable weight gain. The blood routine test and liver and kidney function parameters did not significantly differ among four groups (all P > 0.05) except that the white blood cell count of mice in the MFS group was increased. H.E. staining of the lung tissues indicated that the alveolar tissue contour in the four groups was normal, and the structures of trachea, bronchi and blood vessels were intact, which did not significantly differ from those in the control group, suggesting that MFS exerts no evident toxic effect upon mice after the model establishment conditions are modified. The increase of white blood cell count in the MFS group is probably correlated with the activation of relevant signaling pathways and the accumulation of inflammatory cells induces the immune response of the host body. The expression levels of TAK1, NFκB p65, and IκB were significantly up-regulated in the MFS group. With the up-regulation of TAK1 expression, NFκB p65 was trans-located into the nucleus, and the expression in the nucleus was up-regulated correspondingly, which led to the up-regulation of the expression level of IL6, indicating that TLR-MyD88-NFκB signaling pathway was activated during the prevention of respiratory tract infection by MFS and resulting in the release of inflammatory cytokines and cell aggregation, such as natural killer cells, and early immune response. However, the expression levels of TAK1, NFκB p65, IκB and IL6 in the MyD88 KO and MyD88 KO + MFS groups were lower than those in the control group, indicating that MFS mainly relies upon the TLR-MyD88-NFκb signaling pathway mediated by MyD88 to achieve the purpose of improving the animal immunity. However, compared with MyD88 KO group, the expression levels of TAK1, NFκB p65, IκB and IL6 in the MyD88 KO + MFS group were slightly higher, which does not the possibility that MFS can activate alternative signaling pathways to function by up-regulating these target genes.