In this study, LPS-induced RAW264.7 macrophages were used as the TLR4-activated cell model to investigate the effect of KCPM on macrophage inflammatory mediators. To determine the optimal concentration of KCPM, CCK-8 assays were conducted. We confirmed that KCPM could inhibit M1 polarization and promote M2 polarization may through the inhibition of TLR4/MyD88/NF-κB signal pathways.
Lung macrophages are important innate immune cells, which play critical roles in lung homeostasis, host defense against pathogens, and resolution of inflammation. These diverse functions of macrophages are achieved by the plasticity of these cells, which, depending on signals present in their microenvironment, can polarize into a plethora of different phenotypes[13, 4]. In infected tissues, macrophages are first polarized to pro-inflammatory M1 phenotype to assist the host against pathogens. Subsequently, macrophages are polarized to form an anti-inflammatory response to the M2 phenotype and repair damaged tissue[15]. Recently, the regulation of macrophage polarization to regulate its immune function has been successfully stimulated. A variety of strategies have been used to modulate macrophage polarization to treat disease.
Chinese herbs are a traditional medicinal resource in China, which has a stimulating activity and regulating effect on the immune system. Macrophages may be one of the target cells for Chinese herbal medicine’s anti-inflammatory effect[16, 17]. KCPM was formulated on the basis of MXSGD, which reduced Glycyrrhiza and added reed root, Houttuynia cordata, big green leaves of Polygonum, stir fried Perilla seed, Tingli seed and stir fried Radish seed, thus the effect of clearing heat and resolving phlegm was enhanced. Modern pharmacology research shows that the traditional Chinese medicine of clearing away heat has anti-inflammatory effect[18, 19]. In our study, we found that KCPM could inhibit the expression of IL-6 and TNF-α, promote the expression of IL-10 and TGF-β. Research about Xiaoer Magan Granule which formulated on the basis of MXSGD, also found that it could reduce the level of inflammatory cytokines such as IL-6, IL-8, TNF-α, and IFN-γ, so as to improve the pneumonia clinical symptoms such as cough, expectoration, fever and rale[20]. The anti-inflammatory effect of Ephedra-Glycyrrhiza showed that it can reduce the inflammatory factors TNF-α and IL-β in mice lung tissue with pneumonia[21]. More studies have found that Maxingshigan decoction can effectively reduce lung inflammation, protect immune organs and regulate cytokine balance. Its mechanism was through inhibiting the activation of TLRs signal pathways, alleviate lung injury caused by infection[22, 23]. So, we inferred that the KCPM play a role in the treatment of pneumonia through inhibiting the secretion of macrophage pro-inflammatory factors .
In the present study, a cell model was established using LPS stimulation in RAW264.7 cells. LPS was demonstrated to promote the secretion of TNF-α and IL-6. The mouse macrophage RAW264.7 cell line is a type of monocyte macrophage in mice with leukemia, which is commonly used in biological experiments investigating inflammation[24]. TNF-α is the earliest endogenous mediator of an inflammatory reaction, and IL-6 is a major pro-inflammatory cytokine that serves an important role in the acute phase response of inflammation[25]. When infections or tissue injuries occur, IL-6 and TNF-α was promptly produced by macrophages and contributes to removal of infectious agents and restoration of damaged tissues through activation of immune, hematological, and acute-phase responses. The TNF-α and IL-6 influence the ability to limit pathogen infection but their over-production might result in inflammatory disorders[26–29]. Thus, the proper expression is very important for host defense. KCPM could inhibit the expression of IL-6 and TNF-α, limit the uncontrolled excessive or persistent pro-inflammatory production, as a result, preventing the further tissue damage of macrophage immune response in the pneumonia.
In our study we also found that KCPM could promote the secretion of TGF-β and IL-10. TGF-β is an evolutionarily conserved pleiotropic factor that regulates a myriad of biological processes including development, tissue regeneration, immune responses, and tumorigenesis[30]. It is necessary for lung organogenesis and homeostasis as evidenced by genetically engineered mouse models[31]. Collectively, TGF-β inhibits the development of immunopathology to self or nonharmful antigens without compromising immune responses to pathogens[32]. IL-10 is a potent anti-inflammatory cytokine that plays a crucial, and often essential, role in preventing inflammatory and autoimmune pathologies[33]. IL-10 emerged before the adaptive immune response and elicit diverse host defense mechanisms, especially from epithelial cells during an infection. It promotes innate immune responses from tissue epithelia that limit the damage caused by both viral and bacterial infections. It also facilitate tissue healing after infection /inflammation[34]. In this regard, IL-10 suppresses pro-inflammatory responses, limiting tissue disruption resulting from an inflammatory response[35]. In our research, KCPM could promote the secretion of TGF-β and IL-10, so as to limit the macrophage immune response, relief the tissue disruption, and ameliorate the prognosis of pneumonia.
The present study found that KCPM limited LPS-induced M1 macrophage polarization, inhibited the expression of iNOS, promoted the expression of Arg-1, and induced macrophage polarization to the M2 phenotype, so as to play an anti-inflammatory role. iNOS, an intracellular marker of M1 subpopulation, has been found supressed by herbs. iNOS converts l-arginine to l-citrulline and nitric oxide that interacts with reactive oxygen species to exert pro-inflammatory effects[36]. Chinese herbs has been found powerful potential in alleviating inflammatory response, they promoted the expression of Arg-1, while the expression of iNOS was decreased, induced the polarization of macrophages toward an M2 phenotype[37–40].
TLR4/MyD88/NF-κB pathways are considered to be pivotal in the inflammatory response. TLR4 can be activated by LPS, it may be the target protein of Chinese herbs, which inhibited LPS-induced inflammation in RAW264.7 cells[41]. Research about Chinese herbs found that Chinese herbs inhibited LPS‑induced TLR4 and MyD88 expressions in RAW264.7 macrophages, as a result, inhibiting the production of inflammatory mediators[42, 43]. Of the several transcriptional factors activated by inflammatory responses during bacterial infections, NF-κB plays a critical role in several signal transduction pathways. It is a key transcription factor that promotes transcription of genes encoding pro-inflammatory cytokines[44]. Many studies reports that inhibition of the NF-κB signaling pathway reduces the production of inflammatory mediators, promotes the development of M2 macrophage polarization, stimulation of the NF-κB signaling pathway can also polarize M2 macrophages into M1 macrophages[45, 46]. Hence, agents that are able to inhibit TLR4/MyD88/NF-κB transcriptional regulation and modulate the inflammatory response may have therapeutic effect. In this study we found that inhibition of the TLR4/MyD88/NF-κB signal pathway was associated with the inhibitory effects of KCPM on inflammatory mediators in LPS-stimulated RAW264.7 cells. We inferred that KCPM inhibit the macrophage immune response though the TLR4/MyD88/NF-κB signal pathway.
In this study, only in vitro studies were carried out, further animal experiments are still needed to verify the above results. Further research about the effect of KCPM on the protein expression of TLR4/MyD88/NF-κB signal pathways were also needed.