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Polysaccharide of Atractylodes Macrocephala Koidz(PAMK) Enhance the Immunomodulatory Activity of RAW264.7 Through TLR4/MyD88/NF-κB Pathway

The Polysaccharide of Atractylodes macrocephala Koidz (PAMK) has been reported to play an important role in improving the immune function of mammals and poultry. But the molecular mechanism in cell is still not unclear. For investigating the mechanism in mice, RAW264.7 were cultured and divided into six groups (cell, NC, siTLR4, NC + PAMK, PAMK, siTLR4 + PAMK). The serum cytokines, mRNA relative expression level of cytokines and TLR4 signal pathway, the protein expression level of TLR4 signal pathway were measured. The results showed PAMK could increase the level of IL-4, IL-6, TNF-α, IFN-γ, and increase the mRNA relative expression level and protein expression level of TLR4, MyD88, IKK, NF-κB. In conclusion, PAMK may regulate the immue activity of RAW264.7 through the TLR4/MyD88/NF-κB pathway.


Introduction
Polysaccharide of Atractylodes macrocephala Koidz (PAMK) as one of the main extracts of Atractylodes macrocephala, which is a traditional medicine and has been utilized for at least 2000 years. PAMK was often used to treat diarrhea, spleen and stomach disease, respiratory infection etc 1 . PAMK has antiin ammatory, anti-cancer activities, maintains the balance of gastrointestinal microbiome and regulates the organism's immunity balance 1 . In addition, PAMK promotes the proliferation of lymphocytes which in spleen and thymus of human, chicken, and mice, cause it could induce T lymphocytes enter S and G2/M phases, and effectively elevates the percentage of CD4 + and CD8 + T cells 4 . PAMK also increases the index of spleen and thymus in geese, upregulates the level of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interferon-γ (IFN-γ), Immunoglobulin G (IgG) in serum of mice and geese 5 ; 6 . However, Xu 7 indicated that PAMK could decrease the high level of Tumor Necrosis Factor-α (TNF-α), interleukin-1 (IL-1), H 2 O 2 caused by heat stress (HS) in geese. Jeong D 8 . suggested that PAMK down regulated the high level of pro-in ammatory cytokines (IL-1β, IL-6 and TNF-α) caused by Lipopolysaccharides (LPS). In our previous study, mice spleen was used to detected the mechanism of PAMK, and nd that PAMK could enhance cytokines secretion by stimulating the TLR4/MyD88/NF-κB signaling pathway 8 . However, the mechanism of PAMK in regulating the immune function of macrophages was still not clear. Therefore, RAW264.7 was used in this experiment.
TLR4 plays an important role in immunomodulatory, which could recognize the pathogen-related molecular patterns, transmit signal molecules to the cytoplasm, and promote the production of proin ammatory factors 10 .There are two ways to active the TLR4/NF-κB signal pathway: the dependent of myeloiddifferentiationfactor8 (MyD88) signal pathway and independent of MyD88 signal pathway. When TLR4 was active, the intracellular TIR region binds to the carboxyl group of MyD88, and its amino group binds to IL-1 amino group to activate IL-1 receptor-associated kinase (IRAK), IRAK activates TNF-receptorassociated factor6 (TRAF6), TRAF6 activates inhibitor of NF-κB kinases (IKKs), and the NF-κB inhibitor phosphorylates and degrades under the action of IKKs compound to activates NF-κB, then induces the expression of speci c cytokines (IL-1,IL-6, IL-8, IL-12) in the nucleus 11 . As some article suggested that polysaccharide regulate the immune function through TLR4 signal pathway. Some researchers indicated that a novel polysaccharide obtained from Craterellus cornucopioides, polysaccharides from Cordyceps gunnii mycelia , polysaccharide from wheat bran could induce lymphocyte proliferation, promote  macrophage activation and phagocytic activity, promote cytokines IL-2, IL-6, IL-1β, TNF-α and IFN-γ   secretion and increase immune function through TLR4 signal pathway [12][13][14] .
Based on previous research, we preliminary con rmed that PAMK promote cytokines secretion by stimulating TLR4/MyD88/NF-κB signal pathway in mice spleen. Thus, we chose RAW264.7 as immune cell model in vitro, and measured serum cytokines of cellular supernatant, mRNA relative expression of cytokines and TLR4 signal pathway, protein expression level of TLR4 signal pathway by ELISA, qPCR, and western blot to further con rm that PAMK play an important role in promote the cytokines through TLR4 signal pathway in RAW264.7.

The effects of different TLR4-siRNAs on TLR4 mRNA relative expression
Total RNA was extracted from RAW 264.7 to measure the mRNA relative expression of TLR4. As presented in Fig. 1, the results showed that si-TLR4-2 at 100 nM has the best inhibitory effect and e cency of inhibiting TLR4 expression was up to 72 %.
PAMK promoted the cytokines production of RAW264.7 cellular supernatant The cellular supernatant of six groups was collected, and then measure cellular supernatant cytokines level. The results in Fig. 2A-Figure2D revealed that all the cytokines were signi cantly (P < 0.05) down regulated by siTLR4 when compared with NC group. cellular supernatant cytokines levels in cell group and NC group showed no signi cant difference, and PAMK group and NC + PAMK group showed no signi cant difference (P > 0.05), which means that lip 3000 has no toxic side effect to RAW264.7. However, the cellular supernatant of all cytokines treated with PAMK were signi cantly (P < 0.05) upregulated when compared with siTLR4 group, which indicated that PAMK could promote cellular supernatant cytokines production.

PAMK promoted the mRNA relative expression of cytokines
Total RNA was extracted from RAW 264.7 to measure the mRNA relative expression of cytokines. Four genes were tested by qPCR. As showed in the Fig. 2E- Figure 2H, the results indicated that the relative mRNA of IL-2, IL-4, TNF-α, and IFN-γ in siTLR4 was signi cantly (P < 0.05) decreased when compared with NC group. The relative mRNA expressions of TNF-α, and IFN-γ were signi cantly (P < 0.05) upregulated and IL-2, IL-4 had the uptrend in siTLR4 + PAMK group compared to siTLR4 group. Compared with cell group and NC + PAMK group respectively, the relative mRNA expression showed no signi cant (P > 0.05) difference in NC group and PAMK group. All the results indicated that PAMK could promote relative mRNA of cytokines expression production.
2.3 PAMK promoted the relative mRNA expression of TLR4 signaling pathway.
Total RNA was extracted from RAW 264.7 to measure the relative mRNA expression of TLR4 signal pathway. The results were showed in Fig. 3. The relative mRNA expressions of TLR4, MyD88, NF-κB, IKK were signi cantly (P < 0.05) down regulated in siTLR4 group compared with NC group. However, when treating with PAMK, NF-κB signi cant (P < 0.05) increased and TLR4, MyD88 and IKK had the uptrend. The difference between cell group and NC group, NC + PAMK group and PAMK group has no signi cant difference(P > 0.05). Thus, the results indicated that PAMK could promote relative mRNA expression of TLR4 signal pathway.

PAMK regulated the relative protein expression in the TLR4 signaling pathway
Proteins were extracted from RAW 264.7 to measure the relative protein level of NF-κB, TLR4, MyD88, IKK, GAPDH. The relative protein expression level of the genes were showed in Fig. 4. The results indicated that in siTLR4 group, the relative protein expression level of all genes were signi cantly (P < 0.05) increased than NC group. The relative protein expression of TLR4 was signi cant upregulated (P < 0.05) and NF-κB, MyD88, IKK has the uptrend in siTLR4 + PAMK group compared with siTLR4 group. The relative protein expression level has the same trend with the relative mRNA expression level. The above results suggested that PAMK could regulate the protein expression level in the TLR4 signaling pathway.

Discussion
PAMK has the function of promoting the lymphocytes active and proliferation, maintaining the stable cell morphology, increasing the secretion of cytokines such as TNF-α, IL-4, IL-6, IL-10, IL-17, IL-17A, IFN-γ, alleviating the immunosuppression caused by CTX, and increasing the index of immune organs, which was considered one of the characteristic of the immune level enhance 15 ; 16 . We all know that cytokines play a very important role in body immunity 15 ; 17 ; 18 and macrophages play an important role in defending the body against invading pathogens 15 ; 17 ; 18 . Increased level of IL-4, IL-2, TNF-α and IFN-γ could enhance the macrophage activity and improve speci c immunity. In our research, we found that PAMK could upregulated the mRNA and protein expression of IL-2, IL-4, TNF-α and IFN-γ caused by siTLR4. IL-2 can enhance the secretion of TNF-α, IL-1, NO and other cytokines from macrophages, enhance the cytotoxic effect of macrophages and increase the killing activity of macrophages. IL-4 can increase the tumor killing ability of bone marrow Mφ and the expression of Fc(II) receptor, and promote the transformation of macrophages from M1 to M2 type. From our research, the results indicated that the relative mRNA expression level andcellular supernatant level of IL-2, IL-4 were signi cant decreased by siTLR4, but it signi cant increased or had increase trend after PAMK treated. Thus, the proliferative activity of macrophages is also increased, and the secretion levels of TNF-α and IFN-γ also increased when treated with PAMK, just as our research showed. According, we suggested that PAMK could enhanced macrophage activity and enhanced the release of TNF-α and IFN-γ to increased macrophage immunomodulation. S, Li, Shi etc found polysaccharide from Auricularia auricula, fungal polysaccharide could promoted the activation of macrophages and induced the secretion of cytokines such as IL-1β, IL-6, NO and TNF-α to enhance the phagocytic activity of macrophages and increase macrophage immunomodulation 19 ; 21 ; 22 . However, Zeng, Ma.G, Wang etc indicated that Lonicerae Japonicae Flos and Lonicerae Flos, polysaccharide from Pleurotus eryngii and Procyanidin A2 could inhibit of NO secretion in RAW264.7, down-regulate of IL-1β, IL-6 and TNF-α release, down-regulate of iNOS protein and COX2, NF-κB (p-IKKα/β, p-IκBα and p-p65) and MAPK (p-p38, p-JNK and p-ERK) protein levels caused by LPS [23][24][25] .
These results were similar to our research. Consequently, we suggested that PAMK could maintain stable immune levels of macrophages and maintain the body's immune balance when the body is stimulated by either LPS or siTLR4, etc.
TLR4, an important member of the TLR family, was the rst upstream gene for NF-kappaB (NF-κB) and has been shown to be involved in immune responses 26 . Some research suggested that when TLR4 is activated, there are two conduction signal pathway: dependent myeloiddifferentiationfactor88 (MyD88) signaling pathway and non-dependent MyD88 signaling pathway. The activated TLR domain of TLR4 binds to PAMK in a complex that can bind to MyD88. Interleukin-1 receptor associated kinase 1 (IRAK1), IRAK4 and TNF receptor associated factor 6 (TRAF6) recruit to the TLR-MyD88 complex, resulting in phosphorylation of IRAK1 and TRAF6. The signal is propagated by the phosphorylated linker molecular complex to downstream MAP kinases (JNK, p38 MAPK), Apetala 1 (AP1) and the nuclear factor kappa-B kinase (IKK) complex NF-κB. Activation of inhibitor of IKKα/β phosphorylates inhibitor of NF-κB (IκB), which is shed from NF-κB and ubiquitinated, allowing NF-κB to be activated by the inhibitory state leading to transcription factor activation 12 ; 27 ; 28 . In our research, we indicated that PAMK could increase the relative mRNA expression level and protein expression level of TLR4, MyD88, IKKα/β, NF-κB in siTLR4 + PAMK group when it compared with siTLR4 group, and we found that the tend of mRNA and protein level was the same. Thus, We suggested that PAMK activate IKK through a MyD88-dependent pathway, which phosphorylates IKB and make it off NF-κB and allow NF-κB to enter the nucleus to exert immunoregulatory effects. When NF-κB signal pathway was activated, cells would secrete cytokines to maintain the immunity balance while the organism is stimulated, such as IL-2, IL-4, TNF-α and IFN-γ,just as our research showed. Li indicated that PAMK could increase the serum level of IL-2, IL-4, TNF-α and IFN-γ, enhance splenocytes immunomodulatoryactivity by TLR4/MyD88/NF-κB signal pathway 9 . Some researchers showed that Polysaccharides from Cordyceps gunnii mycelia could increase the spleen thymus index, up-regulate the serum level of IL-2, IL-12, IFN-γ, IgG and other cytokines secretion levels, protect immunosuppressed mice through TLR4/TRAF6/NF-κB signaling pathway 29 . These ndings were consistent with previous research. However, Zheng, He and Jun suggested that Diethyl Blechnic Exhibits, an inulin-type fructan from Codonopsis pilosula, a novel polysaccharide from Acorus tatarinowii could inhibit release of pro-in ammatory factors caused by LPS such as prostaglandin E2 (PGE2) and relative mRNA expression of TNF-α, IL-1β, IL-6 and myeloperoxidase (MPO) through TLR4/MyD88/NF-κB in RAW264.7 30-33 . That also indicated that polysaccharide regulate RAW264.7 immune function by TLR4/MyD88/NF-κB signal pathway.
In conclusion, our research indicated that PAMK could increase the relative mRNA expression level and serum cytokines level of IL-2, IL-4, TNF-α and IFN-γ, enhance the immunomodulatory activity of RAW264.  (Table 1), and PAMK was added, and cells were collected 24 h later for mRNA relative expression detection.

Quantitative reserve-transcription PCR analysis
Total RNA was extracted from the RAW264.7 with TRIzol reagent (Thermo Fisher, USA) and reversetranscribed was performed by the Reverse Transcription Kit (Takara, Japan). The primers ( Table 2) of related genes were designed by using Primer Premier 5.0 software (Premier Biosoft International, USA). An ABI PRISM 7500 detection system (Applied Biosystems, Foster City, CA) was used to measure the mRNA relative expression of genes.

Western blot analysis
The total protein was extracted by RIPA (Biyuntian Biological Technology Co., Ltd., Shanghai, China), adjusted the concentration with 5 × simple buffer (Biyuntian Biological Technology Co., Ltd., Shanghai, China), and denatured by heating at 100 ℃ for 10 min. We analyzed it by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditio 12 % gels were used to transfer to PVDF membranes using a tank transfer system at 200 mA in Tris-glycine buffer containing 20 % methanol for 70-90 min at 4℃. The membranes were then blocked with 5% skim milk at 37 ℃ for 60 min. Next, the membranes were incubated with diluted primary antibodies overnight; the diluted concentrations and antibody sources are shown in Table 3. We incubated peroxidase-conjugated secondary antibodies against rabbit IgG at room temperature for 1 h (1:10000, Beijing Boaosen Biotechnology Co., Ltd, China). Chemiluminescence Detection Reagents (Applygen, Beijing) were incubated for 1 min to determine the ratios of target protein signals on a Chemiluminescence Imaging System (Tianneng, Shanghai) to signals of control proteins. GAPDH (Abcam) served as internal controls, and the relative expression levels of the proteins were expressed as the ratios of gray value of each of these proteins to that of GAPDH.

Statistical analysis
The data were analysed using SPSS for Windows (version 18, SPSS Inc, Chicago, IL, USA) and expressed as the means ± standard deviations (SD). All the qPCR assays were repeated in triplicate, and the relative expression levels were measured in terms of threshold cycle (Ct) values and normalized via the formula 2 −∆∆Ct . Differences between groups were compared using one-way analysis of variance (ANOVA) followed by Tukey's honestly signi cant difference test. P < 0.05 was considered statistically signi cant.

Con ict of interest statement
There is no con ict of interest in this manuscript.The content has not been published or submitted for publication elsewhere.All authors are in agreement with the content of the manuscript and submission in this journal.  Figure 1 The effects of different TLR4-siRNAs on TLR4 mRNA relative expression. Data were expressed as the means ± SD, n = 3. *P < 0.05, **P < 0.01, compared with the NC group. . SD, n = 6. Means with different letters represent statistically signi cant difference (P < 0.05); the bars with the same letter are not signi cantly difference (P > 0.05).

Figure 3
Effects of PAMK on the TLR4 signal pathway in mice RAW264.7 treated with siTLR4. Relative mRNA expression of (A) TLR4; (B) MyD88; (C) NF-κB; (D) IKK; Data were expressed as the means ± SD, n = 6. Data were expressed as the means ± SD, n = 6. Means with different letters represent statistically signi cant difference (P < 0.05); the bars with the same letter are not signi cantly difference (P > 0.05).