| Plantago asiatica is a traditional Chinese medicine which has the effect of detoxification, expectorant and diuretic. It’s first recorded in "Shen Nong’s Herbal Classic" and now contained in "Chinese Pharmacopoeia(2015)". Plantago asiatica contains a variety of biological active components, including polysaccharides, flavonoids, glycosides and ursolic acid, etc. Recent studies have found the Plantago asiatica polysaccharides(hereinafter refer to as PLP) have significant activity of enhancing immunity, but the immunomodulatory mechanism of PLP is unknown. Autophagy plays an important role in the innate and adaptive immunity, including pathogen elimination, inflammation and antigen presentation. It has been known that m TOR and Ⅲ-PI3 K signaling pathways are regulatory pathways of autophagy, and they are also targets of immune molecules and immunomodulatory drugs to regulate immunity. Therefore, we investigated the regulation effect of PLP on autophagy, and shed some light on the molecule mechanism of PLP in regulation of autophagy and crosstalk between autophagy pathway and TLR4/NF-κB signaling pathway in order to figure out the underlying immune pharmacological targets of PLP and provide experimental basis for PLP to be a novel immunomodulator. The results and final conclusions are as follows:1. The effect of PLP on LPS-induced autophagy in macrophageExperiment researches were aim at investigating the regulation effect of PLP on autophagy. PLP at 25, 50, 100μg/m L was used to interfere with autophagy of Raw264.7 macrophage that was stimulated with lipopolysaccharide(LPS). The autophagosomes accumulation was observed under fluorescent microscope by monodansylcadaverine(MDC) staining, the protein and m RNA expression of LC3 and Beclin-1 were detected by Western-blot and RT-PCR respectively. The results showed that the treatment of PLP increased fluorescence accumulation of autophagosomesand the protein or m RNA expression of LC3 and Beclin-1 in LPS-stimulated macrophage. The ratio of LC3Ⅱ/Ⅰ in PLP(50μg/m L) group was significantly higher than that in LPS group at 20h(P<0.01), the m RNA expression of LC3 in PLP(50μg/m L) group was significantly higher than that in LPS group at 2h, 16 h, 20 h, and 24h(P<0.05). The ratio of LC3Ⅱ/Ⅰ in PLP(100μg/m L) group was significantly higher than that in LPS group at 16 h, 20 h and 24h(P<0.05 or P<0.01), the m RNA expression of LC3 in PLP(100μg/m L) group was significantly higher than that in LPS group at 2h, 16 h, 20 h, and 24h(P<0.05). The protein expression of Beclin-1 in PLP(50μg/m L) group was significantly higher than that in LPS group at 16 h, 20 h and 24h(P<0.05 or P<0.01), the m RNA expression of Beclin-1 in PLP(50μg/m L) group was significantly higher than that in LPS group at 16h(P<0.01). The protein expression of Beclin-1 in PLP(100μg/m L) group was significantly higher than that in LPS group at 16 h, 20 h and 24h(P<0.01), the m RNA expression of Beclin-1 in PLP(100μg/m L) group was significantly higher than that in LPS group at 2h, 8h, 16 h and 20h(P<0.05 or P<0.01). These results indicate that PLP promoted LPS-induced autophagy in macrophage by increasing accumulation of autophagosomes and the protein or m RNA expression of LC3 and Beclin-1.2. The effect of PLP on m TOR and Ⅲ-PI3 K signaling pathway in LPS-induced autophagy in macrophageThe autophagy regulatory mechanism of PLP was investigated by forward and reverse tests to m TOR and III-PI3 K pathway. In this experiment, LPS was used to induced autophagy, inhibitors as rapamycin and 3-MA were respectively used to block m TOR and III-PI3 K pathways, and then Western-blot and RT-PCR were used to detect protein and m RNA expression level of m TOR, ULK1, Ⅲ-PI3 K, Beclin-1. The results showed that the protein and m RNA expression level of LC3, ULK1, Ⅲ-PI3 K, and Beclin-1 in LPS group were higher than these in control group, the protein and m RNA expression level of m TOR in LPS group were lower than these in control group. On the basis of the LPS group, PLP increased the ratio of LC3Ⅱ/Ⅰ, and the protein or m RNA expression level of ULK1, Ⅲ-PI3 K, Beclin-1. When m TOR was inhibited by rapamycin, the ratio of LC3Ⅱ/Ⅰ and the m RNA expression level of LC3 were significantly increased compared with the LPS group(P<0.05 or P<0.01), moreover, LPS+Rapa+PLP group was lower than LPS+Rapa group(P<0.05 or P<0.01), and the protein expression level of ULK1 in LPS+Rapa+PLP group was significantly higher than that in LPS+Rapa group(P<0.05). When Ⅲ-PI3 K was inhibited by 3-MA, the ratio of LC3Ⅱ/Ⅰ and the protein or m RNA expression level of LC3 and Beclin-1 were significantly decreased compared with the LPS group(P<0.05 orP<0.01), moreover, there were no significant differences between LPS+3-MA group and LPS+3-MA+PLP group at the ratio of LC3Ⅱ/Ⅰ and the m RNA expression level of LC3(P>0.05), and the protein expression level of Beclin-1 in LPS+3-MA+PLP group was significantly lower than LPS+3-MA group(P<0.05). These results indicate that PLP upregulated expression of ULK1 and Beclin-1, and then promoted autophagy by activating Ⅲ-PI3 K pathway.3. The regulatory mechanism of PLP in crosstalk between TLR4/NF-κB and autophagy pathwayIn this syudy, the regulatory mechanism of PLP in crosstalk between TLR4/NF-κB and autophagy pathway was investigated by forward and reverse tests to these pathways. The autophagy of Raw264.7 cells were stimulated by LPS, at the same time, inhibitors as rapamycin and 3-MA were respectively used to block m TOR and Ⅲ-PI3 K pathway, and then m RNA expression level of TLR4, My D88, TRIF, NF-κB were detected by RT-PCR, the phosphorylation level of NF-κB p65 was detected by Western-blot. On the other hand, the inhibitor BAY11-7082 was used to block NF-κB pathway, and then protein and m RNA expression level of LC3、Beclin-1、Ⅲ-PI3K、m TOR were detected by Western-blot and RT-PCR respectively. The results showed that PLP increased the m RNA expression level of TLR4, My D88, TRIF, NF-κB and the phosphorylation level of NF-κB p65 on the basis of the LPS group. When m TOR was inhibited by rapamycin, the phosphorylation level of NF-κB p65 was significantly decreased compared with LPS group(P<0.01), the m RNA expression level of TRIF was significantly increased compared with LPS group(P<0.05). There was no significant difference between LPS+Rapa group and LPS+Rapa+PLP group at the phosphorylation level of NF-κB p65(P>0.05), and the m RNA expression level of TRIF in LPS+Rapa+PLP group was significantly lower than that in LPS+Rapa group(P<0.05). When Ⅲ-PI3 K was inhibited by 3-MA, the m RNA expression level of NF-κB was significantly increased compared with LPS group(P<0.01), and there was no difference between LPS+3-MA group and LPS+3-MA+PLP group(P>0.05). When NF-κB was inhibited by BAY11-7082, the ratio of LC3Ⅱ/Ⅰ and the m RNA expression level of LC3 were significantly decreased compared with LPS group, and there was no difference between LPS+BAY+PLP group and LPS+BAY group(P>0.05). The protein and m RNA expression of m TOR were significantly increased compared with LPS group when NF-κB was inhibited(P<0.05 or P<0.01), and there were no differences between LPS+BAY+PLP group and LPS+BAY group(P>0.05).The protein or m RNA expression of Beclin-1 and Ⅲ-PI3 K were significantly decreased compared with LPS group when NF-κB was inhibited, moreover, the protein expression of Beclin-1 inLPS+BAY+PLP was significantly lower than that in LPS+BAY group(P<0.01), there was no difference between LPS+BAY+PLP group and LPS+BAY group at the m RNA expression of Ⅲ-PI3K(P>0.05). These results indicate that PLP promoted LPS-induced autophagy through synergistic activation of TLR4/NF-κB signaling pathway. In the crosstalk between autophagy pathway and TLR4/NF-κB pathway, those key molecules in Ⅲ-PI3 K and TLR4/NF-κB pathway are potential targets of PLP to regulate autophagy and innate immunity. |
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