Protein Expression Levels Of MyD88 Research Articles

Effect of silkworm peptide on inducting M1 type polarization and Th1 activation via TLR2-induced MyD88-dependent pathway.

The aim of this study was to explore immune activity and molecular mechanism of silkworm peptide. The cell subsets induced by silkworm peptides were detected by flow cytometry. The IFN‐γ and IL‐4 levels in CD4+ cells were measured by ELISA. TLR2 mRNA expression in mouse CD4+ T cells was detected by qRT‐PCR. Western blot was used to detect the protein expression levels of MyD88 and p‐IκB. The growth rate of Lewis lung cancer xenografts in mice of the medium‐dose group was significantly reduced, and the tumor volume was significantly smaller than that of the control group on the 14th day. The relative vitality values of spleen lymphocytes in the medium‐dose and high‐dose groups were higher than the control group. The IFN‐γ levels in the medium‐dose and high‐dose groups were significantly higher than the control group. The levels of IL‐4 were no significant change among different groups. Compared with the control group, different doses of silkworm peptide groups could increase the levels of NO, IL‐6, IL‐12, and IL‐1β. Compared with the control group, the protein expression levels of MyD88 and p‐IκB in 10 μg/ml group and 20 μg/ml groups were significantly increased compared with the control group. Silkworm peptide could induce Th1 activation and M1 type polarization, which was dose‐dependent and was relative to the effect of silkworm peptide on inhibiting tumor growth. Silkworm peptide could directly induce M1 type polarization and Th1 activation via TLR2‐induced MyD88‐dependent pathway in vitro.

TLR4/MyD88 signaling determines the metastatic potential of breast cancer cells

The influence of Toll-like receptor (TLR)4/myeloid differentiation factor (MyD)88 signaling on the invasion and metastasis of cancer cells has been previously reported. The purpose of the present study was to determine the role of TLR4/MyD88 in breast cancer cell migration and invasion, and to discover novel therapeutic targets for breast cancer treatment. TLR4, MyD88 and high mobility group box 1 (HMGB1) mRNA expression levels were assessed in highly invasive human MDA-MB-231 breast cancer cells, breast cancer cells with a low rate of invasion (MCF-7) and normal human MDA-Kb2 mammary gland cells by reverse transcription-quantitative polymerase chain reaction. The protein expression levels of these markers were detected by western blotting and immunofluorescence. Randomly selected breast cancer and paracarcinoma tissues were used to measure TLR4 and MyD88 protein expression levels by immunohistochemistry. The mRNA and protein expression levels of TLR4 and MyD88 were significantly higher in MDA-MB-231 cells compared with either MCF-7 cells or MDA-Kb2 cells. The mRNA and protein expression levels of HMGB1 were comparable in the two breast cancer cell lines, with no statistical difference (P>0.05). TLR4 and MyD88 protein expression levels were also significantly higher in breast cancer tissues compared with paracarcinoma tissues (P<0.05). TLR4 and MyD88 protein expression levels were positively correlated with axillary lymph node metastasis and histological grade (P<0.05). TLR4/MyD88 expression levels were positively correlated with the metastasis of breast cancer cells. TLR4/MyD88 may be useful as a novel biomarker to evaluate the prognosis and treatment of patients with breast cancer.

Effects of ligustilide on the extracellular recombinant human heat shock protein 60 induced inflammatory reactions in the THP-1 cells and the related mechanisms

Objective: To investigate the effects of ligustilide(LIG) on extracellular recombinant human heat shock protein 60 (HSP60) induced inflammatory reactions in the THP-1 cells and the related mechanisms. Methods: THP-1 cells were differentiated to macrophages by incubation with phorbol-12-myristate-13-acetate (PMA). The immunofluorescence method was used to screen the optimum transfection concentration of MyD88 siRNA.The macrophages were divided into six groups(n=3), including blank control(siRNA transfection reagent), model(siRNA transfection reagent+ HSP60 10 mg/L), negative control(MyD88 negative control+ HSP60 10 mg/L), LIG group(siRNA transfection reagent+ HSP60 10 mg/L+ LIG 20 mg/L), RNA interfering(RNAi) group(MyD88 siRNA+ HSP60 10 mg/L) and RNAi+ LIG group(MyD88 siRNA+ HSP60 10 mg/L+ LIG 20 mg/L). The protein expression level of MyD88 and phospho-nuclear factor-κB(p-NF-κB) in macrophages and the level of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in the culture supernatant were assessed by Western blot analyses or ELISA, respectively. Results: (1)The protein expression levels of MyD88 (1.196±0.125 vs. 0.341±0.063, P<0.01) and p-NF-κB(0.817±0.034 vs. 0.312±0.046, P<0.01) were significantly higher in the model group than those in the blank control group.The protein expression levels of MyD88(0.554±0.043) and p-NF-κB(0.538±0.063) in the RNAi group were significantly lower than those in the model group (all P<0.01) but significantly higher than those in the blank control group (all P<0.05). The protein expression levels of MyD88(0.694±0.087, P<0.05) and p-NF-κB(0.669±0.043, P<0.01)in the LIG group were markedly lower than those in the model group, but higher than those in the RNAi group (P<0.05) and the blank control group (P<0.01). The protein expression levels of MyD88(0.409±0.069) and p-NF-κB(0.395±0.046) in the RNAi+ LIG group were significantly lower than in the model group (all P<0.01) and in the LIG group(P<0.05 or 0.01), and were similar to the blank control group(P>0.05). The expression level of p-NF-κB in the RNAi+ LIG group was significantly lower than in the RNAi group (P<0.05). (2) The contents of TNF-α((312.24±28.69) ng/L vs. (5.99±1.03) ng/L, P<0.01) and IL-6((233.45±57.77) ng/L vs. (2.25±0.67) ng/L, P<0.01) were significantly higher in the model group than in the blank control group. The contents of TNF-α((235.66±25.12) ng/L) and IL-6((131.59±13.99) ng/L) were significantly lower in the RNAi group than in the model group (P<0.01). The contents of TNF-α((258.13±44.80) ng/L) and IL-6((175.92±28.27) ng/L) were also significantly lower in the LIG group than in the model group(P<0.05) while the content of IL-6 was significantly higher in the LIG group than in the RNAi group(P<0.01). The contents of TNF-α((88.57±16.10) ng/L) and IL-6((59.99±10.31) ng/L) were significantly lower in the RNAi+ LIG group than those in the model group, the RNAi group and the LIG group(P<0.05 or 0.01). Conclusions: The MyD88/NF-κB signaling pathway is one of the key signaling pathways of human HSP60 induced inflammation in THP-1 cells. Ligustilide could exhibit the anti-inflammatory effect probably by inhibiting the MyD88/NF-κB signaling pathway.

Anti-Inflammatory Activity of Tanshinone IIA in LPS-Stimulated RAW264.7 Macrophages via miRNAs and TLR4-NF-κB Pathway.

Inflammation is a physiological response to infection or injury and involves the innate and adaptive immune system. Tanshinone IIA (Tan IIA) is a well-known flavonoid that elicits an important therapeutic effect by inhibiting inflammatory response. In this study, we examined whether Tan IIA exerts anti-inflammatory activity and investigated the possible mechanisms, including Toll-like receptor 4 (TLR4)-MyD88-nuclear factor kappa B (NF-κB) signaling pathway and microRNA expression in lipopolysaccharide (LPS)-induced RAW264.7 cells. Tan IIA could attenuate the inflammatory reaction via decreasing cytokine, chemokine, and acute-phase protein production, including GM-CSF, sICAM-1, cxcl-1, MIP-1α, and tumor necrosis factor alpha (TNF-α), analyzed by Proteome profile array in LPS-induced RAW264.7 cells. Concurrently, the messenger RNA (mRNA) expressions of IL-1β, TNF-α, and COX-2 were also significantly reduced by Tan IIA. Additionally, Tan IIA decreased LPS-induced NF-κB activation and downregulated TLR4 and MyD88 protein expression levels. We also observed reduced microRNA-155, miR-147, miR-184, miR-29b, and miR-34c expression levels, while LPS-induced microRNA-105, miR-145a, miR-194, miR-383, miR-132, and miR-451a expression levels were upregulated using microRNA (miRNA) qPCR array. Our results indicate that Tan IIA could exert an anti-inflammatory effect on LPS-induced RAW264.7 cells by decreasing TLR4-MyD88-NF-κB signaling pathway and regulating a series of cytokine production and miRNA expression.