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

Abstract

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.