Effects of ligustilide on the extracellular recombinant human heat shock protein 60 induced inflammatory reactions in the THP-1 cells and the related mechanisms
10.3760/cma.j.issn.0253-3758.2016.09.012
- VernacularTitle:藁本内酯对重组人热休克蛋白60诱导的THP-1源性巨噬细胞炎症反应的作用及机制
- Author:
Ying GAO
1
;
Rui CHEN
;
Ning GU
;
Xiaoli HE
;
Zhijun YANG
;
Keke ZUO
Author Information
1. 210046,南京中医药大学博士后流动站
- Keywords:
Ligustilide;
Inflammation;
Heat-shock proteins
- From:
Chinese Journal of Cardiology
2016;44(9):793-798
- CountryChina
- Language:Chinese
-
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 phosphonuclear 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 ofTNF-α((312.24±28.69) ng/Lvs.(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.
