Alkannin reverses lipopolysaccharides-induced inflammatory responses by suppressing mitogen-activated protein kinase and nuclear factor kappa-B signalling

Abstract

Rheumatoid arthritis (RA) is an inflammatory disease that seriously affects human health worldwide. Meanwhile, inflammation in RAW264.7 cells could lead to the progression of RA. Alkannin (ALK) is derived from Alkanna tinctoria and is known to exert anti-tumor effects. However, the function of ALK in inflammation of RAW264.7 cells remains unclear. Thus, this research sought to investigate the detailed function of ALK in inflammatory responses of RAW264.7 cells. To induce an inflammatory response, RAW264.7 cells were exposed to lipopolysaccharide (LPS). MTT assay was applied to examine cell viability. Enzyme-linked immunosorbent assay (ELISA) was used to assess the levels of inflammatory cytokines. Furthermore, the mechanism underlying ALK function in inflammatory responses was investigated using RT-qPCR and western blotting. The data revealed that LPS significantly increased the expression of cyclooxygenase 2 (COX-2), Interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and IL-6, whereas ALK reversed this effect. ALK also restored LPS-induced nuclear factor kappa-B (NF-κB) activation by inhibiting the downregulation of p-inhibitor kappa B alpha (IκBα). LPS elevated p-extracellular regulated protein kinases 1/2 (ERK1/2), phosphorylated p38 (p-p38), and phosphorylated -c-Jun N-terminal kinase (p-JNK) levels, which were markedly decreased in the presence of ALK. In summary, Alkannin attenuated LPS-induced inflammation by inhibiting NF-κB and MAPK signaling. Thus, our research might provide a new theoretical basis for exploring new strategies against RA.

Keywords: Alkannin; MAPK; NF-κB; arthritis; inflammatory response.

Graphical abstract

Figure 1.

ALK had limited cytotoxicity in RAW 264.7 cells. (a) the chemical structure of ALK was presented. (b) RAW264.7 cells were exposed to 1, 3, 5, 10 or 20 μM ALK for 24 h. The cell viability was assessed by MTT assay.

Figure 2.

ALK reverses LPS-induced inflammation in RAW 264.7 cells. RAW264.7 cells were exposed to LPS, LPS + DMSO, LPS+1 μM ALK, LPS+5 μM ALK, LPS+10 μM ALK. The mRNA levels of (a) IL-1β, (b) IL-6 and (c) TNF-α in RAW264.7 cells were detected by RT-qPCR. GADPH was used as an internal control. (d) IL-1β, (e) IL-6 and (f) TNF-α levels in supernatants of RAW264.7 cells were measured by ELISA kits. (g) the protein levels of IL-1β, IL-6 and TNF-α in RAW264.7 cells were measured by RAW 264.7 cells. **P<0.01 compared to control. ^##P<0.01 compared to 1 μg/ml LPS.

Figure 3.

ALK suppresses iNOS and COX-2 levels in LPS-stimulated RAW 264.7 cells. The levels of (a) iNOS and (b) COX2 in RAW 264.7 cells were detected by RT-qPCR. (c) iNOS and COX-2 levels were detected by western blot. (d) the relative expression was quantified by normalizing to GAPDH. **P<0.01 compared to control. #P<0.05, ^##P<0.01 compared to 1 μg/ml LPS.

Figure 4.

ALK reverses LPS-induced inflammation via inactivation of NF-κB signaling. (a) Western blot was applied to determine the expressions of p-IκBα and IκBα in RAW 264.7 cells. (b) the relative expression was quantified by normalizing to GAPDH. (c) Western blot was applied to determine the expressions of p-p65 and p65. (d) the relative expression was quantified by normalizing to GAPDH. **P<0.01 compared to control. #P<0.05, ^##P<0.01 compared to 1 μg/ml LPS.

Figure 5.

ALK attenuates LPS-caused inflammatory responses via inactivation of MAPK signaling. (a, b, c, d, e, f) the protein levels of p38, p-p38, p-ERK1/2, ERK1/2, p-JNK and JNK in RAW 264.7 cells were assessed by western blot. The relative expression was quantified by normalizing to GAPDH. **P<0.01 compared to control. #P<0.05, ^##P<0.01 compared to 1 μg/ml LPS.