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. 2017 Jun 8;22(6):951.
doi: 10.3390/molecules22060951.

Anti-Inflammatory Activities and Liver Protection of Alisol F and 25-Anhydroalisol F through the Inhibition of MAPK, STAT3, and NF-κB Activation In Vitro and In Vivo

Affiliations

Anti-Inflammatory Activities and Liver Protection of Alisol F and 25-Anhydroalisol F through the Inhibition of MAPK, STAT3, and NF-κB Activation In Vitro and In Vivo

Xiaoxu Bi et al. Molecules. .

Abstract

Alisol F and 25-anhydroalisol F isolated from Alisma orientale, were proved to exhibit anti-inflammatory potential in our previous work. In the current study, the anti-inflammatory effects and action mechanisms of alisol F and 25-anhydroalisol F were investigated in vitro. Moreover, the pharmacological effects of alisol F in lipopolysaccharide (LPS)/d-galactosamine (d-gal)-induced acute liver-injured mice were evaluated. The results demonstrated that alisol F and 25-anhydroalisol F could suppress LPS-induced production of nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β), as well as inhibit the mRNA and protein levels of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2). In addition, we investigated the role of alisol F and 25-anhydroalisol F in mediating mitogen-activated protein kinases (MAPKs), signal transducers, and activators of transcription 3 (STAT3) and nuclear factor κB (NF-κB) pathways involved in the inflammation process of LPS-stimulated RAW 264.7 cells. The phosphorylation of ERK, JNK, p38, and STAT3, and the NF-κB signaling pathway, were obviously suppressed in alisol F and 25-anhydroalisol F treated cells. Results obtained from in vitro experiments suggested alisol F obviously improved liver pathological injury by inhibiting the production of TNF-α, IL-1β, and IL-6, and significantly decreasing the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in LPS/d-gal-induced mice. Furthermore, the reduction of phosphorylation of ERK and JNK, as well as suppression of the NF-κB signaling pathway, were also observed in liver tissues of the alisol F-treated mice model. Alisol F and 25-anhydroalisol F may serve as potential leads for development of anti-inflammatory agents for acute liver failure treatment.

Keywords: ">d-gal-induced acute liver injured mice; 25-anhydroalisol F; LPS/; NF-κB, MAPKs, STAT3 signaling pathways; RAW 264.7 macrophages; alisol F; anti-inflammatory.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The effect of alisol F and 25-anhydroalisol F on cell viability of RAW 264.7 cells. The structures of alisol F and 25-anhydroalisol F are shown in (A,B); the cell viability was measured by MTT assay (C). Cells were pretreated with the different concentrations of alisol F (0, 3.3, 11, 33 and 100 μM) and 25-anhydroalisol F (0, 3.3, 11, 33 and 100 μM) for 2 h and then stimulated with or without LPS (1 μg/mL) for 24 h. The data represent the means ± S.E. values of three independent experiments.
Figure 2
Figure 2
The effect of alisol F and 25-anhydroalisol F on the expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the various concentrations of alisol F (0, 3.3, 11 and 33 μM) and 25-anhydroalisol F (0, 3.3, 11 and 33 μM) for 2 h and then stimulated with or without LPS (1 μg/mL) for 24 h. The expression of iNOS and COX-2 protein levels were analyzed by Western blot (A,B); Cells were pretreated in the same way for 2 h and then stimulated with or without LPS (1 μg/mL) for 4 h. The expression of iNOS and COX-2 mRNA levels were measured by quantitative real-time PCR (C,D). The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05, ** p < 0.001 vs. LPS group).
Figure 3
Figure 3
The effects of alisol F and 25-anhydroalisol F on LPS-induced expression and production of TNF-α, IL-6 and IL-1β in RAW 264.7 cells. The cells were pretreated with the different concentrations of alisol F (0, 3.3, 11 and 33 μM) and 25-anhydroalisol F (0, 3.3, 11 and 33 μM) for 2 h and then stimulated with or without LPS (1 μg/mL) for 4 h. The expression of TNF-α (A); IL-6 (C); and IL-1β (E) were determined by quantitative real-time PCR. The culture media were collected after 24 h LPS treatment, the production of TNF-α (B); IL-6 (D); and IL-1β (F) were measured using the ELISA kits. The dexamethasone (DXM) was used as the positive control. The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05; ** p < 0.001 vs. LPS group).
Figure 4
Figure 4
The effect of alisol F and 25-anhydroalisol F on LPS-induced activation of the NF-κB pathway in RAW264.7 cells. Cells were pretreated with the various concentrations of alisol F (0, 3.3, 11 and 33 μM) and 25-anhydroalisol F (0, 3.3, 11 and 33 μM) for 2 h and then stimulated with or without LPS (1 μg/mL) for 30 min. The expression of phospho-p65, p65, phospho-IκB-α and IκB-α were analyzed by Western blot (AD); Cytosolic (C) and nuclear (N) protein were extracted to determine the expression of p65 by Western blot (E,F). The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05; ** p < 0.001 vs. LPS group).
Figure 5
Figure 5
The effect of alisol F and 25-anhydroalisol F on the translocation of the NF-κB p65 subunit into the nucleus of LPS-simulated RAW 264.7 cells. Cells were pretreated with the alisol F (33 μM) and 25-anhydroalisol F (33 μM) for 2 h, and then stimulated with or without LPS (1 μg/mL) for 30 min. The translocation of NF-κB p65 was determined by immunocytochemistry. Hoechst 33342 was used to stain the nucleus of cells (blue). The cytoplasm was marked by the anti-NF-κB p65 antibody followed by fluorescein isothiocyanate (FITC)-conjugated anti-rabbit lgG (green).
Figure 6
Figure 6
The effect of alisol F and 25-anhydroalisol F on LPS-induced activation of the MAPK signaling pathway. RAW 264.7 cells were pretreated with the different concentrations (0, 3.3, 11 and 33 μM) of alisol F (A) and 25-anhydroalisol F (B) for 2 h, then stimulated with or without LPS (1 μg/mL) for 30 min. The expressions of phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, and JNK proteins were analyzed by Western blot. The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05; ** p < 0.001 vs. LPS group).
Figure 6
Figure 6
The effect of alisol F and 25-anhydroalisol F on LPS-induced activation of the MAPK signaling pathway. RAW 264.7 cells were pretreated with the different concentrations (0, 3.3, 11 and 33 μM) of alisol F (A) and 25-anhydroalisol F (B) for 2 h, then stimulated with or without LPS (1 μg/mL) for 30 min. The expressions of phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, and JNK proteins were analyzed by Western blot. The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05; ** p < 0.001 vs. LPS group).
Figure 7
Figure 7
The effect of alisol F and 25-anhydroalisol F on LPS-induced activation of stat3 in RAW264.7 cells. Cells were pretreated with the different concentrations (0, 3.3, 11 and 33 μM) and of alisol F (A) and 25-anhydroalisol F (B) for 2 h and then stimulated with or without LPS (1 μg/mL) for 6 h. The expression of phospho-stat3 and stat3 proteins were analyzed by Western blot. The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05, ** p < 0.001 vs. LPS group).
Figure 8
Figure 8
Alisol F alleviated LPS/d-gal-induced hepatotoxicity. C57BL/6 mice were pretreated with alisol F (20 mg/kg), DXM (5 mg/kg) or vehicle for three days. After 30 min of the last treatment, mice exposed to LPS/d-gal (LPS 40 μg/kg, d-gal 700 mg/kg) except for control group. Serum were harvested for measured the production of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 5 h after LPS/d-gal exposure (A,B); Serum were harvested at 2 h after LPS/d-gal treatment for measured the production of TNF-α (C), IL-6 (D), and IL-1β (E) by using ELISA kits. The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05, ** p < 0.001 vs. LPS/d-gal group).
Figure 9
Figure 9
Alisol F improved histological alterations of LPS/d-gal-induced liver injury. The liver samples stained with hematoxylin-eosin (H&E) stain and acquired the image under the magnification (AD) original magnification 200× and E-H: original magnification 400×. (A,E) mice treated with vehicles; (B,F) exposed to LPS/d-gal; (C,G) treated with Alisol F (20 mg/kg) and exposed to LPS/d-gal; (D,H) treated with DXM (5 mg/kg) and exposed to LPS/d-gal.
Figure 10
Figure 10
The effect of Alisol F on LPS/d-gal-induced activation of MAPKs and NF-κB signaling pathways in vivo. The mice were pretreated with Alisol F (20 mg/kg) and DXM (5 mg/kg) for three days, and induced with LPS/d-gal (40 μg/kg, 700 mg/kg). Liver samples were harvested at 5 h after LPS/d-gal treatment. The protein expression of phospho-ERK phospho-JNK and IκB-α were detected by Western blot. (A). Densitometry analysis of phospho-ERK (B), phospho-JNK (C) and IκB-α (D) were shown. The data represent the means ± S.E. of three independent experiments. (## p < 0.001 vs. control group; * p < 0.05, ** p < 0.001 vs. LPS/d-gal group).

References

    1. Nathan C. Points of control in inflammation. Nature. 2002;420:846–852. doi: 10.1038/nature01320. - DOI - PubMed
    1. McCulloch C.A., Downey G.P., El-Gabalawy H. Signalling platforms that modulate the inflammatory response: New targets for drug development. Nat. Rev. Drug Discov. 2006;5:864–876. doi: 10.1038/nrd2109. - DOI - PubMed
    1. Woolbright B.L., Jaeschke H. Role of the inflammasome in acetaminophen-induced liver injury and acute liver failure. J. Hepatol. 2017;66:836–848. doi: 10.1016/j.jhep.2016.11.017. - DOI - PMC - PubMed
    1. Fatkhullina A.R., Peshkova I.O., Koltsova E.K. The role of cytokines in the development of atherosclerosis. Biochemistry (Moscow) 2016;81:1358–1370. doi: 10.1134/S0006297916110134. - DOI - PMC - PubMed
    1. Zhou H.Y., Shin E.M., Guo L.Y., Youn U.J., Bae K., Kang S.S., Zou L.B., Kim Y.S. Anti-inflammatory activity of 4-methoxyhonokiol is a function of the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-kappaB, JNK and p38 MAPK inactivation. Eur. J. Pharmacol. 2008;586:340–349. doi: 10.1016/j.ejphar.2008.02.044. - DOI - PubMed

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