β-Hydroxyisovalerylshikonin regulates macrophage polarization via the AMPK/Nrf2 pathway and ameliorates sepsis in mice

Figures & data

Table 1. PCR primers for the indicated genes.

Gene	Forward	Reverse
TNF-α	5′-CATCTTCTCAAAATTCGAGTGAC-3′	5′-TGGGAGTAGACAAGGTACAACCC-3′
IL-1β	5′-TGGAAAAGCGGTTTGTCTTC-3′	5′-TACCAGTTGGGGAACTCTGC-3′
CD206	5′-CTTCGGGCCTTTGGAATAAT-3′	5′-TAGAAGAGCCCTTGGGTTGA-3′
Arginase-1	5′-GTGAAGAACCCACGGTCTGT-3′	5′-GCCAGAGATGCTTCCAACTG-3′
HO-1	5′-GAGATAGAGCGCAACAAGCAG-3′	5′-CTTGACCTCAGGTGTCATCTC-3′
NQO-1	5′-GCCTGAGCCCAGATATTGTG-3′	5′-GGAAAGGACCGTTGTCGT-3′
GAPDH	5′-GGCCTTCCGTGTTCCTAC-3′	5′-TGTCATCATATCTGGCAGGTT-3′

Figure 4. β-HIVS regulates macrophage polarization in primary murine BMDMs via AMPK/Nrf2 pathway. (A,B) Primary murine BMDMs were incubated with β-HIVS at the indicated doses for 24 h. Then, MTT assay (A) and Trypan blue staining (B) were employed to assess the survival and death rates of BMDMs, respectively. *p < 0.05 compared with the control group. (C) The BMDMs were treated with 0.5 μM β-HIVS for different time periods, and the levels of p-AMPKα/AMPKα and nuclear Nrf2 were measured by Western blot analysis. *p < 0.05 compared with the ‘0 h’ group. (D,E) The BMDMs were pre-treated with 0.5 μM β-HIVS alone or combined with CC (10 μM, 30 min earlier) for 1 h, followed by 100 ng/mL LPS for an additional 3 h (D) or 24 h (E). After treatment, Western blot analysis was conducted to detect the levels of p-AMPKα/AMPKα and nuclear Nrf2 (D). ^$p < 0.05, ^#p < 0.05, ^△p < 0.05, and *p < 0.05 vs. the control, β-HIVS alone, LPS alone, and LPS + β-HIVS groups, respectively. In the meantime, the mRNA levels of M1 and M2 marker genes were detected using real-time PCR assay (E). ^$p < 0.05, ^#p < 0.05, and *p < 0.05 vs. the control, LPS alone, and LPS + β-HIVS groups, respectively.

Figure 5. β-HIVS ameliorates LPS-induced mouse sepsis. (A) Survival rates of mice. *p < 0.05 vs. the LPS alone group. (B) The levels of TNF-α and IL-1β in serum. (C) The mRNA expression of TNF-α and IL-1β detected in lung tissues. (D) The levels of TNF-α and IL-1β detected in BALF. (E) The percentage of F4/80⁺ CD86⁺ M1 alveolar macrophages and F4/80⁺ CD206⁺ M2 alveolar macrophages in BALF was analyzed by flow cytometry. (F) The lung tissue sections with H&E staining. Original magnification, ×200. ^#p < 0.05 and *p < 0.05 vs. the control and LPS alone groups, respectively.