Fifteen unreported compounds in Anemarrhena asphodeloides, iriflophene (3), hostaplantagineoside C (7), tuberoside G (8), spicatoside B (9), platycodin D (14), platycoside A (15), platycodin D2 (16), polygalacin D2 (17), platycodin D3 (18), isovitexin (20), vitexin (21), 3,4-dihydroxyallylbenzene-3-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside (22), iryptophan (24), adenosine (25), α-d-Glucose monoallyl ether (26), together with eleven known compounds (1, 2, 4–6, 10–13, 19 and 23), were isolated from the rhizomes of Anemarrhena asphodeloides. The chemical structures of these compounds were characterized using HRMS and NMR. The anti-inflammatory activities of the compounds were evaluated by investigating their ability to inhibit LPS-induced NO production in N9 microglial cells. Timosaponin BIII (TBIII) and trans-hinokiresinol (t-HL) exhibited significant inhibitory effects on the NO production in a dose-dependent manner with IC₅₀ values of 11.91 and 39.08 μM, respectively. Immunoblotting demonstrated that TBIII and t-HL suppressed NO production by inhibiting the expressions of iNOS in LPS-stimulated N9 microglial cells. Further results revealed that pretreatment of N9 microglial cells with TBIII and t-HL attenuated the LPS-induced expression tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) at mRNAs and protein levels. Moreover, the activation of nuclear factor-κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways were inhibited by TBIII and t-HL, respectively. Our findings indicate that the therapeutic implication of TBIII and t-HL for neurogenerative disease associated with neuroinflammation.

The accumulation of microglia around senile plaques is one of the pathological features of Alzheimer’s disease (AD). Chemerin is an adipokine with immune-modulating properties. Our previous study showed that chemokine-like receptor 1 (CMKLR1), the receptor for chemerin, is also a functional receptor of Aβ. However, it remains unclear whether and how the chemerin/CMKLR1 axis affects the migration of microglia. The impact of CMKLR1 on microglial activation and recruitment toward Aβ deposits was examined in APP/PS1 mice mated with CMKLR1 knockout (CMKLR1^−/−) mice. CMKLR1 deficiency reduced the number of microglia around Aβ deposits in aged APP/PS1-CMKLR1^−/− mice compared with APP/PS1 mice. Chemerin expression was significantly decreased in the hippocampus and cortex of aged APP/PS1 mice compared with WT mice. In vitro assays demonstrated that activation of the chemerin/CMKLR1 axis promoted the migration of primary cultures of microglia and murine microglial N9 cells. Mechanistic studies found that chemerin/CMKLR1 induced polarization and protrusion formation of microglia by promoting the remodeling of actin filaments and microtubules, and Golgi apparatus reorientation. The inhibition of p38 MAPK attenuated the promotion of the chemerin/CMKLR1 axis on microglial migration and polarization. In addition, chemerin inhibited Aβ-induced microglial clustering. The inhibition of p38 MAPK alleviated the suppressive effect of chemerin on Aβ-induced microglial aggregation. Our data indicate that the chemerin/CMKLR1 axis is involved in the migration and recruitment of microglia to senile plaques via the p38 MAPK pathway. Modulation of the chemerin/CMKLR1 axis is a potential new strategy for AD therapy.