The root bark of Illicium henryi has been used in traditional Chinese medicine to treat various diseases. Its ethanol extract (EEIH) was found to contain a large number of phenols and possess in vitro antioxidant activities. The present study aimed to investigate its protective effect against lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice. BALB/c mice were intraperitoneally pretreated with EEIH for five days, and then LPS injection was applied to induce AKI. Blood samples and kidney tissues were collected and used for histopathology, biochemical assay, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analyses. EEIH not only significantly dose-dependently attenuated histological damage and reduced renal myeloperoxidase (MPO) activity (from 9.77 ± 0.73 to 0.84 ± 0.30 U/g tissue) but also decreased serum creatinine (from 55.60 ± 2.70 to 27.20 ± 2.39 µmol/L) and blood urea nitrogen (BUN) (from 29.95 ± 1.96 to 16.12 ± 1.24 mmol/L) levels in LPS-treated mice. EEIH also markedly dose-dependently inhibited mRNA expression and production of TNF-α (from 140.40 ± 5.15 to 84.74 ± 5.65 pg/mg), IL-1β (from 135.54 ± 8.20 to 77.15 ± 5.34 pg/mg), IL-6 (from 168.74 ± 7.23 to 119.16 ± 9.35 pg/mg), and COX-2 in renal tissue of LPS-treated mice via downregulating mRNA and protein expressions of toll-like receptor 4 (TLR4) and phosphorylation of nuclear factor-κB (NF-κB) p65. Moreover, EEIH significantly dose-dependently reduced malondialdehyde (MDA) (from 5.43 ± 0.43 to 2.80 ± 0.25 nmol/mg prot) and NO (from 1.01 ± 0.05 to 0.24 ± 0.05 µmol/g prot) levels and increased superoxide dismutase (SOD) (from 22.32 ± 2.92 to 47.59 ± 3.79 U/mg prot) and glutathione (GSH) (from 6.57 ± 0.53 to 16.89 ± 0.68 µmol/g prot) levels in renal tissue induced by LPS through upregulating mRNA expression of nuclear factor erythroid 2 related factor 2 (Nrf2). Furthermore, EEIH inhibited LPS-induced intracellular reactive oxygen species (ROS) production from RAW264.7 cells in a concentration-dependent manner. These results suggest that EEIH has protective effects against AKI in mice through regulating inflammation and oxidative stress.

Platycodin D (PD) is a potent adjuvant with dual Th1 and Th2 potentiating activity, but its mechanisms of action remain unclear. Here, the C2C12 myoblast cell line and mice were used as in vitro and in vivo models to identify potential signaling pathways involved in the adjuvant activity of PD. PD induced a transient cytotoxicity and inflammatory response in the C2C12 cells and in mouse quadricep muscles. A comparative analysis of microarray data revealed that PD induced similar gene expression profiles in the C2C12 cells and in the quadricep muscles, and triggered rapid regulation of death, immune, and inflammation-related genes, both in vivo and in vitro. It was further demonstrated that caspase-1-dependent pyroptosis was involved in the PD-induced cytotoxicity and inflammatory response in the C2C12 cells via the Ca²⁺–c-jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK)–NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. Consistently, the in vivo analysis revealed that a local blockage of NLRP3 and caspase-1 inhibited PD-induced cytokine production and immune cell recruitment at the injection site, and impaired the adjuvant activity of PD on antigen-specific immune responses to model antigen ovalbumin (OVA) in mice. These findings identified the caspase-1-dependent adjuvanticity of PD and expanded the current knowledge on the mechanisms of action of saponin-based adjuvants.

3β-hydroxy-12-oleanen-27-oic acid (ATA), a cytotoxic oleanane triterpenoid with C14-COOH isolated from the rhizome of Astilbe chinensis, has been previously proven to possess antitumor activity and may be a promising antitumor agent. However, its molecular mechanisms of antitumor action were still unclear. This study explored the underlying mechanisms of cytotoxicity and potential target of ATA against human colorectal cancer HCT116 cells via integrative analysis of transcriptomics and network pharmacology in combination with in vitro and in vivo experimental validations. ATA significantly inhibited the proliferation of HCT116 cells in a concentration- and time-dependent manner and induced the cell cycle arrest at the G0/G1 phase, apoptosis, autophagy, and ferroptosis. Transcriptomic analysis manifested that ATA regulated mRNA expression of the genes related to cell proliferation, cell cycle, and cell death in HCT116 cells. The integrated analysis of transcriptomics, network pharmacology, and molecular docking revealed that ATA exerted cytotoxic activity via interactions with FDFT1, PPARA, and PPARG. Furthermore, FDFT1 was verified to be an upstream key target mediating the antiproliferative effect of ATA against HCT116 cells. Of note, ATA remarkably suppressed the growth of HCT116 xenografts in nude mice and displayed an apparent attenuation of FDFT1 in tumor tissues accompanied by the alteration of the biomarkers of autophagy, cell cycle, apoptosis, and ferroptosis. These results demonstrate that ATA exerted in vitro and in vivo antiproliferative effects against HCT116 cells through inducing cell apoptosis, autophagy, and ferroptosis via targeting FDFT1.

Albizia julibrissin saponin active fraction (AJSAF), is a prospective adjuvant with dual Th1/Th2 and Tc1/Tc2 potentiating activity. Its adjuvant activity has previously been proven to be strictly dependent on its spatial co-localization with antigens, highlighting the role of local innate immunity in its mechanisms. However, its potential targets and pathways remain unclear. Here, its intracellular molecular mechanisms of innate immune response were explored using mouse C2C12 myoblast by integrative analysis of the in vivo and in vitro transcriptome in combination with experimental validations. AJSAF elicited a temporary cytotoxicity and inflammation towards C2C12 cells. Gene set enrichment analysis demonstrated that AJSAF regulated similar cell death- and inflammatory response-related genes in vitro and in vivo through activating second messenger–MAPK–CREB pathways. AJSAF markedly enhanced the Ca²⁺, cAMP, and reactive oxygen species levels and accelerated MAPK and CREB phosphorylation in C2C12 cells. Furthermore, Ca²⁺ chelator, CREB inhibitor, and MAPK inhibitors dramatically blocked the up-regulation of IL-6, CXCL1, and COX2 in AJSAF-treated C2C12 cells. Collectively, these results demonstrated that AJSAF induced innate immunity via Ca²⁺–MAPK–CREB pathways. This study is beneficial for insights into the molecular mechanisms of saponin adjuvants.