Effects and mechanisms of frehmaglutin D and rehmaionoside C improve LPS-induced acute kidney injury through the estrogen receptor-mediated TLR4 pathway in vivo and in vitro

Sepsis-induced acute kidney injury (S-AKI) is an inflammatory disease with sex differences and there has no effective drugs to cure it. Frehmaglutin D (Fre D) and rehmaionoside C (Reh C) are two violetone compounds with estrogenic activity isolated from Rehmannia glutinosa. However, whether these two drugs exert protective effects on S-AKI through their estrogen-like activity are unclear. This study aimed to explore the effects and mechanisms of Fre D and Reh C on lipopolysaccharide (LPS)-induced S-AKI through the estrogen receptor pathway in vivo and in vitro and to explore the interaction between ER and TLR4 for the first time. The LPS-induced female BALB/c mice S-AKI mouse model was established by adding the estrogen receptor antagonist ICI182,780. Renal function, inflammation, oxidative stress, apoptosis, immune cells, and expression of key proteins of the ER-TLR4-IL-1β pathway were tested. The affinity of Fre D and Reh C for the ER was investigated by molecular docking. Then, an in vitro S-AKI model was established, and ERα/ERβ antagonists (MPP/PHTPP) were added and combined with gene overexpression techniques. The interaction between ER and TLR4 was further explored by Co-IP, GST pull-down and SPR techniques. Fre D and Reh C ameliorated LPS-induced renal damage, inflammation in mice, regulated the immune cells, decreased ROS levels, increased ERα and ERβ protein expression, and decreased TLR4, caspase 11 and IL-1β protein expression. These effects were blocked by ICI182,780. Molecular docking results showed that Fre D and Reh C bound ERα and ERβ with similar potency. The results of in vitro suggested that Fre D and Reh C reduced the levels of inflammation, ROS and apoptosis, TLR4, caspase 11, and IL-1β protein expression and increased ERα/ERβ protein expression in cells. All of these effects were reversed by the addition of MPP/PHTPP and further enhanced after ERα/ERβ gene overexpression with no significant difference in effects. Moreover, there was an indirect or direct interaction between ER and TLR4, and the binding of ERα and ERβ to TLR4 was concentration dependent. Fre D and Reh C may improve S-AKI through the ER-TLR4-IL-1β pathway and may act on both ERα and ERβ receptors. Moreover, ERα and ERβ may interact directly or indirectly with TLR4, which was studied for the first time.