Militarine, a natural glucosyloxybenzyl 2-isobutylmalate, isolated from Bletilla striata, was reported with a prominent neuroprotective effect recently. The limited information on the metabolism of militarine impedes comprehension of its biological actions and pharmacology. This study aimed to investigate the metabolite profile and the distribution of militarine in vivo, which help to clarify the action mechanism further. A total of 71 metabolites (57 new metabolites) in rats were identified with a systematic method by ultra-high-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). The proposed metabolic pathways of militarine include hydrolyzation, oxidation, glycosylation, esterification, sulfation, glucuronidation and glycine conjugation. Militarine and its metabolites were distributed extensively in the treated rats. Notably, six metabolites of militarine were identified in cerebrospinal fluid (CSF), which were highly consistent with the metabolites after oral administration of gastrodin in rats. Among the metabolites in CSF, five of them were not reported before. It is the first systematic metabolic study of militarine in vivo, which is very helpful for better comprehension of the functions and the central nervous system (CNS) bioactivities of militarine. The findings will also provide an essential reference for the metabolism of other glucosylated benzyl esters of succinic, malic, tartaric and citric acids.

Nanomaterial-assisted chemodynamic cancer therapy has received considerable attention in recent years. It outperforms other modalities for its unique pattern of reactive oxygen species (ROS) production via the non-exogenous stimulant. However, the treatment outcome is limited by the insufficient content of endogenous hydrogen peroxide (H2O2). Herein, a biodegradable H2S-releasing MnS@HA-DOX nanocluster (MnS@HA-DOX NC) was constructed by in situ biomineralization from hyaluronic acid, to amplify ROS cascade and boost Mn2+-based chemodynamic therapy (CDT). The acid-responsive NCs could quickly degrade after internalization into endo/lysosomes, releasing Mn2+, H2S gas, and anticancer drug doxorubicin (DOX). The Fenton-like reaction catalyzed by Mn2+ was amplified by both H2S and DOX, producing a mass of cytotoxic ·OH radicals. Through the combined action of gas therapy (GT), CDT, and chemotherapy, oxidative stress would be synergistically amplified, inducing irreversible DNA damage and cell cycle arrest, eventually resulting in cancer cell apoptosis.

Depleting tumoral lactate is a promising strategy to enhance the immune response and thereby suppressing tumorigenesis. However, the direct use of lactate oxidase (LOx), the most straightforward lactate-eliminating agent, faces several issues including low stability, no targeting capacity. To solve these problems, a “carrier-free” nanodrug LOx@manganese sulfide (LOx@MnS) was facilely prepared via biomineralization. This way, all the constituents integrated in the nanodrug, including LOx, Mn2+, and hydrogen sulfide (H2S), could be readily delivered into tumor cells and exert their effects. LOx combined with Mn2+ to convert lactate to cytotoxic reactive oxygen species (ROS) through the cascade reaction, meanwhile the crosstalk between H2S and ROS induced metabolism suppression to further augment the therapeutic efficacy. Consequently, the multi-mode therapeutic modalities led to effective immune activation and tumor suppression, suggesting the great potential for tumor treatment. We believe this strategy opens a new avenue to construct functional materials from frangible biomolecules and expand their applications.

Hydrogen sulfide (H2S) plays crucial inflammatory modulating roles, representing a promising candidate for anti-inflammatory therapies. However, current H2S delivery approaches lack sufficient specificity against inflammatory response. Herein, regarding the overexpressed aminopeptidase N (APN) at the inflammation sites, an APN-activated self-immolative carbonyl sulfide (COS)/H2S donor (AlaCOS) was developed for inflammatory response-specific H2S delivery. The compound showed sustained H2S generation upon APN activation in the presence of carbonic anhydrase (CA), and the responsiveness could be well regulated by modulating the amino acid sequence. Due to the inflammatory response-specific sustained H2S delivery, AlaCOS provided potent anti-inflammatory capability, which was further validated by RNA sequencing. In vivo experiments on a full-thickness cutaneous wound murine model also showed the strong promoting effect on wound healing, mainly due to the regulation of the inflammatory response by AlaCOS. By introducing a caged coumarin fluorophore to the molecular architecture, self-reporting fluorescence could be generated accompanied with APN-mediated COS/H2S release, which achieved the visualization of H2S delivery in vitro and in vivo. This work not only offers a useful tool for studying the bioactivity of H2S on inflammation, but also provides new insights for developing novel therapies to cope with inflammation-associated diseases.