Baicalein

Hypoxia is the key pathobiological trigger of tubular oxidative stress and cell death that drives the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). The mitochondrial-rich proximal tubular epithelial cells (PTEC) are uniquely sensitive to hypoxia and thus, are pivotal in propagating the sustained tubular loss of AKI-to-CKD transition. Here, we examined the role of PTEC-derived small extracellular vesicles (sEV) in propagating the 'wave of tubular death'. Ex vivo patient-derived PTEC were cultured under normoxia (21 % O₂) and hypoxia (1 % O₂) on Transwell inserts for isolation and analysis of sEV secreted from apical versus basolateral PTEC surfaces. Increased numbers of sEV were secreted from the apical surface of hypoxic PTEC compared with normoxic PTEC. No differences in basolateral sEV numbers were observed between culture conditions. Biological pathway analysis of hypoxic-apical sEV cargo identified distinct miRNAs linked with cellular injury pathways. In functional assays, hypoxic-apical sEV selectively induced ferroptotic cell death (↓glutathione peroxidase-4, ↑lipid peroxidation) in autologous PTEC compared with normoxic-apical sEV. The addition of ferroptosis inhibitors, ferrostatin-1 and baicalein, attenuated PTEC ferroptosis. RNAse A pretreatment of hypoxic-apical sEV also abrogated PTEC ferroptosis, demonstrating a role for sEV RNA in ferroptotic 'wave of death' signalling. In line with these in vitro findings, in situ immunolabelling of diagnostic kidney biopsies from AKI patients with clinical progression to CKD (AKI-to-CKD transition) showed evidence of ferroptosis propagation (increased numbers of ACSL4⁺ PTEC), while urine-derived sEV (usEV) from these 'AKI-to-CKD transition' patients triggered PTEC ferroptosis (↑lipid peroxidation) in functional studies. Our data establish PTEC-derived apical sEV and their intravesicular RNA as mediators of tubular lipid peroxidation and ferroptosis in hypoxic kidney injury. This concept of how tubular pathology is propagated from the initiating insult into a 'wave of death' provides novel therapeutic check-points for targeting AKI-to-CKD transition.

San Huang Pill (SHP) is a prescription in Dunhuang Ancient Medical Prescription, which has the efficacy of heat-clearing and dampness-drying, and is a traditional formula for the treatment of gastrointestinal diseases. However, its efficacy and mechanism in treating ulcerative colitis (UC) are still unclear.

To investigate the protective effects of SHP and its bioactive compounds against Dextran Sulfate Sodium (DSS)-induced intestinal damage using the Drosophila melanogaster model, and to detect the molecular mechanism of SHP in the treatment of UC.

Survival rate, locomotion, feeding, and excretion were used to explore the anti-inflammatory effects of SHP. The pharmacotoxicity of SHP was measured using developmental analysis. Intestinal integrity, intestinal length, intestinal acid-base homeostasis, and Tepan blue assay were used to analyze the protective effect of SHP against DSS-induced intestinal damage. The molecular mechanism of SHP was detected using DHE staining, immunofluorescence, real-time PCR, 16 S rRNA gene sequencing, and network pharmacology analysis. Survival rate, intestinal length, and integrity analysis were used to detect the protective effect of bioactive compounds of SHP against intestinal damage.

SHP supplementation significantly increased the survival rate, restored locomotion, increased metabolic rate, maintained intestinal morphological integrity and intestinal homeostasis, protected intestinal epithelial cells, and alleviated intestinal oxidative damage in adult flies under DSS stimulation. Besides, administration of SHP had no toxic effect on flies. Moreover, SHP supplementation remarkably decreased the expression levels of genes related to JAK/STAT, apoptosis, and Toll signaling pathways, increased the gene expressions of the Nrf2/Keap1 pathway, and also reduced the relative abundance of harmful bacteria in DSS-treated flies. Additionally, the ingredients in SHP (palmatine, berberine, baicalein, wogonin, rhein, and aloeemodin) had protection against DSS-induced intestinal injury, such as prolonging survival rate, increasing intestinal length, and maintaining intestinal barrier integrity.

SHP had a strong anti-inflammatory function, and remarkably alleviated DSS-induced intestinal morphological damage and intestinal homeostatic imbalance in adult flies by regulating JAK/STAT, apoptosis, Toll and Nrf2/Keap1 signaling pathways, and also gut microbial homeostasis. This suggests that SHP may be a potential complementary and alternative medicine herb therapy for UC, which provides a basis for modern pharmacodynamic evaluation of other prescriptions in Dunhuang ancient medical prescription.