Lobetyolin Alleviates Ferroptosis of Skeletal Muscle in 5/6 Nephrectomized Mice via Activation of Hedgehog-GLI1 Signaling.

BACKGROUND: Muscle wasting increases morbidity and mortality and is related to chronic kidney disease (CKD) and dialysis. It is still unclear whether ferroptosis occurs during this progression and whether it is a potential intervention target for the treatment of CKD-related muscle injury. PURPOSE: The objective is to identify potential compounds for treating ferroptosis and muscle wasting and explore the potential mechanisms in vivo/in vitro. METHODS: Initially, we explored whether ferroptosis is present in the skeletal muscle of 5/6 nephrectomized (NPM) mice via RNA-Seq analysis, TUNEL staining, Oil red O staining, MDA/GSH/GSSG level detection and real-time quantitative PCR (qPCR). Subsequently, utilizing our established molecular phenotyping strategy, we screened potential traditional Chinese herb-derived compounds for alleviation of muscle wasting and ferroptosis. HE staining, Oil red O staining, TUNEL staining, immunofluorescence staining, MDA/GSH/GSSG level detection, Fe level detection, western blotting and qPCR were applied to assess the effects of the identified compound on muscle wasting and ferroptosis and explore the potential mechanism. Furthermore, RNA-Seq analysis, ChIP-Seq analysis and further experiments in vitro were performed to determine the role of Hedgehog signaling in the effect of Lobetyolin (LBT) on ferroptosis. RESULTS: In NPM mice, skeletal muscle dysfunction, lipogenesis, reduced GSH/GSSG ratio, decreased GSH content, increased MDA production and and higher levels of ferroptosis markers were observed. LBT treatment (30 mg/kg or 50 mg/kg) significantly alleviates skeletal muscle injury by inhibiting ferroptosis. Additionally, in an in vitro investigation, C2C12 cells exposed to Indolyl sulfate (IS) induced ferroptosis and LBT treatment (20 µM and 50 µM) protected C2C12 from such injury, consistent with the results from the in vivo analysis. Furthermore, it was found LBT increased the levels of protein involving Hedgehog signaling pathway (SMO and GLI1), and rescue analysis revealed that this pathway played a crucial role in the regulation of ferroptosis. Further experiments demonstrated that LBT upregulated a series of suppressors of ferroptosis by activating Gli1 transcription. CONCLUSION: LBT alleviates CKD-induced muscle injury by inhibiting ferroptosis through activation of the Hedgehog signaling pathway.

A molecular phenotypic screen reveals that lobetyolin alleviates cardiac dysfunction in 5/6 nephrectomized mice by inhibiting osteopontin.

BACKGROUND: Cardiovascular diseases are the major cause of mortality in patients with advanced chronic kidney diseases. The predominant abnormality observed among this population is cardiac dysfunction secondary to myocardial remodelings, such as hypertrophy and fibrosis, emphasizing the need to develop potent therapies that maintain cardiac function in patients with end-stage renal disease. AIMS: To identify potential compounds and their targets as treatments for cardiorenal syndrome type 4 (CRS) using molecular phenotyping and in vivo/in vitro experiments. METHODS: Gene expression was assessed using bioinformatics and verified in animal experiments using 5/6 nephrectomized mice (NPM). Based on this information, a molecular phenotyping strategy was pursued to screen potential compounds. Picrosirius red staining, wheat germ agglutinin staining, Echocardiography, immunofluorescence staining, and real-time quantitative PCR (qPCR) were utilized to evaluate the effects of compounds on CRS in vivo. Furthermore, qPCR, immunofluorescence staining and flow cytometry were applied to assess the effects of these compounds on macrophages/cardiac fibroblasts/cardiomyocytes. RNA-Seq analysis was performed to locate the targets of the selected compounds. Western blotting was performed to validate the targets and mechanisms. The reversibility of these effects was tested by overexpressing Osteopontin (OPN). RESULTS: OPN expression increased more remarkably in individuals with uremia-induced cardiac dysfunction than in other cardiomyopathies. Lobetyolin (LBT) was identified in the compound screen, and it improved cardiac dysfunction and suppressed remodeling in NPM mice. Additionally, OPN modulated the effect of LBT on cardiac dysfunction in vivo and in vitro. Further experiments revealed that LBT suppressed OPN expression via the phosphorylation of c-Jun N-terminal protein kinase (JNK) signaling pathway. CONCLUSIONS: LBT improved CRS by inhibiting OPN expression through the JNK pathway. This study is the first to describe a cardioprotective effect of LBT and provides new insights into CRS drug discovery.

[Responses of the Benthic Diatom Community to Nutrients and the Identification of Nutrient Thresholds in Three Aquatic Ecoregions of the Huntai River, Northeast China].

Benthic diatom communities and nutrient gradients were investigated from 287 sampling sites in three aquatic ecoregions (AE) of the Huntai River to characterize the spatial distribution of nutrients and the benthic diatom communities. Locally weighted scatterplot smoothing (LOWESS) was used to analyze the thresholds for nitrogen and phosphorus. The results showed that:① The concentration of ammonia nitrogen and total phosphorus significantly differs in the three AEs, and shows a tendency of AEⅠ < AE Ⅱ < AE Ⅲ. ② To reveal the structure of benthic diatom communities, various benthic diatom indexes, including the sportive diatom percentage, sensitive diatom percentage, stipitate diatom percentage, Pielou evenness index, specific polluosensitivity index (IPS), biological diatom index (IBD), and generic diatom index (IDG) were analyzed. The sportive diatom percentage varied significantly in AEI, AEⅡ, and AE Ⅲ, with the highest percentage observed in AEⅢ and the lowest in AEⅠ. However, the other six indexes exhibited an opposite trend. All revealed that AE Ⅲ has been seriously damaged, while AEⅠ is less disturbed than AEⅡ and AE Ⅲ. ③ LOWESS fitting curves show thresholds for ammonia nitrogen (NH4+ -N) in the three aquatic ecoregions as 0.13, 0.30, and 1.98 mg·L-1, respectively and for total phosphorous (TP) were 0.04, 0.06, and 0.20 mg·L-1. All results were tested by independent-sample T tests. This study will provide assistance for effectively protecting the benthic diatom community in different aquatic ecoregions and also provide a theoretical basis for water management.