This study explores the role and underlying molecular mechanisms of fucoidan sulfate(FPS) in regulating heme oxygenase-1(Hmox1)-mediated ferroptosis to ameliorate myocardial injury in diabetic cardiomyopathy(DCM) through in vivo and in vitro experiments and network pharmacology analysis. In vivo, a DCM rat model was established using a combination of "high-fat diet feeding + two low-dose streptozotocin(STZ) intraperitoneal injections". The rats were randomly divided into four groups: normal, model, FPS, and dapagliflozin(Dapa) groups. In vitro, a cellular model was created by inducing rat cardiomyocytes(H9c2 cells) with high glucose(HG), using zinc protoporphyrin(ZnPP), an Hmox1 inhibitor, as the positive control. An automatic biochemical analyzer was used to measure blood glucose(BG), serum aspartate aminotransferase(AST), serum lactate dehydrogenase(LDH), and serum creatine kinase-MB(CK-MB) levels. Echocardiography was used to assess rat cardiac function, including ejection fraction(EF) and fractional shortening(FS). Pathological staining was performed to observe myocardial morphology and fibrotic characteristics. DCFH-DA fluorescence probe was used to detect reactive oxygen species(ROS) levels in myocardial tissue. Specific assay kits were used to measure serum brain natriuretic peptide(BNP), myocardial Fe~(2+), and malondialdehyde(MDA) levels. Western blot(WB) was used to detect the expression levels of myosin heavy chain 7B(MYH7B), natriuretic peptide A(NPPA), collagens type Ⅰ(Col-Ⅰ), α-smooth muscle actin(α-SMA), ferritin heavy chain 1(FTH1), solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), 4-hydroxy-2-nonenal(4-HNE), and Hmox1. Immunohistochemistry(IHC) was used to examine Hmox1 protein expression patterns. FerroOrange and Highly Sensitive DCFH-DA fluorescence probes were used to detect intracellular Fe~(2+) and ROS levels. Transmission electron microscopy was used to observe changes in mitochondrial morphology. In network pharmacology, FPS targets were identified through the PubChem database and PharmMapper platform. DCM-related targets were integrated from OMIM, GeneCards, and DisGeNET databases, while ferroptosis-related targets were obtained from the FerrDb database. A protein-protein interaction(PPI) network was constructed for the intersection of these targets using STRING 11.0, and core targets were screened with Cytoscape 3.9.0. Molecular docking analysis was conducted using AutoDock and PyMOL 2.5. In vivo results showed that FPS significantly reduced AST, LDH, CK-MB, and BNP levels in DCM model rats, improved cardiac function, decreased the expression of myocardial injury proteins(MYH7B, NPPA, Col-Ⅰ, and α-SMA), alleviated myocardial hypertrophy and fibrosis, and reduced Fe~(2+), ROS, and MDA levels in myocardial tissue. Furthermore, FPS regulated the expression of ferroptosis-related markers(Hmox1, FTH1, SLC7A11, GPX4, and 4-HNE) to varying degrees. Network pharmacology results revealed 313 potential targets for FPS, 1 125 targets for DCM, and 14 common targets among FPS, DCM, and FerrDb. Hmox1 was identified as a key target, with FPS showing high docking activity with Hmox1. In vitro results demonstrated that FPS restored the expression levels of ferroptosis-related proteins, reduced intracellular Fe~(2+) and ROS levels, and alleviated mitochondrial structural damage in cardiomyocytes. In conclusion, FPS improves myocardial injury in DCM, with its underlying mechanism potentially involving the regulation of Hmox1 to inhibit ferroptosis. This study provides pharmacological evidence supporting the therapeutic potential of FPS for DCM-induced myocardial injury.
Animals ; Ferroptosis/drug effects* ; Rats ; Diabetic Cardiomyopathies/physiopathology* ; Male ; Rats, Sprague-Dawley ; Polysaccharides/pharmacology* ; Heme Oxygenase-1/genetics* ; Myocytes, Cardiac/metabolism* ; Myocardium/pathology* ; Humans ; Cell Line ; Heme Oxygenase (Decyclizing)

Objective:To investigate the expression of cGAS-STING signaling pathway molecules in labial gland specimen of primary Sj?gren′s syndrome (pSS) patients and its correlation with disease characteristics.Methods:Clinical data and labial gland specimens were collected from 19 patients diagnosed with pSS who visited Xuanwu Hospital, Capital Medical University, from January 2021 to December 2021, along with a control group of 7 gender-and age-matched individuals with isolated xerostomia.We performed immunofluorescence staining for cGAS on labial gland specimens, and Western blot analysis was further utilized to confirm the expression levels of cGAS-STING signaling pathway-related molecules (cGAS, pSTING, pIRF3 and pTBK1) in the labial gland tissues of both groups. For normally distributed data, t test was employed for comparison between groups, while the Mann-Whitney U test was used for non-normally distributed data. And Spearman's correlation analysis was utilized to explore the correlation between clinical characteristics and molecular expression levels. P value<0.05 was considered statistically significant. Results:Immun-ofluorescence analysis showed significant infiltration of cGAS-positive cells in the labial gland tissues of the pSS group, while the control group exhibited no or minimal expression of cGAS-positive cells and there was a statistical difference between the two groups ( t=3.87, P=0.001). Moreover, the expression level of cGAS in labial gland tissues of pSS patients had a positive correlation with the focus scores ( r=0.55, P=0.014). Western blot analysis revealed statistically significant differences in the expression of cGAS and pSTING between the pSS group and the control group in labial gland tissues (0.73±0.39 vs. 0.18±0.05, t=2.38, P=0.049 and 0.91±0.17 vs. 0.23±0.10, t=2.17, P=0.043). Additionally, a correlation between the expression levels of cGAS and pSTING in labial gland tissues was found according to Spearman correlation analysis ( r=0.823, P=0.001). However, there was no correlation between the expression level of cGAS in labial gland tissues and clinical features, serological indicators (IgG level, titer of rheumatoid factor, C3 level and autoantibody positive rate) or disease activity (EULAR Sj?gren′s syndrome disease activity index scores). Conclusion:The cGAS-STING signaling pathway is activated in the labial gland tissue of pSS patients, which may be involved in its pathogenesis.