THBru attenuates diabetic cardiomyopathy by inhibiting RAGE-dependent inflammation

Abstract

Diabetic cardiomyopathy (DCM) is a complication of diabetes mellitus characterized by heart failure and cardiac remodeling. Previous studies show that tetrahydroberberrubine (THBru) retrogrades cardiac aging by promoting PHB2-mediated mitochondrial autophagy and prevents peritoneal adhesion by suppressing inflammation. In this study we investigated whether THBru exerted protective effect against DCM in db/db mice and potential mechanisms. Eight-week-old male db/db mice were administered THBru (25, 50 mg·kg^-1·d^-1, i.g.) for 12 weeks. Cardiac function was assessed using echocardiography. We showed that THBru administration significantly improved both cardiac systolic and diastolic function, as well as attenuated cardiac remodeling in db/db mice. In primary neonatal mouse cardiomyocytes (NMCMs), THBru (20, 40 μM) dose-dependently ameliorated high glucose (HG)-induced cell damage, hypertrophy, inflammatory cytokines release, and reactive oxygen species (ROS) production. Using Autodock, surface plasmon resonance (SPR) and DARTS analyses, we revealed that THBru bound to the domain of the receptor for advanced glycosylation end products (RAGE), subsequently leading to inactivation of the PI3K/AKT/NF-κB pathway. Importantly, overexpression of RAGE in NMCMs reversed HG-induced inactivation of the PI3K/AKT/NF-κB pathway and subsequently counteracted the beneficial effects mediated by THBru. We conclude that THBru acts as an inhibitor of RAGE, leading to inactivation of the PI3K/AKT/NF-κB pathway. This action effectively alleviates the inflammatory responses and oxidative stress in cardiomyocytes, ultimately leading to ameliorated DCM.

Keywords: RAGE; diabetic cardiomyopathy; inflammation; tetrahydroberberrubine.

Fig. 1. THBru improves cardiac dysfunction in db/db mice.

a Representative echocardiographic images showing the echocardiograms and ratio of the E/A ratio for assessment of cardiac function in db/db mice. b Statistical results for EF, an indicator of cardiac systolic function in mice. n = 5. c Statistical results for FS, an indicator of cardiac systolic function in mice. n = 5. d Statistical results of E/A ratio, an indicator of cardiac diastolic function in mice. n = 5. e Statistical results for IVRT,an indicator of cardiac diastolic function in mice. n = 5. f Histopathological changes and collagen deposition were assessed by H&E (magnification 200×, Scale bar:100 μm), Masson trichrome (magnification 200×, Scale bar:100 μm), WGA (magnification 200×, Scale bar:100 μm) and electron microscopy. The green arrow indicates normal sarcomeres, yellow arrows indicate normal sarcomeres, green asterisk indicates normal mitochondria, yellow asterisk indicates mitochondrial swelling. (magnification 2500×, Scale bar: 10 μm) (g) Statistical results for HW/TL. (Scale bar: 0.5 cm). n = 5. h Statistical results for fibrosis as revealed by Masson staining. n = 5. i qPCR verification of BNP. n = 5. j qPCR verification of Col1a. ^###P < 0.001 vs. control; *P < 0.05, **P < 0.01, ***P < 0.001 vs. db/db. The data are expressed as the mean ± SEM.

Fig. 2. THBru directly binds to RAGE and modulates the expression of RAGE protein.

a KEGG enrichment of THBru banding protein. b SPR quantification analysis of THBru with RAGE. c, d The protein expression changes of RAGE in DARTS (Drug Affinity Responsive Target Stability) analysis. n = 3. ***P < 0.001 vs. THBru 0. e Western blot analysis of the protein level of RAGE in mice heart. n = 5. ^###P < 0.001 vs. control; **P < 0.01, ***P < 0.001 vs. db/db. f The binding pocket of THBru on RAGE. g The key residues with the lowest binding energy. h RMSD of the docked complex reflects the stability of the protein. i The distance between the residues and THBru. The data are expressed as the mean ± SEM.

Fig. 3. THBru reduces inflammatory responses in db/db mice by inhibiting PI3K/AKT/NF-κB pathway.

a Multiplex mouse Cytokine ELISA Kit to detect mouse serum inflammatory factors. b Statistical results of changed inflammatory factors. c–i Western blot analysis of the protein level of phosphorylation (p-) and total PI3K, AKT, and NF-κB in mice heart. n = 5. j IL-1β levels in serum tested by Elisa. n = 5. k TNF-α levels in serum tested by Elisa. n = 5. ^###P < 0.001 vs. control; ^*P < 0.01, ^**P < 0.01, ^***P < 0.001 vs. db/db. The data are expressed as the mean ± SEM.

Fig. 4. THBru ameliorates cardiomyocyte damage and inflammatory response induced by high glucose.

a CCK8 was used to detect cell viability of NMCMs after THBru treatment for 24 h. n = 5. b Representative imagies of ROS production in NMCMs. (magnification 100×, scale bar:20 μm). n = 5. c Immunofluorescence staining of NMCMs for α-actinin (green). (magnification 400×, scale bar:20 μm). n = 5. d Statistical results for cell area. n = 5. e TNF-α levels in cell culture medium tested by Elisa. n = 5. f IL-1β levels in cell culture medium tested by Elisa. n = 5. ^###P < 0.001 vs. control; **P < 0.01, ***P < 0.001 vs. HG. The data are expressed as the mean ± SEM.

Fig. 5. THBru Inhibits RAGE-dependent signaling pathway in cardiomyocytes exposed to HG.

a Immunofluorescence staining of NMCMs for RAGE (red). (magnification 400×, scale bar:10 μm). n = 5. b Statistical results for RAGE staining. n = 5. c Western blot analysis of the protein level of RAGE in NMCMs. n = 5. d–j Western blot analysis of the protein level of phosphorylation (p-) and total PI3K, AKT, and NF-κB with RAGE overexpression. n = 5. ^###P < 0.001 vs. control; *P < 0.01, **P < 0.01, ***P < 0.001 vs. HG. The data are expressed as the mean ± SEM.

Fig. 6. Overexpression of RAGE reversed the protective effects of THBru in HG-treated cardiomyocyte.

a Immunofluorescence staining of NMCMs for α-actinin (green). (magnification 400×, scale bar:20 μm). n = 5. b Statistical results for cell area. n = 5. c Representative imagies of ROS production with RAGE overexpression in NMCMs. (magnification 200×, Scale bar:50 μm). n = 5. d Statistical results of ROS production in NMCMs. n = 5. e–k Western blot analysis of the protein level of phosphorylation (p-) and total PI3K, AKT, and NF-κB with RAGE overexpression. n = 5. ^***P < 0.001 vs. HG; ^&&&P < 0.001 vs. +HG+THBru. The data are expressed as the mean ± SEM.