SGLT1 Knockdown Attenuates Cardiac Fibroblast Activation in Diabetic Cardiac Fibrosis

Figures

• 

  FIGURE 1. Changes of blood glucose level and histopathology in DCM rats. SD rats were fed a high-fat diet (HFD) for 4 weeks before streptozotocin (STZ) was injected intraperitoneally at a dose of 60 mg/kg, and rats were fed for another 12 weeks. (A) Intraperitoneal glucose tolerance tests (IPGTTs) were used to identify insulin resistance in the two groups. (B) The FBG levels of the two groups measured at indicated times. (C) The body weight of the two groups was monitored at the indicated time. The paraffin sections of rat left ventricular tissues after (D) hematoxylin and eosin (H&E) staining and (E) Masson’s trichrome staining. (F) Quantitative analysis of interstitial Masson’s trichrome staining (n = 6 per group).

• 

  FIGURE 2. SGLT1 is downregulated in the experimental diabetic cardiac fibrosis model and high-glucose–induced CFs. (A) The mRNA level of SGLT1 was detected using RT‐qPCR (n = 6). (B) The protein level of SGLT1 was analyzed using western blot analysis (n = 6). (C) The protein levels of SGLT1, collagen I, and collagen III were evaluated using immunohistochemical staining. Relative expression of SGLT1 (D) and collagen I and collagen III (E) was found using semiquantitative analysis based on immunohistochemistry. (F) The morphology of CFs with or without high-glucose stimulation was imaged under an optical microscope. (G) The protein level of SGLT1 was analyzed by immunofluorescent staining in high-glucose–induced CFs. (H) The protein levels of SGLT1, collagen I, and collagen III were analyzed using western blot analysis in high-glucose–induced CFs (n = 3).

• 

  FIGURE 3. Knockdown of SGLT1 inhibited high-glucose–induced CF activation. CFs were transfected with SGLT1 siRNAs, and SGLT1 mRNA and protein levels were detected using RT‐qPCR (A) and western blotting, respectively (B). CCK-8 assay was used to detect the proliferation of CFs under high-glucose condition with or without SGLT1 inhibition. (D, E) The representative images of the wound-healing assay were obtained at 0 and 24 h after knockdown of SGLT1, and the migrative ability of CFs was compared. (F–H) ELISA was used to detect the levels of collagen-synthesis–related markers, including TGF-β1, collagen I, and collagen III in the cell supernatant (n = 6). (I) Western blotting analysis was performed to investigate the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 in CFs under high-glucose condition with or without SGLT1 inhibition.

• 

  FIGURE 4. SGLT1 overexpression promotes collagen release via the p38 and ERK1/2 signaling pathway in CFs. CFs were transfected with an SGLT1 plasmid or vector, and SGLT1 expression levels were detected using RT‐qPCR (A) or western blotting analysis (B). (C) After overexpression of SGLT1 in CFs, CCK-8 assay was used to detect the proliferation of CFs under normal or high-glucose condition (n = 6). (D–F) After overexpressing SGLT1 in CFs, the collagen-synthesis–related markers, including TGF-β1, collagen I, and collagen III, in the cell supernatant were measured using ELISA (n = 6). (G) After overexpressing SGLT1 in CFs, the protein levels of phosphorylated p38 and p-ERK1/2 were analyzed using western blotting in CFs both under normal or high-glucose condition (n = 3).

• 

  FIGURE 5. SGLT1 inhibition alleviated myocardial fibrosis and apoptosis in diabetic rat hearts (n = 6). (A, B) Knockdown of SGLT1 was achieved by continuous intravenous injection into the tail vein with specific SGLT1 siRNA in DCM rats, and the knockdown efficiency was identified by detecting SGLT1 expression in myocardial tissues using RT-qPCR and western blotting analysis. (C) SGLT1 protein levels in myocardial tissue were measured by immunohistochemistry staining. (D) Masson’s trichrome staining of myocardial tissue shows interstitial fibrosis in DCM rats treated with si-NC or si-SGLT1. (E, F) Relative protein levels of collagen I and collagen III in myocardial tissue were measured using immunohistochemical staining and western blotting analysis (n = 6). (G) Wheat germ agglutinin staining and quantitation of myocardial cell size in DCM rats treated with si-NC or si-SGLT1 (n = 6).