Image_1_Ginsenoside Rg3 Mitigates Atherosclerosis Progression in Diabetic apoE–/– Mice by Skewing Macrophages to the M2 Phenotype.TIF

<p>Atherosclerosis (AS) in diabetic patients is often associated with low stability, which might be largely attributed to unfavorable macrophage polarization and increased inflammatory response induced by hyperglycaemia. Ginsenoside Rg3 is one of the main active principles of Panax Ginseng, which has been reported to be a natural ligand of peroxisome proliferator-activated receptor-gamma (PPARγ), a key nuclear transcriptional factor involved in inflammation and macrophage differentiation. However, it remains unclear if Rg3 could exert protective effects on plaque stability in diabetes. In this study, we investigated the role of ginsenoside 20(S)-Rg3 in macrophage polarization and AS plaque stability using advanced glycation end products-treated macrophages and diabetic AS mice models. In vitro, advanced glycation end products (AGEs) treatment promoted the expression of proinflammatory molecules and M1 surface markers, whereas 20(S)-Rg3 could reverse the M1 polarization to the M2 phenotype. In vivo, the administration of 20(S)-Rg3 promoted AS lesion stability and reduced the plaque burden, accompanied by increased M2 macrophages and reduced M1 macrophages. In addition, PPARγ antagonist GW9662 co-administration mostly blocked these effects, suggesting the important role of PPARγ pathways in mediating 20(S)-Rg3 effects in macrophage polarization and atherosclerosis progression. Together, these results demonstrated an immunomodulatory role of ginsenoside 20(S)-Rg3 in promoting macrophages to a profile of the M2 type through PPARγ-dependent mechanisms, and indicated a potential role of 20(S)-Rg3 in the prevention and treatment of diabetic atherosclerosis.</p

Sp1 Mediates a Therapeutic Role of MiR-7a/b in Angiotensin II-Induced Cardiac Fibrosis via Mechanism Involving the TGF-β and MAPKs Pathways in Cardiac Fibroblasts

<div><p>MicroRNA-7a/b (miR-7a/b) protects cardiac myocytes from apoptosis during ischemia/reperfusion injury; however, its role in angiotensin II (ANG II)-stimulated cardiac fibroblasts (CFs) remains unknown. Therefore, the present study investigated the anti-fibrotic mechanism of miR-7a/b in ANG II-treated CFs. ANG II stimulated the expression of specific protein 1 (Sp1) and collagen I in a dose- and time-dependent manner, and the overexpression of miR-7a/b significantly down-regulated the expression of Sp1 and collagen I stimulated by ANG II (100 nM) for 24 h. miR-7a/b overexpression effectively inhibited MMP-2 expression/activity and MMP-9 expression, as well as CF proliferation and migration. In addition, miR-7a/b also repressed the activation of TGF-β, ERK, JNK and p38 by ANG II. The inhibition of Sp1 binding activity by mithramycin prevented collagen I overproduction; however, miR-7a/b down-regulation reversed this effect. Further studies revealed that Sp1 also mediated miR-7a/b-regulated MMP expression and CF migration, as well as TGF-β and ERK activation. In conclusion, miR-7a/b has an anti-fibrotic role in ANG II-treated CFs that is mediated by Sp1 mechanism involving the TGF-β and MAPKs pathways.</p></div

Effect of miR-7a/b mimics on proliferation, migration, and MMP-2 and MMP-9 expression/activity in CFs.

<p>A: MTT assay. B-C: CFs on the external surface of the Transwell were dyed with Crystal Violet and photographed under a microscope. D-F: Western blot analysis of MMP-2 (D, E) and MMP-9 (D, F) protein levels. G-H: Media were harvested for gelatin zymography analysis of MMP-2 activity; no MMP-9 activity was detected in the media. Con: normal untreated CFs; NC: negative control siRNA; *p, < 0.05 compared with control; and #p, < 0.05 compared with NC.</p

Effect of different concentrations of mithramycin on the regulation of Sp1, collagen Iexpression and signal transduction in CFs.

<p>A-F: Western blots and analysis of mithramycin on the regulation of Sp1 (A), collagen I (B), TGF-β (C), p-ERK (D), p-JNK (E) and p-p38 (F). Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; and #p < 0.05, compared with NC.</p

Sp1 mediates miR-7a/b-regulated proliferation migration, and MMP-2 and MMP-9 expression/activity in CFs.

<p>A-C: Western blot analysis of MMP-2 (A, B) and MMP-9 (A, C) protein levels. D-E: Media were harvested for gelatin zymography analysis of MMP-2 activity; no MMP-9 activity was detected in media. F: MTT assay. G-H: CFs on the external surface of the Transwell were dyed with Crystal Violet and photographed under a microscope. Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; #p < 0.05, compared with NC; and ◆p < 0.05, compared with mithramycin.</p

Effect of miR-7a/b mimics on TGF-β and MAPK pathways.

<p>Western blots and analysis of TGF-β (A), p-ERK (B), p-JNK (C), and p-p38 (D) expression. Con: normal untreated CFs; NC: negative control siRNA; *p < 0.05, compared with control; and #p, < 0.05 compared with NC.</p

Effect of miR-7a/b mimics on the regulation of Sp1 and collagen Iexpression and localization in CFs.

<p>A: Western blots of Sp1 and collagen I in untreated CFs and ANG II-treated CFs that were treated with NC siRNA or miR-7a/b mimics. B: Immunofluorescent staining of Sp1 and collagen I location in normal untreated CFs and ANG II-treated CFs that were treated with NC siRNA or miR-7a/b mimics. Con: normal untreated CFs; NC: negative control siRNA. *p < 0.05, compared with control; and #p < 0.05, compared with NC.</p

Sp1 mediates miR-7a/b-regulated collagen I expression and signal transduction in CFs.

<p>A-D: Western blots and analysis of Sp1-mediated miR-7a/b-regulated expression of Sp1 (A), collagen I (B), TGF-β (C) and p-ERK (D). Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; and #p, < 0.05 compared with NC; and ◆p < 0.05, compared with mithramycin.</p

ANG II stimulated Sp1 and collagen I expression in CFs.

<p>A: Protein expression of Sp1 and collagen I in 100 nM ANG II-treated CFs at different time points. B: Protein expression of Sp1 and collagen I treated with different concentrations of ANG II for 24 h. C: normal untreated CFs. *p < 0.05, compared with control.</p