MicroRNA‑24 attenuates diabetic vascular remodeling by suppressing the NLRP3/caspase‑1/IL‑1β signaling pathway

Abstract

Vascular remodeling plays an important role in the pathogenesis of diabetic cardiovascular complications. Previous published research has indicated that microRNA‑24 (miR‑24) is involved in diabetic vascular remodeling, but the underlying molecular mechanisms have yet to be fully elucidated. The aim of the present study was to investigate whether adenovirus‑mediated miR‑24 overexpression can suppress the NOD‑like receptor family pyrin domain‑containing 3 (NLRP3)‑related inflammatory signaling pathway and attenuate diabetic vascular remodeling. The carotid arteries of diabetic rats were harvested and prepared for analysis. Reverse transcription‑quantitative PCR and western blotting assays were used to detect the expressions of related mRNAs and proteins. Morphological examinations, including hematoxylin and eosin, immunohistochemical and Masson's trichrome staining, were also performed. The results of the present study demonstrated that miR‑24 upregulation suppressed neointimal hyperplasia and accelerated reendothelialization in the injured arteries, lowered the expression of NLRP3, apoptosis‑associated speck‑like protein, caspase‑1, proliferating cell nuclear antigen, CD45, interleukin (IL)‑1β, IL‑18 and tumor necrosis factor‑α, and increased the expression of CD31, smooth muscle (SM) α‑actin and SM‑myosin heavy chain. These data indicated that miR‑24 overexpression can attenuate vascular remodeling in a diabetic rat model through suppressing the NLRP3/caspase‑1/IL‑1β signaling pathway.

Figure 1

Schematic representation of the experimental protocol. The diagram illustrates the grouping of experimental animals. STZ, streptozotocin; miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 2

Overexpression of miR-24 reduces NLRP3 protein expression. (A) Reverse transcription-quantitative PCR was used to detect miR-24 levels following gene transfer into the injured arteries in diabetic rats. (B) Western blotting was performed to measure the NLRP3 protein expression level. Values are presented as the mean ± standard deviation (n=5/group). ^*P<0.05 vs. sham group; ^#P<0.05 vs. saline group; and ^&P<0.05 vs. Ad-NC group. miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 3

Overexpression of miR-24 attenuates intimal hyperplasia. (A) Hematoxylin and eosin-stained sections of the carotid arteries. Magnification, ×200. Areas of the (B) intima and (C) media were analyzed. (D) The ratio of intima to media was also calculated. Values are presented as the mean ± standard deviation (n=8/group). ^*P<0.05 vs. sham group; ^#P<0.05 vs. saline; and ^&P<0.05 vs. Ad-NC group. A, adventitia; M, media; Ni, neointima; miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 4

Overexpression of miR-24 reduces the expression of PCNA in the neointima. Immunohistochemical staining of PCNA was performed and PCNA-positive cells were stained brown and yellow. (A) Representative microphotograph of section stained immunohistochemically for PCNA. Magnification, ×100. (B) The percentage of PCNA-positive cells in the neointima. Values are presented as the mean ± standard deviation (n=8/group). ^*P<0.05 vs. Ad-NC group. PCNA, proliferating cell nuclear antigen; miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 5

Overexpression of miR-24 decreases collagen deposition. Masson’s trichrome staining was performed to evaluate the collagen deposition. Blue staining indicates collagen fibers. (A) Representative microphotograph of Masson’s trichrome staining. Magnification, ×200. (B) Percentage of collagen area in the neointima. Values are presented as the mean ± standard deviation (n=8/group). ^*P<0.05 vs. Ad-NC group. miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 6

Overexpression of miR-24 attenuates phenotypic transformation of VSMCs by enhancing the expression levels of contractile markers. (A) Representative western blots of SM-MHC and SMα-actin. (B) Corresponding densitometric analyses of SM-MHC protein expression. (C) Corresponding densitometric analyses of SMα-actin protein expression. Values are presented as the mean ± standard deviation (n=6/group). ^*P<0.05 vs. Ad-NC group. VSMCs, vascular smooth muscle cells; SM, smooth muscle; MHC, myosin heavy chain; miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 7

Overexpression of miR-24 enhances reendothelialization. Immunohistochemical staining of the endothelial cell marker CD31 was performed and positive cells were stained brown and yellow. Magnification, ×400. Arrows indicate CD31-positive cells. Compared with the saline and Ad-NC groups, infection with Ad-miR-24 increased the expression of CD31 and the positively-stained area was larger and more continuous. A, adventitia; M, media; Ni, neointima; miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 8

Overexpression of miR-24 reduces the inflammatory response. (A) Immunohistochemistry was used to detect neutrophil cell marker CD45-positive cells in the neointima (arrows). Magnification, ×400. Expression levels of (B) IL-1β, (C) IL-18 and (D) TNF-α were detected by ELISA. Values are presented as the mean ± standard deviation (n=6 per group). ^*P<0.05 vs. sham group; ^#P<0.05 vs. saline; and ^&P<0.05 vs. Ad-NC group. A, adventitia; M, media; Ni, neointima. IL, interleukin; TNF, tumor necrosis factor; miR, microRNA; Ad, adenovirus; NC, GFP sequence.

Figure 9

Overexpression of miR-24 inhibits NLRP3-mediated inflammation. miR-24 upregulation inhibited ASC and caspase-1 mRNA and protein expression. (A) mRNA expression levels of ASC and caspase-1 were detected by RT-qPCR. (B) Representative western blots of ASC and caspase-1. (C) Corresponding densitometric analyses of ASC and caspase-1 protein expression. Values are presented as the mean ± standard deviation (n=6/group). ^*P<0.05 vs. sham group; ^#P<0.05 vs. saline; and ^&P<0.05 vs. Ad-NC group. ASC, apoptosis-associated speck-like protein; miR, microRNA; Ad, adenovirus; NC, GFP sequence.