Exosomes Derived from Endothelial Cells Inhibit Neointimal Hyperplasia Induced by Carotid Artery Injury in Rats via ROS-NLRP3 Inflammasome Pathway.

This study attempted to investigate whether exosomes derived from rat endothelial cells (EC-Exo) attenuate intimal hyperplasia after balloon injury using hematoxylin and eosin staining, immunohistochemistry, immunofluorescence staining, Evans blue staining, and Western blotting. The results indicated that EC-Exo inhibited intimal hyperplasia in the carotid artery after balloon injury, promoted re-endothelialization, and reduced vascular inflammation and ROS-NLRP3-mediated cell pyroptosis. Thus, EC-Exo can inhibit neointimal hyperplasia after carotid artery injury in rats presumably by inhibiting the ROS-NLRP3 inflammasome and phenotypic transformation of vascular smooth muscle cells.

[Impact and related mechanism on the improvement of hyperglycemia-induced pyroptosis in H9c2 cells by mircoRNA-214].

Objective: To investigate whether microRNA(miR)-214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase-1. Methods: H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37 ℃ with 5%CO(2), repeat passage was made after cell density reached about 80%, The 5(th) to 8(th) generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR-214 on pyroptosis and caspase-1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimic-negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR-214 mimic-negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti-pyroptosis effect of miR-214 was mediated by targeted inhibition on caspase-1, cells overexpressing caspase-1 were used in the rescue experiment. The cells overexpressing caspase-1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimics+hyperglycosis+recombinant adenovirus (Ad-caspase-1-EGFP) group with caspase-1 gene and EGFP green fluorescent protein expression (mimics+HG+Ad-caspase-1-EGFP group, H9c2 cells were transfected with caspase-1-green fluorescent protein-carrying adenovirus for 48 hours, followed by transfection of miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR-214 mimics+HG+Ad-EGFP empty virus group (mimics+HG+Ad-EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA-214 and caspase-1 in cells were detected by real-time quantitative PCR. The expression and localization of caspase-1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase-1, cleaved caspase-1, NLRP3 and ACS with ß-actin as internal reference. The secretion of IL-1ß and IL-18 in cell culture medium was detected by ELISA. The correlation between miR-214 and caspase-1 was detected by double luciferase reporter gene. Results: (1) The mRNA expression levels of miR-214 and caspase-1 in each group: the mRNA expressions of miR-214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR-214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase-1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase-1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase-1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL-1ß and IL-18 in the cell culture medium of each group: the content of IL-1ß and IL-18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL-1ß and IL-18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR-214 and caspase-1: miR-214 specifically binds to caspase-1 3 'UTR. Meanwhile, Western blot results showed that cleaved caspase-1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase-1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase-1 expression among groups (P>0.05). (6) The expression levels of procaspase-1, cleaved caspase-1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase-1 in each group (P>0.05). Cleaved caspase-1, ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase-1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+ Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL-1ß and IL-18 in rescue experiment: the secretions of IL-1ß and IL-18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). Conclusion: miR-214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase-1.

[Effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit(+) cardiac stem cells apoptosis].

Objective: To explored the effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit(+) cardiac stem cells apoptosis. Methods: C-kit(+) cardiac stem cells were isolated from SD rats by the methods of enzyme digestion and magnetic bead. Cells were divided into the following experimental groups: (1) negative control mimics (NCM)group: cells were transfected with negative control miRNA-21 mimics for 48 hours; (2)mimics group: cells were transfected with miRNA-21 mimics for 48 hours; (3) NCM+ H(2)O(2) group: negative control miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 µmol H(2)O(2) for 2 hours; (4)mimics+ H(2)O(2) group: miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 µmol H(2)O(2) for 2 hours. mRNA of miRNA-21 was detected by RT-PCR. The apoptosis rate of C-kit(+) cardiac stem cells was determined using the annexin V-FITC/PI staining assay. Western blot was employed to measure the expression of apoptosis related proteins(Caspase-3, Bax, and Bcl-2). Results: (1) Compared with the NCM group, the mRNA expression level of miRNA-21 was significantly up-regulated in mimics group, while obviously down-regulated in NCM+ H(2)O(2) group(all P<0.05). Compared with the mimics group, the mRNA expression levels of miRNA-21 in mimics+ H(2)O(2) group was significantly downregulated (P<0.05), but remarkably upregulated compared with the NCM+ H(2)O(2) group(P<0.05). (2) Flow cytometry results indicated that the early apoptosis rates were similar between the NCM group and mimics group ((4.57±3.45)% vs. (5.13±3.21)%, P>0.05). Compared with the NCM group, the early apoptosis rates were remarkably increased ((79.07±5.75)% vs.(4.57±3.45)%, P<0.05) in NCM+ H(2)O(2) group. Compared with the mimics group, the early apoptosis was significantly up-regulated in the mimics+ H(2)O(2) group ((30.27±1.36)% vs.(5.13±3.21)%, P<0.05), which were further down-regulated in mimics+ H(2)O(2) group compared with the NCM+ H(2)O(2) group ((30.27±1.36)% vs.(79.07±5.75)%, P<0.05). (3) Western blot results showed similar protein expression of Caspase-3, Bax and Bcl-2 between NCM group and mimics group(all P>0.05). Compared with the NCM group, the Caspase-3 and Bax protein expression was significantly increased in NCM+ H(2)O(2) group (all P<0.05), but the protein expression level of Bcl-2 was similar between the 2 groups(P>0.05). The Caspase-3 and Bax protein expression was markedly decreased, while Bcl-2 apparently increased in the mimics+ H(2)O(2) group compared with the NCM+ H(2)O(2) group(all P<0.05). Conclusion: Overexpression of miRNA-21 protects the C-kit(+) cardiac stem cells from apoptosis caused by oxidative stress through downregulating proapoptotic and upregulating the antiapoptotic proteins.

[The effect and mechanism investigation of intermedin on the apoptosis of bone marrow derived mesenchymal stem cells induced by hydrogen peroxide].

Objective: To investigate the effect and mechanism of intermedin (IMD)on the apoptosis of BMSCs induced by H2O2 and its mechanism. Methods: BMSCs being cultured in vitro. After 30 min pretreatment with 10-6mol/L IMD, cells were cultured in presence of 100 µmol/L H2O2 for 2 hour, cell apoptosis was determined by flow cytometry; Apoptosis related protein expression was determined by Western blotting and immunofluorescence; Protein and phosphorylation of ERK1/2 was detected by Western blotting and observe the influence of IMD after pretreatment with inhibitor of ERK1/2. Results: IMD could increase phosphorylation of ERK1/2 pathway of BMSCs (P<0.01). Pretreatment of IMD could decrease BMSCs apoptosis induced by H2O2, and up regulation expression of Caspase-3 and Bax, down regulation expression of Bcl-2 (P<0.01), and the effect was abrogated by U0126. Conclusion: IMD can activate ERK1/2 signaling pathway of BMSCs, and can regulate the expression of apoptosis related proteins. Furthermore, it can protect BMSCs from apoptosis induced by H2O2. Activation of ERK1/2 pathway was involved in the effect of IMD on apoptosis.