[Connexin 43 (Cx43) inhibits autophagy of rat thoracic aortic smooth muscle cells induced by ox-LDL].

Objective To investigate the role of connexin 43 (Cx43) in the autophagy of rat thoracic aortic vascular smooth muscle cells (VSMCs) induced by oxidized low-density lipoprotein (ox-LDL). Methods The primary VSMCs were identified by immunofluorescence cytochemical staining of α-smooth muscle actin (α-SMA). After ox-LDL treatment, the foam cells were identified by oil red O staining; the expression of microtubule associated protein 1 light chain 3 (LC3) protein in VSMCs treated with 0, 40, 80, 160 µg/mL ox-LDL for 0, 6, 12, 24 hours and the expression of Cx43 protein treated with 80 µg/mL ox-LDL for 24 hours were detected by Western blotting. VSMCs were randomly divided into control group, ox-LDL group, and ox-LDL combined with Cx43 specific antagonist Gap26 group to detect the expressions of LC3 and beclin 1 by Western blotting. Results The positive rate of α-SMA in the isolated cells was more than 95%. The oil red O positive cells in ox-LDL treated cells significantly increased, ox-LDL decreased the ratio of LC3II/LC3I and the expression of beclin 1 protein in a concentration- and time-dependent manner, and the expression of Cx43 protein was significantly increased. After administration of Gap26, the ratio of LC3II/LC3I and the expression of beclin 1 protein were up-regulated. Conclusion Cx43 inhibits autophagy of VSMCs induced by ox-LDL. Cx43 inhibits ox-LDL induced autophagy.

Molecular Mechanism of Naringenin Against High-Glucose-Induced Vascular Smooth Muscle Cells Proliferation and Migration Based on Network Pharmacology and Transcriptomic Analyses.

Although the protective effects of naringenin (Nar) on vascular smooth muscle cells (VSMCs) have been confirmed, whether it has anti-proliferation and anti-migration effects in high-glucose-induced VSMCs has remained unclear. This study aimed to clarify the potential targets and molecular mechanism of Nar when used to treat high-glucose-induced vasculopathy based on transcriptomics, network pharmacology, molecular docking, and in vivo and in vitro assays. We found that Nar has visible anti-proliferation and anti-migration effects both in vitro (high-glucose-induced VSMC proliferation and migration model) and in vivo (type 1 diabetes mouse model). Based on the results of network pharmacology and molecular docking, vascular endothelial growth factor A (VEGFA), the proto-oncogene tyrosine-protein kinase Src (Src) and the kinase insert domain receptor (KDR) are the core targets of Nar when used to treat diabetic angiopathies, according to the degree value and the docking score of the three core genes. Interestingly, not only the Biological Process (BP), Molecular Function (MF), and KEGG enrichment results from network pharmacology analysis but also transcriptomics showed that phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) is the most likely downstream pathway involved in the protective effects of Nar on VSMCs. Notably, according to the differentially expressed genes (DEGs) in the transcriptomic analysis, we found that cAMP-responsive element binding protein 5 (CREB5) is a downstream protein of the PI3K/Akt pathway that participates in VSMCs proliferation and migration. Furthermore, the results of molecular experiments in vitro were consistent with the bioinformatic analysis. Nar significantly inhibited the protein expression of the core targets (VEGFA, Src and KDR) and downregulated the PI3K/Akt/CREB5 pathway. Our results indicated that Nar exerted anti-proliferation and anti-migration effects on high-glucose-induced VSMCs through decreasing expression of the target protein VEGFA, and then downregulating the PI3K/Akt/CREB5 pathway, suggesting its potential for treating diabetic angiopathies.

Inhibition of Connexin 43 reverses ox-LDL-mediated inhibition of autophagy in VSMC by inhibiting the PI3K/Akt/mTOR signaling pathway.

Background: Oxidized low-density lipoproteins (ox-LDL) may induce foam cell formation from the vascular smooth muscle cell (VSMC) by inhibiting VSMC autophagy. This process accelerates the formation of atherosclerosis (AS). Connexin 43 (Cx43), which is the most widely distributed connexin in VSMC is associated with autophagy. However, the mechanism of action and the involvement of Cx43 in ox-LDL-inhibited VSMC autophagy remain unclear. Methods: The primary VSMC were obtained and identified, before primary VSMC were pretreated with an inhibitor (Cx43-specific inhibitor Gap26 and PI3K inhibitor LY294002) and stimulated with ox-LDL. Results: Ox-LDL not only inhibited autophagy in VSMC via downregulation of autophagy-related proteins (such as Beclin 1, LC3B, p62), but also increased Cx43 protein levels. Then we added Gap26 to VSMC in the ox-LDL+Gap26 group, in which autophagy-related proteins were increased and the accumulation of lipid droplets was reduced. These result suggested that an enhanced level of autophagy and an alleviation of lipid accumulation might be caused by inhibiting Cx43 in VSMC. The phosphorylation levels of PI3K, AKT, mTOR were increased by ox-LDL, thus down-regulating autophagy-related proteins. However, this situation was partially reversed by the Gap26. Moreover, Cx43 expression were decreased by LY294002 in ox-LDL-induced VSMCs. Conclusion: Inhibiting Cx43 may activate VSMC autophagy to inhibit foam cell formation by inhibiting the PI3K/AKT/mTOR signaling pathway.

Adiponectin Attenuates Lipopolysaccharide-induced Apoptosis by Regulating the Cx43/PI3K/AKT Pathway.

Cardiomyocyte apoptosis is a crucial factor leading to myocardial dysfunction. Adiponectin (APN) has a cardiomyocyte-protective impact. Studies have shown that the connexin43 (Cx43) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathways play an important role in the heart, but whether APN plays a protective role by regulating these pathways is unclear. Our study aimed to confirm whether APN protects against lipopolysaccharide (LPS)-induced cardiomyocyte apoptosis and to explore whether it plays an important role through regulating the Cx43 and PI3K/AKT signaling pathways. In addition, our research aimed to explore the relationship between the Cx43 and PI3K/AKT signaling pathways. In vitro experiments: Before H9c2 cells were treated with LPS for 24 h, they were pre-treated with APN for 2 h. The cytotoxic effect of APN on H9c2 cells was evaluated by a CCK-8 assay. The protein levels of Bax, Bcl2, cleaved caspase-3, cleaved caspase-9, Cx43, PI3K, p-PI3K, AKT and p-AKT were evaluated by Western blot analysis, and the apoptosis rate was evaluated by flow cytometry. APN attenuated the cytotoxicity induced by LPS. LPS upregulated Bax, cleaved caspase-3 and cleaved caspase-9 and downregulated Bcl2 in H9c2 cells; however, these effects were attenuated by APN. In addition, LPS upregulated Cx43 expression, and APN downregulated Cx43 expression and activated the PI3K/AKT signaling pathway. LPS induced apoptosis and inhibited PI3K/AKT signaling pathway in H9c2 cells, and these effects were attenuated by Gap26 (a Cx43 inhibitor). Moreover, the preservation of APN expression was reversed by LY294002 (a PI3K/AKT signaling pathway inhibitor). In vivo experiments: In C57BL/6J mice, a sepsis model was established by intraperitoneal injection of LPS, and APN was injected into enterocoelia. The protein levels of Bax, Bcl2, cleaved caspase-3, and Cx43 were evaluated by Western blot analysis, and immunohistochemistry was used to detect Cx43 expression and localization in myocardial tissue. LPS upregulated Bax and cleaved caspase-3 and downregulated Bcl2 in sepsis; however, these effects were attenuated by APN. In addition, the expression of Cx43 was upregulated in septic myocardial tissue, and APN downregulated Cx43 expression in septic myocardial tissue. In conclusion, both in vitro and in vivo, the data demonstrated that APN can protect against LPS-induced apoptosis during sepsis by modifying the Cx43 and PI3K/AKT signaling pathways.

A role of AMPK and connexin 43 in the suppression of CoCl2-induced apoptosis of spiral modiolar artery smooth muscle cells by adiponectin.

AIMS: Adiponectin (APN) is a protein hormone secreted mainly by adipose tissue that exhibits biological functions such as anti-inflammatory, anti-atherosclerotic, anti-apoptotic, hearing-protective and microcirculation-regulating functions. In this study, we explored whether APN could attenuate damage caused by CoCl2-induced hypoxic conditions in smooth muscle cells (SMCs) of the spiral modiolar artery (SMA). MAIN METHODS: We first cultured and identified primary SMCs of the SMA. Afterward, the SMCs were pre-treated with APN and then stimulated with CoCl2. KEY FINDINGS: Compared with the control group, the group treated with CoCl2 for 24 h exhibited significantly decreased cell viability, significantly increased apoptosis rates and Malondialdehyde (MDA) levels, and decreased Superoxide Dismutase (SOD) activity. In addition, the expression levels of Bax and cleaved caspase-3 were upregulated, while those of Bcl2 were downregulated evidently. Compared with the CoCl2 group, the group pre-treated with APN before receiving CoCl2 treatment had increased cell viability and SOD activity but decreased MDA levels and apoptosis rates. The expression levels of Bcl2, p-AMPKα and Cx43 were evidently increased, while those of Bax and cleaved caspase-3 were decreased, in the group pre-treated with APN compared to the CoCl2 group. The protective effect of APN was blocked by the AMPK inhibitor Compound C and the Cx43 inhibitor Gap19. SIGNIFICANCE: Our study demonstrated that APN protected SMCs against CoCl2-induced hypoxic injury via the AMPK signalling pathway and regulated the expression of Cx43 in cells. Therefore, APN might be a promising treatment for diseases related to circulation disturbances of the inner ear.