FOXO3a Induces Myocardial Fibrosis by Upregulating Mitophagy.

BACKGROUND: Atrial fibrillation is one of the most common cardiac arrhythmias. Myocardial fibrosis is closely associated with atrial remodeling, which leads to heightened risk of atrial fibrillation. This study aimed to explore whether forkhead box protein O3 (FOXO3a) impacts myocardial fibrosis incidence by regulating mitophagy. METHODS: Cell viability was assessed by cell counting kit-8 (CCK-8) assays. The expression of vimentin and cytochrome C was detected by immunofluorescence assays. Quantitative real-time polymerase chain reaction (PCR) was used to analyze the relative mRNA level of FOXO3a. Expression of FOXO3a, phosphorylated FOXO3a, Collagen I, Collagen III, alpha-smooth muscle actin (α-SMA), matrix metalloprotease 9 (MMP9), light chain 3 (LC3), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), Parkin, and sequestosome-1 (p62) proteins were determined by western blotting. 5-ethynyl-2'-deoxyuridine (EDU) incorporation was employed to measure cell proliferation. Changes in mitochondrial membrane potential were determined by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining. A wound healing assay was used to examine cell migration, and the levels of reactive oxygen species were determined by flow cytometry. RESULTS: The expression of FOXO3a was upregulated in cardiac fibroblasts treated with angiotensin II (AngII), while the expression of phosphorylated FOXO3a was downregulated under these conditions. FOXO3a knockdown significantly inhibited the proliferation, migration and collagen secretion of cardiac fibroblasts treated with AngII. The ratio of LC3 II/I as well as expression of PINK1 and Parkin was increased, and the expression of p62 was decreased, in cardiac fibroblasts treated with AngII. Moreover, these effects were limited by FOXO3a knockdown. Finally, the mitophagy inducer everolimus (RAD001) attenuated the suppressive effect of FOXO3a knockdown on cardiac fibroblast activation. CONCLUSIONS: FOXO3a promotes the progress of myocardial fibrosis by triggering mitophagy in cardiac fibroblasts.

Circ_ROBO2/miR-149 Axis Promotes the Proliferation and Migration of Human Aortic Smooth Muscle Cells by Activating NF-κB Signaling.

Atherosclerosis is the leading global cause of mortality. The occurrence of coronary artery disease (CAD) is regulated by a diversity of pathways, including circRNAs. However, the potential mechanisms of circRNAs in CAD remain unclear. Here, qRT-PCR was used to examine the expressions of miR-149 and circ_ROBO2. Their influences on cell proliferation, migration, and apoptosis were measured by CCK-8, trans-well, and flow cytometry assays, respectively. The protein levels of p-IκBα and NF-κB p65 were examined using western blot. The molecular interactions were validated using dual luciferase reporter and RNA pull-down assays. The expression patterns of circ_ROBO2 and miR-149 in CAD patients and PDGF-BB-treated human aortic smooth muscle cells (HASMCs) were upregulated and downregulated, respectively. Knockdown of circ_ROBO2 could markedly inhibit the capabilities of proliferation and migration, enhance the apoptotic rate, and suppress NF-κB signaling in PDGF-BB-treated HASMCs. Mechanistically, circ_ROBO2 acted as a sponge of miR-149 to activate TRAF6/NF-κB signaling. Rescue studies demonstrated that neither silencing miR-149 nor activation of NF-κB signaling obviously abolished the biological roles of circ_ROBO2 knockdown in PDGF-BB treated-HASMCs. This discovery elucidated a functional mechanism of circ_ROBO2 in CAD, suggesting that circRNAs serve a vital role in the progression of CAD.

LncRNA-TCONS_00034812 is upregulated in atherosclerosis and upregulates miR-21 through methylation in vascular smooth muscle cells.

BACKGROUND: LncRNA-TCONS_00034812 is a critical player in the proliferation of aortic smooth muscle cells. It is known that artery injury plays an important role in atherosclerosis. However, the potential implication of LncRNA-TCONS_00034812 in atherosclerosis remains unclear. In this study, we collected artery specimens from patients with atherosclerosis and healthy controls to investigate the involvement of LncRNA-TCONS_00034812 in atherosclerosis. METHODS: Sixty patients with atherosclerosis and 60 controls, admitted at The First Hospital of Changsha (Changsha, China), between March 2017 and March 2019, were included. An artery biopsy was performed on all participants to obtain the artery specimens. Real-time quantitative PCR were performed to quantify the relative expression level of LncRNA-TCONS_00034812. Its role in atherosclerotic lesion was evaluated in (high fat diet) HFD-induced ApoE-/- mice. Moreover, human aortic smooth muscle cells (HAOSMCs) was employed to study functional role of LncRNA-TCONS_00034812 overexpression and knockdown by methylation-specific PCR and cell proliferation assay. RESULTS: Overexpression of TCONS_00034812 resulted in miR-21 upregulation and a decrease of miR-21 gene methylation. In contrast, silencing of TCONS_00034812 caused miR-21 downregulation and an increase of miR-21 gene methylation. Cell proliferation analysis indicated that the overexpression of TCONS_00034812 and miR-21 promoted cell proliferation, while silencing of TCONS_00034812 played an opposite role. Moreover, miR-21 overexpression weakened the effects of silencing TCONS_00034812 on cell proliferation. CONCLUSIONS: In summary, LncRNA-TCONS_00034812 is upregulated in atherosclerotic samples, and its overexpression upregulates miR-21 through methylation in human aortic smooth muscle cells (HAOSMCs). Our study indicates that LncRNA-TCONS_00034812 could serve as a potential biomarker for diagnosis of atherosclerosis.

Effects of varying degrees of ligation in a neuropathic pain model induced by chronic constriction injury.

AIM: Ligature tightness of chronic constriction injury (CCI) model remains inconsistent and controversial, presenting barriers for researchers. METHODS: We summarized the different ligation criteria in literature and attempted to clarify their effects. To assess constriction under different criteria, we calculated the radial strain (εR) of ligated nerves from digital photographs. The mechanical withdrawal thresholds (MWT), thermal withdrawal latency (TWL) and sciatic functional index (SFI) were observed in rats of different groups to assess the state of model. Changes of myelin sheath were detected by pathological staining and immunohistochemistry. RESULTS: The median εR values in the Loose, Medium and Tight groups were 13.6%, 15.2% and 21.7%, respectively. Ligated groups had lower MWT than Sham group and the TWL of rats in the Loose approached to rats with sham operation, while that of the Tight group was higher than Medium group 14 days after surgery. Medium and Tight groups showed more abnormal in SFI, compared with the other two groups 14 days. Pathological staining revealed demyelination in three CCI groups, especially in the sciatic nerves. Myelin protein zero levels decreased in the sciatic nerves as the degree of constriction increased, but myelin basic protein of the Medium group was lowest abundant in the spinal cords of all rats. CONCLUSIONS: Our study demonstrated that the surrounding muscles briefly twitched when the diameter of the sciatic nerves was constricted by approximately 14-15%, which may provide a reference for other researchers for establishing CCI models.

Proteomic Analysis of Emodin Treatment in Neuropathic Pain Reveals Dysfunction of the Calcium Signaling Pathway.

BACKGROUND: Neuropathic pain (NP) is a syndrome of pain mediated by distinct pathophysiological processes, and current treatments are not fully satisfactory. Emodin is an effective component of Chinese traditional medicine and has an alleviating effect on NP, but the pharmacological mechanism is not clear. METHODS: We used isobaric tags for relative and absolute quantitation (iTRAQ) technique integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify potential targets of emodin in a rat peripheral nerve chronic constriction injury (CCI) model. RESULTS: A total of 177 differentially expressed proteins were identified among the sham group, CCI group, and emodin group, with a threshold of 1.2-fold change and a P value ≤ 0.05. Among them, 100 differentially expressed proteins (51 up-regulated and 49 down-regulated) were identified in the CCI group compared with sham group. Moreover, 108 differentially expressed proteins (65 up-regulated and 43 down-regulated) were identified in the emodin group with the CCI group as reference. The enrichment analysis of Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed an important role of calcium signaling pathway, neurotransmitter regulation, and long-term potentiation (LTP) in emodin-treated CCI model. Real-time quantitative fluorescence PCR (qRT-PCR) and Western blot analysis revealed that emodin decreased expression of calcium signaling related proteins, including calmodulin (CaM) dependent protein kinase II (CaMK II), phospholipase Cß1 (PLCß1), protein kinase C (PKC), protein kinase C (PKA), and tropomyosin-related kinase B (TrkB), compared with the CCI group. CONCLUSION: Overall, these findings indicated that emodin might alleviate NP by regulating the calcium signaling pathway.

Identification of Slc6a19os and SOX11 as Two Novel Essential Genes in Neuropathic Pain Using Integrated Bioinformatic Analysis and Experimental Verification.

The aim of this study was to identify critical genes associated with neuropathic pain. We also used the competing endogenous RNA (ceRNA) hypothesis to identify related long non-coding RNAs (lncRNAs) and messenger RNAs (miRNAs) with potential regulatory roles. We downloaded GSE107180 from the Gene Expression Omnibus database, screened differentially expressed genes (DEGs) using R software, performed comprehensive bioinformatic analyses, and validated the expression of lncRNA Slc6a19os, miR-125a-5p, miR-125b-5p, miR-351-5p, and Sox11 by qRT-PCR and Western blots. We identified 620 DEGs in spared nerve injury (SNI) mice compared with sham (control) mice, including 309 mRNAs and 311 non-coding RNAs. The up-regulated mRNAs were enriched primarily in several inflammation-related GO biological processes and KEGG signaling pathways. A ceRNA network was constructed that included 82 mRNAs, 4 miRNAs, and 2 lnRNAs. An ingenuity pathway analysis (IPA)-based interaction network for mRNAs differentially expressed in the ceRNA identified several biological processes, including "cellular development, connective tissue development and function, tissue development." Compared with sham mice, lncRNA Slc6a19os and Sox11 expression were significantly up-regulated in dorsal root ganglion (DRG) samples from SNI mice detected using qRT-PCR and Western blots (P < 0.05). MiR-125a-5p, miR-125b-5p, and miR-351-5p expression were down-regulated in DRG samples from SNI mice detected using qRT-PCR (P < 0.05). We concluded that Sox11 and lncRNA Slc6a19os were novel essential genes in the pathogenesis and progression of neuropathic pain and speculated that these two genes were regulated by miR-125a-5p, miR-125b-5p, and miR-351-5p.

[Effects of different materials for partial sciatic nerve ligation on glial cell activation in rat models of chronic constriction injury].

OBJECTIVE: To compare the effects of different materials for partial sciatic nerve ligation on glial cell activation in the spinal cord in a rat model of chronic constriction injury (CCI). METHODS: SD rats were randomly divided into the sham group (n=15), silk suture CCI group (n=15) and chromic catgut CCI group (n=14). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of the rats were detected at 3, 7, 11 and 15 days after the operation. The changes in the sciatic nerve, the activation of spinal cord glial cells and the expression of inflammatory factors were observed using Western blotting and RT-PCR. RESULTS: At 3 to 15 days after the surgery, MWT and TWL of the rats were significantly lower in silk suture group and chromic catgut group than in the control group (P < 0.05), and was significantly lower in chromic catgut group than in the silk suture group (P < 0.05) at 3 days after the surgery. The results of sciatic nerve myelin staining showed that the sciatic nerve was damaged and demyelinated in both the ligation groups. The expressions of CD11b, GFAP, IL-1ß and TNF-α in the two ligation groups were similar and all significantly higher than those in the control group (P < 0.05). IL-6 mRNA level was significantly higher in chromic catgut group than in the silk suture group (P < 0.05). CONCLUSIONS: The CCI models established by partial sciatic nerve ligation with silk suture and chromic catgut all show glial activation, and the inflammatory response is stronger in chromic catgut group.