miR-92b-3p protects retinal tissues against DNA damage and apoptosis by targeting BTG2 in experimental myopia.

BACKGROUND: Myopia is one of the eye diseases that can damage the vision of young people. This study aimed to explore the protective role of miR-92b-3p against DNA damage and apoptosis in retinal tissues of negative lens-induced myopic (LIM) guinea pigs by targeting BTG2. METHODS: Biometric measurements of ocular parameters, flash electroretinogram (FERG), and retinal thickness (RT) were performed after miR-92b-3p intravitreal injection in LIM guinea pigs. The apoptotic rate was detected by Annexin V-FITC/PI double staining, and the change in mitochondrial membrane potential was measured by JC-1 staining. Retinal apoptosis and expression of p53, BTG2, and CDK2 were explored by TdT-mediated dUTP-biotin nick labeling (TUNEL) and immunofluorescence staining assays, respectively. BTG2 and its upstream and downstream molecules at gene and protein levels in retinal tissues were measured by real-time quantitative PCR (qPCR) and Western blotting. RESULTS: Compared with normal controls (NC), the ocular axial length of LIM guinea pig significantly increased, whereas refraction decreased. Meanwhile, dMax-a and -b wave amplitudes of ERG declined, retinal thickness was decreased, the number of apoptotic cells and apoptotic rate in LIM eyes was exaggerated, and the mitochondrial membrane potential significantly decreased. In addition, results of qPCR and Western blot assays showed that the expression levels of p53, BTG2, CDK2, and BAX in LIM guinea pigs were higher than the levels of the NC group, whereas the BCL-2 expression level was decreased. By contrast, the miR-92b-3p intravitreal injection in LIM guinea pigs could significantly inhibit axial elongation, alleviate DNA damage and apoptosis, and thus protect guinea pigs against myopia. CONCLUSION: In conclusion, p53 and BTG2 were activated in the retinal tissue of myopic guinea pigs, and the activated BTG2 could elevate the expression of CDK2 and BAX, and attenuate the expression of BCL-2, which in turn promote apoptosis and eventually lead to retinal thinning and impaired visual function in myopic guinea pigs. The miR-92b-3p intravitreal injection can attenuate the elongation of ocular length and retinal thickness, and inhibit the CDK2, BAX, and p53 expression by targeting BTG2, thereby ameliorating DNA damage and apoptosis in LIM guinea pigs and protecting ocular tissues.

Electroacupuncture alleviates ciliary muscle cell apoptosis in lens-induced myopic guinea pigs through inhibiting the mitochondrial signaling pathway.

AIM: To investigate the effect of electroacupuncture (EA) on the mitochondria-dependent apoptotic signaling pathway in the ciliary muscle of guinea pigs with negative lens-induced myopia (LIM). METHODS: Guinea pigs were randomly divided into normal control (NC) group, LIM group, LIM+SHAM acupoint (LIM+SHAM) group, and LIM+EA group. Animals in the NC group received no intervention, while those in other three groups were covered with -6.0 diopter (D) lenses on right eyes. Meanwhile, animals in the LIM+EA group received EA at Hegu (LI4) combined with Taiyang (EX-HN5) acupoints, while those in the LIM+SHAM group were treated at sham points. After treatments for 1, 2, and 4wk, morphological changes in ciliary muscles were observed with hematoxylin and eosin (H&E) staining and nick end labeling (TUNEL), and the expression of the mitochondrial apoptotic signaling pathway-related molecules in ciliary muscles was measured by real-time quantitative polymerase chain reaction (qPCR) and Western blot. Additionally, the adenosine triphosphate (ATP) contents were also determined in ciliary muscles. RESULTS: Axial length increased significantly in the LIM and LIM+SHAM groups and decreased in the LIM+EA group. The ciliary muscle fibers were broken and destroyed in both LIM and LIM+SHAM groups, whereas those in the LIM+EA group improved significantly. TUNEL assay showed the number of apoptotic cells increased in the LIM and LIM+SHAM groups, whereas reduced in the LIM+EA group. ATP contents showed a significant decrease in the LIM and LIM+SHAM groups, whereas increased after EA treatment. Compared with the NC group, the dynamin-related protein 1 (DRP1), Caspase3, and apoptotic protease activator 1 (APAF1) levels were significantly increased in the LIM group and decreased in the LIM+EA group. CONCLUSION: The results provide evidence of EA inhibiting the development of myopia by regulating the mitochondrial apoptotic signaling pathway.

Suppressive effect of nitric oxide synthase (NOS) inhibitor L-NMMA acetate on choroidal fibrosis in experimental myopic guinea pigs through the nitric oxide signaling pathway.

Myopia is one of the most prevalent eye diseases that seriously threaten the eyesight of children and adolescents worldwide. However, the pathogenesis is still unclear, and effective drugs are still scarce. In the present study, the guinea pigs were randomly divided into a normal control (NC) group, a lens-induced myopia (LIM) group, a NOS inhibitor (L-NMMA) injection group, and a NOS inhibitor solvent phosphate-buffered saline (PBS) group and the animals received relevant treatments. After 2- and 4-week different treatments, we noted that the refraction and choroidal thickness in the LIM group decreased compared with the NC group, whereas the ocular axial length increased significantly, and the choroid showed a fibrotic trend. The expression of NOS1, NOS3, TGF-ß1, COLI, and α-SMA at gene and protein levels was increased significantly in the choroid (all P < 0.05). After intravitreal injection of NOS inhibitor L-NMMA, we found that compared with the LIM group, the refraction and the choroidal thickness significantly increased, whereas the axial length reduced significantly, accompanied by an increase of choroidal thickness and an improvement of choroidal fibrosis. The expression levels of choroidal NOS1, NOS3, TGF-ß, COLI, and α-SMA were significantly reduced (all P < 0.05). In conclusion, the trend of choroidal fibrosis in LIM guinea pigs is positively correlated with the increase in axial length. The NOS inhibitor L-NMMA can alleviate the process of choroidal fibrosis in myopic guinea pigs by inhibiting NO signaling pathway.

Elevated retinal fibrosis in experimental myopia is involved in the activation of the PI3K/AKT/ERK signaling pathway.

OBJECTIVE: This study aimed to investigate the regulatory role of the PI3K/AKT/ERK signaling pathway in retinal fibrosis in -6.0 diopter (D) lens-induced myopic (LIM) guinea pigs. METHODS: Biological measurements of eye tissues were performed on guinea pigs to obtain their refraction, axial length, retinal thickness, physiological function, and fundus retinal status. In addition, Masson staining and immunohistochemical (IHC) assay were further done to explore the changes in retinal morphology after myopic induction. Meanwhile, hydroxyproline (HYP) content was measured to evaluate the degree of retinal fibrosis. Moreover, the levels of the PI3K/AKT/ERK signaling pathway and fibrosis-related molecules in retinal tissues including matrix metalloproteinase 2(MMP2), collagen type I (Collagen I), and α-smooth muscle actin (α-SMA) were detected by real-time quantitative PCR (qPCR) and Western blot. RESULTS: The LIM guinea pigs showed a significant myopic shift in refractive error and an increase in axial length compared with those of the normal control (NC) group. Masson staining, hydroxyproline content determination, and IHC showed an increase in retinal fibrosis. After myopic induction, qPCR and western blot analyses showed that phosphatidylinositol-3-kinase catalytic subunit α (PIK3CA), protein kinase B (AKT), extracellular regulated protein kinase 1/2 (ERK1/2), MMP2, Collagen I, and α-SMA were consistently elevated in the LIM group than those in the NC group. CONCLUSION: The PI3K/AKT/ERK signaling pathway was activated in the retinal tissues of myopic guinea pigs, which exaggerated fibrotic lesions and reduced retinal thickness, ultimately leading to retinal physiological dysfunctions in myopic guinea pigs.

Inhibitory effect of miR-138-5p on choroidal fibrosis in lens-induced myopia guinea pigs via suppressing the HIF-1α signaling pathway.

Myopia is one of the most common eye diseases in children and adolescents worldwide. Currently, there is no effective treatment in clinical practice. Ocular tissue fibrosis is involved in the development of myopia and this study aimed to investigate the effect of miR-138-5p on choroidal fibrosis in myopic guinea pigs via regulating the HIF-1α signaling pathway. First, guinea pigs were randomly divided into a normal control (NC) group, a lens-induced myopia (LIM) group, a LIM + miR-138-5p-carried Lentivirus treatment (LV) group, and a LIM + miR-138-5p-Vector treatment (VECTOR) group. All animals were induced experimental myopia with a -6.0 diopter lens except those in the NC group. Meanwhile, animals in the LV group were supplemented with 5 µl of miR-138-5p-carried Lentivirus, while those in the VECTOR group were only supplemented with the same volume of miR-138-5p-Vector. After myopia induction for 2 and 4 weeks, the refractive status and other ocular parameters of the guinea pigs were measured. Further, the expression of hypoxia-inducible factor (HIF)-1α, transforming growth factor (TGF)-ß, collagen I, hydroxyproline (HYP), interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and a-smooth muscle actin (α-SMA) in choroidal tissues was investigated. Results showed that the refraction and axial length of the experimental myopic guinea pigs increased, and choroid fibrosis aggravated after experimental myopic induction. miR-138-5p can efficiently decrease the refraction and ocular length, and ameliorate the choroidal fibrosis of the experimental myopic guinea pigs via downregulating the fibrosis-related TGF-ß1, collagen I, HYP, IL-1ß, TNF-α, and α-SMA expression through inhibiting the HIF-1α signaling pathway. Our results provide new insight into controlling myopic development using microRNAs in clinical practice.

Enhanced Apoptosis in Choroidal Tissues in Lens-Induced Myopia Guinea Pigs by Activating the RASA1 Signaling Pathway.

Purpose: This study aimed to explore the role of the RAS p21 protein activator 1 (RASA1) signaling pathway in apoptosis in choroid tissues from guinea pigs with negative lens-induced myopia (LIM). Methods: Biometric measurements were performed to examine refractive status, ocular parameters, and choroidal thickness (ChT) after myopia induction. The choroidal morphology was observed by hematoxylin and eosin (H&E) staining and TUNEL assay. The expression of the RASA1 signaling pathway at the mRNA and protein levels in choroidal tissues was measured by real-time quantitative PCR (qPCR) and western blot assays. Results: Compared with the normal control (NC) group, the ocular length of the guinea pigs in LIM increased remarkably, as did the myopic refraction. ChT decreased after myopia induction. H&E staining showed that the thickness and laxity of the choroidal tissues in LIM were strikingly reduced. The number of apoptotic cells in the LIM eyes was increased. Moreover, qPCR and western blot assays showed that the expression levels of both RASA1 and BCL-2-associated agonist of cell death (BAD) were higher in the LIM group than in the NC group, whereas the expression level of B-cell lymphoma 2 (BCL-2) was decreased after 2 weeks of experimental myopia. However, the trend of RASA1, BAD, and BCL-2 expression was reversed after 4 weeks of experimental myopia compared with levels after 2 weeks of experimental myopia. Conclusions: Results showed that the RASA1 signaling pathway is activated in choroid tissues in myopic guinea pigs. Activated RASA1 signaling induces high BAD expression and low BCL-2 expression, which in turn promotes apoptosis and ultimately causes ChT thinning in myopic guinea pigs.

Zinc oxide nanoparticle-triggered oxidative stress and autophagy activation in human tenon fibroblasts.

Glaucoma is a leading cause of irreversible blindness worldwide due to elevated intraocular pressure, and filtering surgery can efficiently control intraocular pressure of glaucoma patients. However, failure of filtering surgery commonly results from scarring formation at the surgical site, in which fibroblast proliferation plays an essential role in the scarring process. Our previous study has demonstrated that zinc oxide (ZnO) nanoparticles could efficiently inhibit human tenon fibroblasts (HTFs) proliferation. The present study aimed to explore the underlying mechanism involved in oxidative stress and autophagy signaling in zinc oxide (ZnO) nanoparticles-induced inhibition of HTFs proliferation. In this study, we investigated the effect of ZnO nanoparticles on HTFs proliferation, mitochondrial function, ATP production and nuclear morphology. Moreover, we also explored the interactions between ZnO nanoparticles and HTFs, investigated the influence of ZnO nanoparticles on the autophagosome formation, the expression of autophagy-related 5 (Atg5), Atg12 and Becn1 (Beclin 1), and the level of light chain 3 (LC3). The results suggested that ZnO nanoparticles can efficiently inhibit HTFs proliferation, disrupt the mitochondrial function, attenuate the adenosine triphosphate (ATP) generation, and damage the nuclear morphology of HTFs. Exposure of HTFs to ZnO nanoparticles can also induce the shifted peak, elevate the expression of Atg5, Atg12 and Becn1, enhance the autophagosome formation, and promote the LC3 expression, and thus activate autophagy signaling. Overall, ZnO nanoparticles can apparently trigger oxidative stress and activate autophagy signaling in HTFs, and thus inhibit HTFs proliferation and mediate HTFs apoptosis.

Longdan Xiegan Decoction alleviates experimental autoimmune uveitis in rats by inhibiting Notch signaling pathway activation and Th17 cell differentiation.

This study aimed to investigate the dynamic effects of the traditional Chinese medicine compound Longdan Xiegan Decoction (LXD) on the inhibition of Notch signaling pathway activation and T helper (Th) cell differentiation in rats with experimental autoimmune uveitis (EAU). Based on a network pharmacology strategy, we conducted protein interaction network analysis to construct an active ingredient-disease treatment network. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were further used to screen out the possible signaling pathways regulated by LXD in the treatment of uveitis. In the subsequent functional studies, we established an EAU rat model and investigated the regulatory role of LXD in the Notch signaling pathway and Th cell differentiation in rats with EAU. Female Lewis rats were randomly divided into a normal control (NC) group, an EAU group, and an LXD group. After the induction of EAU, the ocular inflammation and pathological changes in the rats in each group were observed; for documentation, a scanning laser ophthalmoscope (SLO) was used to observe fundus inflammation on day 12 after immunization. Additionally, quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of Notch1, DLL4, IL-10 and IL-17A in the spleen, lymph nodes and ocular tissues of each group at 0, 6, 9, 12, 15 and 18 days after immunization. In addition, the dynamic frequencies of the CD4+, CD8+, Th17 and Treg cell subsets in the spleen, lymph nodes and ocular tissues were measured by flow cytometry. We found that the Notch signaling pathway was activated and the Th17 frequency was elevated in rats with EAU, leading to disrupted CD4+/CD8+ and Th17/Treg balance. The expression of Notch1, DLL4 and IL-17 mRNA and proteins in the EAU and LXD groups reached a peak on day 12, and then gradually decreased (all P < 0.05), and the ratios of the CD4+/CD8+ and Th17/Treg also peaked on day 12. However, after treatment with LXD, the expression of Notch1, DLL4 and IL-17 mRNA and proteins was significantly decreased (all P < 0.05), and the CD4+/CD8+ and Th17/Treg ratios significantly gradually returns to balance. LXD can efficiently inhibit Th17 cell differentiation, decrease inflammatory cytokine expression, and restore the CD4+/CD8+ and Th17/Treg balance by inhibiting the activation of the Notch signaling pathway in rats with EAU, thus effectively alleviating eye inflammation, protecting eye tissue structures, and positively regulating the immune state of the whole body and the intraocular microenvironment.