In this study, we demonstrated that treatment with PLGA-icariin preserved the visual function and RGC density by inhibiting RGC apoptosis after ON infarct. In addition, the ON edema and the macrophage infiltration were attenuated by icariin treatment. Moreover, we found that icariin bound the transcriptional factor, CEBP-β, to induce endogenous G-CSF production in the retinal cells. The endogenous G-CSF expression promoted noncanonical NF-κB activation to further activate PI3K/AKT1 survival pathway and M2 macrophage polarization.
The visual function was protected by treatment with PLGA-icariin in the rAION model. This protective effect on visual function indicated that treatment with PLGA-icariin suppressed RGC apoptosis after the ischemic insult, which was supported by the evidence of RGC density and TUNEL assay. Baojun Liu et al., reported that icariin treatment reduced the corticosterone-induced apoptosis in rat hippocampal neurons by p38 MAPK inhibition . One recent study demonstrated that the NF-κB mediated apoptosis in fetal rat hippocampal neurons was attenuated by treatment with icariin . These evidence supported our hypothesis that intravitreal injection of icariin provides neuroprotective effects in the rAION model. Herein, we provided the first evidence that intravitreal injection of icariin inhibited RGC apoptosis and preserved the visual function. However, the protective mechanisms of icariin need further investigation.
The infarct at the ONH causes a breakdown of the BONB, which facilitates the increase in vascular permeability and causes infiltration of the macrophages at the ONH. This further reduces the oncotic pressure gradient, and the hydrostatic pressure in the capillaries of the ON forces out more water, increasing fluid production in the tissue. Thus, the increase in tissue fluid in the ON results in edema at the ON head[2, 3]. In addition, maximum ON edema was observed on day 1 after infarct, but the resolution of ON edema occurred 1 week after ON infract. This implies that ON edema is primarily related to damage to the RGCs in the acute stage of ON ischemia. Contrarily, after treatment with PLGA-icariin, ON edema was found to be decreased on day 1. Therefore, the early relief of swelling pressure on the axons at the ONH may also have contributed to the RGC survival.
After ON infarct, the infiltration of blood-borne macrophages is the major event of inflammation in the ONH . BONB stabilization reduces the infiltration of macrophages and the degree of inflammation, hence reduce RGC death in rAION . The increased ED1-positive macrophages in the ON of the PBS-treated group caused increased local inflammation, whereas fewer macrophages observed at the ONH in the PLGA-icariin-treated group suggest the stabilization of the BONB and post-treatment reduction in macrophage infiltration. This could have also contributed to the resolution of neuroinflammation in the acute stage. Taken together, we suggested that icariin can reduce the breakdown of BONB to attenuate ON edema and macrophage infiltration after ON infarct.
Driven by a binding simulation, we found that icariin has a good binding affinity with CEBP-β. CEBP-β is a known regulator of the G-CSF promoter and is generally present in the system for G-CSF production [23, 24]. The binding of icariin to the C-terminal of CEBP-β, which is known to regulate DNA binding in CEBP-β [25, 26], could have increased its DNA-binding ability and hence promote the increased production of G-CSF. Moreover, CEBP-β regulated transcription coactivator 2 and 3 (CTRC2/3) inhibit the G-CSF production, whereas their depletion is followed by increased STAT3 and G-CSF production via activating the CEBP-β [27, 28]. Our findings demonstrated that intravitreal injection of PLGA-icariin highly induced G-CSF expression in the ganglion cell layer and the retinal pigmented epithelium layer. In addition, our in vitro experiment proved the dose-response relationship between icariin and G-CSF in the human RPE cell line (Figure S2). We suggested that Icariin could block the binding of CTRC2/3 to enhance G-CSF production in the retinal cells. G-CSF is a potent neuroprotective agent through anti-inflammation and anti-apoptosis in the experimental model of optic nerve ischemia. Thus, we considered that icariin may trigger endogenous G-CSF expression to modulate the neuroinflammation after ON infarct.
NAION is an inflammatory disease similar to many other neurodegenerative diseases [29–31]. Hence, we expected increased levels of NF-kB (p65) in PBS treated group but not in PLGA-icariin treated group. but to our surprise, the levels of NF-κB remained high after treatment with PLGA-icariin. To gain resolution on this situation, we checked the protein levels of FAS ligand in all the groups. FAS ligand is known to upregulate under the canonical NF-κB(p65) condition but downregulates while in the noncanonical progression of NF-κB(p52) . FAS ligand was found to be upregulated in the PBS-treated group while downregulated after PLGA-icariin treatment. This suggested the possibility that although NF-κB (p65) may have been upregulated in the PLGA-icariin-treated group, it may not have translocated to the nucleus for the transcription of its downstream products. These doubts were confirmed by the downregulation of the protein levels of p65 in the nucleus in reference to its levels in the cytoplasm. On accessing the p52 levels in the retina, increased translocation of p52 to the nucleus with respect to the cytoplasm was observed in the PLGA-icariin-treated group compared with the PBS-treated group, which corresponds to noncanonical NF-κB progression in the PLGA-icariin-treated group. The upregulation of the noncanonical NF-κB pathway was further confirmed by the increase in the IKK-α levels observed after PLGA-icariin treatment compared with the PBS-treated group . Hence, we considered that PLGA-icariin treatment could promote a switch from the canonical to noncanonical NF-κB pathway. A similar trend was observed after PEG-G-CSF treatment, in which an increase in IKK-α and p52 levels was noted compared to the PBS treated group. This demonstrates that PLGA-icariin treatment could promote noncanonical NF-κB progression via the G-CSF-mediated pathway. The noncanonical NF-κB pathway is antiapoptotic in nature and has been reported to regulate inflammation . It also provides anti-inflammatory benefits, whereas its deregulation could promote inflammation . Although the benefits of noncanonical NF-κB are overwhelming, little is known about the mechanism that regulates its switch .
With the aim of investigating the interactions that favor the switch to the noncanonical NF-κB pathway, we observed that the upregulated p65 was unable to translocate to the nucleus after PLGA-icariin treatment, which could be explained by the inability of IKK-β to phosphorylate p65 . We also observed that PTEN is phosphorylated rather than being downregulated. The PI3K/AKT pathway is favored by downregulated PTEN, which otherwise gets phosphorylated by scavenging the phosphates from PI3K and inhibits the phosphorylation of PIP2, hence inhibiting the PI3K/AKT pathway . Therefore, PTEN phosphorylation and PI3K/AKT progression are generally mutually exclusive, indicating that another kinase must have acted on the PTEN instead. A previous study demonstrated the ability of the NF-κB pathway to modulate the PTEN activity via the observation of decreased PTEN levels in IKK-β(+,+) cells but not in the IKK-β(-,-) cells . There have also been reports suggesting that the NF-κB pathway has an inhibitory effect on the PTEN in the form of a positive feedback loop [39, 40]. Based on the observations and arguments stated above, we suspected the possibility of IKK-β’s alternative role in the phosphorylation of PTEN. On assessing the situation via kinase assay, we confirmed that IKK-β has the potential for PTEN phosphorylation. In addition, in-silico evidence also indicated the ability of human IKK-β to phosphorylate the human PTEN sequence. This explains the unavailability of IKK-β for p65 phosphorylation as well as the presence of the upregulated p-PTEN in the presence of the upregulated PI3K/AKT pathway. This finding suggests that the phosphorylation target of IKK-β plays a decisive role in the progression of the NF-κB pathway. The phosphorylation of p65 by IKK-β leads to the canonical whereas the phosphorylation of PTEN by IKK-β to the noncanonical progression of NF-κB. There is also a possibility that the phosphorylation of PTEN is not the sole alternative target for IKK-β but is rather an important target, as the noncanonical NF-κB pathway also requires the upregulated PI3K/AKT pathway for its functioning . NF-κB (p65) progression via the canonical pathway increases inflammation in rAION and promotes neurodegeneration. Conversely, its switch to the noncanonical pathway after PLGA-icariin treatment, owing to its anti-inflammatory nature, is desired over the canonical progression . The mechanism of this switch via the exhaustion of the availability of IKK-β for phosphorylation of p65 could provide a new approach for targeting inflammation.
RGC survival along with G-CSF upregulation suggested that G-CSF influenced the underlying mechanism. The PI3K/AKT survival pathway mediated by the upregulated G-CSF elucidated the rescue of the RGCs along with upregulated PTEN phosphorylation in the retina. Here we report that the upregulation of the PI3K /AKT survival pathway via AKT 1 upregulation is sufficient to bring about considerable RGC survival, whereas AKT2 has no role in this process, which is consistent with the findings of a previous study .
Macrophages assume either M1 or M2 polarization and previous studies have proved that IKK-α activation inhibits M1 phenotype of the macrophages whereas inactivation of IKK-α is known to increase inflammation in mice, which suggested the noncanonical NF-κB signaling pathway could regulate the macrophage polarization. When analyzed by the C206 marker, increased M2 polarized macrophages were recorded at the ON head after treatment with PLGA-icariin. While increased M2 macrophage polarization was found to be promoted via the upregulation of phosphorylated STAT3(p-STAT3) [44, 45]. p-STAT3 upregulation is downstream of JAK activation, which is known to be activated upon G-CSF–G-CSF receptor activation . Owing to their high phagocytic ability and ability to produce type 1 inflammatory cytokines, such as IL-1β and tumor necrosis factor–α, M1-type macrophages are inflammatory in nature [47, 48]. As M2 polarization is anti-inflammatory in nature, its benefits were analyzed at the site of primary inflammation, the ON head . Increased IL-1β after rAION represents an upregulated inflammatory response, whereas decreased IL 1-β expression after treatment with PLGA-icariin indicates its anti-inflammatory effects [10, 49]. We also observed the M2 macrophage/microglia polarization in the retina after PLGA-icariin treatment. As the canonical NF-κB progression drives M1 macrophage polarization ; based on our observation, the noncanonical NF-κB progression regulates M2 macrophage/microglia polarization and contributes to the anti-inflammatory phenomenon exerted by M2 polarized macrophages/microglia. The degree to which the progression of NF-κB regulates macrophage polarization requires further study.