Kaempferol reversals retinal ischemia/reperfusion (IR) injury through activating of PI3K/Akt/mTOR signaling pathway

Purpose Retinal ischemia/reperfusion (IR) injury is associated with many ocular diseases, including acute glaucoma, diabetic retinopathy, and retinal vascular occlusion. However, currently there are no effective medications to prevent the development ofretinal IR injury.Kaempferol is a kind of plant extract which has showed an excellent ability to inhibit the inammation.. Materials and Methods In this study, both in vitro and in vivo retinaloxidative damage models were established.Cell viability was assessed by Cell Counting Kit-8 assay. Apoptosis was examined using ow cytometry analysis.Atherosclerotic lesion analysis was performed using hematoxylin-eosin staining,The expressions of Inammatory cytokines were detected by quantitative real-time PCR and ELISA respectively.The effect of expression of apoptosis (cid:0) utophagy and the PI3K/Akt/mTOR signaling pathway related pathway was evaluated by Western blot. Results We found kaempferol was able to protect the viability of ARPE-19 cells against oxidative damage by reducing its apoptosis. In addition, it also kept structurally complete epithelium, stroma and endothelium of cornea after oxidative damage. Moreover, it also able to reduce the expression of inammatory cytokines and increased the expression of anti-inammatory cytokines.Kaempferol was able to enhanced the expression of anti-apoptotic genes BCL-2, and reduced the expression of autophagy gene Beclin 1 and increased the expression of anti-autophagy gene LC-3,was also able to enhance the expression PI3K and the phosphorylation ofAkt andmTOR. Conclusion Kaempferolreversals retinal ischemia/reperfusion (IR) injury through activating of PI3K/Akt/mTOR signaling pathway.


Introduction
Retinal ischemia / reperfusion (IR) injury involves many ophthalmic diseases, such as diabetic retinopathy, ocular ischemic syndrome, glaucoma and ischemic optic neuropathy, which can lead to irreversible neuronal cell damage. 1, 2Studies speculate that oxidative stress may be related to IR-induced retinal damage. 3, 4 Oxidative stress is de ned as an increase in the physiological value of intracellular reactive oxygen species (ROS) concentration. ROS includes molecules such assinglet oxygen, hydroxyl radical,hydrogen peroxide, peroxy radical, nitric oxide and superoxide anion. 5 When the endogenous activity of antioxidant enzymes (such as glutathione,catalase, superoxide dismutase, glutathione,metallothionein and SOD) and / or the concentration of certain vitamins changes, this will happen. 6 A large amount of evidence indicates that ROS is the initial cause of IR damage. 7 Thus, lots of studies focused on reducing the ROS. It is well known that the ROS was able to induce in ammatory responses. So, our study is trying to targeting the in ammation.
Lots of plant extracts have showed great potential on anti-in ammation. Kaempferol, 3,4′,5,7tetrahydroxy avone, is a natural avonol, a type of avonoid, found in a variety of plants and plantderived foods including kale, beans, tea, spinach and broccoli. 8 Kong et al. reported kaempferol showed an anti-in ammatory effect on early atherosclerosis in high cholesterol fed rabbits. 9 Similarly, Park et al. revealed the kaempferol also had anti-In ammatory effect inaged kidney tissues by inhibiting thenuclear factor-κB via nuclear factor-inducing kinase/IκBkinase and mitogen-activated protein kinase pathways.
10 However, the kaempferol doesn't use for the treatment of oxidative stress injury of the retina. Our hypothesis that the kaempferol could used for the treatment of oxidative stress injury of the retina by reducing in ammatory responses. In this study, both in vitro and in vivo oxidative stress injury model was established. The protective effects of kaempferol were explored in this study, and we also explored the potential mechanism.

H&E staining
After the hearts were harvested, samples were washed with PBS and xed in 4% PFA. Tissues were dehydrated in a series of graded ethanol solutions (70-100%) and embedded in para n. Five-micrometer sections were prepared and stained with either Hematoxylin and Eosin (H&E) staining according to the standard procedures.

ELISA assay
The tissues were washed with PBS twice and tissue homogenate was made according to the instructions of manufacturer at 4 O C, then centrifuged for 10 min at 13,000 rpm at 4°C.The supernatant was used for ELISA detection. The secretion level of VEGF, IL-10 and IFN-γ were detected using a commercially available enzyme-linked immunosorbent assay (ELISA) system (Lichen, Shanghai, China) following kit instructions.

RT-PCR
Total RNAs were isolated from cell lysis solution by using a commercial RNA extraction kit, and the RNA concentration was measured by nanodrop. Fifteen microliters of RNA (50ng/ul) was used as a template to prepare for the cDNA. The relative expression of IL-1, TNF-α, TLR-4, NK-kB was quanti ed using SYBR green reagents on the ABI 7500 sequence detection system. PCR was performed with the following thermocycling conditions: An initial 5 min at 95˚C, followed by 40 cycles of 95˚C for 30 sec, 55˚C for 30 sec and 72˚C for 30 sec. The primers were purchased from Funengbio Co. (Shanghai, China).
Housekeeping gene β-actin was used as an internal reference to normalize the results. For the analysis of the results, the 2 -ΔΔCt method was performed to calculate the relative expression.

Western blotting
TheARPE-19 cells were lysed directly for 5 minutes in the Lysis buffer. Lysates were separated by centrifugation (13,000×g, 30 min, 4 °C) and 50 µg of total proteins was electrophoresed on a 10% SDS-PAGE, which was transferred onto polyvinylidene di uoride (PVDF) membranes in a transfer tank using transfer buffer (195 mM glycine, 25 mM Tris-HCl and 20% (v/v) methanol). The rst stained membrane was con rmed the transfer e ciency with Ponceau S. Then the PVDF membranes were blocked for 1 h at RT with 3% (w/v) bovine serum albumin (BSA) in Tris-buffered saline (50 mM Tris, pH 8.0, 150 mM NaCl) with 0.05% Tween 20 (TBS-T). Membranes were incubated by primary antibody diluted 1:1000, then followed by secondary antibody conjugated with horseradish peroxidase at 1:2000 dilutions. Positive band intensities were shown by utilizing a gel documentation system (LAS-3000 Fuji lm).

Statistical analysis
In this study, all results were exhibited as the mean ± S.D. And the statistical analysis was performed using GraphPad Prism 8.0 software. Differences between two groups were evaluated using student T-test, and differences among the groups were assessed using one-way ANOVA. A p-value <0.05 was considered statistically signi cant.

Kaempferol protects the viability of ARPE-19 cells against oxidative injury
The chemical formula of kaempferol was shown in Figure 1A. The viability of ARPE-19 cells was signi cantly reduced after treated with H 2 O 2 . However, the viability of H 2 O 2 pretreated ARPE-19 cells gradually increased with the treatment of increased kaempferol from 1mM to 10mM ( Figure 1B). Suggesting the kaempferol has protective role on ARPE-19 cells against oxidative injury.

Kaempferol reduces the apoptosis of ARPE-19 cells against oxidative injury
Following, the apoptosis behaviors of ARPE-19 cells with different treatment were explored. As shown in Figure 2, the apoptotic rate of ARPE-19 cells treated with H2O2 was signi cantly increased to 13.73% compared to the control 2.65%. The apoptotic rate of ARPE-19 cells was reduced to 8.17% with the treatment of 1mM kaempferol. And it further decreased to 6.22% and 4.59% with the treatment of 5 mM and 10 mM kaempferol respectively. This result revealed that kaempferol couldreduces the apoptosis of ARPE-19 cells after oxidative injury.
Kaempferol keeps structural and organizational integrity of cornea after oxidative injury.
As shown in Figure 3, structurally complete epithelium, stroma and endothelium of cornea was exhibitedin the control group. However, the epithelium was disappeared and the structure of stroma became loose after the cornea treated with H 2 O 2 . Fortunately, the epithelium came back after treated with 50 mg/kg kaempferol. The treatment of 12.5 mg/kg and 25 mg/kg kaempferol failed to regenerated the damaged cornea.
The anti-in ammatory effects of kaempferol Then we explored the in ammatory responses after kaempferol treatment. As shown in Figure 4A-4C, the secretion of VEGF and IFN-γ of cornea treated with H 2 O 2 was signi cantly higher than the control group.
And it gradually decreased with the treatment of kaempferol, and the higher concentration of kaempferol showed lower secretion of VEGF and IFN-γ. On the contrary, the secretion of IL-10 of cornea treated with H 2 O 2 was signi cantly reducedcompared to the control group. And it gradually increased with the treatment of kaempferol, and the higher concentration of kaempferol showed higher secretion of IL-10. Moreover, the RT-PCR results showed that the relative expression of in ammatory cytokines, including IL-1α, IL-6 and TNF-α were signi cantly reduced in kaempferol treated groups. The pro-in ammatory effects of kaempferol was in a does depend manner. While the relative expression of IL-10 was gradually increased with the increased concentration of kaempferol ( Figure 4D).
The potential mechanism of the protective effects of kaempferol Finally, we also explored the potential mechanism of the protective effects of kaempferol on cornea against oxidative injury.As shown in Figure 5, rstly, we examined the expression of apoptosis and autophagy related gene expression. We found kaempferol was able to enhanced the expression of antiapoptotic genes BCL-2, and reduced the expression of autophagy gene Beclin 1 and increased the expression of anti-autophagy gene LC-3. In addition, we also found kaempferol was also able to enhance the expression PI3K and the phosphorylation ofAkt andmTOR. Suggesting the protective effects of kaempferol against oxidative injury via activating the PI3K/Akt/mTOR signaling pathway.

Discussion
The PI3K/AKT/mTOR pathway is an intracellular signaling pathway important in regulating the cell cycle.
Therefore, it is directly related to cellular quiescence, proliferation, cancer, and longevity. 11 PI3K activation phosphorylates and activates AKT, localizing it in the plasma membrane. 12AKT can have a number of downstream effects such as activating CREB, 13 inhibiting p27, localizing FOXO in the cytoplasm, 14 activating PtdIns-3ps, and activating Mtorwhich can affect transcription of p70 or 4EBP1. 14, 15In many cancers, this pathway is overactive, thus reducing apoptosis and allowing proliferation. This pathway is necessary, however, to promote growth and proliferation over differentiation of adult stem cells, neural stem cells speci cally. 13 It is the di culty in nding an appropriate amount of proliferation versus differentiation that researchers are trying to determine in order to utilize this balance in the development of various therapies. 13Additionally, this pathway has been found to be a necessary component in neural long-term potentiation. 16 In this study, we demonstrated that kaempferol protected cornea against oxidative injury via activating the PI3K/Akt/mTOR signaling pathway.
In our study, we rst demonstrated that the kaempferol was able to reverse the viability of ARPE-19 cells after oxidative damage by reducing cell apoptosis. Kimura et al. reported glucagon-like peptide-1 (GLP-1) protects against methylglyoxal-induced PC12 cell apoptosis through enhancing the PI3K/Akt/mTOR signaling pathway, 17 which was consistent with our study, the PI3K/Akt/mTOR signaling pathway could prevent cells from apoptosis. Moreover, we also found the kaempferol was capable of reducing the expression of in ammatory cytokines and increasing the expression of anti-in ammatory cytokines. Wu et al. revealed that gambogic acid suppresses in ammation in rheumatoid arthritis rats via up-regulating the PI3K/Akt/mTOR signaling pathway. 18 Our results were consistent with this study, the up-regulating the PI3K/Akt/mTOR signaling pathway could decreased the in ammation. According to the abovementioned information, our study provides a promising strategy to protect the eyes against from oxidative injury.

Conclusion
Kaempferolreversals retinal ischemia/reperfusion (IR) injury through activating of PI3K/Akt/mTOR signaling pathway. Availability of data and materials The data and materials used in the current study are all available from the corresponding author upon reasonable request.
Ethics approval and consent to participate Not applicable.

Consent for publication
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