Effect of Combined Treatment With Fallopia Japonica and Prunella Vulgaris Extract on Myopia Progress

The increased global incidence of myopia requires the establishment of therapeutic approaches. Previous studies have suggested that inammation plays an important role in the development and progression of myopia. This study aimed to investigate the effect of Fallopia Japonica (FJ) and Prunella Vulgaris (PV) extract on myopia caused by monocular form deprivation (MFD) and elucidate the underlying mechanisms. hamster model myopia MFD with right eyelid fusion for 21 days . The were sutured with 6-0 PROLENE nonabsorbable sutures, blue monolament (W8706, ETHICON, on day 21 after birth. control hamsters were randomly each group different topical The present work was undertaken to evaluate in vitro anti-inammatory activity of FJE, PVE, and FJE + PVE. FJE showed strong anti-inammatory activity at a concentration of 30 ng/ml. In addition, topical application of FJE + PVE treatment inhibited inammation to a greater extent when compared to the group treated with either FJE or PVE alone. AKT and NF-κB signaling pathway have an important role in the inammation. It has been reported that a high level of cytokines increases the expression of AKT and NF-κB in RPE 23, 24 . Consistent with these reports, results from the present study indicated that cytokine (IL-6 + TNF-α) treatment increased the phosphorylation AKT and NF-κB in ARPE-19 cells. FJE and PVE suppressed IL-6 + TNF-α-induced AKT and NF-κB expression. Therefore, FJE and PVE treatment improved inammation induced by these cytokines via suppressing AKT and NF-κB signaling pathway.


Abstract Background
The increased global incidence of myopia requires the establishment of therapeutic approaches. Previous studies have suggested that in ammation plays an important role in the development and progression of myopia. This study aimed to investigate the effect of Fallopia Japonica (FJ) and Prunella Vulgaris (PV) extract on myopia caused by monocular form deprivation (MFD) and elucidate the underlying mechanisms.

Methods
We used human retinal pigment epithelial cell to study the molecular mechanisms on how FJE and PVE lowering the in ammation of the eye. The effect of FJE and PVE in MFD induced hamster model and explore the role of in ammation cytokines in myopia.

Conclusion
Overall, these results suggest that FJE + PVE may have a therapeutic effect on myopia and be used as a potential treatment option.

Background
Myopia is one of the most common refractive disorders in humans and its prevalence has been increasing over the past decades. By 2050, it is expected that approximately 4.8 billion people will be myopic 1,2 . Most of the patients with myopia have the excessive elongation of the vitreous chamber, which largely accounts for the increase in eye elongation, loss of scleral tissue, and degenerative changes, such as atrophy of the retina and choroid 3 . Myopia is also a major risk factor for severe sight-threatening pathologies including cataract, glaucoma, choroidal neovascularization, and macular and retinal complications 2,4 .
Although the underlying molecular mechanisms of myopia progression are not fully understood, accumulated evidence has demonstrated that in ammation plays an important role in the pathogenesis of myopia [4][5][6][7] . Several studies indicated a role for in ammation in myopia progression and increased prevalence of myopia in children with an in ammatory disease, such as type 1 and 2 diabetes, systemic lupus erythematosus, uveitis, allergic diseases, and Kawasaki disease [4][5][6] . Our recent animal model showed that in ammatory markers, such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, transforming growth factor-beta (TGF)-β, and nuclear factor kappa-light-chin-enhancer of activated B (NF-κB) are upregulated in myopic eyes. Increasing myopia prevalence and subsequent consequences pose a major public health concern. Although spectacle correction can improve vision, myopia is associated with an increased risk of retinal detachment, myopic macular degeneration, cataract, and glaucoma. There are currently no pharmaceutical agents approved by the FDA in the US for use in myopia treatment, although researchers and clinicians are searching for better therapies.
Plants provide an abundant source of primary compounds for a variety of diseases. Fallopia Japonica (FJ) and Prunella vulgaris (PV) are used in traditional Chinese medicine for the treatment of various in ammatory diseases and are widely distributed in China, Kapan, Korea, and Europe 8-11 . FJ and PV belong to the group of polyphenols. Main constituents of FJ include resveratrol, polydatin, emodin, physcion, chrysophanol, and rhein 9, 12 . Resveratrol is a naturally occurring anti-in ammatory compound typically associated with red wine and is also present in FJ 12 . PV is a perennial herb also known as the self-heal herb and is a standard medicinal material in the Chinese Pharmacopoeia. Ursolic acid, a pentacyclic triterpene acid, is found in PV, which exhibits many bioactivities, including anti-in ammation, anti-hyperglycemia, and anti-tumor effects 13,14 .
We hypothesize that FP and PV extracts can improve myopia progression via decreasing in ammation.
This study was designed to investigate the effect of FJ and PV in MFD-induced hamster model and explore the role of in ammatory cytokines in myopia. We demonstrated that FJ and PV extracts may attenuate myopia progression via inhibiting in ammation. Our study provides insight into myopia and supports the potential therapeutic value of FJ and PV in myopia treatment. Western blot analysis Cells were lysed in RIPA lysis buffer (10 mM Tris-Cl, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na 4 P 2 O 7 , 2 mM Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, and 0.5% separated into four groups (n = 10 animals each), each group receiving a different topical treatment. The three groups were: (1) control (hamsters received phosphate-buffered saline (PBS)); (2) 150 ng/ml FJE (3) 150 ng/ml PVE (4) 150 ng/ml FJE +PVE. Hamsters were raised with a right eyelid fusion for 21 days. Eye drops (10 μl) were applied topically to both eyes of the hamsters twice a day (8 AM, 5 PM) until they were euthanized. All animals were sacri ced in a CO 2 chamber. Before the animals were anesthetized by CO 2 gas and sacri ced, the axial lengths of the hamsters were measured. The axial length of the eye was de ned as the distance from the front of the cornea to the back of the sclera. The axial lengths of each left and right eye were measured by A-scan ultrasonography (PacScan Plus, New Hyde Park NY, USA), and the axial lengths of three independent measurements were averaged.

Software and statistical analysis
Each result was expressed as mean ± standard deviation (SD). Unpaired independent t-test and one-way ANOVA analysis of variance were performed to compare the differences between the two groups using the GraphPad prism software. A P-value <0.05 was considered to be signi cant.
Pro-in ammatory cytokines induce the expression of IL-6, IL-8, and TNF-α in ARPE-19 cells Immuno uorescence results showed increased TNF-α expression levels in RPE 4, 15 of the MFD eye, and that the FJE + PVE treatment resulted in the attenuation of TNF-α levels in the myopic (right) eyes compared to the control group (Right eye MFD) (Supplementary Fig. S1). Therefore, we used human retinal pigment epithelial cell to study the molecular mechanisms on how FJE and PVE lowered the in ammation of the eye. To explore treatment on ARPE-19 cells response of pro-in ammatory cytokines was investigated. ARPE-19 cells were treated with 5 ng/ml of different cytokines, as well as combinations of cytokines (IL-6, TNF-α, and IL-6 + TNF-α), for 16 hours. Cell-free supernatants were collected and stored at -80°C until further use. ELISA analysis treated cells demonstrated the IL-6 + TNF-α administration signi cantly increased the levels of IL-6, IL-8, and TNF-α ( Fig. 2A-C). IL-6 administration exhibited a lower effect on in ammatory cytokine production. However, when IL-6 and TNF-α were combined, it showed the highest level of in ammatory cytokine production. A synergistic effect was found between IL-6 and TNFα.

FJE and FJE + PVE inhibit the in ammatory response in stressed ARPE-19 and human RPE cells
The anti-in ammatory effect of FJE was assessed by measuring the production of IL-6 and IL-8. ARPE-19 cells were treated with 5 ng/mL of IL-6 + TNF-α cytokines for 2 hours. Treatment media were subsequently removed and fresh media with or without FJE (10, 20, and 30 ng/ml) were applied and incubated for 6 hours. When ARPE-19 cells were stimulated with IL-6 + TNF-α, IL-6 and IL-8 production was induced massively. However, cells treated with FJE showed less production of IL-6 and IL-8 compared with those treated with IL-6 + TNF-α, and this inhibitory effect exhibited a dose-dependent manner (Fig.   3A-B). To examine whether the combination of FJE and PVE have additive effects in suppressing IL-6, IL-8, and TNF-α production in IL-6 + TNF-α-stimulated ARPE-19 cells, we applied different concentrations (10,20, and 30 ng/ml) of FJE + PVE. The levels of IL-6, IL-8, and TNF-α were further reduced when combined treatment of FJE and PVE was applied (Fig. 3C-E). Since IL-6, IL-8, and TNF-α levels were barely detectable when FJE and PVE were combined were at a concentration of over 30 ng/ml, we used a combination of FJE + PVE treatments up to 30 ng/ml in the subsequent experiment. A similar decrease of IL-6, IL-8, and TNF-α levels was also observed in H-RPE cells treated with FJE + PVE (Fig. 4A-C).
FJE and PVE regulate the phosphorylation of AKT and NF-κB in ARPE-19 cells Next, we determined the pathway through which FJE and PVE interfered with the production of proin ammatory cytokines by measuring the effects of these compounds on the activity of AKT and NF-κB in IL-6 + TNF-α-treated ARPE-19 cells. ARPE-19 cells were treated with IL-6 + TNF-α, for 2 hours. Treatment media were subsequently removed and fresh media with or without FJE and PVE (30 ng/ml) were applied and incubated for 6 hours. Results showed that after 6 hours of FJE and PVE treatment, phosphorylation levels of AKT and NF-κB decreased markedly compared to cells treated with cytokines (Fig. 5). This result indicated that FJE and PVE inhibited in ammation through downregulation of the AKT and NF-κB pathways.

FJE + PVE inhibited myopia progression through modulating the in ammatory response in the eyes
To understand the molecular factors of FJE and PVE on myopia progression, in ammatory proteins were examined in the hamster's eye. As shown in Fig. 6 B, IL-6, IL-8, and TNF-α expressions increased after treatment right eye MFD for 21 days, whereas FJE, PVE, and FJE + PVE treatment reduced their expression. But, the PVE treatment resulted in a no signi cant (P < 0.05) attenuation of IL-8 levels in myopic eyes compared to the MFD group. An elongation of the eye axial length was accompanied with scleral remodeling, such as suppression of collagen production, and TGF-β and MMP-2 activity. Thus, we assessed TGF-β, MMP-2, and collagen expression using IHC (Fig. 6 C). Our results showed that the FJE, PVE, and FJE + PVE treatment resulted in a signi cant (P < 0.05) decrease in TGF-β and MMP-2 expression and an increase in type collagen expression compared to the MFD group. To further con rm the involvement of in ammatory signaling pathways in myopia progression, the expression of NF-κB was determined using IHC. Expression levels of NF-κB in retinas were lower in FJE-, PVE-, and FJE + PVEtreated eye compared to the MFD groups (Fig. 6 D). Taken together, it was suggested that myopic stimuli induced IL-6, IL-8, TNF-α, TGF-β, MMP-2, and NF-κB overexpression, and decreased collagen expression, and that FJE and PVE reversed these effects.
Treatment media were subsequently removed and fresh media with or without resveratrol, ursolic acid, and resveratrol + ursolic acid (1000 ng/ml) were applied and incubated for 6 hours. ELISA results indicated that expression levels of IL-6, IL-8, and TNF-α were signi cantly increased in IL-6 + TNF-αstimulated ARPE-19 and H-RPE cells compared with those in untreated cells. Treatment with resveratrol + ursolic acid combination signi cantly reduced expression levels of IL-6, IL-8, and TNF-α compared to resveratrol and ursolic acid alone. (Fig. 7D-I). These results suggested that resveratrol + ursolic acid could attenuate the production of retinal in ammatory disease-related mediators' expression levels.

Discussion
Here, we aimed to investigate the effect of FJ and PV in MFD-induced hamster model and explore the role of in ammatory cytokines in myopia and revealed that FJ and PV extracts attenuated myopia progression via inhibiting in ammation.
In 2020, 3366 million people (42.6% of the population worldwide) had myopia. Patients with myopia carry higher risks of cataract, glaucoma, choroidal neovascularization, and macular and retinal complications, which may result in an irreversible vision loss 16 . Emerging evidence indicated that in ammation has a key role in the pathogenesis of several ocular diseases, including uveitis, age-related macular (AMD), dry eye, and myopia 4,17 . In ammation is part of the body's defense mechanism against infection or injury, which results in cell activation and the release of various mediators responsible for the in ammatory response 18 . In myopia animal model, expressions of IL-6, IL-8, and TNF-α increase when compared with control eyes 4 . IL-6, IL-8, and TNF-α are pro-in ammatory factors involved in the communication between cells and their overexpression has been implicated in the pathogenesis of various in ammatory and ocular diseases 19,20 . In the present study, we demonstrated that FJE + PVE suppressed myopia in a hamster MFD model. This was concomitant with the inhibition of MFD-induced increase in IL-6, IL-8, and TNF-α expression, TGF-β, MMP-2, and NF-κB activation, and a decrease in collagen in the NFD hamster model.
The use of Chinese herb extracts to treat ophthalmic conditions dates back for hundreds of years. FP and PV are traditional Chinese medicine for the treatment of various in ammatory diseases, like hepatitis and tumors, and are o cially listed in the Chinese Pharmacopoeia 21,22 . In addition, these herbs have antiin ammatory, anti-oxidant, anti-allergic, anti-bacterial, and anti-viral effects 8, 10, 21 .
The present work was undertaken to evaluate in vitro anti-in ammatory activity of FJE, PVE, and FJE + PVE. FJE showed strong anti-in ammatory activity at a concentration of 30 ng/ml. In addition, topical application of FJE + PVE treatment inhibited in ammation to a greater extent when compared to the group treated with either FJE or PVE alone. AKT and NF-κB signaling pathway have an important role in the in ammation. It has been reported that a high level of cytokines increases the expression of AKT and NF-κB in RPE 23,24 . Consistent with these reports, results from the present study indicated that cytokine (IL-6 + TNF-α) treatment increased the phosphorylation AKT and NF-κB in ARPE-19 cells. FJE and PVE suppressed IL-6 + TNF-α-induced AKT and NF-κB expression. Therefore, FJE and PVE treatment improved in ammation induced by these cytokines via suppressing AKT and NF-κB signaling pathway.
It has been indicated that compounds with various biological activities are commonly produced by plants. Resveratrol and ursolic acid are the secondary metabolites in FJ and PV and have various pharmacological effects on different diseases 9,11 . Previous studies reported the anti-in ammatory activity of resveratrol and ursolic acid species 25,26 . The present study demonstrated that resveratrol + ursolic acid showed the highest anti-in ammatory activity and no cytotoxicity. Collectively, the current data suggested that combined treatment of FJE + PVE and resveratrol + ursolic acid are more effective inhibitors of in ammation than FJE, PVE, resveratrol, or ursolic acid alone. The mechanism for this greater inhibition appeared to be multi-faceted.
Subsequently, we examined the therapeutic effect of FJE, PVE, and FJE + PVE against myopia progression in vivo. Utilizing MFD-induced hamster myopia, we observed that axial elongation changes were suppressed upon FJE, PVE, and FJE + PVE treatments. In our animal models, we demonstrated that FJE + PVE suppressed myopia. This was concomitant with the inhibition of MFD-induced increase in IL-6, IL-8, and TNF-α expressions, TGF-β, MMP-2, and NF-κB activation, and a decrease in collagen expression in the hamster MFD model. Previous studies have shown that expressions of IL-6, IL-8, and TNF-α were higher in uveitis 27 . TNF-α induces the expression of IL-6, and IL-8 expression can be upregulated by TNFα and IL-6 28, 29 . In our study, we found that IL-6, IL-8, and TNF-α expressions were higher in the myopia eye. In contrast, IL-6, IL-8, and TNF-α expression decreased in the FJE + PVE-treated group. The development of myopia occurs mainly because of excessive axial length rather than changes in cornea or lens power. In animal models of myopia, there is a loss of extracellular matrix (ECM), which may cause axial elongation. Induction of myopia leads to increased TGF-β expression and continues to activate MMP-2 expression. MMP-2 is an enzyme that cleaves collagen and capable of triggering the decomposition of scleral ECM components. Furthermore, in hamsters with myopia, an increased expression of MMP-2 was induced by TGF-β through NF-κB activation. In this study, we demonstrated that while MFD enhanced TGF-β and MMP-2 activity, FJE + PVE reversed this effect.

Conclusions
In conclusion, the current study shows the e cacy of the combination of FJE + PVE in the inhibition of myopia progression in hamster eyes for the rst time (Fig. 8). This combination resulted in greater inhibition of myopia progression compared to FJE or PVE treatment alone. In addition, resveratrol and ursolic acid are the secondary metabolites in FJ and PV and they also have inhibitory effects on in ammation. Emerging evidence suggests that combinations of phytochemicals may have more effective anti-in ammatory effects than single agents 26 . Hence, FJE + PVE are considered bene cial to prevent myopia development in humans.

Competing interests
The authors have no con icts of interest to declare.   FJE + PVE protects against cytokine-induced in ammation in ARPE-19 cells. ARPE-19 cells were pretreated with 5 ng/ml of IL-6 + TNF-α for 2 hours and then FJE was added (10, 20, and 30 ng/ml) and cells were incubated for 6 hours. Expressions of IL-6 (A) and IL-8 (B) in ARPE-19 cells were measured using the ELISA Ready-Set-Go kit. ARPE-19 cells were pretreated with 5 ng/ml of IL-6 + TNF-α for 2 hours and then FJE + PVE was added (10, 20, and 30 ng/ml) and the cells were incubated for 6 hours.
Expression of IL-6 (C) and IL-8 (D) in ARPE-19 cells were measured using the ELISA Ready-Set-Go kit.
ARPE-19 cells were pretreated with 5 ng/ml of IL-6 + TNF-α for 2 hours and then 30 ng/ml of FJE, 30 ng/ml of PVE, and 30 ng/ml of FJE + PVE were added and the cells were incubated for 6 hours.
Expressions of TNF-α (E) in ARPE-19 cells were measured using the ELISA Ready-Set-Go kit. The data are expressed as the mean ± SD of three independent experiments. * p < 0.05 compared with the control.  MFD eyes (Right eye). Quanti cation of NF-κB of accumulation in the retinas of the right eye using Image J software. ANOVA was used for paired comparisons between control, atropine (1%), and diacerein (10 mM). Original IHC are presented in Supplementary Fig. S3. The IHC data are expressed as the mean ± SD of three independent experiments. * p < 0.05 compared to the control. ng) were applied for 72 hours. Cell viability was determined using MTS assay. (C) Different concentrations of resveratrol + ursolic acid (0, 100, 500, 1000, 2000, and 4000 ng/ml) were administered for 72 hours. Cell viability was determined using the MTS assay. (D, E, and F) ARPE-19 cells were pretreated with 5 ng/ml of IL-6 + TNF-α for 2 hours and then treated with resveratrol, ursolic acid, and resveratrol + ursolic acid (1000 ng/ml) and incubated for 6 hours. Expressions of IL-6, IL-8, and, TNF-α in ARPE-19 cells were measured using the ELISA Ready-Set-Go kit. (G, H, and I) H-RPE cells were pretreated with 5 ng/ml of IL-6 + TNF-α for 2 hours and then administered resveratrol, ursolic acid, and resveratrol + ursolic acid (1000 ng/ml) and incubated for 6 hours. Expressions of IL-6, IL-8, and TNF-α in H-RPE cells were measured using the ELISA Ready-Set-Go kit. The data are expressed as the mean ± SD of three independent experiments. * p < 0.05 compared with the control.

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