Abstract

   Retinitis pigmentosa (RP) is a group of visual disorders caused by
   mutations in over 70 genes. RP is characterized by initial degeneration
   of rod cells and late cone cell death, regardless of genetic
   abnormality. Rod cells are the main consumers of oxygen in the retina,
   and after the death of rod cells, the cone cells have to endure high
   levels of oxygen, which in turn leads to oxidative damage and cone
   degeneration. Gypenosides (Gyp) are major dammarane-type saponins of
   Gynostemma pentaphyllum that are known to reduce oxidative stress and
   inflammation. In this project we assessed the protective effect of Gyp
   against cone cell death in the rpgrip1 mutant zebrafish, which
   recapitulate the classical pathological features found in RP patients.
   Rpgrip1 mutant zebrafish were treated with Gyp (50 µg/g body weight)
   from two-months post fertilization (mpf) until 6 mpf. Gyp treatment
   resulted in a significant decrease in cone cell death compared to that
   of untreated mutant zebrafish. A markedly low level of reactive oxygen
   species and increased expression of antioxidant genes were detected in
   Gyp-incubated mutant zebrafish eyes compared to that of untreated
   mutant zebrafish. Similarly, the activities of catalase and superoxide
   dismutase and the level of glutathione were significantly increased in
   Gyp-treated mutant zebrafish eyes compared to that of untreated mutant
   zebrafish. Gyp treatment also decreased endoplasmic reticulum stress in
   rpgrip1 mutant eyes. Expression of proinflammatory cytokines was also
   significantly decreased in Gyp-treated mutant zebrafish eyes compared
   to that of untreated mutant zebrafish. Network pharmacology analysis
   demonstrated that the promotion of cone cell survival by Gyp is
   possibly mediated by multiple hub genes and associated signalling
   pathways. These data suggest treatment with Gyp will benefit RP
   patients.

   Keywords: retinitis pigmentosa, gypenosides, cone cell death, rpgrip1,
   zebrafish, network pharmacology

1. Introduction

   Retinitis pigmentosa (RP) is a group of inherited retinal disorders,
   affecting approximately 1 in 4000 adults worldwide [[38]1]. Most RP
   cases, including the autosomal dominant, autosomal recessive and
   X-linked forms, are non-syndromic with dysfunction only occurring in
   the retina. However, some RP cases, such as Usher syndrome and
   Bardet-Biedl syndrome, are syndromic with defects occurring in other
   tissues [[39]2]. Mutations in over 70 genes have been reported to cause
   RP (RetNet; [40]https://sph.uth.edu/retnet/, accessed on 20 April
   2021). The retinitis pigmentosa GTPase regulator (RPGR) gene is the
   most common X-linked RP-causing gene. The RPGR protein directly or
   indirectly interacts with other ciliary proteins to form a protein
   complex, which promotes ciliary protein trafficking in the
   photoreceptors [[41]3]. One of the RPGR interacting partners, the RPGR
   interacting protein 1 (RPGRIP1) gene is also known to mutate in
   patients with RP [[42]4], cone-rod dystrophy [[43]5] or Leber
   congenital amaurosis (LCA) [[44]6,[45]7]. Loss of RPGRIP1 protein
   causes defects in ciliogenesis in the human retinal pigment epithelial
   (RPE) cell line (RPE1) and both abnormal development of the rod outer
   segments and early retinal degeneration in mice and zebrafish
   [[46]8,[47]9,[48]10].

   RP is characterized by initial rod cell death and late cone cell
   degeneration. RP patients experience night blindness at an early stage;
   with tunnel vision developing as a result of rod cell death. Central
   vision loss develops at a later stage as a result of cone cell
   degeneration. The products of RP-associated genes demonstrate diverse
   functions, which involve the visual cycle, phototransduction,
   maintenance of photoreceptors and regulation of transcription. As a
   result, the death of rod cells is linked to distinct processes
   associated with light damage, endoplasmic reticulum stress, metabolic
   stress, ciliary protein trafficking defect and abnormal mRNA processing
   [[49]11]. Cone cells start to degenerate when almost all rod cells are
   dead. This degeneration is thought to result from cone cells being
   subjected to an increasing production of reactive oxygen species (ROS),
   due to the high levels of oxygen being delivered from choroidal
   capillary circulation. This results from the choroidal vessels being
   unable to autoregulate the blood supply when photoreceptor oxygen
   consumption is decreased due to the death of the rod cells [[50]2].
   High levels of oxidative damage have also been reported in the cone
   cells of the transgenic RP pig model following rod degeneration
   [[51]12]. These findings suggest that cone cell degeneration could be
   reduced by limiting oxidative damage. Several studies have also shown
   that administration of antioxidants preserve cone function and slow
   cone death in RP mouse models (Rd1, Rd10 and RHO^Q344ter transgenic
   line) [[52]13,[53]14,[54]15]. NRF2 is the master regulator that
   controls expression of antioxidant proteins and triggers the protective
   response against oxidative damage. Overexpression of NRF2 using an
   adeno-associated virus in the retina of three RP mouse models (Rd1,
   Rd10 and Rho^−/−) results in decreased oxidative stress, survival of
   cone cells and improved visual function [[55]16]. These data suggest
   antioxidant therapy may help preserve cone function in RP patients.

   Gypenosides (Gyp) are major functional saponins in Gynostemma
   pentaphyllum, which has been widely used as a traditional herbal
   medicine to treat different types of diseases in China and other Asian
   countries [[56]17]. Gyp have been shown to have anti-oxidative stress,
   anti-inflammation, anti-apoptotic, anti-diabetic, anti-hyperlipidemic,
   anti-atherosclerotic and anti-cancer properties in cell lines and in
   animal models [[57]17]. The protective or toxic effect presented by Gyp
   is dependent upon the type of disease model and is mediated by
   different signaling pathways [[58]17]. For example, Gyp demonstrates
   protective effects against oxidative stress and inflammation at low
   concentrations, predominantly through the NRF2 and NF-κB pathways
   [[59]18]. In contrast Gyp also has a toxic effect against cancer at
   high concentrations by inducing apoptosis and cell cycle arrest and
   through the inhibition of proliferation and DNA repair [[60]17]. Recent
   studies have shown Gyp inhibited H[2]O[2]-induced oxidative damage and
   inflammation in the retinal pigment epithelial (RPE) and the ganglion
   cells [[61]18,[62]19]. Gyp also reduces inflammation, demyelination and
   axonal loss in the optic nerve of the optic neuritis mouse model
   [[63]20]. In a previous study we utilized an rpgrip1 mutant zebrafish
   model, which showed early retinal degeneration with only a few rod
   cells remaining at one-month of age, followed by the death of cone
   cells [[64]9]. The rpgrip1 mutant zebrafish mirrors many of the
   clinical features of RP and the phenotypes of rodent RP models
   [[65]2,[66]9,[67]10,[68]13,[69]14,[70]15]. In a subsequent paper we
   found that Gyp inhibited oxidative damage and inflammation and
   attenuated rod cell death in rpgrip1 mutant zebrafish [[71]21],
   however, it remains uncertain as to whether Gyp has a protective effect
   upon cone cell death. In the current study, we examine whether Gyp
   protects against cone cell death in the rpgrip1 mutant zebrafish.

2. Materials and Methods

2.1. Zebrafish Treatment with Gyp

   All zebrafish work were approved by the UK Home Office (Project licence
   PPL. 70/8697 and ethical protocol code 03/GMA/XS). Zebrafish were
   maintained in a ZebTec stand-alone system ([72]www.tecniplas.it,
   accessed on 20 April 2021) at 28 °C. Gyp, containing several gypenoside
   components, was ordered from Xi’an Jiatian Biotech Co. Ltd., Xi’an,
   China (98% purity). For oral administration of Gyp, zebrafish were
   anesthetized by immersion in 0.016% tricaine solution. The anesthetized
   zebrafish were vertically propped in an E3 medium-soaked sponge. Gyp
   was dispensed into the zebrafish mouth using a Hamilton syringe with a
   22-gauge needle. The dose of Gyp given for each individual zebrafish
   was based on their body weight. To determine the appropriate dosage for
   treatment of zebrafish, wildtype and rpgrip1 mutant zebrafish at
   two-months post fertilization (mpf) were orally treated with Gyp
   dissolved in phosphate-buffered saline (PBS) at concentrations of 50,
   100, 150 or 200 µg/g body weight once/week for three weeks). The health
   and wellbeing (such as swimming behavior and food intake) of the fish
   were monitored daily. At the end of the initial toxicity test, an ideal
   dose was chosen for further treatment. Three groups of zebrafish at 2
   mpf were setup: wildtype zebrafish, rpgrip1 mutant zebrafish and
   rpgrip1 mutant zebrafish orally treated with Gyp (50 µg/g body weight,
   once/week for 16 weeks). Gyp were dissolved in PBS, so the wildtype
   zebrafish group and the rpgrip1 control group were orally treated with
   PBS. After the treatment, zebrafish were sacrificed and samples were
   collected for histology, immunohistochemistry and biochemical analyses.

2.2. Histology and Immunostaining

   Zebrafish eyes were fixed in 4% paraformaldehyde (PFA) solution for two
   hours at room temperature and then washed with PBS twice, followed by
   dehydration with gradient concentrations of ethanol. After dehydration,
   the samples were immersed in Histo-clear and then in molten paraffin
   wax. The paraffin embedded eyes were sectioned with a thickness of 7 µm
   using a microtome. The sections were dewaxed and hematoxylin and eosin
   stained. Images were captured using a light microscope. For
   quantification of photoreceptor layer thickness, five eyes from five
   individual zebrafish from wildtype, rpgrip1 mutant and Gyp-treated
   rpgrip1 mutant group, were sectioned with two retinal sections of each
   eye chosen for imaging. The thickness of the photoreceptor layer was
   measured at the location 0.4 mm from the optic nerve head using ImageJ
   software.

   For immunostaining, PFA fixed zebrafish eyes were immersed in 5% (2 h),
   15% (2 h) and 30% (2 h) sucrose, embedded in an optimal cutting
   temperature compound and cryosectioned at 8 µm thickness. The sections
   were blocked for 30 min at room temperature with 2% bovine serum
   albumin (BSA) in PBS and then incubated with Zpr-1 antibody (1:200,
   [73]www.zfin.org, accessed on 20 April 2021), which specifically
   targets red and green cone arrestin 3a [[74]22] at 4 °C overnight. The
   sections were washed three times with PBS (5 min each) and were
   incubated for 1 h at room temperature with a secondary antibody (1:500,
   FITC-labelled goat anti-mouse IgG, Sigma). The sections were washed
   with PBS (5 times for 5 min/each) and mounted with Vectashield medium
   containing 4′,6-diamidino-2-phenylindole (DAPI) to stain the nuclei.
   Images were taken under confocal microscopy. Signals for cone cells
   were quantified using a method described in detail elsewhere [[75]23].
   Briefly, 10 retinal sections from 5 eyes (2 sections per eye) of the
   wildtype zebrafish, rpgrip1 mutants or Gyp-treated rpgrip1 mutants were
   chosen. Fluorescent signals in one region (10 µm × 10 µm, under 400×
   magnification) in the superior side (0.4 mm from the optic nerve head)
   of the eye were then quantified with ImageJ software.

2.3. ROS Measurement

   The zebrafish eyes were collected and lenses were taken out. The eye
   samples were homogenized in a cold buffer (pH 7.4) containing sucrose
   (0.32 mM), HEPES (20 mM), MgCl[2] (1 mM) and phenylmethyl
   sulfonylfluoride (PMSF, 0.5 mM), and centrifuged for 20 min at 15,000×
   g (4 °C). The supernatants were collected and 20 µL of each sample was
   transferred into 96-well plates, followed by incubation with 20 μg/mL
   DCFH-DA (6-Carboxy2′,7′-Dichlorofluorescin diacetate, 8.3 µL/well) for
   30 min at 37 °C in the dark. The fluorescent signals were detected
   using a microplate reader (Fluostar Optima, BMG-labtech) at 485 nm
   (excitation) and 525 nm (emission).

2.4. TUNEL Assay

   Cell death in zebrafish retinas was detected using TUNEL assay. The
   paraffin sections were dewaxed with Histo-clear and rehydrated with
   decreasing concentrations of ethanol. The sections were permeabilized
   with proteinase K solution (20 µg/mL) for 10 min, washed with PBS for 5
   min, then fixed again with 4% PFA for 5 min, followed by washing with
   PBS for 5 min. The sections were equilibrated using equilibration
   buffer (100 µL) at room temperature (RT) for 10 min and labelled with
   the rTdT reaction mix for an hour in a dark humidified incubator (37
   °C). The reaction was stopped with 2 × saline-sodium citrate (SSC) and
   the sections were co-stained with DAPI. Images were captured using
   confocal microscopy. To quantify cell death, six sections from six eyes
   (one section per eye) of the wildtype zebrafish, rpgrip1 mutants or
   Gyp-treated rpgrip1 mutants were used. In each section, TUNEL positive
   photoreceptors in one region (50 µm × 20 µm, long × wide, under 400×
   magnification) in the superior side (0.4 mm from the optic nerve head)
   were counted and are presented as a percentage of total photoreceptor
   number in the same region.

2.5. Gene Expression

   Total RNA was extracted from zebrafish eye samples with the TRIzol
   reagent and cDNA was synthesized using a cDNA synthesis kit (Applied
   Biosystems, Warrington, UK) following the manufacturer’s guidance.
   Expression of target genes was measured by quantitative real-time PCR
   (qRT-PCR) with SYBR Green reagent (Thermo Fisher Scientific, Paisley,
   UK). The relative expression of candidate genes was calculated using a
   formula of 2^−ΔΔCT. The sequences for primers used in qRT-PCR are
   listed in [76]Table S1.

2.6. Biochemical Assays

   To measure activities of catalase and superoxide dismutase (SOD) and to
   quantify levels of glutathione (GSH) and malondialdehyde (MDA) in
   zebrafish eye samples, OxiSelect Superoxide Dismutase Activity Assay
   (STA-340), OxiSelect Catalase Activity Assay (STA-341), OxiSelect Total
   Glutathione (GSSG/GSH) Assay (STA-312) and OxiSelect TBARS (MDA
   Quantitation) Assay (STA-330) were performed according to the
   manufacturer’s protocols.

2.7. Network Pharmacological Analysis

2.7.1. Potential Targets or Genes Screening

   For screening active components in herbs, “Gynostemmae pentaphylli” was
   used as keywords searched against the Traditional Chinese Medicine
   Systems Pharmacology Database and Analysis Platform
   ([77]https://tcmspw.com/tcmsp.php, accessed on 20 April 2021) [[78]24].
   Active components were collected by name and ADME (absorption,
   distribution, metabolism and excretion) properties, namely, gypenoside,
   drug-likeness (DL) ≥0.18 and oral bioavailability (OB) ≥30%
   [[79]25,[80]26]. The effective targets were fished via on-line tool,
   Swiss Target Prediction ([81]http://swisstargetprediction.ch/, accessed
   on 20 April 2021), by using active components as baits [[82]27].

   Retinitis pigmentosa (RP)-associated genes were collected from DisGeNET
   ([83]http://www.disgenet.org/, accessed on 20 April 2021) [[84]28] and
   Online Mendelian Inheritance in Man (OMIM, [85]https://www.omim.org/,
   accessed on 20 April 2021) by using “retinitis pigmentosa” as
   researching keyword.

   In view of oxidative stress and inflammation having an important role
   in the pathogenesis of RP, genes related to oxidative stress or
   inflammation were gathered from DisGeNET ([86]http://www.disgenet.org/,
   accessed on 20 April 2021) [[87]28] or GeneCards (The Human Gene
   Database, [88]https://www.genecards.org/, accessed on 20 April 2021) by
   using “inflammation”, “inflammation disorder”, “oxidative stress”,
   “oxidative damage” and “oxidative injury” as researching keywords. All
   sources used for target screening or disease-related gene prediction
   are listed in [89]Table S2. All targeting gene identifiers were
   converted into official gene symbols by using the bitr function from
   the R package clusterProfiler (v 3.17.0) [[90]29] and only human genes
   were included.

2.7.2. Interaction Network and Enrichment Analysis

   To construct a compound-target-disease network, common targets among
   active compounds, RP and inflammation or oxidative stress were focused
   on, and the interactions between them were obtained from the STRING (v
   11.0, [91]https://string-db.org/, accessed on 20 April 2021) database.
   The protein interaction was mapped using the STRING database including
   high-through lab experiments, automated text mining, previous knowledge
   of the database and co-expression with a minimum required interaction
   score of 0.40 (medium confidence). To evaluate the centrality of
   network nodes, topological analysis was performed by NetworkAnalyzer (a
   plugin for Cytoscape). This obtains parameters of degree, betweenness,
   closeness and identifies hub genes as potential key genes for Gyp
   interfering with RP via anti-inflammatory or anti-oxidative capacity.
   The visualization of the network was fulfilled by Cytoscape 3.8.0
   software ([92]https://cytoscape.org/, accessed on 20 April 2021).

   To identify the typical biological functions and pathways of potential
   targets for Gyp treating RP, both gene ontology (GO) functional
   enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathway analysis were performed using the enrichGO and enrichKEGG
   functions from R package clusterProfiler (v 3.17.0) [[93]29]. The
   hypergeometric test was conducted for identifying significant
   enrichment. Terms or pathways with p < 0.05 were considered as
   statistically significant.

2.8. Statistical Analysis

   Data were collected from three independent experiments and analyzed
   using the GraphPad Prism software. Statistical comparisons between
   individual groups were performed using one-way analysis of variance
   (ANOVA) followed by the Bonferroni test. Data were presented as mean ±
   SEM (standard error of the mean). p < 0.05 was considered to be
   significant.

3. Results

3.1. Initial Toxicity Test of Gyp in Zebrafish

   To determine the appropriate dosage of Gyp for this study, 2 mpf
   wildtype and rpgrip1 mutant zebrafish were orally treated with Gyp at
   concentration of 50, 100, 150 or 200 µg/g body weight for three weeks.
   All zebrafish treated with 150 µg/g body weight died within six days
   while those treated with 200 µg/g body weight died within four days.
   All zebrafish treated with 100 µg/g body weight were dead within 2
   weeks. Zebrafish treated with 50 µg/g Gyp lived for three weeks without
   any abnormality. Therefore, Gyp at 50 µg/g bodyweight was chosen for
   further treatment of rpgrip1 mutants. Rpgrip1 mutant zebrafish were
   administrated Gyp (50 µg/g body weight) orally every week for 16 weeks.
   At the end of the treatment period, histology, immunochemistry, gene
   expression and biochemical assays were performed.

3.2. Cone Cell Death Was Decreased in Gyp-Treated rpgrip1 Mutant Zebrafish

   Rpgrip1 mutant zebrafish at 3 mpf demonstrate that almost all rod cells
   were degenerated, cone cells are morphologically normal though they are
   not tightly aligned. Degeneration of cone cells was obvious in rpgrip1
   mutant zebrafish at 6 mpf [[94]9]. To examine the protective impact of
   Gyp on cone cell death, rpgrip1 mutant zebrafish at 2 mpf, before cone
   cell death starts, were orally treated for 16 weeks till 6 mpf. H&E
   staining showed that the thickness of the cone photoreceptor layer was
   significantly reduced by 32.67% in the untreated rpgrip1 mutant retinas
   compared to that of wildtype retinas; the thickness of cone
   photoreceptor layer was significantly increased by 14.74% in rpgrip1
   mutant zebrafish treated with Gyp when compared with that of rpgrip1
   mutants; however, the cone photoreceptor layer in Gyp-treated rpgrip1
   mutants was still significantly thinner than that of wildtype zebrafish
   ([95]Figure 1).

Figure 1.

   [96]Figure 1
   [97]Open in a new tab

   (A) Hematoxylin and eosin-stained image of retinal sections of wildtype
   (WT), rpgrip1 mutant and Gyp-treated rpgrip1 mutant zebrafish at 6 mpf.
   (B) Thickness of photoreceptor layer of the three zebrafish groups.
   Statistical comparisons between individual groups were carried out
   using one-way ANOVA followed by Bonferroni’s test. Data are displayed
   as mean ± SEM (n = 5 animals of each group). **** p < 0.0001. INL,
   inner nuclear layer; GCL, ganglion cell layer; ONL, outer nuclear
   layer; RPE, retinal pigment epithelial cells.

   Cone cell degeneration was evaluated by immunostaining zebrafish
   retinas using the Zpr-1 antibody to detect both red and green cone
   photoreceptors. The retinas of untreated rpgrip1 mutant zebrafish
   demonstrated a significant decrease in the green signal (for both red
   and green cones) by 64.33% compared to that of wildtype zebrafish
   retinas; however, in rpgrip1 mutant zebrafish retinas treated with Gyp,
   there was a significant increase in the green signal by 29.2% compared
   to that of untreated rpgrip1 mutant retinas; green signals in
   Gyp-treated rpgrip1 mutant zebrafish eyes was still notably less than
   that of wildtype zebrafish ([98]Figure 2).

Figure 2.

   [99]Figure 2
   [100]Open in a new tab

   The effect of Gyp treatment on cone degeneration. (A) Immunostaining of
   retinal sections of wildtype, rpgrip1 mutant and Gyp-treated rpgrip1
   mutant zebrafish at 6 mpf using zpr-1 antibody where zpr1 stained the
   cones in green and DAPI labelled nuclei in blue. (B) Intensity
   fluorescence signals (the corrected total cell fluorescence, CTCF) were
   measured using ImageJ. Statistical comparisons between individual
   groups were carried out using one-way ANOVA followed by Bonferroni’s
   test. Data are displayed as mean ± SEM (n = 5 animals of each group).
   Ns, no significance; * p < 0.05, *** p < 0.001. INL, inner nuclear
   layer; ONL, outer nuclear layer.

   Death of cone cells was also detected by TUNEL staining, which can
   detect apoptotic and non-apoptotic cell death. The results demonstrated
   a significantly increased cell death in the photoreceptor layer of
   untreated rpgrip1 mutant zebrafish compared to that of wildtype
   zebrafish; Gyp treatment led to a significant decrease in cell death
   compared to that of untreated rpgrip1 mutants; however, there was
   significant higher cone cell death in GYP-treated rpgrip1 mutants than
   that of wildtype zebrafish ([101]Figure 3). The apoptotic cell death
   pathways involve caspase-3 and caspase-9 [[102]30,[103]31,[104]32], so
   we performed qRT-PCR to detect expression of the two caspase genes.
   Both caspase3 and caspase 9 had markedly increased expression in
   rpgrip1 mutant eyes, compared to that of wildtype zebrafish; Gyp
   treatment resulted in a significant decrease in expression of both
   genes, compared to that of untreated rpgrip1 mutants; however,
   expression of both genes in Gyp-treated rpgrip1 mutant eyes remained
   higher than that of wildtype zebrafish ([105]Figure S1).

Figure 3.

   Figure 3
   [106]Open in a new tab

   Significant decreases were found in cone apoptosis in rpgrip1 mutant
   zebrafish treated with Gyp. (A) Retinal sections of wildtype (WT),
   untreated (UT) rpgrip1 mutant and Gyp-treated rpgrip1 mutant zebrafish
   at 6 mpf were stained with TUNEL reagents. Nuclei of apoptotic cone
   cells were stained in green. DAPI labelled nuclei were stained in blue.
   (B) Quantification of apoptotic cells in above retinal sections were
   compared between the wildtype, rpgrip1 mutant and Gyp-treated rpgrip1
   mutant zebrafish. Statistical comparisons between individual groups
   were carried out using one-way ANOVA followed by Bonferroni’s test.
   Data are displayed as mean ± SEM (n = 6 animals of each group). ** p <
   0.01, **** p < 0.0001. INL, inner nuclear layer; ONL, outer nuclear
   layer.

3.3. Gyp Treatment Decreased ROS Production and Increased Antioxidant
Capacity in rpgrip1 Mutant Zebrafish

   It has been suggested that rod degeneration leads to increased ROS in
   the outer retina [[107]2]. Similarly, there was a significant increase
   in ROS production in rpgrip1 mutant eyes compared to that of wildtype
   zebrafish; Gyp treatment significantly reduced ROS production compared
   to that of untreated rpgrip1 mutants, though ROS level in Gyp-treated
   eyes higher than that of wildtype zebrafish ([108]Figure 4A).

Figure 4.

   Figure 4
   [109]Open in a new tab

   Effects of Gyp on antioxidant capacity in rpgrip1 mutant zebrafish
   eyes. (A) ROS generation in the eyes of wildtype (WT), untreated (UT)
   and Gyp-treated rpgrip1 mutant zebrafish at 6 mpf. (B–H) Expression of
   catalase, sod1, sod2, gpx1, gclm, nqo-1, gclm and nrf2 was determined
   using qRT-PCR. Statistical comparisons between individual groups were
   carried out using one-way ANOVA followed by Bonferroni’s test. Data are
   displayed as mean ± SEM (n = 6 animals of each group). ** p < 0.01,
   **** p < 0.0001.

   The effect of Gyp treatment on the expression of the antioxidant genes
   was assessed by qRT-PCR. In rpgrip1 mutant zebrafish eyes, catalase
   expression was significantly reduced compared to that of wildtype
   zebrafish; in rpgrip1 mutant zebrafish treated with Gyp, catalase
   expression was significantly increased compared to that of untreated
   rpgrip1 mutants; however, Gyp-treated rpgrip1 mutant eyes had lower
   expression of catalase than that of wildtype zebrafish ([110]Figure
   4B). Similarly, expression of sod1, sod2, gpx1, gclm, nqo1 and nrf2 was
   significantly reduced in rpgrip1 mutant zebrafish eyes compared to that
   of wildtype zebrafish; while Gyp treatment significantly increased
   expression of these genes compared to that of untreated mutant
   zebrafish, though Gyp-treated mutant zebrafish still had lower
   expression of the examined genes than that of wildtype zebrafish
   ([111]Figure 4C–H).

   Activities of SOD and catalase and levels of GSH and MDA were also
   measured in the eyes of wildtype, GYP-treated and untreated rpgrip1
   mutant zebrafish. Rpgrip1 mutant zebrafish had significantly decreased
   activities of SOD and catalase compared to that of wildtype zebrafish;
   GYP treatment resulted in significantly increased activities of SOD and
   catalase, compared to untreated rpgrip1 mutants; however, GYP-treated
   mutants had lower activities of SOD and catalase compared to wildtype
   zebrafish ([112]Figure 5A,B). In untreated rpgrip1 mutant zebrafish
   eyes, GSH level was significantly reduced compared to that of wildtype
   zebrafish; Gyp-treated zebrafish had significantly increased GSH levels
   compared to that of untreated mutant zebrafish, though it was
   significantly lower than that of wildtype zebrafish ([113]Figure 5C).
   MDA level was significantly increased in rpgrip1 mutant zebrafish eyes
   compared to that of wildtype zebrafish; Gyp treatment led to a
   significant decrease in MDA level compared to that of untreated rpgrip1
   mutant zebrafish; Gyp-treated rpgrip1 mutants had higher MDA levels
   than that of wildtype zebrafish ([114]Figure 5D).

Figure 5.

   Figure 5
   [115]Open in a new tab

   Sod (A) and catalase (B) activities, and GSH level (C) were markedly
   increased but MDA level (D) was significantly decreased in Gyp-treated
   rpgrip1 mutant zebrafish eye samples. WT, wildtype zebrafish; UT,
   untreated rpgrip1 mutant zebrafish. Statistical comparisons between
   individual groups were carried out using one-way ANOVA followed by
   Bonferroni’s test. Data are displayed as mean ± SEM (n = 6 animals of
   each group). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

3.4. Gyp Treatment Suppressed Inflammation in rpgrip1 Mutant Zebrafish Eyes

   Oxidative stress is linked to inflammation, in that oxidative stress
   modifies substrates to induce expression of proinflammatory cytokines
   [[116]18,[117]33]. Expression of inflammatory-related genes in eyes of
   wildtype, rpgrip1 mutant and Gyp-treated mutant zebrafish was evaluated
   using qRT-PCR. In rpgrip1 mutant zebrafish eyes, expression of il-1β,
   il-6 and tnfα was significantly increased, compared to wildtype
   zebrafish; Gyp-treated rpgrip1 mutants had a significant decrease in
   expression of il-1β, il-6 and tnfα, compared to untreated rpgrip1
   mutants; When compared to wildtype zebrafish, Gyp-treated rpgrip1
   mutants had higher expression of the three cytokine genes ([118]Figure
   6).

Figure 6.

   [119]Figure 6
   [120]Open in a new tab

   Gyp treatment reduced the expression of inflammation associated genes
   in rpgrip1 mutant zebrafish eye samples. Expression of il-1β, il-6,
   tnfα, rip1 and rip3 in eyes of wildtype (WT), untreated (UT) and
   Gyp-treated rpgrip1 mutant zebrafish at 6 mpf was determined by
   qRT-PCR. Statistical comparisons between individual groups were carried
   out using one-way ANOVA followed by Bonferroni’s test. Data are
   displayed as mean ± SEM (n = 6 animals of each group). **** p < 0.0001.

   Receptor-interacting serine/threonine protein kinases (RIP) 1 and RIP3
   play a critical role in regulating inflammation and cell death
   [[121]34]. We also found that expression of both rip1 and rip3 was
   markedly increased compared to that of wildtype zebrafish;
   Gyp-treatment resulted in significantly decreased expression of rip1
   and rip3 compared to that of untreated rpgrip1 mutants; however,
   expression of rip1 and rip3 in Gyp-treated rpgrip1 mutants was higher
   than that of wildtype zebrafish ([122]Figure 6).

3.5. Gyp Attenuated Expression of Endoplasmic Reticulum (ER) Stress
Associated Genes in rpgrip1 Mutant Eyes

   ROS production and ER stress are two interactive events, associated
   with a wide range of human diseases [[123]35,[124]36]. ER stress
   activates the unfolded protein response (UPR), containing PERK, IRE1
   and ATF6 signaling pathways. We examined expression of the UPR
   components including bip, atf4, atf6 and xbp1 in eyes of wildtype,
   rpgrip1 mutant and Gyp-treated mutant zebrafish. Expression of bip,
   atf4, atf6, xbp1t (total) and xbp1s (spliced variant) was markedly
   increased in rpgrip1 mutant eyes compared to that of wildtype
   zebrafish; Gyp-treatment led to significantly decreased expression of
   these genes compared to that of untreated rpgrip1 mutants; expression
   of bip and xbp1s (but not atf4, atf6 or xbp1t) remained higher in
   Gyp-treated mutants compared to that of wildtype zebrafish ([125]Figure
   S2).

3.6. Network Pharmacological Analysis

   To identify the potential active compounds in herbs, firstly,
   “Gynostemmae Pentaphylli” was searched against the TCMSP database.
   Based on the ADME parameters of DL ≥ 0.18 and OB ≥ 30%, a total of 24
   active compounds were identified. Among these, 9 gypenosides were
   included. The main properties of Gyp are listed in [126]Table S3.
   Subsequently, potential targets were predicted using Swiss Target
   prediction, and the top 15 targets of each gypenoside were kept and
   merged. After unifying the gene symbols and dereplication, a total of
   63 targets of gypenosides were finally screened out ([127]Figure S3).
   The RP-related genes were gathered from DisGeNET and OMIM. After
   normalizing the gene symbols and removing duplicates, a total of 745
   RP-related genes were finally identified. Furthermore, 1132 and 745
   unique genes related to oxidative stress and inflammation were
   collected from GeneCards and DisGeNET database, respectively.

   To explore the link between Gyp and RP in oxidative stress and
   inflammatory aspects, 7 and 11 common targets among potential targets
   of gypenosides, RP-related genes, inflammation-related genes or
   oxidative stress-related genes were extracted, respectively.
   Subsequently, two PPI networks ([128]Figure 7) were constructed by
   employing online tool STRING. Among the common targets, SIGMAR1 was not
   displayed in [129]Figure 7, because it was not connected with the
   network. The shared targets among gypenosides, RP and inflammation were
   MMP2, IL-6, FGF2, MMP9, MAPK14, HSP90AA1 and SLC2A1, are shown in
   [130]Figure 7A. The connection of Gyp, RP and oxidative stress is
   illustrated in [131]Figure 7B. Among these mapped interactions,
   MMP2-HSP90AA1 and MAPK14-HSP90AA1 were taken from a verified
   experimental source ([132]Figure 7A). Similarly, FGF2-INSR and
   SLC2A1-INSR belong to verifiable experimental interactions ([133]Figure
   7B). According to topological network analysis ([134]Tables S4 and S5),
   both MMP2 and IL-6 had the highest degree, and were identified as hub
   genes. In addition, FGF2, SLC2A1, MMP9 and MAPK14 were also suggested
   to be crucial nodes in the networks. Owing to the common targets shared
   by gypenosides, RP and inflammation being fully included in that shared
   by gypenosides, RP and oxidative stress, all common targets were
   combined into one set.

Figure 7.

   [135]Figure 7
   [136]Open in a new tab

   PPI network between common targets. (A) Inflammation, (B) oxidative
   stress. The nodes represented targets and edges represented interaction
   among targets. The node size and color were represented with degree,
   while edge size and color were represented with combined score.

   To investigate the multiple functions of the common targets
   ([137]Figure 7), GO molecular function enrichment analysis was
   conducted. The top 6 significantly enriched GO terms (p < 0.05) were
   predominantly involved in protein binding (GO:0005515), identical
   protein binding (GO:0042802), metallopeptidase activity (GO:0008237)
   and protein tyrosine kinase activity (GO:0004713) ([138]Table S6). In
   addition to GO enrichment, the KEGG pathway was also analyzed to
   clarify the potential mechanism of gypenosides treating RP via
   anti-oxidative stress or anti-inflammation. The candidate common
   targets were principally enriched in pathways in cancer (hsa05200 and
   hsa05205), the HIF-1 signaling pathway (hsa04066), the TNF signaling
   pathway (hsa04668), the PI3K-Akt signaling pathway (hsa04151) and
   leukocyte transendothelial migration (hsa04670) ([139]Table S7). In
   addition, the hub genes identified from GO enrichment analysis are
   similar to those in KEGG signalling pathways, including FGF2, IL-6,
   MAPK14, MMP2, MMP9 and SLC2A1 ([140]Tables S6 and S7).

4. Discussion

   The retinal pathology of rpgrip1 mutant zebrafish resembles that of RP
   patients who show initial rod cell death and subsequent cone cell
   death. So rpgrip1 mutant zebrafish is an excellent platform for the
   development of new treatments for RP. Here we have demonstrated that
   Gyp administration reduces cone cell death, enhances antioxidant
   capacity, suppresses inflammation and inhibits ER stress in rpgrip1
   mutant zebrafish. Network pharmacological analysis further suggests
   that the protective effects of Gyp are mediated by inhibition of
   oxidative stress and inflammation.

   Early studies demonstrate that exposure to high oxygen levels for
   several weeks results in mouse photoreceptor degeneration. Increased
   photoreceptor death was found in the central retinal region, where the
   highest amount of oxygen is supplied from the choroidal blood flow
   [[141]37]. In RP, the progressive death of rod cells leads to decreased
   oxygen utilization and a resultant increase in oxygen tension in the
   outer retina. In the Royal College of Surgeons (RCS) rats, rod
   photoreceptors are completely degenerated by postnatal (P) 30. Oxygen
   tension in the outer retina was increased from P25 and after P30 the
   cones endured oxygen tension as high as four times compared to that of
   the normal rats [[142]38]. Similarly, significantly higher oxygen
   tension has been reported in the outer retinas of P23H rats and of an
   RP cat model [[143]39,[144]40]. Exposure to higher levels of oxygen
   tension is likely to cause oxidative damage to cone cells, resulting in
   the gradual death of cone cells. Acrolein and 4-hydroxynonenal, the
   biomarkers for oxidative damage, have been detected at higher levels in
   the cone cells of the RHODOPSINPro347Leu transgenic pigs at 10 months
   old, when all the rod cells have completely degenerated [[145]12].
   Acrolein has also been detected in the cone cells of rd1 mice at P35,
   about two weeks after degeneration of most rod cells [[146]13]. Here we
   also found a significantly higher level of ROS in rpgrip1 mutant eyes
   at 6 mpf ([147]Figure 4A), when only cones were survived and
   degeneration of cone cells was evident [[148]9]. The antioxidant
   defense system was also compromised in rpgrip1 mutant eyes,
   demonstrating a marked decrease in expression of antioxidant genes and
   in activities of antioxidant enzymes ([149]Figure 4 and [150]Figure 5).
   Previous studies have shown Gyp’s protection against oxidative damage
   in vitro and in vivo
   [[151]18,[152]19,[153]21,[154]41,[155]42,[156]43,[157]44]. Here we have
   demonstrated that Gyp treatment can suppress ROS production,
   upregulated antioxidant gene expression and increased antioxidant
   enzyme activities and GSH level in rigrip1 mutant eyes ([158]Figure 4
   and [159]Figure 5). Similar to the other antioxidants which protect
   against cone death in RP models [[160]12,[161]13,[162]14,[163]15], Gyp
   treatment also attenuated cone death in rpgrip1 mutant zebrafish
   ([164]Figure 2 and [165]Figure 3).

   It is well known that oxidative stress triggers inflammation in chronic
   diseases such as RP [[166]45]. RP patients have shown significantly
   high levels of proinflammatory cytokines and chemokines in the vitreous
   cavity and aqueous humor [[167]46]; previous studies also reported
   increased expression of proinflammatory cytokines (e.g., IL-1β and
   TNFα) and microglial activation in the retinas of RP mouse models
   [[168]23,[169]47]. In rd1 mice at P40, when only cone cells are
   survived, the retinas had significantly high expression of Il-1α.
   Il-1β, Tnfα, C1qα and Tmem119 (a marker for microglial activation);
   however, rd1 mice treated with PLX5622, which eliminates microglia,
   showed similar levels of expression of those genes in the retinas as
   that of wildtype mice, indicating activated microglia mediated
   expression of these genes [[170]48]. We also detected higher expression
   of proinflammatory genes (il-1β, il-6 and tnfα) in rpgrip1 mutant eyes
   at 6 mpf, when only cone cells are survived. Gyp treatment
   significantly reduced expression of il-1β, il-6 and tnfα. NF-κB pathway
   plays an important role retinal inflammation and is involved in
   RP-associated cone cell death [[171]49]. We, and others, have shown
   that Gyp suppresses inflammation via inhibiting activation of the NF-κB
   pathway [[172]50,[173]51]. So downregulation of proinflammatory gene
   expression by Gyp in rpgrip1 mutant eyes is possibly mediated by
   inactivation of NF-κB pathway.

   We further utilized network pharmacology to clarify the possible
   mechanisms of gypenosides against photoreceptor death in RP. Based on
   the predicted gene associations from the PPI network, IL-6, MMP2, MMP9,
   MAPK14, SLC2A1 and FGF2 were identified as main hub genes, which are
   potentially involved in the treatment of RP using Gyp via an
   anti-oxidative stress or anti-inflammation route. IL-6 plays important
   roles in inflammation, infection responses, immunity and disease
   [[174]52]. Increased IL-6 expression has been detected in intraocular
   fluid of RP patients and in the retinas of RP animal models
   [[175]46,[176]53,[177]54]. We also detected a high level of il-6
   expression in rpgrip1 mutant eye, suggesting IL-6 contributes to the
   pathogenesis of RP. IL-6 can enhance the generation of matrix
   metalloproteinases (MMPs) [[178]55]. MMP-2 and MMP-9 were members of
   gelatinase family and zinc-dependent enzymes, with capability of
   decomposing the extracellular matrix and basement membrane components.
   MMP2 has been detected at significantly high levels in the aqueous
   humor of RP patients [[179]56] and inhibition of MMP9 has been found to
   suppress rod cell death in a rat RP model [[180]57]. However, the
   underlying MMP2 and MMP9 associated mechanisms with RP are not clear
   and need further attention. Mitogen-activated protein kinases (MAPKs)
   belonged to the family of evolutionarily conserved serine/threonine
   kinases, which mediated the regulation of multiple cell processes,
   including cell growth, differentiation, autophagy and apoptosis. The
   MAPK signaling pathway has been involved in RP pathogenesis. A recent
   study demonstrated that inhibition of histone deacetylases attenuated
   secondary cone death via regulation of MAPK and PI3K-Akt signaling
   pathways [[181]58]. Solute carrier family 2 member 1 (SLC2A1), also
   known as glucose transporter 1 (GLUT1), is closely associated with
   cellular energy metabolism. The protection of rod-derived cone
   viability factor (RdCVF) against cone death in RP mouse models is
   mediated by promotion of GLUT1 transport of glucose into cone cells and
   stimulation of aerobic glycolysis [[182]59]. FGF2, as a pleiotropic
   heparin-binding factor, has angiogenic, mitogenic and immunomodulatory
   activities. Early studies have demonstrated that FGF2 delays
   photoreceptor degeneration in RP rat models
   [[183]60,[184]61,[185]62,[186]63]. Recently, Di Pierdomenico reported
   that FGF2 suppressed cone cell death in RP rats [[187]64].

   The GO functional analysis uncovered the associated functions of common
   targets. These GO terms gathered core hub targets ([188]Table S6) which
   were shared by Gyp, RP, oxidative stress and inflammation. The pathway
   enrichment analysis demonstrated that the HIF, TNF and PI3K-AKT
   signaling pathways were the most enriched and related to RP and
   Gyp-mediated protection ([189]Table S7). The TNF signaling pathway is
   particularly critical in photoreceptor death. TNFα mediates
   photoreceptor apoptosis, necroptosis and pyroptosis in RP [[190]65]. It
   is also reported that TNFα knockdown in RP mouse models promotes cone
   survival [[191]66]. The data from network pharmacology analysis suggest
   that the protection of Gyp against photoreceptor death is mediated by
   the main hub genes-associated signaling pathways. However, the
   underlying mechanisms remain elusive.

   Our data were mainly gathered from molecular biology, biochemistry and
   histology approaches. Ideally, we would combine retinal
   biochemical/histological changes with retinal visual functions. The
   data from network pharmacology analysis will need to be validated in RP
   animal models. Additionally, the protection of Gyp against rod and cone
   degeneration requires further evaluated in other RP models. Data from
   preclinical studies will guide future clinic trials of Gyp-treating RP
   patients.

5. Conclusions

   Gyp decreased ROS production, increased antioxidant capacity and
   suppressed ER stress and inflammation; resulting in increased cone
   survival in rpgrip1 mutant zebrafish. The delay of cone degeneration by
   Gyp is possibly mediated by multiple signaling pathways. Gyp has
   therapeutic potential for RP patients.

Acknowledgments