International Journal of
Molecular Sciences Article
Genipin Derivatives Protect RGC-5 from Sodium Nitroprusside-Induced Nitrosative Stress Rikang Wang 1,† , Jiaqiang Zhao 2,† , Lei Zhang 2 , Lizhi Peng 2 , Xinyi Zhang 2 , Wenhua Zheng 3, * and Heru Chen 2,4, * Received: 20 October 2015; Accepted: 8 January 2016; Published: 19 January 2016 Academic Editor: Katalin Prokai-Tatrai 1 2
3 4
* †
National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China; [email protected] Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China; [email protected] (J.Z.); [email protected] (L.Z.); [email protected] (L.P.); [email protected] (X.Z.) Faculty of Health Sciences, University of Macao, Macao, China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Guangzhou 510632, China Correspondence: [email protected] (W.Z.); [email protected] (H.C.); Tel.: +853-8822-4919 (W.Z.); +86-20-3837-5299 (H.C.); Fax: +853-8822-2314 (W.Z.); +86-20-8522-1559 (H.C.) These authors contributed equally to this work.
Abstract: CHR20 and CHR21 are a pair of stable diastereoisomers derived from genipin. These stereoisomers are activators of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In the rat retinal ganglion (RGC-5) cell model these compounds are non-toxic. Treatment of RGC-5 with 750 µM of sodium nitroprusside (SNP) produces nitrosative stress. Both genipin derivatives, however, protect these cells against SNP-induced apoptic cell death, although CHR21 is significantly more potent than CHR20 in this regard. With Western blotting we showed that the observed neuroprotection is primarily due to the activation of protein kinase B (Akt)/eNOS and extracellular signal-regulated kinase (ERK1/2) signaling pathways. Therefore, LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or PD98059 (a MAPK-activating enzyme inhibitor) abrogated the protective effects of CHR20 and CHR21. Altogether, our results show that in our experimental setup neuroprotection by the diasteromeric pair is mediated through the PI3K/Akt/eNOS and ERK1/2 signaling pathways. Further studies are needed to establish the potential of these compounds to prevent ntric oxide (NO)-induced toxicity commonly seen in many neurodegenerative diseases. Keywords: NO neurotoxicity; neuroprotection; neurodegeneration; apoptosis; genipin; nitric oxide synthase
1. Introduction Nitric oxide (NO), a Janus-faced molecule with pleiotropic effects in different tissues, is physiologically produced through the L-arginine/NO synthase (NOS) pathway. NOS is a family of four major types of enzymes including endothelial (eNOS), neuronal (nNOS), inducible (iNOS) and mitochondrial (mtNOSs). On the one hand, endogenous NO takes part in various physiological processes in the central nervous system, such as neuro-survival, neuro-differentiation, neuro-modulation, neuro-transmission and synaptic plasticity [1–3]. On the other hand, overproduction of NO produces oxidative and nitrosative stress [4–7]. Excess NO also induces nitrotyrosination, leading to the inhibition of tyrosine phosphorylation and blockade of the signal transduction pathways of growth factors [8]. Solid evidence supports that all
Int. J. Mol. Sci. 2016, 17, 117; doi:10.3390/ijms17010117
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Int. J. Mol. Sci. 2016, 17, 117
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transduction pathways Int. J. Mol. Sci. 2016, 17, 117
of growth factors [8]. Solid evidence supports that all neurodegenerative 2 of 16 diseases are closely related to the overproduction of NO [9–13]. For example, when nNOS is activated with persistent stimulation of excitatory amino acid receptors mediating glutamate toxicity, as well as neurodegenerative diseasesbyarediverse closely related the endotoxin overproduction of NO [9–13]. example, when when iNOS is induced stimuli tolike or cytokines, NO For may probably be nNOS is activated with persistent stimulation of excitatory amino acid receptors mediating glutamate overproduced [9]. In these cases, the role of NO changes from a physiological neuromodulator to a toxicity, as well as when iNOS is induced by diverse stimuli like endotoxin or cytokines, NO may probably neurotoxic factor. be overproduced [9]. In these cases,ofthe roleinduces of NO changes from a physiological a No doubt, overproduction NO oxidative stress. That is aneuromodulator risk factor for to the neurotoxic factor. development of neurodegenerative diseases including glaucomatous visual field deterioration doubt, overproduction of NO induces stress. That is a risk factor for the development [5–7].NoUnfortunately, retinal ganglion cells oxidative (RGCs) are susceptible to oxidative stress that may of neurodegenerative diseases including glaucomatous visual field deterioration [5–7]. Unfortunately, trigger cell apoptosis [7,14,15]. In a variety of retinal degenerative diseases, such as glaucoma and retinal ganglion cells (RGCs) are susceptible to oxidative stress that may cell apoptosis [7,14,15]. diabetic retinopathy, this oxidative stress-induced RGC apoptosis is trigger a fundamental pathogenesis In a variety of retinal degenerative diseases, such as glaucoma and diabetic retinopathy, this oxidative [15–17]. Although the rat retinal ganglion (RGC-5) cell line is actually misidentified [18], we believe stress-induced RGC apoptosis is a fundamental pathogenesis [15–17]. Although the rat retinal that it still possesses the fundamental properties of the retinal neuronal precursor cells andganglion hence is (RGC-5) cellour linestudies. is actually misidentified that still the possesses the fundamental suitable for In this way, RGC-5[18], cellswe canbelieve be used to itstudy pathogenesis process of properties of the retinal neuronal precursor cells and hencenitric is suitable our studies. In this way, oxidative/nitrosative stress induced by an exogenous oxideforagent, such as, sodium RGC-5 cells can be used to study the pathogenesis process of oxidative/nitrosative stress induced by nitroprusside (SNP). an exogenous oxide agent, such as, sodium nitroprusside (SNP).of nNOS for the treatment of Recently,nitric application of several types of selective inhibitors Recently, application of several types of selective inhibitors of nNOS for the based treatment of central central nervous system (CNS) disorders have been reported [19–22]. Evidences on the rodent nervous disorders have been reportedpain [19–22]. basedmodel on theofrodent model model ofsystem dural (CNS) inflammation relevant to migraine and Evidences the rat Chung neuropathic of dural inflammation relevant to migraine pain and the rat model of neuropathic pain pain confirmed that some 3,4-dihydroquinolin-2(1H)-one andChung 1,2,3,4-tetrahydroquinoline-based confirmed that some 3,4-dihydroquinolin-2(1H)-one and 1,2,3,4-tetrahydroquinoline-based derivatives derivatives had significant efficacy to relieve the pains [22]. As a matter of fact, our research group had efficacy to relievegardenamide the pains [22].AAs a matter of was fact, confirmed our research has synthesized has significant synthesized and identified (GA), which asgroup an activator of nNOS and gardenamide A (GA), which was confirmed as an activator nNOSserum and eNOS [23]. and identified eNOS [23]. Quite interestingly, GA showed neuroprotective effects of against deprived Quite interestingly, GA cells showed effects against serum in PC12 impairments in PC12 andneuroprotective H2O2-induced insults in RGC-5 cellsdeprived [24,25]. impairments The neuroprotective cells and H2 Oinvolved insults in RGC-5 [24,25]. The neuroprotective mechanisms 2 -inducedare mechanisms closely relatedcells to the activations of ERK1/2 and PI3K/Akt involved signaling are closely related the activations ERK1/2 and PI3K/Akt signaling pathways. pathways. Anothertotwo new stable ofgenipin derivatives, CHR20 (1-R isomer) and Another CHR21 two (1-S new stable genipin CHR20 (1-R isomer) and CHR21 (1-S isomer), which are a pair of isomer), which are aderivatives, pair of diastereoisomers, and confirmed as nNOS and eNOS activators have diastereoisomers, and confirmed as nNOS and eNOS activators have also been synthesized by us [26] also been synthesized by us [26] (Figure 1). They protected PC12 cells from apoptosis induced by (Figure 1). They protected PC12oxidative cells fromstress apoptosis induced SNP. SNP wasincrease found to raise oxidative SNP. SNP was found to raise in PC12 cells by and CHR20/21 mRNA levels of stress in PC12 cells and CHR20/21 increase mRNA levelsand of glutamate-cysteine ligase catalytic subunit glutamate-cysteine ligase catalytic subunit (GCLC) superoxide dismutase 1 (SOD1) in a (GCLC) and superoxide dismutase 1 (SOD1) in a time-dependent manner [26]. time-dependent manner [26].
Figure 1.1.Chemical Chemical structures of genipin and CHR20/21. “a” is ina the symbol the IUPAC’ Figure structures of genipin and CHR20/21. “a” is a symbol IUPAC’innomemclature nomemclature rule to represent the first atom linked to the outside ring. rule to represent the first atom linked to the outside ring.
In the present study we wished to investigate whether CHR20 and CHR21 can prevent RGC-5 In the present study we wished to investigate whether CHR20 and CHR21 can prevent RGC-5 cell death brought about by SNP-produced oxidative/nitrosative insults. cell death brought about by SNP-produced oxidative/nitrosative insults. 2. Results 2. Resultsand andDiscussion Discussion 2.1. Effects Effects of of SNP SNP on on RGC-5 RGC-5 Cells Cells 2.1. As shown shown in in Figure Figure 2, 2, SNP SNP dose-dependently dose-dependently influenced influenced RGC-5 RGC-5 viability. viability. When When RGC-5 RGC-5 cells cells As were treated with SNP at a concentration between 62.5 to 250 μmol/L, their MTT activities increased were treated with SNP at a concentration between 62.5 to 250 µmol/L, their MTT activities increased with SNP SNPconcentration, concentration,in inwhich whichMTT MTTmeans means3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium tetrazolium with bromide. This maximal at at 250 250 µmol/L. μmol/L. Because MTT activity activity is is bromide. This increasing increasing effect effect went went to to the the maximal Because MTT dependent on NADPH-dependent oxido-reductase enzymes largely in the cytosolic compartment of
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dependent on NADPH-dependent oxido-reductase enzymes largely in the cytosolic compartment the cell [27,28], underunder defined conditions, it may it reflect number presentofviable cells.viable Therefore, ofdependent the cell [27,28], defined conditions, maythe reflect the of number present cells. on NADPH-dependent oxido-reductase enzymes largely in the cytosolic compartment SNP at these concentrations showed no cytotoxicity to RGC-5 cells. Under these conditions, cell Therefore, SNP at these concentrations showed no cytotoxicity to RGC-5 cells. Under these conditions, of the cell [27,28], under defined conditions, it may reflect the number of present viable cells. viability seems to increase withwith increasing SNP concentration. However, atathigh concentrations cell viability seems to increase increasing concentration. However, high Therefore, SNP at these concentrations showed noSNP cytotoxicity to RGC-5 cells. Under theseconcentrations conditions, (750 and 1000 µmol/L), SNP was cytotoxic; exposure to 750 µmol/L of SNP induced injury to (750 and 1000 μmol/L), SNP was cytotoxic; exposure to 750 μmol/L of SNP induced injury to the the cells. cells. cell viability seems to increase with increasing SNP concentration. However, at high concentrations The MTT activity was only 41.0% ˘ 4.8% compared with that of the control group. The MTT activity was only 41.0% ± 4.8% compared with that of the control group. (750 and 1000 μmol/L), SNP was cytotoxic; exposure to 750 μmol/L of SNP induced injury to the cells. The MTT activity was only 41.0% ± 4.8% compared with that of the control group.
Figure2.2. The The neuro-effects of SNP on RGC-5 cells. Cells were exposed totodifferent concentrations of Figure SNP on onRGC-5 RGC-5cells. cells.Cells Cells were exposed different concentrations Figure 2. Theneuro-effects neuro-effects of of SNP were exposed to different concentrations of SNP asas indicated forfor 24 h. Cell viability was evaluated byby MTT assay. The values were expressed as of SNP indicated Cell viability was evaluated MTT assay. values expressed SNP as indicated for 2424h.h.Cell viability was evaluated by MTT assay. The The values werewere expressed as percentage of control, which is set to 100%. The percentage of MTT activity was presented as mean ± as percentage percentageofof control, which is to set100%. to 100%. The percentage MTT was activity was presented control, which is set The percentage of MTTof activity presented as mean ± as SD for six replicates. * p < 0.05 vs. control group. mean ˘ SD six replicates. * pvs.< control 0.05 vs.group. control group. SD for six for replicates. * p < 0.05
It isisquite interesting that low concentrationofofSNP SNP (≤250μM) μM) stimulates RGC-5 cell viability. quiteinteresting interestingthat thatlow low concentration concentration RGC-5 cellcell viability. It It is quite of SNP(≤250 (ď250 µM)stimulates stimulates RGC-5 viability. We did observe cell proliferation under microscopy. microscopy. However,the the exact mechanism needs to be observecell cellproliferation proliferationunder under needs to to be WeWe diddid observe microscopy. However, However, theexact exactmechanism mechanism needs be further investigated. further investigated. further investigated. 2.2. Cytotoxicity and MTT Activity Enhancement CHR20/21tototoRGC-5 RGC-5 Cells 2.2. Cytotoxicityand andMTT MTTActivity ActivityEnhancement Enhancement of Cells 2.2. Cytotoxicity ofofCHR20/21 CHR20/21 RGC-5 Cells To examine the cellular tolerance toCHR20/CHR21, CHR20/CHR21,RGC-5 RGC-5cells cells were cultured inRPMI-1640 RPMI-1640 To examinethe thecellular cellulartolerance tolerance to to CHR20/CHR21, were cultured inin RPMI-1640 To examine RGC-5 cells were cultured mediumwith with10% 10%fetal fetal bovine bovine serum (FBS) or serum (0.2% ofofFBS) forfor 2424 h. h. Then RGC-5 cellscells medium (FBS) orlow low serum (0.2% FBS) Then RGC-5 medium with 10% fetal bovine serum (FBS) or low serum (0.2% of FBS) for 24 h. Then RGC-5 cells with withlow lowserum serum were were treated treated with the test compounds atatconcentrations of of 3, 3, 10 10 andand 30 30 μM,μM, with the test compounds concentrations low serum were treated with the test compounds at concentrations of 3, 10 and 30 µM, respectively, respectively, for 24 h. As shown in Figure 3, CHR20 and CHR21 did not show any toxicity at at respectively, for 24in h. As shown in Figure 3, CHR20 and CHR21 did notatshow any toxicity for 24 h. As shown Figure 3, CHR20 and CHR21 did not show any toxicity concentrations up to concentrations up to 30 μM. On the contrary, MTT activity of RGC-5 cells increased in the presence concentrations up to 30MTT μM. activity On the contrary, activity ofinRGC-5 cells increased in and the presence 30 µM. On the contrary, of RGC-5MTT cells increased the presence of CHR20 CHR21, ofCHR20 CHR20and andCHR21, CHR21, respectively, respectively, at aa dose of 30 μM. This evidence suggests that each of these of at dose of 30 μM. This evidence suggests that each of these respectively, atcould a doseexert of 30a µM. This evidence suggests that each of these compounds could exert a compounds neuroprotective effect and stimulated cell survival under low serum compounds could exert astimulated neuroprotective effectunder and stimulated cell survival under lowfurther serum neuroprotective effect and cell survival low serum environment. However, environment.However, However, further investigations investigations are totoconfirm interpretation of this environment. are required required confirmthethe interpretation of this investigations are requiredfurther to confirm the interpretation of this finding. finding. finding.
Figure 3. Cytotoxicity and MTT activity of CHR20 and CHR21 on RGC-5 cells. Cells that were Figure 3. Cytotoxicity and MTT activity of CHR20 and CHR21 on RGC-5 cells. Cells that were cultured cultured RPMI-1640 and medium low serum (0.2% of FBS) for 24 h were set ascells. the control group. Figure 3. in Cytotoxicity MTTwith activity of CHR20 and Cells that were in RPMI-1640 medium with low serum (0.2% of FBS) for 24CHR21 h were on set RGC-5 as the control group. RGC-5 RGC-5 cells with 0.2% medium FBS werewith treated with CHR20 (A) and CHR21 (B) at concentrations of 3,group. 10 cultured in RPMI-1640 low serum (0.2% of FBS) for 24 h were set as the control cells with 0.2% FBS were treated with CHR20 (A) and CHR21 (B) at concentrations of 3, 10 and 30 µM, and 30cells μM,with respectively for 24 h. Cell viability was determined by MTT and presented as RGC-5 FBS were treated with CHR20 (A) and (B)assay at concentrations 10 respectively for 24 0.2% h. Cell viability was determined by MTT assayCHR21 and presented as mean ˘ SDof for3,six mean ± SD for six replicates. * p < 0.05 vs. control group. and 30 μM, respectively for 24 h. Cell viability was determined by MTT assay and presented as replicates. * p < 0.05 vs. control group. mean ± SD for six replicates. * p < 0.05 vs. control group.
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Although it is not obvious from data shown in Figure 3 that CHR21 is more potent than CHR20, we previously that CHR21 more that CHR21 had Although it is not found obvious from data was shown in potent Figure than 3 thatCHR20, CHR21and is more potent than greater effects impairments in and PC12that cells [25]. had Therefore, CHR20,neuroprotective we previously found thatagainst CHR21SNP-induced was more potent than CHR20, CHR21 greater CHR21 was used as the representative compound in some of the following experiments in was the neuroprotective effects against SNP-induced impairments in PC12 cells [25]. Therefore, CHR21 current used as study. the representative compound in some of the following experiments in the current study. 2.3. Effects Effects of of CHR20/CHR21 CHR20/CHR21 after after SNP-Induced SNP-Induced Cytotoxicity at Different Duration 2.3. To investigate whether CHR20/CHR21 can protect RGC-5 cells after SNP-induced oxidative oxidative To damage at RGC-5 cells were pre-treated with with SNP at a concentration of 750 µmol/L damage at different differentdurations, durations, RGC-5 cells were pre-treated SNP at a concentration of 750 for 0.5, 1,for 2, and respectively. cells were co-treated at various concentrations of μmol/L 0.5, 41,h, 2, and 4 h, Afterwards, respectively. the Afterwards, the cells were co-treated at various CHR20/CHR21 of for CHR20/CHR21 another 24 h, respectively. Then activity was evaluated by the MTT assay. concentrations for another 24the h, MTT respectively. Then the MTT activity was As shown in 4, it assay. was found that either CHR20 attenuated the SNP-induced injury evaluated byFigure the MTT As shown in Figure 4, or it CHR21 was found that either CHR20 or CHR21 in a dose-dependent mannerinjury after exposure to SNP for 0.5, 1, 2, and 4 h, respectively. For each attenuated the SNP-induced in a dose-dependent manner after exposure to SNP for 0.5,dose, 1, 2, CHR21 betterFor neuroprotection than CHR20. effect of CHR21 was statistically and 4 h, showed respectively. each dose, CHR21 showed The better neuroprotection than CHR20. significant The effect at the concentration of 10 µmol/L (p < 0.05) andconcentration maximal neuroprotection was(pobserved at 30maximal µmol/L. of CHR21 was statistically significant at the of 10 μmol/L < 0.05) and At this concentration, we also observed significant mitochondrial stimulation. the duration of neuroprotection was observed at 30 μmol/L. At this concentration, we also When observed significant SNP exposure increased, theWhen neuroprotective effects of CHR20 and CHR21 decreased. Milder effect mitochondrial stimulation. the duration of SNP exposure increased, the neuroprotective was observed after exposed SNP for 4 Milder h. effects of CHR20 and CHR21todecreased. effect was observed after exposed to SNP for 4 h.
Figure 4. Effects Effectsof of CHR20/21 against SNP–induced in cells. RGC-5 cells.cells RGC-5 were Figure 4. CHR20/21 against SNP–induced insultsinsults in RGC-5 RGC-5 were cells pre-treated pre-treated with SNP at a concentration of 750 μmol/L for 0.5, 1, 2, and 4 h, respectively, then with SNP at a concentration of 750 µmol/L for 0.5, 1, 2, and 4 h, respectively, then exposed to exposed to various concentrations of and CHR20 (A) and CHR21 (B), for respectively, 24 h. various concentrations of CHR20 (A) CHR21 (B), respectively, another 24for h. another MTT activity MTT activity was assay. of The percentage of MTT activity was presented as was determined bydetermined MTT assay. by TheMTT percentage MTT activity was presented as mean ˘ SD for six ## p < 0.01 vs. control group, ** p < 0.01 vs. SNP-treated group. mean ± SD for six replicates. ## replicates. p < 0.01 vs. control group, ** p < 0.01 vs. SNP-treated group.
It was interesting to find that the MTT activity of RGC-5 cells increased after a short duration It was interesting to find that the MTT activity of RGC-5 cells increased after a short duration of of treatment with 750 μM SNP (0.5 and 1 h, respectively) followed by treatment with 30 μM of treatment with 750 µM SNP (0.5 and 1 h, respectively) followed by treatment with 30 µM of CHR21. CHR21. We propose that short duration of 750 μM SNP following the treatment by 30 μM CHR21 We propose that short duration of 750 µM SNP following the treatment by 30 µM CHR21 may have may have synergystic effects to stimulate the RGC-5 cells’ mitochondria activity. However, this synergystic effects to stimulate the RGC-5 cells’ mitochondria activity. However, this observation observation requires further investigation. requires further investigation. 2.4. 2.4. CHR20 CHR20 and and CHR21 CHR21 Attenuated Attenuated SNP-Induced SNP-Induced Apoptosis Apoptosis of of RGC-5 RGC-5 Cells Cells As shown in inFigure Figure5A, 5A,cells cells to groups (c) groups looked normal. However, 750SNP μM(dSNP (d As shown in in (a)(a) to (c) looked normal. However, 750 µM group) group) did induce apoptosis in RGC-5 cells. Interestingly, treatment with either CHR21 or CHR20 did induce apoptosis in RGC-5 cells. Interestingly, treatment with either CHR21 or CHR20 (30 µM), (30 μM), repectively, attenuated nuclear fragmentation, chromatin condensation, and cell apoptosis repectively, attenuated nuclear fragmentation, chromatin condensation, and cell apoptosis induced induced by SNP. CHR20 decreased apoptosis rate from 50% ± 2.3% to 25% ± 3.2%; for this CHR21, this by SNP. CHR20 decreased apoptosis rate from 50% ˘ 2.3% to 25% ˘ 3.2%; for CHR21, rate was rate 4.5%(Figure ± 0.6% 5B). (Figure 5B). 4.5%was ˘ 0.6% To confirm that CHR20/CHR21 did protect apoptosis, caspase-3 activity was To confirm that CHR20/CHR21 did protect againstagainst apoptosis, caspase-3 activity was measured. measured. Our result showed that SNP increased the amount of cleaved caspase-3 (active Our result showed that SNP increased the amount of cleaved caspase-3 (active caspase-3), while caspase-3), while pretreatment ofsignificantly CHR20/CHR21 significantly attenuated pretreatment of CHR20/CHR21 attenuated this effect (Figure this 5C).effect (Figure 5C).
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Figure 5. CHR20 and CHR21 protected RGC-5 cells from apoptosis induced by SNP. RGC-5 cells Figure 5. CHR20 and CHR21 protected RGC-5 cells from apoptosis induced by SNP. RGC-5 cells were pretreated with/without CHR20/CHR21 for 2 h, respectively, after treatment of 750 μM SNP were pretreated with/without CHR20/CHR21 for 2 h, respectively, after treatment of 750 µM SNP for 24 h and the cells were stained with Hoechst 33258 as described in Materials and Methods. (A) for 24 h and the cells were stained with Hoechst 33258 as described in Materials and Methods. Morphological changes shown by fluorescence microscope (200×) image analysis. (a) control group; (A) Morphological changes shown by fluorescence microscope (200ˆ) image analysis. (a) control group; (b) 30 μM CHR20 group; (c) 30 μM CHR21 group; (d) 750 μM SNP group; (e) 750 μM SNP + 30 μM (b) 30 µM CHR20 group; (c) 30 µM CHR21 group; (d) 750 µM SNP group; (e) 750 µM SNP + 30 µM CHR20 group; (f) 750 μM SNP + 30 μM CHR21 group. The arrow indicates nuclear fragmentation or CHR20 group; (f) 750 µM SNP + 30 µM CHR21 group. The arrow indicates nuclear fragmentation chromatin condensation; (B) (B) Histogram showing the the apoptosis raterate in RGC-5 cells. TheThe number of or chromatin condensation; Histogram showing apoptosis in RGC-5 cells. number 4 cells/well in 25 visual fields in 48-well culture plate, counted by high apoptic cells was about 1.5 × 10 4 of apoptic cells was about 1.5 ˆ 10 cells/well in 25 visual fields in 48-well culture plate, counted by content screening system (ArrayScanVTI, Thermo Fisher Scientific, Waltham, high content screening system (ArrayScanVTI, Thermo Fisher Scientific, Waltham,MA, MA,USA). USA).The The percentage of apoptotic cells was calculated as ratio of apoptotic cells and the total number percentage of apoptotic cells was calculated as ratio of apoptotic cells and the total numberof ofcells cells ## counted. counted.Data Dataare aregiven givenas as mean mean ±˘SD SDfor forthree threeindividual individualexperiments. experiments. ##pp
Molecular Sciences Article
Genipin Derivatives Protect RGC-5 from Sodium Nitroprusside-Induced Nitrosative Stress Rikang Wang 1,† , Jiaqiang Zhao 2,† , Lei Zhang 2 , Lizhi Peng 2 , Xinyi Zhang 2 , Wenhua Zheng 3, * and Heru Chen 2,4, * Received: 20 October 2015; Accepted: 8 January 2016; Published: 19 January 2016 Academic Editor: Katalin Prokai-Tatrai 1 2
3 4
* †
National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China; [email protected] Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China; [email protected] (J.Z.); [email protected] (L.Z.); [email protected] (L.P.); [email protected] (X.Z.) Faculty of Health Sciences, University of Macao, Macao, China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Guangzhou 510632, China Correspondence: [email protected] (W.Z.); [email protected] (H.C.); Tel.: +853-8822-4919 (W.Z.); +86-20-3837-5299 (H.C.); Fax: +853-8822-2314 (W.Z.); +86-20-8522-1559 (H.C.) These authors contributed equally to this work.
Abstract: CHR20 and CHR21 are a pair of stable diastereoisomers derived from genipin. These stereoisomers are activators of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In the rat retinal ganglion (RGC-5) cell model these compounds are non-toxic. Treatment of RGC-5 with 750 µM of sodium nitroprusside (SNP) produces nitrosative stress. Both genipin derivatives, however, protect these cells against SNP-induced apoptic cell death, although CHR21 is significantly more potent than CHR20 in this regard. With Western blotting we showed that the observed neuroprotection is primarily due to the activation of protein kinase B (Akt)/eNOS and extracellular signal-regulated kinase (ERK1/2) signaling pathways. Therefore, LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or PD98059 (a MAPK-activating enzyme inhibitor) abrogated the protective effects of CHR20 and CHR21. Altogether, our results show that in our experimental setup neuroprotection by the diasteromeric pair is mediated through the PI3K/Akt/eNOS and ERK1/2 signaling pathways. Further studies are needed to establish the potential of these compounds to prevent ntric oxide (NO)-induced toxicity commonly seen in many neurodegenerative diseases. Keywords: NO neurotoxicity; neuroprotection; neurodegeneration; apoptosis; genipin; nitric oxide synthase
1. Introduction Nitric oxide (NO), a Janus-faced molecule with pleiotropic effects in different tissues, is physiologically produced through the L-arginine/NO synthase (NOS) pathway. NOS is a family of four major types of enzymes including endothelial (eNOS), neuronal (nNOS), inducible (iNOS) and mitochondrial (mtNOSs). On the one hand, endogenous NO takes part in various physiological processes in the central nervous system, such as neuro-survival, neuro-differentiation, neuro-modulation, neuro-transmission and synaptic plasticity [1–3]. On the other hand, overproduction of NO produces oxidative and nitrosative stress [4–7]. Excess NO also induces nitrotyrosination, leading to the inhibition of tyrosine phosphorylation and blockade of the signal transduction pathways of growth factors [8]. Solid evidence supports that all
Int. J. Mol. Sci. 2016, 17, 117; doi:10.3390/ijms17010117
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Int. J. Mol. Sci. 2016, 17, 117
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transduction pathways Int. J. Mol. Sci. 2016, 17, 117
of growth factors [8]. Solid evidence supports that all neurodegenerative 2 of 16 diseases are closely related to the overproduction of NO [9–13]. For example, when nNOS is activated with persistent stimulation of excitatory amino acid receptors mediating glutamate toxicity, as well as neurodegenerative diseasesbyarediverse closely related the endotoxin overproduction of NO [9–13]. example, when when iNOS is induced stimuli tolike or cytokines, NO For may probably be nNOS is activated with persistent stimulation of excitatory amino acid receptors mediating glutamate overproduced [9]. In these cases, the role of NO changes from a physiological neuromodulator to a toxicity, as well as when iNOS is induced by diverse stimuli like endotoxin or cytokines, NO may probably neurotoxic factor. be overproduced [9]. In these cases,ofthe roleinduces of NO changes from a physiological a No doubt, overproduction NO oxidative stress. That is aneuromodulator risk factor for to the neurotoxic factor. development of neurodegenerative diseases including glaucomatous visual field deterioration doubt, overproduction of NO induces stress. That is a risk factor for the development [5–7].NoUnfortunately, retinal ganglion cells oxidative (RGCs) are susceptible to oxidative stress that may of neurodegenerative diseases including glaucomatous visual field deterioration [5–7]. Unfortunately, trigger cell apoptosis [7,14,15]. In a variety of retinal degenerative diseases, such as glaucoma and retinal ganglion cells (RGCs) are susceptible to oxidative stress that may cell apoptosis [7,14,15]. diabetic retinopathy, this oxidative stress-induced RGC apoptosis is trigger a fundamental pathogenesis In a variety of retinal degenerative diseases, such as glaucoma and diabetic retinopathy, this oxidative [15–17]. Although the rat retinal ganglion (RGC-5) cell line is actually misidentified [18], we believe stress-induced RGC apoptosis is a fundamental pathogenesis [15–17]. Although the rat retinal that it still possesses the fundamental properties of the retinal neuronal precursor cells andganglion hence is (RGC-5) cellour linestudies. is actually misidentified that still the possesses the fundamental suitable for In this way, RGC-5[18], cellswe canbelieve be used to itstudy pathogenesis process of properties of the retinal neuronal precursor cells and hencenitric is suitable our studies. In this way, oxidative/nitrosative stress induced by an exogenous oxideforagent, such as, sodium RGC-5 cells can be used to study the pathogenesis process of oxidative/nitrosative stress induced by nitroprusside (SNP). an exogenous oxide agent, such as, sodium nitroprusside (SNP).of nNOS for the treatment of Recently,nitric application of several types of selective inhibitors Recently, application of several types of selective inhibitors of nNOS for the based treatment of central central nervous system (CNS) disorders have been reported [19–22]. Evidences on the rodent nervous disorders have been reportedpain [19–22]. basedmodel on theofrodent model model ofsystem dural (CNS) inflammation relevant to migraine and Evidences the rat Chung neuropathic of dural inflammation relevant to migraine pain and the rat model of neuropathic pain pain confirmed that some 3,4-dihydroquinolin-2(1H)-one andChung 1,2,3,4-tetrahydroquinoline-based confirmed that some 3,4-dihydroquinolin-2(1H)-one and 1,2,3,4-tetrahydroquinoline-based derivatives derivatives had significant efficacy to relieve the pains [22]. As a matter of fact, our research group had efficacy to relievegardenamide the pains [22].AAs a matter of was fact, confirmed our research has synthesized has significant synthesized and identified (GA), which asgroup an activator of nNOS and gardenamide A (GA), which was confirmed as an activator nNOSserum and eNOS [23]. and identified eNOS [23]. Quite interestingly, GA showed neuroprotective effects of against deprived Quite interestingly, GA cells showed effects against serum in PC12 impairments in PC12 andneuroprotective H2O2-induced insults in RGC-5 cellsdeprived [24,25]. impairments The neuroprotective cells and H2 Oinvolved insults in RGC-5 [24,25]. The neuroprotective mechanisms 2 -inducedare mechanisms closely relatedcells to the activations of ERK1/2 and PI3K/Akt involved signaling are closely related the activations ERK1/2 and PI3K/Akt signaling pathways. pathways. Anothertotwo new stable ofgenipin derivatives, CHR20 (1-R isomer) and Another CHR21 two (1-S new stable genipin CHR20 (1-R isomer) and CHR21 (1-S isomer), which are a pair of isomer), which are aderivatives, pair of diastereoisomers, and confirmed as nNOS and eNOS activators have diastereoisomers, and confirmed as nNOS and eNOS activators have also been synthesized by us [26] also been synthesized by us [26] (Figure 1). They protected PC12 cells from apoptosis induced by (Figure 1). They protected PC12oxidative cells fromstress apoptosis induced SNP. SNP wasincrease found to raise oxidative SNP. SNP was found to raise in PC12 cells by and CHR20/21 mRNA levels of stress in PC12 cells and CHR20/21 increase mRNA levelsand of glutamate-cysteine ligase catalytic subunit glutamate-cysteine ligase catalytic subunit (GCLC) superoxide dismutase 1 (SOD1) in a (GCLC) and superoxide dismutase 1 (SOD1) in a time-dependent manner [26]. time-dependent manner [26].
Figure 1.1.Chemical Chemical structures of genipin and CHR20/21. “a” is ina the symbol the IUPAC’ Figure structures of genipin and CHR20/21. “a” is a symbol IUPAC’innomemclature nomemclature rule to represent the first atom linked to the outside ring. rule to represent the first atom linked to the outside ring.
In the present study we wished to investigate whether CHR20 and CHR21 can prevent RGC-5 In the present study we wished to investigate whether CHR20 and CHR21 can prevent RGC-5 cell death brought about by SNP-produced oxidative/nitrosative insults. cell death brought about by SNP-produced oxidative/nitrosative insults. 2. Results 2. Resultsand andDiscussion Discussion 2.1. Effects Effects of of SNP SNP on on RGC-5 RGC-5 Cells Cells 2.1. As shown shown in in Figure Figure 2, 2, SNP SNP dose-dependently dose-dependently influenced influenced RGC-5 RGC-5 viability. viability. When When RGC-5 RGC-5 cells cells As were treated with SNP at a concentration between 62.5 to 250 μmol/L, their MTT activities increased were treated with SNP at a concentration between 62.5 to 250 µmol/L, their MTT activities increased with SNP SNPconcentration, concentration,in inwhich whichMTT MTTmeans means3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium tetrazolium with bromide. This maximal at at 250 250 µmol/L. μmol/L. Because MTT activity activity is is bromide. This increasing increasing effect effect went went to to the the maximal Because MTT dependent on NADPH-dependent oxido-reductase enzymes largely in the cytosolic compartment of
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dependent on NADPH-dependent oxido-reductase enzymes largely in the cytosolic compartment the cell [27,28], underunder defined conditions, it may it reflect number presentofviable cells.viable Therefore, ofdependent the cell [27,28], defined conditions, maythe reflect the of number present cells. on NADPH-dependent oxido-reductase enzymes largely in the cytosolic compartment SNP at these concentrations showed no cytotoxicity to RGC-5 cells. Under these conditions, cell Therefore, SNP at these concentrations showed no cytotoxicity to RGC-5 cells. Under these conditions, of the cell [27,28], under defined conditions, it may reflect the number of present viable cells. viability seems to increase withwith increasing SNP concentration. However, atathigh concentrations cell viability seems to increase increasing concentration. However, high Therefore, SNP at these concentrations showed noSNP cytotoxicity to RGC-5 cells. Under theseconcentrations conditions, (750 and 1000 µmol/L), SNP was cytotoxic; exposure to 750 µmol/L of SNP induced injury to (750 and 1000 μmol/L), SNP was cytotoxic; exposure to 750 μmol/L of SNP induced injury to the the cells. cells. cell viability seems to increase with increasing SNP concentration. However, at high concentrations The MTT activity was only 41.0% ˘ 4.8% compared with that of the control group. The MTT activity was only 41.0% ± 4.8% compared with that of the control group. (750 and 1000 μmol/L), SNP was cytotoxic; exposure to 750 μmol/L of SNP induced injury to the cells. The MTT activity was only 41.0% ± 4.8% compared with that of the control group.
Figure2.2. The The neuro-effects of SNP on RGC-5 cells. Cells were exposed totodifferent concentrations of Figure SNP on onRGC-5 RGC-5cells. cells.Cells Cells were exposed different concentrations Figure 2. Theneuro-effects neuro-effects of of SNP were exposed to different concentrations of SNP asas indicated forfor 24 h. Cell viability was evaluated byby MTT assay. The values were expressed as of SNP indicated Cell viability was evaluated MTT assay. values expressed SNP as indicated for 2424h.h.Cell viability was evaluated by MTT assay. The The values werewere expressed as percentage of control, which is set to 100%. The percentage of MTT activity was presented as mean ± as percentage percentageofof control, which is to set100%. to 100%. The percentage MTT was activity was presented control, which is set The percentage of MTTof activity presented as mean ± as SD for six replicates. * p < 0.05 vs. control group. mean ˘ SD six replicates. * pvs.< control 0.05 vs.group. control group. SD for six for replicates. * p < 0.05
It isisquite interesting that low concentrationofofSNP SNP (≤250μM) μM) stimulates RGC-5 cell viability. quiteinteresting interestingthat thatlow low concentration concentration RGC-5 cellcell viability. It It is quite of SNP(≤250 (ď250 µM)stimulates stimulates RGC-5 viability. We did observe cell proliferation under microscopy. microscopy. However,the the exact mechanism needs to be observecell cellproliferation proliferationunder under needs to to be WeWe diddid observe microscopy. However, However, theexact exactmechanism mechanism needs be further investigated. further investigated. further investigated. 2.2. Cytotoxicity and MTT Activity Enhancement CHR20/21tototoRGC-5 RGC-5 Cells 2.2. Cytotoxicityand andMTT MTTActivity ActivityEnhancement Enhancement of Cells 2.2. Cytotoxicity ofofCHR20/21 CHR20/21 RGC-5 Cells To examine the cellular tolerance toCHR20/CHR21, CHR20/CHR21,RGC-5 RGC-5cells cells were cultured inRPMI-1640 RPMI-1640 To examinethe thecellular cellulartolerance tolerance to to CHR20/CHR21, were cultured inin RPMI-1640 To examine RGC-5 cells were cultured mediumwith with10% 10%fetal fetal bovine bovine serum (FBS) or serum (0.2% ofofFBS) forfor 2424 h. h. Then RGC-5 cellscells medium (FBS) orlow low serum (0.2% FBS) Then RGC-5 medium with 10% fetal bovine serum (FBS) or low serum (0.2% of FBS) for 24 h. Then RGC-5 cells with withlow lowserum serum were were treated treated with the test compounds atatconcentrations of of 3, 3, 10 10 andand 30 30 μM,μM, with the test compounds concentrations low serum were treated with the test compounds at concentrations of 3, 10 and 30 µM, respectively, respectively, for 24 h. As shown in Figure 3, CHR20 and CHR21 did not show any toxicity at at respectively, for 24in h. As shown in Figure 3, CHR20 and CHR21 did notatshow any toxicity for 24 h. As shown Figure 3, CHR20 and CHR21 did not show any toxicity concentrations up to concentrations up to 30 μM. On the contrary, MTT activity of RGC-5 cells increased in the presence concentrations up to 30MTT μM. activity On the contrary, activity ofinRGC-5 cells increased in and the presence 30 µM. On the contrary, of RGC-5MTT cells increased the presence of CHR20 CHR21, ofCHR20 CHR20and andCHR21, CHR21, respectively, respectively, at aa dose of 30 μM. This evidence suggests that each of these of at dose of 30 μM. This evidence suggests that each of these respectively, atcould a doseexert of 30a µM. This evidence suggests that each of these compounds could exert a compounds neuroprotective effect and stimulated cell survival under low serum compounds could exert astimulated neuroprotective effectunder and stimulated cell survival under lowfurther serum neuroprotective effect and cell survival low serum environment. However, environment.However, However, further investigations investigations are totoconfirm interpretation of this environment. are required required confirmthethe interpretation of this investigations are requiredfurther to confirm the interpretation of this finding. finding. finding.
Figure 3. Cytotoxicity and MTT activity of CHR20 and CHR21 on RGC-5 cells. Cells that were Figure 3. Cytotoxicity and MTT activity of CHR20 and CHR21 on RGC-5 cells. Cells that were cultured cultured RPMI-1640 and medium low serum (0.2% of FBS) for 24 h were set ascells. the control group. Figure 3. in Cytotoxicity MTTwith activity of CHR20 and Cells that were in RPMI-1640 medium with low serum (0.2% of FBS) for 24CHR21 h were on set RGC-5 as the control group. RGC-5 RGC-5 cells with 0.2% medium FBS werewith treated with CHR20 (A) and CHR21 (B) at concentrations of 3,group. 10 cultured in RPMI-1640 low serum (0.2% of FBS) for 24 h were set as the control cells with 0.2% FBS were treated with CHR20 (A) and CHR21 (B) at concentrations of 3, 10 and 30 µM, and 30cells μM,with respectively for 24 h. Cell viability was determined by MTT and presented as RGC-5 FBS were treated with CHR20 (A) and (B)assay at concentrations 10 respectively for 24 0.2% h. Cell viability was determined by MTT assayCHR21 and presented as mean ˘ SDof for3,six mean ± SD for six replicates. * p < 0.05 vs. control group. and 30 μM, respectively for 24 h. Cell viability was determined by MTT assay and presented as replicates. * p < 0.05 vs. control group. mean ± SD for six replicates. * p < 0.05 vs. control group.
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Although it is not obvious from data shown in Figure 3 that CHR21 is more potent than CHR20, we previously that CHR21 more that CHR21 had Although it is not found obvious from data was shown in potent Figure than 3 thatCHR20, CHR21and is more potent than greater effects impairments in and PC12that cells [25]. had Therefore, CHR20,neuroprotective we previously found thatagainst CHR21SNP-induced was more potent than CHR20, CHR21 greater CHR21 was used as the representative compound in some of the following experiments in was the neuroprotective effects against SNP-induced impairments in PC12 cells [25]. Therefore, CHR21 current used as study. the representative compound in some of the following experiments in the current study. 2.3. Effects Effects of of CHR20/CHR21 CHR20/CHR21 after after SNP-Induced SNP-Induced Cytotoxicity at Different Duration 2.3. To investigate whether CHR20/CHR21 can protect RGC-5 cells after SNP-induced oxidative oxidative To damage at RGC-5 cells were pre-treated with with SNP at a concentration of 750 µmol/L damage at different differentdurations, durations, RGC-5 cells were pre-treated SNP at a concentration of 750 for 0.5, 1,for 2, and respectively. cells were co-treated at various concentrations of μmol/L 0.5, 41,h, 2, and 4 h, Afterwards, respectively. the Afterwards, the cells were co-treated at various CHR20/CHR21 of for CHR20/CHR21 another 24 h, respectively. Then activity was evaluated by the MTT assay. concentrations for another 24the h, MTT respectively. Then the MTT activity was As shown in 4, it assay. was found that either CHR20 attenuated the SNP-induced injury evaluated byFigure the MTT As shown in Figure 4, or it CHR21 was found that either CHR20 or CHR21 in a dose-dependent mannerinjury after exposure to SNP for 0.5, 1, 2, and 4 h, respectively. For each attenuated the SNP-induced in a dose-dependent manner after exposure to SNP for 0.5,dose, 1, 2, CHR21 betterFor neuroprotection than CHR20. effect of CHR21 was statistically and 4 h, showed respectively. each dose, CHR21 showed The better neuroprotection than CHR20. significant The effect at the concentration of 10 µmol/L (p < 0.05) andconcentration maximal neuroprotection was(pobserved at 30maximal µmol/L. of CHR21 was statistically significant at the of 10 μmol/L < 0.05) and At this concentration, we also observed significant mitochondrial stimulation. the duration of neuroprotection was observed at 30 μmol/L. At this concentration, we also When observed significant SNP exposure increased, theWhen neuroprotective effects of CHR20 and CHR21 decreased. Milder effect mitochondrial stimulation. the duration of SNP exposure increased, the neuroprotective was observed after exposed SNP for 4 Milder h. effects of CHR20 and CHR21todecreased. effect was observed after exposed to SNP for 4 h.
Figure 4. Effects Effectsof of CHR20/21 against SNP–induced in cells. RGC-5 cells.cells RGC-5 were Figure 4. CHR20/21 against SNP–induced insultsinsults in RGC-5 RGC-5 were cells pre-treated pre-treated with SNP at a concentration of 750 μmol/L for 0.5, 1, 2, and 4 h, respectively, then with SNP at a concentration of 750 µmol/L for 0.5, 1, 2, and 4 h, respectively, then exposed to exposed to various concentrations of and CHR20 (A) and CHR21 (B), for respectively, 24 h. various concentrations of CHR20 (A) CHR21 (B), respectively, another 24for h. another MTT activity MTT activity was assay. of The percentage of MTT activity was presented as was determined bydetermined MTT assay. by TheMTT percentage MTT activity was presented as mean ˘ SD for six ## p < 0.01 vs. control group, ** p < 0.01 vs. SNP-treated group. mean ± SD for six replicates. ## replicates. p < 0.01 vs. control group, ** p < 0.01 vs. SNP-treated group.
It was interesting to find that the MTT activity of RGC-5 cells increased after a short duration It was interesting to find that the MTT activity of RGC-5 cells increased after a short duration of of treatment with 750 μM SNP (0.5 and 1 h, respectively) followed by treatment with 30 μM of treatment with 750 µM SNP (0.5 and 1 h, respectively) followed by treatment with 30 µM of CHR21. CHR21. We propose that short duration of 750 μM SNP following the treatment by 30 μM CHR21 We propose that short duration of 750 µM SNP following the treatment by 30 µM CHR21 may have may have synergystic effects to stimulate the RGC-5 cells’ mitochondria activity. However, this synergystic effects to stimulate the RGC-5 cells’ mitochondria activity. However, this observation observation requires further investigation. requires further investigation. 2.4. 2.4. CHR20 CHR20 and and CHR21 CHR21 Attenuated Attenuated SNP-Induced SNP-Induced Apoptosis Apoptosis of of RGC-5 RGC-5 Cells Cells As shown in inFigure Figure5A, 5A,cells cells to groups (c) groups looked normal. However, 750SNP μM(dSNP (d As shown in in (a)(a) to (c) looked normal. However, 750 µM group) group) did induce apoptosis in RGC-5 cells. Interestingly, treatment with either CHR21 or CHR20 did induce apoptosis in RGC-5 cells. Interestingly, treatment with either CHR21 or CHR20 (30 µM), (30 μM), repectively, attenuated nuclear fragmentation, chromatin condensation, and cell apoptosis repectively, attenuated nuclear fragmentation, chromatin condensation, and cell apoptosis induced induced by SNP. CHR20 decreased apoptosis rate from 50% ± 2.3% to 25% ± 3.2%; for this CHR21, this by SNP. CHR20 decreased apoptosis rate from 50% ˘ 2.3% to 25% ˘ 3.2%; for CHR21, rate was rate 4.5%(Figure ± 0.6% 5B). (Figure 5B). 4.5%was ˘ 0.6% To confirm that CHR20/CHR21 did protect apoptosis, caspase-3 activity was To confirm that CHR20/CHR21 did protect againstagainst apoptosis, caspase-3 activity was measured. measured. Our result showed that SNP increased the amount of cleaved caspase-3 (active Our result showed that SNP increased the amount of cleaved caspase-3 (active caspase-3), while caspase-3), while pretreatment ofsignificantly CHR20/CHR21 significantly attenuated pretreatment of CHR20/CHR21 attenuated this effect (Figure this 5C).effect (Figure 5C).
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Figure 5. CHR20 and CHR21 protected RGC-5 cells from apoptosis induced by SNP. RGC-5 cells Figure 5. CHR20 and CHR21 protected RGC-5 cells from apoptosis induced by SNP. RGC-5 cells were pretreated with/without CHR20/CHR21 for 2 h, respectively, after treatment of 750 μM SNP were pretreated with/without CHR20/CHR21 for 2 h, respectively, after treatment of 750 µM SNP for 24 h and the cells were stained with Hoechst 33258 as described in Materials and Methods. (A) for 24 h and the cells were stained with Hoechst 33258 as described in Materials and Methods. Morphological changes shown by fluorescence microscope (200×) image analysis. (a) control group; (A) Morphological changes shown by fluorescence microscope (200ˆ) image analysis. (a) control group; (b) 30 μM CHR20 group; (c) 30 μM CHR21 group; (d) 750 μM SNP group; (e) 750 μM SNP + 30 μM (b) 30 µM CHR20 group; (c) 30 µM CHR21 group; (d) 750 µM SNP group; (e) 750 µM SNP + 30 µM CHR20 group; (f) 750 μM SNP + 30 μM CHR21 group. The arrow indicates nuclear fragmentation or CHR20 group; (f) 750 µM SNP + 30 µM CHR21 group. The arrow indicates nuclear fragmentation chromatin condensation; (B) (B) Histogram showing the the apoptosis raterate in RGC-5 cells. TheThe number of or chromatin condensation; Histogram showing apoptosis in RGC-5 cells. number 4 cells/well in 25 visual fields in 48-well culture plate, counted by high apoptic cells was about 1.5 × 10 4 of apoptic cells was about 1.5 ˆ 10 cells/well in 25 visual fields in 48-well culture plate, counted by content screening system (ArrayScanVTI, Thermo Fisher Scientific, Waltham, high content screening system (ArrayScanVTI, Thermo Fisher Scientific, Waltham,MA, MA,USA). USA).The The percentage of apoptotic cells was calculated as ratio of apoptotic cells and the total number percentage of apoptotic cells was calculated as ratio of apoptotic cells and the total numberof ofcells cells ## counted. counted.Data Dataare aregiven givenas as mean mean ±˘SD SDfor forthree threeindividual individualexperiments. experiments. ##pp
