Genipin Derivatives Protect RGC-5 from Sodium Nitroprusside-Induced Nitrosative Stress

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; apoptosis; genipin; neurodegeneration; neuroprotection; nitric oxide synthase.

Figure 1

Chemical structures of genipin and CHR20/21. “a” is a symbol in the IUPAC’ nomemclature rule to represent the first atom linked to the outside ring.

Figure 2

The neuro-effects of SNP on RGC-5 cells. Cells were exposed to different concentrations of SNP as indicated for 24 h. Cell viability was evaluated by MTT assay. The values were expressed as percentage of control, which is set to 100%. The percentage of MTT activity was presented as mean ± SD for six replicates. * p < 0.05 vs. control group.

Figure 3

Cytotoxicity and MTT activity of CHR20 and CHR21 on RGC-5 cells. Cells that were cultured in RPMI-1640 medium with low serum (0.2% of FBS) for 24 h were set as the control group. RGC-5 cells with 0.2% FBS were treated with CHR20 (A) and CHR21 (B) at concentrations of 3, 10 and 30 µM, respectively for 24 h. Cell viability was determined by MTT assay and presented as mean ± SD for six replicates. * p < 0.05 vs. control group.

Figure 4

Effects of CHR20/21 against SNP–induced insults in RGC-5 cells. RGC-5 cells were pre-treated with SNP at a concentration of 750 µmol/L for 0.5, 1, 2, and 4 h, respectively, then exposed to various concentrations of CHR20 (A) and CHR21 (B), respectively, for another 24 h. MTT activity was determined by MTT assay. The percentage of MTT activity was presented as mean ± SD for six replicates. ^## p < 0.01 vs. control group, ** p < 0.01 vs. SNP-treated group.

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 for 24 h and the cells were stained with Hoechst 33258 as described in Materials and Methods. (A) Morphological changes shown by fluorescence microscope (200×) 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 CHR20 group; (f) 750 μM SNP + 30 μM CHR21 group. The arrow indicates nuclear fragmentation or chromatin condensation; (B) Histogram showing the apoptosis rate in RGC-5 cells. The number of apoptic cells was about 1.5 × 10⁴ cells/well in 25 visual fields in 48-well culture plate, counted by high content screening system (ArrayScanVTI, Thermo Fisher Scientific, Waltham, MA, USA). The percentage of apoptotic cells was calculated as ratio of apoptotic cells and the total number of cells counted. Data are given as mean ± SD for three individual experiments. ^## p < 0.05 vs. control group. ** p < 0.05 vs. SNP group; (C) Effects of CHR20/CHR21 on cleaved caspase-3. The expression of cleaved caspase-3 was determined by western blotting as described in Materials and Methods.

Figure 6

CHR20/CHR21 increase phosphorylation levels in PI3K/Akt and MEK/ERK1/2. RGC-5 cells were treated with 10 μM CHR20 and CHR21, repectively for 5–80 min or 0.3–30 μM CHR20/CHR21 for 40 min. By applying Western blotting with antibodies including anti-phospho-Akt (Ser473), anti-phospho-ERK1/2, anti-phospho-and eNOS (Ser1177), respectively, the phosphorylation of the relevant proteins were determined. (A) CHR20 and CHR21 time-dependently increased the phosphorylation levels of Akt and ERK1/2 in RGC-5 cells; (B) CHR20 and CHR21 dose-dependently induced the phosphorylation of Akt and ERK1/2 in RGC-5 cells; (C) Effects of pathway inhibitors on the phosphorylation of Akt, eNOS and ERK1/2 affected by CHR21. The density of the blot in each lane was presented as mean ± standard deviation. Each data was calculated based on at least three individual experiments. * p < 0.05, ** p < 0.01 vs. control group. ^# p < 0.05, ^## p < 0.01 vs. CHR21 pre-treated group. Blots were quantified using ImageJ software. pd: PD98059; ly: LY294002; p-eNOS: phosphorylated protein level of eNOS; pAkt: phosphorylated protein level of Akt; pErk: phosphorylated protein level of ERK; CHR: abbreviation of Chen HeRu; T-Erk: total protein level of ERK; p-NOS: phosphorylated protein level of NOS; T-NOS: total protein level of NOS.

Figure 6

CHR20/CHR21 increase phosphorylation levels in PI3K/Akt and MEK/ERK1/2. RGC-5 cells were treated with 10 μM CHR20 and CHR21, repectively for 5–80 min or 0.3–30 μM CHR20/CHR21 for 40 min. By applying Western blotting with antibodies including anti-phospho-Akt (Ser473), anti-phospho-ERK1/2, anti-phospho-and eNOS (Ser1177), respectively, the phosphorylation of the relevant proteins were determined. (A) CHR20 and CHR21 time-dependently increased the phosphorylation levels of Akt and ERK1/2 in RGC-5 cells; (B) CHR20 and CHR21 dose-dependently induced the phosphorylation of Akt and ERK1/2 in RGC-5 cells; (C) Effects of pathway inhibitors on the phosphorylation of Akt, eNOS and ERK1/2 affected by CHR21. The density of the blot in each lane was presented as mean ± standard deviation. Each data was calculated based on at least three individual experiments. * p < 0.05, ** p < 0.01 vs. control group. ^# p < 0.05, ^## p < 0.01 vs. CHR21 pre-treated group. Blots were quantified using ImageJ software. pd: PD98059; ly: LY294002; p-eNOS: phosphorylated protein level of eNOS; pAkt: phosphorylated protein level of Akt; pErk: phosphorylated protein level of ERK; CHR: abbreviation of Chen HeRu; T-Erk: total protein level of ERK; p-NOS: phosphorylated protein level of NOS; T-NOS: total protein level of NOS.

Figure 7

Effects of NOS on the action of CHR21. (A) Endothelial nitrioxide synthase (eNOS) mediates the action of CHR21 on MTT activity in RGC-5 cells. Cells pre-exposed to 7-nitroindazole (7-NI, 50 μM, 30 min), an specific inhibitor for neuronal nitric oxide synthase (nNOS), N(5)-(-iminoethyl)-l-ornithine (l-NIO, 100 μM, 30 min), an inhibitor of endothelial NOS (eNOS), and compound 1400-W (20 μM, 30 min), a potent inhibitor of inducible NOS (iNOS), respectively, were treated with CHR20/CHR21. The MTT activity of cells determined by MTT assay. Only l-NIO attenuated the protective effects of CHR20/21 on RGC-5 cells with statistically significant; while 7-NIO and 1400 W had no statistically significant effect. ^&& p < 0.01 vs. control; ^## p < 0.01 vs. SNP group; * p < 0.05 vs. CHR20 + SNP or CHR21 + SNP (n = 3); (B) Effects of 7-NI, l-NIO and 1400 W on the NOS activities in RGC-5 cells. RGC-5 cells were treated with 7-NI, l-NIO and 1400 W, respectively. By applying Typed NOS Detection Kit, determination of the activities of nNOS, eNOS (endothelial NOS) and iNOS was carried out. One nmol of NO formed per mL cell lysate in one minute was defined as U/mL. With * p < 0.05 vs. control group, the difference is considered statistically significant. All values are expressed as mean ± SD (n = 3).

Figure 8

Both the PI3K/Akt/eNOS pathway and the ERK1/2 pathways were involved in the neuroprotective effects of CHR20/21. Pre-incubation of LY294002, Akt VIII inhibitor, and PD98059 at a dose of 10 µM, respectively blocked the neuroprotective effects of CHR20 (A) and CHR21 (B) in RGC-5 cells. MTT activity was determined by MTT assay. Each experiment was performed in triplicate. ^## p < 0.01 vs. SNP group; ** p < 0.01 vs. CHR20/CHR21 + SNP group; ^& p < 0.05, ^&& p < 0.01 vs. CHR20/CHR21 + PD group, or CHR20/CHR21 + LY group, respectively; (C) SNP inhibited the phosphorylations of Akt, eNOS and ERK1/2, respectively. And LY294002 and PD98059 reversed the effects of CHR21. The density of each lane was presented as mean ± standard deviation for three individual experiments. * p < 0.05 vs. control group, ^&& p < 0.01 vs. SNP group, ^## p < 0.01 vs. CHR21 + SNP group. Blots were quantified using Image J software. Similar results were obtained with CHR20 (data not shown).

Figure 8

Both the PI3K/Akt/eNOS pathway and the ERK1/2 pathways were involved in the neuroprotective effects of CHR20/21. Pre-incubation of LY294002, Akt VIII inhibitor, and PD98059 at a dose of 10 µM, respectively blocked the neuroprotective effects of CHR20 (A) and CHR21 (B) in RGC-5 cells. MTT activity was determined by MTT assay. Each experiment was performed in triplicate. ^## p < 0.01 vs. SNP group; ** p < 0.01 vs. CHR20/CHR21 + SNP group; ^& p < 0.05, ^&& p < 0.01 vs. CHR20/CHR21 + PD group, or CHR20/CHR21 + LY group, respectively; (C) SNP inhibited the phosphorylations of Akt, eNOS and ERK1/2, respectively. And LY294002 and PD98059 reversed the effects of CHR21. The density of each lane was presented as mean ± standard deviation for three individual experiments. * p < 0.05 vs. control group, ^&& p < 0.01 vs. SNP group, ^## p < 0.01 vs. CHR21 + SNP group. Blots were quantified using Image J software. Similar results were obtained with CHR20 (data not shown).