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Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase

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ABSTRACT

Background: Retinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats.

Methods: High intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120 mmHg for 60 min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7 days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4 % paraformaldehyde and 30 % sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3 mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2 g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4 μL of 0.5 mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15 min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised.

Results: The ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2 g/kg/day of CJDHW (Group 4; ERG: P < 0.001 on I/R day 7; HE stain: P < 0.001 on I/R day 7; TUNEL: P = 0.05 on I/R day 7; retrograde labeling: P = 0.007 on I/R day 7; Thy-1 mRNA: P = 0.02; MMP-9 mRNA: P < 0.001; Bcl-2 protein: P = 0.02; HO-1 protein: P = 0.03; P-p38 MAPK protein: P < 0.001; MMP-9 protein: P = 0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38 ± 0.04 vs. 0.81 ± 0.03), increased inner retinal thickness (45.08 ± 2.85 vs. 67.98 ± 5.48 μm), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40 ± 0.55 vs. 0.60 ± 0.55), and more numerous retinal ganglion cells (887.73 ± 158.18 vs. 1389.02 ± 53.20). Moreover, increased Thy-1 (0.31 ± 0.15 vs. 0.78 ± 0.32) and decreased MMP-9 mRNA levels were found (4.44 ± 0.84 vs. 1.13 ± 0.34), respectively. Furthermore, the Bcl-2 protein level (0.78 ± 0.08 vs. 1.80 ± 0.34) was increased while the HO-1 (0.99 ± 0.20 vs. 4.15 ± 2.08), P-p38 MAPK (1.12 ± 0.18 vs. 0.57 ± 0.18) and MMP-9 levels were decreased (0.70 ± 0.23 vs. 0.39 ± 0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5 mM SB203580 (P-p38 MAPK: 1.12 ± 0.18 vs. 0.18 ± 0.07, P < 0.001; MMP-9: 0.70 ± 0.23 vs. 0.21 ± 0.07, P = 0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03 ± 1.57 vs. 1.59 ± 0.47, P = 0.002; Group 2 vs. 5: 5.03 ± 1.57 vs. 1.35 ± 0.41, P = 0.001).

Conclusion: Treatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.

Electronic supplementary material: The online version of this article (doi:10.1186/s13020-016-0109-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Fluorogold-labeling. The microscopic images show the density of retinal ganglion cells (RGCs) a and 7 days after the sham-procedure (f; Group 1), or b and 7 days of reperfusion following ischemia (I/R) with preadministered vehicle (g; Group 2) or Chi-Ju-Di-Huang-Wan at 2.8 g/kg/day (Group 3; on I/R day 1, c; on I/R day 7, h) and at 4.2 g/kg/day (Group 4; on I/R day 1, d; on I/R day 7, i). Each bar indicates the mean ± SD (e; j; n = 3). **indicates significant difference (P < 0.01; Group 1 vs. 2 on I/R day 1 or 7); †indicates significant difference (P < 0.05; Group 2 vs. 4 on I/R day 1 or 7). Scale bars = 25 μm
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Fig5: Fluorogold-labeling. The microscopic images show the density of retinal ganglion cells (RGCs) a and 7 days after the sham-procedure (f; Group 1), or b and 7 days of reperfusion following ischemia (I/R) with preadministered vehicle (g; Group 2) or Chi-Ju-Di-Huang-Wan at 2.8 g/kg/day (Group 3; on I/R day 1, c; on I/R day 7, h) and at 4.2 g/kg/day (Group 4; on I/R day 1, d; on I/R day 7, i). Each bar indicates the mean ± SD (e; j; n = 3). **indicates significant difference (P < 0.01; Group 1 vs. 2 on I/R day 1 or 7); †indicates significant difference (P < 0.05; Group 2 vs. 4 on I/R day 1 or 7). Scale bars = 25 μm

Mentions: As shown in Fig. 5 and Table 5 (n = 3), the RGC densities were 1952.16 ± 125.29 (sham 1 day) and 1737.23 ± 151.94 cells/mm2 (sham 7 day) 1 and 7 days after the sham procedure (Group 1). In contrast, the RGC densities 1 and 7 days after retinal ischemia pretreated with vehicle decreased significantly to 929.01 ± 135.00 (Group 2 on I/R day 1; P < 0.001) and 887.73 ± 158.18 (Group 2 on I/R day 7; P = 0.003), respectively. Notably, this decrease was alleviated in a dose-dependent manner (with a small effect at 2.8 g/kg/day; Group 3 on I/R day 1: 1112.65 ± 164.10 cells/mm2, P = 0.21; Group 3 on I/R day 7: 941.89 ± 38.91, P = 0.60). The extent of this alleviation effect was significantly and much more pronounced after pretreating rats with a high dose of CJDHW (Group 4 on I/R day 1: 1691.36 ± 237.57, P = 0.008; Group 4 on I/R day 7: 1389.02 ± 53.20, P = 0.007).Fig. 5


Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase
Fluorogold-labeling. The microscopic images show the density of retinal ganglion cells (RGCs) a and 7 days after the sham-procedure (f; Group 1), or b and 7 days of reperfusion following ischemia (I/R) with preadministered vehicle (g; Group 2) or Chi-Ju-Di-Huang-Wan at 2.8 g/kg/day (Group 3; on I/R day 1, c; on I/R day 7, h) and at 4.2 g/kg/day (Group 4; on I/R day 1, d; on I/R day 7, i). Each bar indicates the mean ± SD (e; j; n = 3). **indicates significant difference (P < 0.01; Group 1 vs. 2 on I/R day 1 or 7); †indicates significant difference (P < 0.05; Group 2 vs. 4 on I/R day 1 or 7). Scale bars = 25 μm
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Fig5: Fluorogold-labeling. The microscopic images show the density of retinal ganglion cells (RGCs) a and 7 days after the sham-procedure (f; Group 1), or b and 7 days of reperfusion following ischemia (I/R) with preadministered vehicle (g; Group 2) or Chi-Ju-Di-Huang-Wan at 2.8 g/kg/day (Group 3; on I/R day 1, c; on I/R day 7, h) and at 4.2 g/kg/day (Group 4; on I/R day 1, d; on I/R day 7, i). Each bar indicates the mean ± SD (e; j; n = 3). **indicates significant difference (P < 0.01; Group 1 vs. 2 on I/R day 1 or 7); †indicates significant difference (P < 0.05; Group 2 vs. 4 on I/R day 1 or 7). Scale bars = 25 μm
Mentions: As shown in Fig. 5 and Table 5 (n = 3), the RGC densities were 1952.16 ± 125.29 (sham 1 day) and 1737.23 ± 151.94 cells/mm2 (sham 7 day) 1 and 7 days after the sham procedure (Group 1). In contrast, the RGC densities 1 and 7 days after retinal ischemia pretreated with vehicle decreased significantly to 929.01 ± 135.00 (Group 2 on I/R day 1; P < 0.001) and 887.73 ± 158.18 (Group 2 on I/R day 7; P = 0.003), respectively. Notably, this decrease was alleviated in a dose-dependent manner (with a small effect at 2.8 g/kg/day; Group 3 on I/R day 1: 1112.65 ± 164.10 cells/mm2, P = 0.21; Group 3 on I/R day 7: 941.89 ± 38.91, P = 0.60). The extent of this alleviation effect was significantly and much more pronounced after pretreating rats with a high dose of CJDHW (Group 4 on I/R day 1: 1691.36 ± 237.57, P = 0.008; Group 4 on I/R day 7: 1389.02 ± 53.20, P = 0.007).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: Retinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats.

Methods: High intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120&nbsp;mmHg for 60&nbsp;min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7&nbsp;days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4&nbsp;% paraformaldehyde and 30&nbsp;% sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3&nbsp;mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2&nbsp;g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4&nbsp;&mu;L of 0.5&nbsp;mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15&nbsp;min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised.

Results: The ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2&nbsp;g/kg/day of CJDHW (Group 4; ERG: P&nbsp;&lt;&nbsp;0.001 on I/R day 7; HE stain: P&nbsp;&lt;&nbsp;0.001 on I/R day 7; TUNEL: P&nbsp;=&nbsp;0.05 on I/R day 7; retrograde labeling: P&nbsp;=&nbsp;0.007 on I/R day 7; Thy-1 mRNA: P&nbsp;=&nbsp;0.02; MMP-9 mRNA: P&nbsp;&lt;&nbsp;0.001; Bcl-2 protein: P&nbsp;=&nbsp;0.02; HO-1 protein: P&nbsp;=&nbsp;0.03; P-p38 MAPK protein: P&nbsp;&lt;&nbsp;0.001; MMP-9 protein: P&nbsp;=&nbsp;0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38&nbsp;&plusmn;&nbsp;0.04 vs. 0.81&nbsp;&plusmn;&nbsp;0.03), increased inner retinal thickness (45.08&nbsp;&plusmn;&nbsp;2.85 vs. 67.98&nbsp;&plusmn;&nbsp;5.48&nbsp;&mu;m), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40&nbsp;&plusmn;&nbsp;0.55 vs. 0.60&nbsp;&plusmn;&nbsp;0.55), and more numerous retinal ganglion cells (887.73&nbsp;&plusmn;&nbsp;158.18 vs. 1389.02&nbsp;&plusmn;&nbsp;53.20). Moreover, increased Thy-1 (0.31&nbsp;&plusmn;&nbsp;0.15 vs. 0.78&nbsp;&plusmn;&nbsp;0.32) and decreased MMP-9 mRNA levels were found (4.44&nbsp;&plusmn;&nbsp;0.84 vs. 1.13&nbsp;&plusmn;&nbsp;0.34), respectively. Furthermore, the Bcl-2 protein level (0.78&nbsp;&plusmn;&nbsp;0.08 vs. 1.80&nbsp;&plusmn;&nbsp;0.34) was increased while the HO-1 (0.99&nbsp;&plusmn;&nbsp;0.20 vs. 4.15&nbsp;&plusmn;&nbsp;2.08), P-p38 MAPK (1.12&nbsp;&plusmn;&nbsp;0.18 vs. 0.57&nbsp;&plusmn;&nbsp;0.18) and MMP-9 levels were decreased (0.70&nbsp;&plusmn;&nbsp;0.23 vs. 0.39&nbsp;&plusmn;&nbsp;0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5&nbsp;mM SB203580 (P-p38 MAPK: 1.12&nbsp;&plusmn;&nbsp;0.18 vs. 0.18&nbsp;&plusmn;&nbsp;0.07, P&nbsp;&lt;&nbsp;0.001; MMP-9: 0.70&nbsp;&plusmn;&nbsp;0.23 vs. 0.21&nbsp;&plusmn;&nbsp;0.07, P&nbsp;=&nbsp;0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03&nbsp;&plusmn;&nbsp;1.57 vs. 1.59&nbsp;&plusmn;&nbsp;0.47, P&nbsp;=&nbsp;0.002; Group 2 vs. 5: 5.03&nbsp;&plusmn;&nbsp;1.57 vs. 1.35&nbsp;&plusmn;&nbsp;0.41, P&nbsp;=&nbsp;0.001).

Conclusion: Treatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.

Electronic supplementary material: The online version of this article (doi:10.1186/s13020-016-0109-6) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus