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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

Electroretinogram (ERG): The effect of CJDHW on retinal ischemia plus reperfusion (I/R). a There was a considerable reduction in the amplitude of the ERG b-wave following pressure-induced retinal ischemia plus reperfusion (I/R) and pretreatment with vehicle in a representative animal of the I/R + Vehicle (Group 2) compared with the sham procedure retina (Group 1). Pretreatment with CJDHW (at 2.8 g/kg/day, I/R + CJDHW2.8, Group 3; at 4.2 g/kg/day, I/R + CJDHW4.2, Group 4) led to a dose-dependent attenuation in this reduction in one rat from each defined Group. b In contrast with the Group 1, there was a significant (**P < 0.01) decrease in the b-wave ratio in the Group 2 1, 3, 5 and 7 days following ischemia. Dose-dependent and significant (††P < 0.01) attenuation of this ischemia-induced decrease was achieved after pretreating rats with 2.8 g/kg/day (Group 3) and 4.2 g/kg/day of CJDHW (Group 4). These results are expressed as the mean ± SD (n = 5). CJDHW Chi-Ju-Di-Huang-Wan
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Fig1: Electroretinogram (ERG): The effect of CJDHW on retinal ischemia plus reperfusion (I/R). a There was a considerable reduction in the amplitude of the ERG b-wave following pressure-induced retinal ischemia plus reperfusion (I/R) and pretreatment with vehicle in a representative animal of the I/R + Vehicle (Group 2) compared with the sham procedure retina (Group 1). Pretreatment with CJDHW (at 2.8 g/kg/day, I/R + CJDHW2.8, Group 3; at 4.2 g/kg/day, I/R + CJDHW4.2, Group 4) led to a dose-dependent attenuation in this reduction in one rat from each defined Group. b In contrast with the Group 1, there was a significant (**P < 0.01) decrease in the b-wave ratio in the Group 2 1, 3, 5 and 7 days following ischemia. Dose-dependent and significant (††P < 0.01) attenuation of this ischemia-induced decrease was achieved after pretreating rats with 2.8 g/kg/day (Group 3) and 4.2 g/kg/day of CJDHW (Group 4). These results are expressed as the mean ± SD (n = 5). CJDHW Chi-Ju-Di-Huang-Wan

Mentions: In the sham retina (Fig. 1a), the ERG b-wave was determined to be 0.98 mV. However, retinal ischemia plus 1 day of reperfusion led to a considerable decrease in the amplitude of the b-wave to 0.11 mV. Notably, pretreating rats with CJDHW (Groups 3 and 4) counteracted this ischemia-induced decrease in the amplitude of the b-wave in a dose-dependent manner, with the amplitude of the b-wave increasing to 0.26 and 0.39 mV, respectively, 1 day after I/R. As shown in Fig. 1b (n = 5), administering I/R following the rats’ pretreatment with the vehicle (Group 1) led to a significant (P < 0.001) decrease in the b-wave ratio on I/R days 1, 3, 5 and 7 compared with the preischemic b-wave ratio (day 0: 0.95 ± 0.05; day 1: 0.44 ± 0.07; day 3: 0.44 ± 0.05; day 5: 0.38 ± 0.04; day 7: 0.38 ± 0.04). Pretreating rats with CJDHW (Group 3 vs. 4) led to a significant (P < 0.001; at 2.8 and 4.2 g/kg/day) dose-dependent decrease in the ischemia-induced b-wave ratio on I/R day 1 (0.66 ± 0.07 vs. 0.72 ± 0.07), day 3 (0.70 ± 0.05 vs. 0.78 ± 0.04), day 5 (0.70 ± 0.03 vs. 0.80 ± 0.04) and day 7 (0.69 ± 0.07 vs. 0.81 ± 0.03) after ischemia. Moreover, the preischemic b-wave ratio (day 0) was recorded at 0.99 ± 0.12 vs. 0.93 ± 0.10 (P = 0.391; Group 3 vs. 4).Fig. 1


Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase
Electroretinogram (ERG): The effect of CJDHW on retinal ischemia plus reperfusion (I/R). a There was a considerable reduction in the amplitude of the ERG b-wave following pressure-induced retinal ischemia plus reperfusion (I/R) and pretreatment with vehicle in a representative animal of the I/R + Vehicle (Group 2) compared with the sham procedure retina (Group 1). Pretreatment with CJDHW (at 2.8 g/kg/day, I/R + CJDHW2.8, Group 3; at 4.2 g/kg/day, I/R + CJDHW4.2, Group 4) led to a dose-dependent attenuation in this reduction in one rat from each defined Group. b In contrast with the Group 1, there was a significant (**P < 0.01) decrease in the b-wave ratio in the Group 2 1, 3, 5 and 7 days following ischemia. Dose-dependent and significant (††P < 0.01) attenuation of this ischemia-induced decrease was achieved after pretreating rats with 2.8 g/kg/day (Group 3) and 4.2 g/kg/day of CJDHW (Group 4). These results are expressed as the mean ± SD (n = 5). CJDHW Chi-Ju-Di-Huang-Wan
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5016920&req=5

Fig1: Electroretinogram (ERG): The effect of CJDHW on retinal ischemia plus reperfusion (I/R). a There was a considerable reduction in the amplitude of the ERG b-wave following pressure-induced retinal ischemia plus reperfusion (I/R) and pretreatment with vehicle in a representative animal of the I/R + Vehicle (Group 2) compared with the sham procedure retina (Group 1). Pretreatment with CJDHW (at 2.8 g/kg/day, I/R + CJDHW2.8, Group 3; at 4.2 g/kg/day, I/R + CJDHW4.2, Group 4) led to a dose-dependent attenuation in this reduction in one rat from each defined Group. b In contrast with the Group 1, there was a significant (**P < 0.01) decrease in the b-wave ratio in the Group 2 1, 3, 5 and 7 days following ischemia. Dose-dependent and significant (††P < 0.01) attenuation of this ischemia-induced decrease was achieved after pretreating rats with 2.8 g/kg/day (Group 3) and 4.2 g/kg/day of CJDHW (Group 4). These results are expressed as the mean ± SD (n = 5). CJDHW Chi-Ju-Di-Huang-Wan
Mentions: In the sham retina (Fig. 1a), the ERG b-wave was determined to be 0.98 mV. However, retinal ischemia plus 1 day of reperfusion led to a considerable decrease in the amplitude of the b-wave to 0.11 mV. Notably, pretreating rats with CJDHW (Groups 3 and 4) counteracted this ischemia-induced decrease in the amplitude of the b-wave in a dose-dependent manner, with the amplitude of the b-wave increasing to 0.26 and 0.39 mV, respectively, 1 day after I/R. As shown in Fig. 1b (n = 5), administering I/R following the rats’ pretreatment with the vehicle (Group 1) led to a significant (P < 0.001) decrease in the b-wave ratio on I/R days 1, 3, 5 and 7 compared with the preischemic b-wave ratio (day 0: 0.95 ± 0.05; day 1: 0.44 ± 0.07; day 3: 0.44 ± 0.05; day 5: 0.38 ± 0.04; day 7: 0.38 ± 0.04). Pretreating rats with CJDHW (Group 3 vs. 4) led to a significant (P < 0.001; at 2.8 and 4.2 g/kg/day) dose-dependent decrease in the ischemia-induced b-wave ratio on I/R day 1 (0.66 ± 0.07 vs. 0.72 ± 0.07), day 3 (0.70 ± 0.05 vs. 0.78 ± 0.04), day 5 (0.70 ± 0.03 vs. 0.80 ± 0.04) and day 7 (0.69 ± 0.07 vs. 0.81 ± 0.03) after ischemia. Moreover, the preischemic b-wave ratio (day 0) was recorded at 0.99 ± 0.12 vs. 0.93 ± 0.10 (P = 0.391; Group 3 vs. 4).Fig. 1

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