Korean J Physiol Pharmacol.  2017 Sep;21(5):475-485. 10.4196/kjpp.2017.21.5.475.

RhGLP-1 (7–36) protects diabetic rats against cerebral ischemia-reperfusion injury via up-regulating expression of Nrf2/HO-1 and increasing the activities of SOD

• Fang Y
• Liu X
• Zhao L
• Wei Z
• Jiang D
• Shao H
• Zang Y
• Xu J
• Wang Q
• Liu Y
• Peng Y
• Yin X

• KMID: 2388693
• DOI: http://doi.org/10.4196/kjpp.2017.21.5.475

Abstract

The present study aimed to explore the neuroprotective effect and possible mechanisms of rhGLP-1 (7-36) against transient ischemia/reperfusion injuries induced by middle cerebral artery occlusion (MCAO) in type 2 diabetic rats. First, diabetic rats were established by a combination of a high-fat diet and low-dose streptozotocin (STZ) (30 mg/kg, intraperitoneally). Second, they were subjected to MCAO for 2 h, then treated with rhGLP-1 (7-36) (10, 20, 40 µg/kg i.p.) at the same time of reperfusion. In the following 3 days, they were injected with rhGLP-1 (7-36) at the same dose and route for three times each day. After 72 h, hypoglycemic effects were assessed by blood glucose changes, and neuroprotective effects were evaluated by neurological deficits, infarct volume and histomorphology. Mechanisms were investigated by detecting the distribution and expression of the nuclear factor erythroid-derived factor 2 related factor 2 (Nrf2) in ischemic brain tissue, the levels of phospho-PI3 kinase (PI3K)/PI3K ratio and heme-oxygenase-1 (HO-l), as well as the activities of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA). Our results showed that rhGLP-1 (7-36) significantly reduced blood glucose and infarction volume, alleviated neurological deficits, enhanced the density of surviving neurons and vascular proliferation. The nuclear positive cells ratio and expression of Nrf2, the levels of P-PI3K/PI3K ratio and HO-l increased, the activities of SOD increased and the contents of MDA decreased. The current results indicated the protective effect of rhGLP-1 (7-36) in diabetic rats following MCAO/R that may be concerned with reducing blood glucose, up-regulating expression of Nrf2/HO-1 and increasing the activities of SOD.

Keyword

Brain ischemia/reperfusion; Diabetic rats; Nrf2/HO-1; rhGLP-1 (7-36)

MeSH Terms

Animals
Blood Glucose
Brain
Diet, High-Fat
Hypoglycemic Agents
Infarction
Infarction, Middle Cerebral Artery
Malondialdehyde
Neurons
Neuroprotective Agents
Phosphotransferases
Rats*
Reperfusion
Reperfusion Injury*
Streptozocin
Superoxide Dismutase
Blood Glucose
Hypoglycemic Agents
Malondialdehyde
Neuroprotective Agents
Phosphotransferases
Streptozocin
Superoxide Dismutase

Figure

• 

  Fig. 1 Neurological deficit scores of MCAO/R rats in different groups.Neurological deficits were scored at 2 h after occlusion and 72 h after administration, rhGLP-1 (7-36) (20, 40 µg/kg ip, tid) and nimodipine (0.5 mg/kg ip, qd) can significantly reduced neurological deficit scores compared with the Model group. Columns represent the Mean±S.E.M (n=12). The intergroup variation was analyzed by nonparametric statistics with a Mann-Whitney U test. ##p<0.01 vs. Sham group; **p<0.01 vs. Model group.

• 

  Fig. 2 Effect of rhGLP-1 (7-36) on the infarction volumes of MCAO/R diabetic rats.(A) Typical TTC stained brain slices of MCAO/R rats in different groups. The normal brain tissue is red and the cerebral ischemia and infarction tissues are white (n=6). A, Sham group; B, Model group; C, Nimodipine group; D, Insulin group; E, Low-rhGLP-1 (7-36) group; F, Middle-rhGLP-1 (7-36) group; G, High-rhGLP-1 (7-36) group. (B) Infarction volume percentage of MCAO/R rats in different groups, columns represent the Mean±S.E.M (n=6). Infarction volumes were measured at 72h after administration, rhGLP-1 (7-36) (20, 40 µg/kg ip, tid) and nimodipine (0.5 mg/kg ip, qd) can significantly reduced Infarction volumes compared to the Model group. The intergroup variation was analyzed by ANOVA followed by LSD test. ##p<0.01 vs. Sham group; *p<0.05, **p<0.01 vs. Model group.

• 

  Fig. 3 Summary of typical pathological sections of MCAO/R rats in the different groups (HE, ×200, n=3).At 72 h after administration, compared with the Model group, the cavitation in each of the rhGLP-1, the nimodipine, and the insulin groups were alleviated, the number of surviving neurons increased and vascular proliferation was enhanced. (A) Sham group; (B) Model group; (C) Nimodipine group; (D) Insulin group; (E) Low-rhGLP-1 (7-36) group; (F) Middle-rhGLP-1 (7-36) group; (G) High-rhGLP-1 (7-36) group.

• 

  Fig. 4 Effect of rhGLP-1 (7-36) on the distribution and expression of Nrf2 in the cerebral cortex of the ischemia-reperfusion damage.(A) Immunohistochemistry of Nrf2 of MCAO/R rats in different groups (× 400, n=3), brown indicates Nrf2 positive. The expression of Nrf2 in cytoplasm and nuclear translocation increased after treatment with nimodipine, insulin, and rhGLP-1 (7-36). A, Sham group; B, Model group; C, Nimodipine group; D, Insulin group; E, Low-rhGLP-1 (7-36) group; F, Middle-rhGLP-1 (7-36) group; G, High-rhGLP-1 (7-36) group. (B) Ratio of Nrf2 nuclear positive cells of MCAO/R rats in different groups, columns represent the Mean±S.E.M (n=3). The Nrf2 nuclear positive cells ratio significantly increased at 72 h after treated with rhGLP-1 (7-36) (10, 20, 40 µg/kg ip, tid) and insulin (0~1 IU/kg ip, bid) compared to the Model group. The intergroup variation was analyzed by ANOVA followed by Dunnett's T3 test. #p<0.05 vs. Sham group; *p<0.05, **p<0.01 vs. Model group.

• 

  Fig. 5 Nrf2 expression in MCAO/R rats.(A) Expression of Nrf2 protein in MCAO/R rats in different groups (n=4) with GAPDH as a loading control. A, Sham group; B, Model group; C, Nimodipine group; D, Insulin group; E, Low-rhGLP-1 (7-36) group; F, Middle-rhGLP-1 (7-36) group; G, High-rhGLP-1 (7-36) group. (B) Nrf2/GAPDH in different groups (Mean±S.E.M, n=4). At 72 h after administration, compared with the Model group, rhGLP-1 (7-36) (10, 20, 40 µg/kg ip, tid), nimodipine (0.5 mg/kg ip, qd) and insulin (0~1 IU/kg ip, bid) can significantly enhance Nrf2 protein expression. The intergroup variation was analyzed by ANOVA followed by LSD test. #p<0.05 vs. Sham group; *p<0.05, **p<0.01 vs. Model group.

• 

  Fig. 6 Effect of rhGLP-1 on the levels of HO-1 expression and P-PI3K/PI3K ratio in the cerebral cortex of the ischemia-reperfusion damage.(A) Expressions of HO-1 and P-PI3K protein in MCAO/R rats in different groups (n=4) with GAPDH as a loading control. A, Sham group; B, Model group; C, Nimodipine group; D, Insulin group; E, Low-rhGLP-1 (7-36) group; F, Middle-rhGLP-1 (7-36) group; G, High-rhGLP-1 (7-36) group. (B) HO-1/GAPDH in different groups (Mean±S.E.M, n=4). At 72 h after administration, compared with the Model group, rhGLP-1 (7-36) (10, 20, 40 µg/kg ip, tid), nimodipine (0.5 mg/kg ip, qd) and insulin (0~1 IU/kg ip, bid) can significantly enhance HO-1 protein expression. The intergroup variation was analyzed by ANOVA followed by LSD test. #p<0.05 vs. Sham group; *p<0.05, **p<0.01 vs. Model group. (C) P-PI3K/PI3K ratio in different groups (Mean±S.E.M, n=4). At 72 h after administration, compared with the Model group, rhGLP-1 (7-36) (10, 20, 40 µg/kg ip, tid) and insulin (0~1 IU/kg ip, bid) can significantly enhance P-PI3K/PI3K ratio. The intergroup variation was analyzed by ANOVA followed by LSD test. ##p<0.01 vs. Sham group; *p<0.05, **p<0.01 vs. Model group.

• 

  Fig. 7 The effect of rhGLP-1 (7-36) on the activities of SOD and the contents of MDA.(A) SOD activities of MCAO/R rats in different groups. (B) MDA contents of MCAO/R rats in different groups. Columns represent the Mean±S. E.M (n=4). At 72 h after administration, compared with the Model group, rhGLP-1 (7-36) (10, 20, 40 µg/kg ip, tid), nimodipine (0.5 mg/kg ip, qd) and insulin (0~1 IU/kg ip, bid) can significantly improved the activities of SOD and decrease MDA contents. The intergroup variation was analyzed by ANOVA followed by LSD test. ##p<0.01 vs. Sham group, *p<0.05; **p<0.01 vs. Model group.

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