Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (19)

N-Palmitoylethanolamide-Oxazoline Protects Against Middle Cerebral Artery Occlusion Injury in Diabetic Rats by Regulating the SIRT1 Pathway

Affiliations

N-Palmitoylethanolamide-Oxazoline Protects Against Middle Cerebral Artery Occlusion Injury in Diabetic Rats by Regulating the SIRT1 Pathway

Roberta Fusco et al. Int J Mol Sci.

Abstract

Diabetes causes various macrovascular and microvascular alterations, often culminating in major clinical complications (first of all, stroke) that lack an effective therapeutic intervention. N-palmitoylethanolamide-oxazoline (PEA-OXA) possesses anti-inflammatory and potent neuroprotective effects. Although recent studies have explained the neuroprotective properties of PEA-OXA, nothing is known about its effects in treating cerebral ischemia.

Methods: Focal cerebral ischemia was induced by transient middle cerebral artery occlusion (MCAo) in the right hemisphere. Middle cerebral artery (MCA) occlusion was provided by introducing a 4-0 nylon monofilament (Ethilon; Johnson & Johnson, Somerville, NJ, USA) precoated with silicone via the external carotid artery into the internal carotid artery to occlude the MCA.

Results: A neurological severity score and infarct volumes were carried out to assess the neuroprotective effects of PEA-OXA. Moreover, we observed PEA-OXA-mediated improvements in tissue histology shown by a reduction in lesion size and an improvement in apoptosis level (assessed by caspases, Bax, and Bcl-2 modulation and a TUNEL assay), which further supported the efficacy of PEA-OXA therapy. We also found that PEA-OXA treatment was able to reduce mast cell degranulation and reduce the MCAo-induced expression of NF-κB pathways, cytokines, and neurotrophic factors.

Conclusions: based on these findings, we propose that PEA-OXA could be useful in decreasing the risk of impairment or improving function in ischemia/reperfusion brain injury-related disorders.

Keywords: PEA-OXA; inflammation; ischemic stroke.

Conflict of interest statement

Salvatore Cuzzocrea is a coinventor on patent WO2013121449 A8 (Epitech Group Srl), which deals with methods and compositions for the modulation of amidases capable of hydrolyzing N-acylethanolamines employable in the treatment of inflammatory diseases. This invention is wholly unrelated to the present study. Moreover, Cuzzocrea is also, with Epitech Group, a coinventor on the patents EP 2 821 083, MI2014 A001495, and 102015000067344, which are unrelated to the study. The remaining authors report no conflicts of interest.

Figures

Figure 1
Figure 1
Efficacy of PEA-OXA on rCBF and the ischemic area induced by transient MCAo: rCBF was controlled to confirm the success of the induction of the transient ischemia. This was monitored at baseline (10 min before), at 10 and 60 min during ischemia, and 5 min after ischemia (A). TTC staining of brain sections was performed 24 h after MCAo. Tissues from vehicle-treated animals displayed the presence of an unstained area (B,D) compared to the sham group (B,C). PEA-OXA administration (B,F) reduced this infarcted area more effectively than did the PEA treatment (B,E). A histological analysis of brain samples from vehicle-treated rats revealed the loss of neurons (H,M) compared to the sham group (G,M), which was ameliorated by PEA administration (I,M). PEA-OXA administration displayed more protective effects compared to the PEA treatment (L,M). For the histology, a 20× magnification is shown (50-µm scale bar). A p-value of less than 0.05 was considered significant: °° p < 0.01 versus vehicle; *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; # p < 0.05 versus PEA; ### p < 0.001 versus PEA.
Figure 2
Figure 2
Efficacy of PEA-OXA on mast cell infiltration and degranulation induced by transient MCAo. An increased number of mast cells were detected in tissues from vehicle-treated animals (B,E) compared to the control (A,E). PEA and PEA-OXA-treated ischemic rats showed fewer cells of this type (CE). An increased expression of tryptase (G,L) was found in sections obtained from vehicle-treated rats compared to the sham-operated animals (F,L). PEA-OXA administration reduced tryptase expression more effectively than did PEA (H,I,L). The number of mast cells was counted in three sections per animal and is presented as the number of positive cells per high-power field. For the mast cells, a 100× magnification is shown (10-µm scale bar). For the immunohistochemistry, a 40× magnification is shown (75-µm scale bar). A p-value of less than 0.05 was considered significant: *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; # p < 0.05 versus PEA.
Figure 3
Figure 3
The efficacy of PEA-OXA on SIRT1 and UCP2 expression, the NAD+/NADH ratio, GSH, and SOD activity (induced by transient MCAo). A western blot analysis of SIRT1 showed basal expression that was significantly increased after MCAo. After PEA-OXA treatment, this expression was significantly upregulated, while after PEA administration, it increased less (A,B). Real-time PCR analysis of the SIRT1 gene displayed the same trend (E). UCP2 expression in vehicle-treated animals was significantly increased compared to the sham-operated animals. PEA-OXA administration was able to significantly restore the basal levels (C,D). PEA displayed less protection. The NAD+/NADH ratio was reduced by PEA-OXA treatment compared to the vehicle-treated animals (E). MCAo reduced GSH (G) and SOD (H) activity in vehicle-treated animals, while PEA-OXA administration restored the basal levels. A p-value of less than 0.05 was considered significant: * p < 0.05 versus sham; ° p < 0.05 versus vehicle; °° p < 0.01 versus vehicle; *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; # p < 0.05 versus PEA; ### p < 0.001 versus PEA.
Figure 4
Figure 4
Efficacy of PEA-OXA on apoptosis induced by transient MCAo. In western blot analyses, a minimal expression of Bax in brain samples taken from sham rats was detected. PEA-OXA was able to decrease Bax expression in the MCAo-induced group (A,B). On the other hand, a basal level of Bcl-2 was present in brain tissue collected from sham rats, while the expression of Bcl-2 was significantly lower in the vehicle group. PEA-OXA treatment was able to increase the expression of Bcl-2 at levels similar to the sham group (C,D). Treatment with PEA at the same dose was less beneficial (AD). A low level of TUNEL-positive staining was detected in the sham group (E,I). PEA-OXA (H,I) administration reduced the number of TUNEL-positive cells compared to the MCAo group (F,I). PEA treatment showed less efficacy (G,I). The number of TUNEL-positive cells was counted in three sections per animal and is presented as the number of positive cells per high-power field. For TUNEL staining, a 100× magnification is shown (10-µm scale bar). A p-value of less than 0.05 was considered significant: ° p < 0.05 versus vehicle; °° p < 0.01 versus vehicle; *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; # p < 0.05 versus PEA; ### p < 0.001 versus PEA.
Figure 5
Figure 5
Efficacy of PEA-OXA on caspase 3 expression and LDH levels (induced by transient MCAo). An immunohistochemical analysis of caspase 3 (E) was performed. A marked increase in its expression was detected in brain samples from animals administered vehicle (B) compared to the sham-treated animals (A). This expression was notably reduced by treatment with PEA-OXA (D), while PEA treatment displayed less efficacy (C). PEA-OXA administration also reduced LDH levels to normal levels (F). For the immunohistochemistry, a 20× magnification is shown (50-µm scale bar). A p-value of less than 0.05 was considered significant: ° p < 0.05 versus vehicle; *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; # p < 0.05 versus PEA; ## p < 0.01 versus PEA.
Figure 6
Figure 6
Efficacy of PEA-OXA on IκB-α degradation, NF-κB translocation, and TGF-β expression (induced by transient MCAo). Samples from vehicle-treated rats subjected to ischemia/reperfusion injury showed increased IκB-α degradation (A,B) and NF-κB translocation into the nucleus (C,D) compared to the sham-operated animals. PEA-OXA administration was able to significantly restore them to basal levels, while PEA showed less efficacy. Real-time PCR analysis of the NF-κB gene displayed the same trend (E). An immunohistochemical analysis TGF-β (F) was performed. A marked increase in its expression was detected in brain samples from animals administered vehicle (F,H) compared to the sham-treated animals (F,G). This expression was notably reduced by treatment with PEA-OXA (F,L). Treatment with PEA at the same dose was less beneficial (F,I). For the immunohistochemistry, a 20× magnification is shown (50-µm scale bar). A p-value of less than 0.05 was considered significant: ° p < 0.05 versus vehicle; ** p < 0.01 versus sham; °° p < 0.01 versus vehicle; *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; # p < 0.05 versus PEA; ## p < 0.01 versus PEA.
Figure 7
Figure 7
Efficacy of PEA-OXA on TNF-α and IL-1β expression (induced by transient MCAo). Twenty-four hours after transient MCAo, an increased expression of TNF-α (B,I) and IL-1β (F,L) was found in sections obtained from vehicle-treated rats compared to the sham-operated animals (A,I; E,L). PEA-OXA administration reduced both TNF-α (D,I) and IL-1β (H,L) expression more effectively than PEA did (C,D; G,L). For the immunohistochemistry, a 20× magnification is shown (50-µm scale bar). A p-value of less than 0.05 was considered significant: ° p < 0.05 versus vehicle; *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; ## p < 0.01 versus PEA; ### p < 0.001 versus PEA.
Figure 8
Figure 8
Efficacy of PEA-OXA on BDNF and GDNF expression (induced by transient MCAo). The immunofluorescence of brain samples showed that PEA-OXA-administered animals exhibited the presence of an increased number of positive cells for BDNF (D,I) and GDNF (H,L) staining compared to the samples from vehicle-treated rats (B,I; F,L). PEA displayed less protection (C,I; G,L). Sham-operated animals showed basal levels of BDNF (A,I) and GDNF (E,L). The number of BDNF- and GDNF-positive cells was counted in three sections per animal and is presented as the number of positive cells per high-power field. For immunofluorescence, a 100× magnification is shown (10-µm scale bar). A p-value of less than 0.05 was considered significant: ° p < 0.05 versus vehicle; °° p < 0.01 versus vehicle; *** p < 0.001 versus sham; °°° p < 0.001 versus vehicle; ### p < 0.001 versus PEA

Similar articles

See all similar articles

References

    1. Emerging Risk Factors C., Sarwar N., Gao P., Seshasai S.R., Gobin R., Kaptoge S., di Angelantonio E., Ingelsson E., Lawlor D.A., Selvin E., et al. Diabetes mellitus, fasting blood glucose concentration, and risk of vascular disease: A collaborative meta-analysis of 102 prospective studies. Lancet. 2010;375:2215–2222. doi: 10.1016/S0140-6736(10)60484-9. - DOI - PMC - PubMed
    1. Bourne R.R., Stevens G.A., White R.A., Smith J.L., Flaxman S.R., Price H., Jonas J.B., Keeffe J., Leasher J., Naidoo K., et al. Vision Loss Expert, G. Causes of vision loss worldwide, 1990-2010: A systematic analysis. Lancet Glob. Health. 2013;1:339–439. doi: 10.1016/S2214-109X(13)70113-X. - DOI - PubMed
    1. Biller J., Love B.B. Diabetes and stroke. Med. Clin. North. Am. 1993;77:95–110. doi: 10.1016/S0025-7125(16)30274-7. - DOI - PubMed
    1. Vinik A., Flemmer M. Diabetes and macrovascular disease. J. Diabetes Complicat. 2002;16:235–245. doi: 10.1016/S1056-8727(01)00212-4. - DOI - PubMed
    1. Iwata N., Takayama H., Xuan M., Kamiuchi S., Matsuzaki H., Okazaki M., Hibino Y. Effects of Etanercept against Transient Cerebral Ischemia in Diabetic Rats. Biomed. Res. Int. 2015;2015:189292. doi: 10.1155/2015/189292. - DOI - PMC - PubMed
Feedback