[Effect of selective cerebral mild hypothermia on p300 and lactylation modification in rats following cerebral ischemic reperfusion injury].
To evaluate the effect of selective cerebral mild hypothermia on p300 and lactylation modification in rats following cerebral ischemic reperfusion injury (CIRI).
Sixty healthy male SD rats aged 6 to 8 weeks of age at SPF grade were selected and divided into sham operation group (Sham group), CIRI group, selective cerebral mild hypothermic intervention group (CIRI+HT group), and normal temperature intervention group (CIRI+NT group) using randomized numerical tables, 15 in each group. A middle cerebral artery occlusion was established using thrombosis, revocation of thrombus after 2 hours of ischemia to achieve reperfusion. Sham group underwent only cervical vascular exposure surgery. Immediately after reperfusion, 20 centigrade (CIRI+HT group) or 37 centigrade (CIRI+NT group) saline was perfused at a constant flow rate of 0.6 mL/min through the left internal carotid artery for 10 minutes, respectively. Brain temperature and rectal temperature of rats were monitored throughout the process. At 24 hours after reperfusion, Modified Neurological Severity Score (mNSS) was used to evaluate the neurological function of rats. Then the rats were sacrificed and brain tissues were obtained. Cerebral infarction was observed by staining with 2,3,5-triphenyltetrazolium chloride (TTC), and the percentage of cerebral infarction volume was calculated. The ischemic penumbra tissue of cerebral cortex was taken. TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to measure neuronal apoptosis rate. Hematoxylin-eosin (HE) staining was employed to observe the morphological changes of nerve cells. Immunofluorescence analysis was used to assess the expression of p300. Western blotting analysis was conducted to assess the level of lactylation modification and p300. In addition, lactate level in the cerebral cortex of the ischemic penumbra area were detected.
Compared with Sham group, mNSS, cerebral infarction volume, neuronal apoptosis rate, lactic acid content, lactic acid modification level and p300 enzyme expression were increased in the CIRI group, CIRI+HT group and CIRI+NT group (all P<0.05). Compared with CIRI group, the CIRI+HT group had decreased mNSS, cerebral infarction volume, and neuronal apoptosis rate, as well as decreased lactate content, lactate modification level, and p300 expression [mNSS: 4.20±1.30 vs. 9.40±1.34, cerebral infarction volume percentage: (31.21±1.20) vs. (41.18±2.33)%, neuronal apoptosis rate: (27.69±2.87)% vs. (48.90±2.08)%, lactate content (mmol/g): 0.44±0.04 vs. 0.63±0.04, lactate modification (lactate modification/β-actin): 0.29±0.03 vs. 0.36±0.03, p300 (p300/β-actin): 0.60±0.02 vs. 0.82±0.02, proportion of p300 positive cells: (46.70±2.97)% vs. (63.80±3.41)%, all P<0.05]. However, there were no significant differences in the above indicators between the CIRI+NT group and the CIRI group (all P>0.05). HE staining showed neurocytes had integrated morphology and clear contours in Sham group, a large amount of cellular edema and irregular nucleus enrichment were observed in CIRI group and CIRI+NT group, and the degree of cell edema and nucleus shrinkage were reduced in CIRI+HT group.
Selective mild hypothermia can alleviates CIRI in rats, and the mechanism may be related to the reduction of lactic acid production, inhibition of p300 expression, and then suppressing lactylation modification level.
Sixty healthy male SD rats aged 6 to 8 weeks of age at SPF grade were selected and divided into sham operation group (Sham group), CIRI group, selective cerebral mild hypothermic intervention group (CIRI+HT group), and normal temperature intervention group (CIRI+NT group) using randomized numerical tables, 15 in each group. A middle cerebral artery occlusion was established using thrombosis, revocation of thrombus after 2 hours of ischemia to achieve reperfusion. Sham group underwent only cervical vascular exposure surgery. Immediately after reperfusion, 20 centigrade (CIRI+HT group) or 37 centigrade (CIRI+NT group) saline was perfused at a constant flow rate of 0.6 mL/min through the left internal carotid artery for 10 minutes, respectively. Brain temperature and rectal temperature of rats were monitored throughout the process. At 24 hours after reperfusion, Modified Neurological Severity Score (mNSS) was used to evaluate the neurological function of rats. Then the rats were sacrificed and brain tissues were obtained. Cerebral infarction was observed by staining with 2,3,5-triphenyltetrazolium chloride (TTC), and the percentage of cerebral infarction volume was calculated. The ischemic penumbra tissue of cerebral cortex was taken. TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to measure neuronal apoptosis rate. Hematoxylin-eosin (HE) staining was employed to observe the morphological changes of nerve cells. Immunofluorescence analysis was used to assess the expression of p300. Western blotting analysis was conducted to assess the level of lactylation modification and p300. In addition, lactate level in the cerebral cortex of the ischemic penumbra area were detected.
Compared with Sham group, mNSS, cerebral infarction volume, neuronal apoptosis rate, lactic acid content, lactic acid modification level and p300 enzyme expression were increased in the CIRI group, CIRI+HT group and CIRI+NT group (all P<0.05). Compared with CIRI group, the CIRI+HT group had decreased mNSS, cerebral infarction volume, and neuronal apoptosis rate, as well as decreased lactate content, lactate modification level, and p300 expression [mNSS: 4.20±1.30 vs. 9.40±1.34, cerebral infarction volume percentage: (31.21±1.20) vs. (41.18±2.33)%, neuronal apoptosis rate: (27.69±2.87)% vs. (48.90±2.08)%, lactate content (mmol/g): 0.44±0.04 vs. 0.63±0.04, lactate modification (lactate modification/β-actin): 0.29±0.03 vs. 0.36±0.03, p300 (p300/β-actin): 0.60±0.02 vs. 0.82±0.02, proportion of p300 positive cells: (46.70±2.97)% vs. (63.80±3.41)%, all P<0.05]. However, there were no significant differences in the above indicators between the CIRI+NT group and the CIRI group (all P>0.05). HE staining showed neurocytes had integrated morphology and clear contours in Sham group, a large amount of cellular edema and irregular nucleus enrichment were observed in CIRI group and CIRI+NT group, and the degree of cell edema and nucleus shrinkage were reduced in CIRI+HT group.
Selective mild hypothermia can alleviates CIRI in rats, and the mechanism may be related to the reduction of lactic acid production, inhibition of p300 expression, and then suppressing lactylation modification level.
Authors
Niu Niu, Liu Liu, Dong Dong, Liu Liu, Dong Dong, Wang Wang, Yuan Yuan, Zhang Zhang
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