Purified herba leonuri and leonurine protect middle cerebral artery occluded rats from brain injury through antioxidative mechanism and mitochondrial protection 4

Chapter 4: Results Chapter Results Department of Pharmacology, YLL School of Medicine 89 Chapter 4: Results 4.1 Results of experiment I: Cerebral Protection of Purified Herba Leonuri Extract on Middle Cerebral Artery Occluded Rats 4.1.1 Pharmacological and functional outcome studies 4.1.1.1 pHL reduced infarct volume resulted from MCAO In order to measure the protective effects of pHL, infarct volumes of each treatment group were measured at seventh day postocclusion. As shown in Figure 4-1, rats from sham and sham treated with pHL underwent same procedure but without the occlusion of MCAO, therefore no infarct area was observed in both groups, indicating that the surgery itself did not cause cerebral injury (Figure 4-1ai, ii). When the animal was subjected to ischemia insult by left MCAO, the infarct area was observed in left temporoparietal cortex and striatum of the ipsilateral hemisphere (Figure 4-1aiii). From Figure 4-1aiv, a reduction of damage area was observed in stroke group treated with pHL. Under the treatment of pHL, the infarct volume was reduced significantly from 20.75 ± 0.03% to 15.19 ± 0.02% (Figure 4-1b). a) i) Sham ii) Sham+pHL Department of Pharmacology, YLL School of Medicine iii) Vehicle iv) Stroke+pHL 90 Chapter 4: Results b) Infarct Volume Measurement in Each Treatment Group Percentage of Infarct Volume 30.00% 25.00% 20.00% ** 15.00% 10.00% 5.00% 0.00% Sham Sham + pHL Vehicle Stroke + pHL Treatment Groups Figure 4-1: Infarct volume measured from each treatment group. a) Images of cerebral sections among the treatment groups. b) Infarct volume was analyzed by image analyzer system (Scion image for windows). Data (n>6) was presented as % respective to the total volume of hemisphere. **p 0.01 vs. vehicle. Department of Pharmacology, YLL School of Medicine 91 Chapter 4: Results 4.1.1.2 pHL ameliorated the neurological outcome of MCAO-induced rats In terms of evaluation of neurological function, neurological deficit grading system was carried out for all the animals. The higher the neurological deficit score, the more severe impairment of motor motion is. The result is shown in Figure 4-2. As rats from sham groups had no cerebral injury as shown from TTC images, they did not exhibit neurological deficit, and therefore throughout the entire study, the animals from both groups had the neurological score of zero. For the rats in vehicle group, they remained highest neurological deficit score after the surgery, indicating the MCAO deteriorates the neurological outcome. This is in agreement with that they had largest infarct volume among the groups (Figure 4-1 and 4-2a). Treatment of pHL could ameliorate the neurological function of rats, therefore a lower deficit score was observed throughout the postocclusion treatment. Chi square test was selected to test the sample distribution between vehicle group and stroke group treated with pHL. Under the treatment of pHL, although neurological deficit score of rats undergone MCAO was lowered as compared to vehicle group at day (Figure 4-2a), it did not reach statistical significant level (Figure 42b). As the treatment continued, rats from stroke group treated with pHL had their neurological deficit score significantly declined, with majority of rats having the score of and (Figure 4-2c), suggesting the functional improvement conferred by pHL. Department of Pharmacology, YLL School of Medicine 92 Chapter 4: Results a) Neurological Score of Each Treatment Group After MCAO Neurological Score 2.5 1.5 Sham Sham + pHL Vehicle Stroke + pHL 0.5 Days after MCAO b) Neurological Deficit Score of Vehicle and pHL (Day 1) Percentage of Number of Rats 60% 50% 40% 30% 20% 10% 0% Vehicle pHL Groups Department of Pharmacology, YLL School of Medicine 93 Chapter 4: Results c) Figure 4-2: Neurological deficit score among treatment groups (n>20). a) A chart showing the neurological deficit score among treatment groups throughout days after surgery. Vehicle group showed the highest neurological deficit score while HL stroke group showed a significant reduction of neurological score at the end of treatment (Day 7). b) Neurological score of vehicle and stroke treated with pHL group at Day 1. c) Neurological score of vehicle and stroke treated with pHL group at Day 7. Department of Pharmacology, YLL School of Medicine 94 Chapter 4: Results 4.1.2 Biochemical, cellular and molecular approaches 4.1.2.1 MCAO decreased plasma antioxidant level and protection of pHL on the plasma antioxidant level To investigate the changes of endogenous antioxidant system under the influence of pHL, total antioxidant concentration was quantified. The kit depends on the antioxidants in the sample inhibiting the oxidation of ABTSTM substrate to ABTSTM·+ product by metmyoglobin. Therefore, the concentration of antioxidant present in the sample is inversely proportional to the amount of ABTSTM·+ to be measured. From obtained result, the concentration of antioxidant of sham group was 0.38 ± 0.08mM (Figure 4-3). Under the treatment of pHL, the antioxidant concentration was increased from 0.38 ± 0.08mM to 0.5 ± 0.2mM in sham treated with pHL group, though it is not statistically significant (Figure 4-3). This might be due to the sensitivity of the system which requires a very accurate time point measurement. As for the stroke insult, the antioxidant concentration was decreased significantly to 0.31 ± 0.03mM (p[...]... School of Medicine 118 Chapter 4: Results 4. 3 Results of experiment III: Leonurine (4- guanidino-nbutyl Syringate) Protects the Middle Cerebral Artery Occluded- Rats through Antioxidant effect and Regulation of Mitochondrial Function 4. 3.1 Effect of Leonurine on functional outcome of MCAO-induced rats Figure 4- 14a showed the infarct area of each treatment group As the rats from sham underwent the similar... production in a dose dependent manner to 64. 29±5. 24% (p . 90 4. 1 Results of experiment I: Cerebral Protection of Purified Herba Leonuri Extract on Middle Cerebral Artery Occluded Rats 4. 1.1 Pharmacological and functional outcome studies 4. 1.1.1. Figure 4- 2. As rats from sham groups had no cerebral injury as shown from TTC images, they did not exhibit neurological deficit, and therefore throughout the entire study, the animals from both. Chapter 4: Results Department of Pharmacology, YLL School of Medicine 103 4. 2 Results of experiment II: Modulation of Mitochondrial ROS Generation and Function by Purified Herba Leonuri