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

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, indica

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)

volume of hemisphere **p 0.01 vs vehicle

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 1 (Figure 4-2a), it did not reach statistical significant level (Figure 4-2b) 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

1 and 2 (Figure 4-2c), suggesting the functional improvement conferred by pHL

c)

Figure 4-2: Neurological deficit score among treatment groups (n>20) a) A chart showing the neurological deficit score among treatment groups throughout 7 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

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<0.05) as compared to sham group (Figure 4-3) It was restored to 0.42 ± 0.05mM (p<0.05 as compared to vehicle) by pHL

treatment in stroke group treated with pHL, level that is even higher than sham group (Figure 4-3)

Figure 4-3: Plasma total antioxidant concentration of each treatment group under the influence the pHL Total antioxidant concentration (mM) was reduced in vehicle group and the level was enhanced in both sham-operated and stroke-operated groups treated

with pHL *p<0.05 vs vehicle; #p<0.05 vs sham (n=6)

4.1.2.2 Increased oxidative stress by MCAO and prevention by pHL

DNA oxidative adducts could be considered as one of the marker of oxidative stress To measure the level of oxidative stress in each treatment group, DNA oxidative damage analysis was carried out using GC/MS As shown in Figure 4-4, pHL treatment did not alter the basal level of DNA oxidative damage as the level of DNA oxidative damage is almost the same in both sham group and sham treated with pHL group, 0.98 ± 0.05 and 1.08 ± 0.06, respectively Vehicle group has the highest level of DNA oxidative damage among all the groups (1.78 ± 0.03), which is almost double of the level as measured from

Total Antioxidant Capacity in Each Treatment Group

sham group, shown in Figure 4-4 pHL had a significant effect on reducing the oxidative damage level as reduction of the DNA oxidative damage to 1.19 ± 0.03 in stroke operated pHL treated group was observed (Figure 4-4), level that is comparable to sham group

Level of DNA Damage in Each Treatment Group (Blood)

4.1.2.3 Enhanced TUNEL nuclear green by MCAO and prevention by pHL

The apoptotic cells were identified through TUNEL staining Although this technique has been questioned since DNA can also be cleaved nonspecifically during necrosis, TUNEL staining has been largely used to evidence the apoptotic nature of neuronal death in global and focal ischemic models In TUNEL staining, apoptotic cells exhibited strong,

nuclear green fluorescence Apoptosis was detected in the infarct area of left cerebral cortex, while no apoptosis was detected in contralateral nonischemic hemisphere In addition, green fluorescence was not detected in sham (Figure 4-5a) and sham-operated pHL treated rats (Figure 4-5b), further confirm that no cell death was caused by the surgery itself Strongest apoptosis marker was observed in vehicle group (Figure 4-5c) This is correlated with the largest infarct volume and most severe neurological impairment pHL-treated group had significant reduction of apoptosis marker in the infarct area (Figure 4-5d)

Figure 4-5: Apoptotic staining in cerebral cortex after 7 days of MCAO (20x magnification) for each treatment group Slides were viewed at 520±20 nm to detect nuclear green fluorescence; and viewed at 460nm to detect DAPI staining Images shown were combined (overlapped) to detect overall morphology of cell population a) Sham group, b) Sham group treated with pHL; c) Stroke group rats; d) Stroke group treated with pHL

4.1.2.4 Apoptosis involvement of stroke and protection of pHL

The localizations of the apoptotic-related proteins were identified by immunohistochemical staining In frozen sections of rat brain after MCAO, the normal architecture was absent in the infarct zone of left cerebral cortex, while no change was observed in left cerebral cortex of the sham operated rats (Figure 4-6a) In the infarct zone, due to the immune cell infiltration, higher density of cell population was observed

in the infarct zone (Figure 4-6b)

Figure 4-6: Light photomicrographs (10x magnification) of cryostat section of the rat left

cerebral cortex a) Architecture of normal rat brain tissue b) Infarct area after MCAO Picture was shown in positive staining of Bax (strong brown signal) The localization of the apoptotic related proteins were identified by IHC

The specificity of the secondary antibody was demonstrated by omitting the primary antibody with otherwise identical experimental conditions No positive staining of any protein product of targeted gene was observed in the negative control from each a)

b)

treatment group (Figure 4-7a) No pro-apoptotic protein BAX staining was observed in both sham group and sham-operated-pHL treated group (Figure 4-7b) In contrast, strongest signal of BAX was observed in vehicle group (Fig 4-7b) Weaker BAX immunoreactivity was observed in stroke operated pHL treated group (Fig 4-7b) These positive staining of BAX was observed merely in infarct area Similar results were observed in each treatment group for FAS, as shown in Figure 4-7c Therefore, pro-apoptotic proteins BAX and FAS had been dramatically increased in the infarct area after MCAO as seen in the vehicle group Furthermore, these pro-apoptotic proteins could only

be detected in infarct area, indicating the up-regulation of these proteins in infarct area during ischemia as compared to undetectable levels of these proteins in non-infarct area For both anti-apoptotic proteins, BCL-2 and BCL-XL (Figure 4-7d, e), strongest positive signals could be observed in both sham and sham operated pHL treated groups A very weak BCL-2 positive staining and no BCL-XL signal were detected in vehicle group (Figure 4-7) throughout the brain sections The positive signals of both BCL-2 and BCL-

XL were restored in stroke operated pHL treated group as seen in Figure 4-7

Figure 4-7: Immunohistochemical staining of pro-apoptotic protein (b: BAX and c: FAS) and anti-apoptotic protein (d: BCL-2 and e: BCL-XL) in cerebral cortex after 7 days of MCAO (40x magnification), with a: negative control The staining showed stronger immunoreactivity of pro-apoptotic proteins and diminished immunoreactivity of anti-apoptotic proteins after stroke induction Following the pHL administration, enhanced immunoreactivity of anti-apoptotic proteins and weaker pro-apoptotic proteins immunoreactivity were shown i) Sham group; ii) Sham group treated with pHL; iii) Vehicle; iv) Stroke group treated with pHL (n=3)

4.2 Results of experiment II: Modulation of Mitochondrial

ROS Generation and Function by Purified Herba Leonuri Extract

4.2.1 Quality of isolated cortical mitochondria preparation

To determine the quality as well as the bioenergetics properties of isolated cortical mitochondria, JC-1 assay was employed to verify that isolated mitochondria are suitable for studies of bioenergetics Mitochondrial probe JC-1 is a cationic carbocyanine dye which uptake into mitochondria is driven by transmembrane potential (Reser et al, 1995) Uptake of JC-1 dye and ADP into the mitochondria was demonstrated by the increase in green fluorescence of monomers as basal conditions without added exogenous substrate, with less intense red fluorescence of the J aggregates (Figure 4-8) Mitochondria were treated with complex I and II substrates, malate/glutamate and succinate, respectively Upon energization of complex I substrate, malate/glutamate, an instant increase of membrane potential was shown by reduction of green fluorescence of J monomers and increase intensity of red fluorescence of J aggregates The situation was reversed by addition of complex I inhibitor rotenone (Figure 4-8), that green fluorescence intensity increased and red fluorescence intensity dropped When the intensity of green J monomer and red J aggregates was stabilized, mitochondria were again energized by complex II substrate, succinate A more drastic increase of J aggregates red fluorescence was observed with the addition of succinate as shown in Figure 4-8, reflecting the increase of membrane potential by succinate which is more drastic compared to malate/glutamate Mitochondrial membrane potential was collapsed by adding complex II inhibitor, TTFA This experiment demonstrated that isolated brain cortical mitochondria is responsive to

both complex I and II substrates, and also inhibitors of the complex I and II, therefore mitochondria preparation is suitable for bioenergetics studies Succinate is suggested to

be a better substrate for energization of cortical mitochondria as compared to malate/glutamate since isolated mitochondria have greater response to it, in the subsequent experiments, succinate was chosen as the substrate to energize isolated mitochondria

Figure 4-8: Quantitative recordings of the membrane potential from isolated cortical mitochondria Isolated mitochondria quality was assessed by JC-1 uptake (n=3) A more drastic increase of membrane potential was observed with the addition of complex II substrate succinate as compared to complex I substrate malate/glutamate

Membrane Potential of Isolated Mitochondria

4.2.2 Mitochondrial ROS production and prevention by pHL in vitro

Effect of pHL on mitochondrial ROS generation was determined by oxidation sensitive dye DCFDA In isolated mitochondria energized by succinate, pHL from 0.05mg/ml to 0.25mg/ml resulted in a dose-dependent reduction in ROS generation over 30 minutes

(Figure 4-9a), yielding a significant (p<0.05) reduction in DCF to 73.71±4.70% by

0.1mg/ml pHL and to 67.19±5.82% by 0.25mg/ml pHL at 30th minute (Figure 4-9b) Next, to investigate the effect of pHL on mitochondrial ROS generation under oxidative stress, another set of experiment was carried out by adding 1mM of H2O2 as ROS inducer

to mitochondria together with succinate for 5 minutes, followed by pHL treatment The

observed results demonstrated that about 4-fold increase (p<0.001) of ROS generation in

mitochondria treated H2O2 as compared to the control that was without H2O2 treatment (Figure 4-9c, d) It was observed that pHL treatment from 0.05mg/ml to 0.25mg/ml caused reduction in the ROS generation over 30 minutes under oxidative stress (Figure 4-

9c) A significant reduction of ROS generation to 46.55±5.11% (p<0.01) by 0.075mg/ml pHL, 43.86±3.20% by 0.1mg/ml pHL (p<0.01) and 35.11±2.20% by 0.25mg/ml pHL (p<0.001) was observed at 30th minute and is shown in Figure 4-9d

a)

Mitochondrial ROS Production in Each Treatment Group

(DCFDA)

0 0.5 1 1.5 2 2.5 3

pH0.075

pHL0.10

pHL0.25

were carried out in the absence (a) or presence (c) of 1mM H2O2 and pHL b, d) Percentage of mitochondrial ROS production at 30th minute was analyzed Data (n>5) are normalized and expressed as % respective to the control-Suc (a, b) and control-suc-

H2O2 (c, d) *p<0.05; **p<0.01; ***p<0.001 vs control-Suc (a, b) and control-suc-H2O2(c, d) ###p<0.001 vs control-suc

4.2.3 Effect of pHL on ATP biosynthesis in isolated mitochondria

To elucidate the effect of pHL on mitochondrial function, mitochondrial ATP biosynthesis assay was carried using luciferin-luciferase reaction Under physiological condition, mitochondria synthesize ATP using ATP precursor ADP through a highly organized respiratory chain driven by ETC substrates In this study, mitochondria were treated with complex II substrate succinate, ADP and pHL The study showed that ATP biosynthesis was suppressed by pHL dose-dependently as seen in Figure 4-10a, to

66.67±4.32% by 0.05mg/ml pHL (p<0.001), 34.24±4.80% by 0.1mg/ml pHL (p<0.001) and 11.86±1.39% by 0.25mg/ml pHL (p<0.001) Similarly to previous experiment,

mitochondria were exposed to 1mM of H2O2, and H2O2 could significantly reduce the

ATP synthesis to 52.61±9.32% (p<0.01) as compared to mitochondria treated with

succinate alone (Figure 4-10b) Results also showed that under condition whereby mitochondria treated with succinate together with H2O2, ATP biosynthesis was further suppressed by pHL The inhibitory effect did not reach statistical significant from 0.05mg/ml to 0.1mg/ml However, mitochondrial ATP biosynthesis was significantly

suppressed to 15.13±0.22% at dosage of by 0.25mg/ml (p<0.05) as compared to

52.61±9.32% by 1mM H2O2 (Figure 4-10b)

pHL0.075

pHL0.1

pHL0.25

and expressed as % respective to the control control-suc *p<0.05; ***p<0.001 vs

control-suc ##p<0.001 vs control-suc

4.2.4 Effect of pHL on mitochondrial respiration and RCR value

To further evaluate the effects of pHL on mitochondrial function, oxygen respiration was assessed using Clark-type oxygen electrode Mitochondrial respiration is normally divided into states or stages In this study, state 2 is referred when mitochondria were energized with succinate alone At this state, respiration rate is relatively low due to the lack of ADP For state 3 respiration, it was initiated with addition of succinate and ADP where drastic increase of O2 consumption was observed, indicating strong coupling of oxidative phosphorylation State 4 respiration is the basal level of respiration which can

be restored by adding oligomycin, a F0F1 ATP synthase inhibitor (Figure 4-11a)

In isolated mitochondria, increasing the pHL dosage from 0.05mg/ml to 0.25mg/ml did not produce any significant changes of metabolic rate as compared to the control group (Figure 4-11b) Although there were slight increase in mitochondrial respiration in groups treated with pHL, particularly to state 4 respiration, which was increased in a dose-dependent manner, it is not significant different from control group (Figure 4-11b) The resultant RCR (state 3 respiration rate/state 4 respiration rate) values were also no difference among the treatment groups (Figure 4-11c)

a)

Mitochondrial Oxygen Consumption In Vitro

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