Chapter 6: Conclusion and Future Perspectives Chapter Conclusion and Future Perspectives Department of Pharmacology, YLL School of Medicine 174 Chapter 6: Conclusion and Future Perspectives 6.1 Conclusion During ischemic cascade, neuronal injury results from the interaction of complex pathophysiological processes such as excitotoxicity, depolarization, apoptosis and inflammation and free radical generation. Mitochondrial dysfunction has also been well documented in the ischemic cascade of stroke. Therefore, oxidative stress intervention and mitochondria protection could be the approaches in the stroke therapy. Previous studies focusing on the antioxidant properties of TCMs have shown its promising therapeutic effects on stroke and mitochondrial protection. These encouraging results has prompted the author to further the studies on purified Herba Leonuri (pHL) and Leonurine which had been demonstrated to have cardioprotective effect in myocardiac infarction-induced rats through its antioxidant effects. Permanent focal ischemia by left middle cerebral artery occlusion (MCAO) in rats was employed in this research. Left MCAO model created in this study has consistently produced results of infarct volume spanning the region of ipsilateral cortex and striatum with severe neurological impairment. Oxidative stress is associated with permanent focal ischemia as the rats suffering from MCAO had reduction of total plasma antioxidant concentration level, cortical SOD and GPx activities, and enhanced cortical MDA level. This is accompanied with the increased level of apoptosis which are scattered throughout the ischemic territory. Mitochondrial dysfunction is also associated with MCAO as left cortical mitochondria isolated from MCAO-induced rat had lowered respiration rate. Department of Pharmacology, YLL School of Medicine 175 Chapter 6: Conclusion and Future Perspectives The pilot study on pHL (experiment I) has achieved the first objective in this thesis that to verify the possible therapeutic potential of pHL on MCAO-induced rats. Treatment of pHL could statistically reduce the infarct volume and neurological deficit score in animal subjected to MCAO. It is believed that the therapeutic effect of pHL acts through antioxidant effects and antiapoptosis by the observation of reduction in oxidative stress level and apoptosis. Subsequently, possible roles of pHL in mitochondrial ROS generation and mitochondrial function were evaluated. In experiment II, to elucidate the effect of pHL on modulation of mitochondrial function (objective 2), cortical mitochondria from Wistar rats. In isolated cortical mitochondria were isolated and a basal level of ROS generation ATP biosynthesis and oxygen consumption were observed upon challenged by succinate. Treatment of pHL was demonstrated to reduce mitochondrial ROS generation in a dose dependent manner, thereby preventing the contribution of mitochondria to oxidative stress under any circumstances. From the reduction of ATP biosynthesis, it is suggested that pHL might have effect of metabolic arrest to the mitochondria, leading to the cytoprotective barrier to the mitochondria in the case of stress condition, such as ischemia. However, this is yet to be confirmed. pHL might also have a mild uncoupling effect to the mitochondria respiration which has been shown to be a cytoprotective strategy, especially in brain, under the condition of oxidative stress (Brookes PS, 2006). A greater extent of ROS generation and suppressed ATP biosynthesis were observed in mitochondria treated with H2O2, reflecting the phenomenon mitochondria are the both contributors and targets of free radicals during oxidative stress. In the presence of pHL, Department of Pharmacology, YLL School of Medicine 176 Chapter 6: Conclusion and Future Perspectives both mitochondrial ROS generation and ATP biosynthesis are suppressed, indicating that effects of pHL could be executed in both physiological and pathological conditions. In vivo experiments showed that treatment of pHL could enhance mitochondrial respiration, indicating that pHL could protect mitochondria from being dysfunctional, which could protect the cell from undergoing apoptosis and challenging with free radicals. GSH level of mitochondria was also balanced by treatment of pHL in vivo. To conclude the findings from both experiments, pHL confers neuroprotective effects and therapeutic effects to ischemic stroke via few parameters: reduction in infarct volume, improvement of neurological deficit score, increase of plasma total antioxidant concentration, reduction of DNA oxidative damage, reduction of mitochondrial ROS generation, inhibition of ATP biosynthesis, improvement of oxygen consumption and balancing the mitochondrial GSH pool. The 3rd objective in this project was achieved in experiment III. Pretreatment of Leonurine for days prior to MCAO was applied to the animals. One day after MCAO, animal was sacrificed to assess the effect of Leonurine. Leonurine treatment was shown to reduce infarct volume and improve neurological function possibly through antioxidant effects. To higher degree of similarity, Leonurine showed much profound protective effect to isolated mitochondria as compared to pHL, with wider therapeutic range as compared to Department of Pharmacology, YLL School of Medicine 177 Chapter 6: Conclusion and Future Perspectives pHL, again confirm that Leonurine is one of the active ingredients in pHL. In vivo experiments also showed that Leonurine could enhance state respiration in mitochondria isolated from rats undergone MCAO. Mitochondrial GSH level could also be balanced by the treatment of Leonurine. The results obtained from experiment III showed that protective outcome of Leonurine is much similar as compared with the protective effects of pHL, whereby dosage and treatment period of Leonurine required were much smaller and shorter as compared to pHL. Mitochondrial studies also showed that mitochondria tolerate Leonurine with much wider range of dosage than pHL, and the effect of Leonurine on mitochondria is much stronger than pHL. Over many years of investigation of Chinese herbs, we tend to believe that the therapeutic effects of Chinese herbs are conferred by the synergistic effects from the herb mixture. With the promising results obtained from Leonurine, we still not exclude this possibility for the therapeutic potential for pHL. However, we hope for the better understanding and identification of detailed intracellular mechanistic pathway of pHL, possibly through Leonurine with known chemical and structural analysis. Department of Pharmacology, YLL School of Medicine 178 Chapter 6: Conclusion and Future Perspectives 6.2 Limitation of study Study in human stroke is particularly difficult and due to the limitation of collecting postmortem tissue at the time points after onset of stroke while brain damage occurs. Therefore, majority of brain ischemia studies and development of stroke therapy has relied on the animal models of ischemic injury. In addition to the animal models, attempts have been made to develop in vitro hypoxic model based on the deprivation of oxygen and glucose, or addition of chemical to induce hypoxia. Nonetheless, none of these models fully reflects the human stroke phenomenon. This contributes to countless failures of the clinical trials on therapeutic agents which have showed potential therapeutic effect on stroke, due to the lack of efficacy, inadequate dosing in human, or safety issue, indicating that a much more complicated ischemic cascade could have happened in human stroke. Therefore, more detailed stroke pathophysiology information is urgently needed. However, this does not undermine how important and valuable the current research is as every single research provides valuable information on specific mechanisms for the understanding of stroke and possible design for stroke therapy. Our group has recently shown that both pHL and Leonurine cross blood brain barrier (BBB) (unpublished data). Similarly, many of antioxidants showing promising therapeutic potentials in animal models of stroke injury have failed to show beneficial effects to humans. A major concern of antioxidants is their ability to cross BBB. Inability of antioxidants to cross BBB results in their inefficacy for stroke therapy. For example, feeding rats with coenzyme Q (CoQ) for months was failed to increase brain CoQ level. In addition, some of the antioxidants might not be able to reach the ROS-generating site, Department of Pharmacology, YLL School of Medicine 179 Chapter 6: Conclusion and Future Perspectives for instances, SOD and catalase not penetrate cell membranes, Vitamin E and CoQ are lipophilic which tends to be situated in the cell membranes (Szeto HH, 2006). Although many of TCMs has shown to have therapeutic effects to various kind of diseases, and contribute substantially to human health, pitfalls and shortcomings in herbal production exist. Standardization and effective manufacturing quality control, and supervision are still not in place. Clinical trials are needed to completely evaluate the effectiveness and efficacy of TCMs. Modern science and technology should be applied in the development, while complete quality control system should be employed in the manufacturing of TCMs. We sincerely hope that TCMs will contribute to public health in future. Department of Pharmacology, YLL School of Medicine 180 Chapter 6: Conclusion and Future Perspectives 6.3 Future perspectives The acute treatment of ischemic stroke to prevent neuronal injury and to improve neurological outcome remains a challenging task. Experimental models of focal ischemia have although provided us valuable insights that cerebral ischemia involves a series of complex signaling which ultimately leading to cell death over time and space. These experiments continually suggest a variety of methods for inhibiting the extension of infarction. In long term research we will still be required to understand the multifaceted molecular processes that contribute to the final cell fate in order to identify and develop novel stroke therapies. In recent years, a tremendous effort has been made to elucidate and further understand the oxidative stress involvement in ischemic stroke. Oxidative stress does not play in isolation, but interplay among these signaling pathways. Although countless failure of clinical trials resulted, investigations in this field have continually inspired an increased interest among researchers for the development of antioxidant stroke therapy. Mitochondrial research is also rapidly emerging as a potential therapeutic avenue for the amelioration of stroke injury. Valuable insights of mitochondrial responses to ischemic stroke have been provided by the investigation on the effects of a specific treatment or genetic manipulation which can modulate the mitochondrial changes. Additional studies should be performed to contribute to the understanding of the mitochondrial involvement in stroke injury. This may lead to the identification of potential agents which could Department of Pharmacology, YLL School of Medicine 181 Chapter 6: Conclusion and Future Perspectives intervene with mitochondrial function, thereby limit the tissue damage and improve the stroke therapy (Sims and Anderson, 2002). Mitochondrial medicine is a current unique discipline owing the advanced technologies and knowledge to the role of mitochondria in various diseases. The unique structural and functional properties of mitochondria allow design of drugs to specifically target at mitochondria. However, this idea is still at the stage of development. The reasons are the lack of knowledge on potential toxic effect of long term usage of these drugs in animals and lack of efficient methods to regulate drug delivery to the tissue of interest. Future development of drug delivery to mitochondria will be expected and it will solve these problems and possibly be the improved therapy for stroke treatment. With antioxidant properties of pHL and Leonurine, we aim at preventing mitochondrial ROS generation and protecting mitochondrial components from ROS-induced damage, to provide an environment with normal function of mitochondrial respiration, inhibit intrinsic pathway of apoptosis, and limit the injury resulted from ischemic insult. The multiple pathways involved in the ischemic cascade of stroke which lead to cell injury suggest us that there is considerable potential for additive or synergistic benefit from combined therapies. We have certainly not reached the stage in this field of investigation. We also hope that with the therapeutic information obtained from Chinese herbs, such as pHL and Leonurine, we could combine the drugs with western drugs to potentiate the therapeutic outcome, possibly minimize the size effects also. We expect to Department of Pharmacology, YLL School of Medicine 182 Chapter 6: Conclusion and Future Perspectives see the identification of several further therapeutic targets and with much more refinement of existing therapeutic agents. Anti-ischemic drug development is at a crossroad. Therapeutic benefits of TCMs have been recognized for centuries. Studies focusing on antioxidant properties of Chinese herbs have been shown its promising therapeutic effects against stroke because oxidative damage is a complex interplay that involves in cellular damage and cell death. Although there is lack of evidence and clarification of the specific ‘pathway’ the TCMs acting on, TCMs are still widely acceptable in Asia and beginning to be accepted by the rest of the world. Identification the therapeutic potential of Leonurine is indeed a breakthrough for the studies of pHL. 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Department of Pharmacology, YLL School of Medicine 200 [...]... in neuronal injury Current Alzheimer Research 2006, 3: 327-337(a) Loh KP, Huang SH, Benny KH, Zhu YZ Cerebral Protection of Purified Herba Leonuri Extract on Middle Cerebral Artery Occluded Rats J Ethnopharmacol 2009, (Epub ahead of print) Loh KP, Low LS, Wong WH, Zhou S, Huang SH, De Silva R, Duan W, Chou WH, Zhu YZ A comparison study of cerebral protection using Ginkgo biloba extract and Losartan... 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V, Martin E, Hoffman GE, Chinopoulos C, Kowaltowski A Protection against ischemic brain injury by inhibition of mitochondrial oxidative stress J Bioenerg Biomembr 2004, 36(4): 347-352 Folbergrová J, Memezawa H, Smith ML, Siesjö BK Focal and perifocal changes in tissue energy state during middle cerebral artery occlusion in normo- and hyperglycemic rats J Cereb Blood Flow Metab 1992, 12(1):25-33 Folbergrová... neuronal death and diminishes brain dysfunction after stroke and brain trauma Nat Med 2003, 9(8): 1062-1068 McKenna DJ, Jones K, Hughes K Efficacy, safety, and use of ginkgo biloba in clinical and preclinical applications Altern Ther Health Med 2001, 7: 70-90 Mehmet, H Stroke treatment enters the Fas lane Cell Death and Differentiation 2001, 8: 659-661 Menzies SA, Hoff JT, Betz Al Middle cerebral artery occlusion... forebrain ischemia in the rat 2 A 2-vessel occlusion model Acta Neurol Scand 1984, 69(6): 385-401 St-Pierre J, Buckingham JA, Roebuck SJ and Brand MD Topology of superoxide production from different sites in the mitochondrial electron transport chain J Biol Chem 2002, 277: 44784-44790 Starkov AA, Chinopoulos C, Fiskum G Mitochondrial calcium and oxidative stress as mediators of ischemic brain injury. .. Sharma SS Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats Life Sci 2004, 74(8): 969-985 Thorne Research Rhodiola rosea Alternative medicine review 2002, 7: 421-423 Touzani O, Young AR, Derlon JM, Baron JC, Mackenzie ET Progressive impairment of brain oxidative metabolism reversed by reperfusion following middle cerebral artery occlusion... extracts: requirement for dATP and cytochrome c Cell 1996, 86: 147-157 Liu XH, Chen PF, Pan LL, Zhu YZ 4-Guanidino-n-butyl Syringate (leonurine) Protects H9c2 Rat Ventricular Cells from Hypoxia-Induced Apoptosis Journal of Cardiovascular Pharmacology In press 2009(a) Liu XH, Xin H, Hou AJ, Zhu YZ Protective Effects of Leonurine in neonatal rat hypoxic cardiomyocytes and rat infarcted heart Clin Exp... 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Cerebral Protection of Purified Herba Leonuri Extract on Middle Cerebral Artery Occluded Rats. . pHL and Leonurine, we aim at preventing mitochondrial ROS generation and protecting mitochondrial components from ROS-induced damage, to provide an environment with normal function of mitochondrial. therapeutic effects on stroke and mitochondrial protection. These encouraging results has prompted the author to further the studies on purified Herba Leonuri (pHL) and Leonurine which had been