Kothanahalli S Raghu1, Bhanuvalli R Shamprasad2, Shama P Kabekkodu3, Puspendu Paladhi4, Manjunath B Joshi5, Marthandavarma S Valiathan6, Kanive P Guruprasad7, Kapaettu Satyamoorthy8. 1. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: raghushivanna@gmail.com. 2. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: shamspbr@gmail.com. 3. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: spbhat81@gmail.com. 4. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: puspendumid@gmail.com. 5. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: manjunath.joshi@manipal.edu. 6. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: msvaliathan@gmail.com. 7. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: guruprasad.kp@manipal.edu. 8. School of Life Sciences, Manipal University, Planetarium Complex, Manipal 576 104, Karnataka, India. Electronic address: ksatyamoorthy@manipal.edu.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Indian traditional medicinal system in Ayurveda suggests several preparations, known as medhya rasayanas, of diverse plant origin to enhance the health in general, reduce stress and improve brain function in particular during ageing. These effects in the context of contemporary knowledge and the underlying mechanisms are not clearly understood. Autophagy and DNA damage induced repair are inter-related quintessential pathways and are significantly altered during stress and ageing. Hence, medhya rasayana prepared from Clitoria ternatea (locally known as shankhpushpi) was used to test these effects in Wistar rat model of various age groups upon stereotaxic mediated kainic acid induced brain injury. MATERIALS AND METHODS: The rodent experiments were carried out in one, twelve and eighteen months old male Wistar rats. The rats were orally fed with medhya rasayana prepared from Clitoria ternatea (3g per kg body weight/day) for 60 days. Stereotaxic mediated kainate stress to the hippocampus was performed on day 61. The rats were sacrificed on 66th day and the brain tissues were analyzed histologically and measured for autophagy, base excision repair and antioxidant enzyme activities. In addition, cognitive functions were analyzed by employing novel object recognition task and Morris water maze tests. The gene expression profile of hippocampus was assessed by microarray hybridization and two genes are validated. RESULTS: Our study showed significant decrease of autophagy by medhya rasayana in both 12 and 18 months old rats. The hippocampal CA3 cellularity were increased in stereotaxic mediated stressed rats by medhya rasayana. There were no significant differences in constitutive base excision repair and antioxidant enzyme activities. Medhya rasayana treatment also significantly increased episodic memory in rats. Microarray experiments for pathway specific gene expression analysis showed altered expression of genes of long-term potentiation, axon guidance, neuroactive ligand-receptor interaction, regulation of autophagy, lysosome, homologous recombination and nucleotide excision repair pathways in adult rats by medhya rasayana. CONCLUSIONS: In the present study, we show that reduction in autophagy is crucial for medhya rasayana induced protection of rat hippocampal cells and that artificially enhanced autophagy protects the brain cell damage by maintaining the selective DNA damage repair pathway and removal of reactive oxygen species to inhibit apoptosis. These findings suggest autophagy directed pathways by medhya rasayana prepared from C. ternatea protects the brain cells from stress induced injury.
ETHNOPHARMACOLOGICAL RELEVANCE: Indian traditional medicinal system in Ayurveda suggests several preparations, known as medhya rasayanas, of diverse plant origin to enhance the health in general, reduce stress and improve brain function in particular during ageing. These effects in the context of contemporary knowledge and the underlying mechanisms are not clearly understood. Autophagy and DNA damage induced repair are inter-related quintessential pathways and are significantly altered during stress and ageing. Hence, medhya rasayana prepared from Clitoria ternatea (locally known as shankhpushpi) was used to test these effects in Wistar rat model of various age groups upon stereotaxic mediated kainic acid induced brain injury. MATERIALS AND METHODS: The rodent experiments were carried out in one, twelve and eighteen months old male Wistar rats. The rats were orally fed with medhya rasayana prepared from Clitoria ternatea (3g per kg body weight/day) for 60 days. Stereotaxic mediated kainate stress to the hippocampus was performed on day 61. The rats were sacrificed on 66th day and the brain tissues were analyzed histologically and measured for autophagy, base excision repair and antioxidant enzyme activities. In addition, cognitive functions were analyzed by employing novel object recognition task and Morris water maze tests. The gene expression profile of hippocampus was assessed by microarray hybridization and two genes are validated. RESULTS: Our study showed significant decrease of autophagy by medhya rasayana in both 12 and 18 months old rats. The hippocampal CA3 cellularity were increased in stereotaxic mediated stressed rats by medhya rasayana. There were no significant differences in constitutive base excision repair and antioxidant enzyme activities. Medhya rasayana treatment also significantly increased episodic memory in rats. Microarray experiments for pathway specific gene expression analysis showed altered expression of genes of long-term potentiation, axon guidance, neuroactive ligand-receptor interaction, regulation of autophagy, lysosome, homologous recombination and nucleotide excision repair pathways in adult rats by medhya rasayana. CONCLUSIONS: In the present study, we show that reduction in autophagy is crucial for medhya rasayana induced protection of rat hippocampal cells and that artificially enhanced autophagy protects the brain cell damage by maintaining the selective DNA damage repair pathway and removal of reactive oxygen species to inhibit apoptosis. These findings suggest autophagy directed pathways by medhya rasayana prepared from C. ternatea protects the brain cells from stress induced injury.