Regulation, Detoxification And Collaterals Of Acute Cerebral Ischemia In Rats Rok¦Á And Pkc¦Ä In / Marcks Signaling Pathway Interaction

BackgroundThe treatment of acute cerebral ischemia injury is based on the Hypothesis of the Injury of Brain Collaterals by Toxins. MARCKS (myristoylated alanine-rich kinase substrate) is a pro minent substrate for protein kinase C (PKC). Previous studies have shown that the medcine with the function of Resolving Toxin and Dredging Collaterals can greatly decrease the activation of PKC/MARCKS signaling pathway in cerebral ischemic injury. Re minding the relevance between the modern biological content of Injury of Brain Collaterals by Toxins and cell signal transduction.The latest research shows that Rho/Rho-kianse (Rho/ROK) signaling pathway is also involved in regulation of MARCKS phosphorylation. Fasudil, a novel and specific ROK inhibitor, inhibited not only MARCKS but also PKCδphosphorylation in vitro. Therefore, it was speculated that PKCδ/MARCKS is downstream of ROKα. The crosstalk between ROKa and PKCδ/MARCKS pathway in vivo has not been reported. Whether KuDieZi injection with the function of Resolving Toxin and Dredging Collaterals could decrease cerebral ischemic injury by inhibiting activation of ROKa and PKC 8/MARCKS in vivo has yet to be clarified.ObjectiveTo investigate the crosstalk between ROKαand PKCδ/MARCKS pathway in vivo, confirm that ROKαand its downstream PKCδ/MARCKS participate in the cerebral ischemic injury. Furthermore, to investigate that KuDieZi injection with the function of Resolving Toxin and Dredging Collaterals plays a role in cerebral protection by decreasing activation of ROKα/PKCδ/MARCKS pathway. To clarify the relevance between ROKα/PKCδ/MARCKS pathway and interior Toxins and pathological Collaterals, and the mechanism of cerebral protection in acute cerebral ischemic by Resolving Toxin and Dredging CollateralsMethods1. The middle cerebral artery occlusion (MCAO) rat model was established by method of improved Zea Longa. To therapy with KuDieZi injection and/or Fasudil hydrochloride injection.2. The Longa 5 level neurological injury score was used to evaluate the model. To investigate changes after therapy.3. HE staining and microscope was used to observe the pathology of rat brain and changes after treatment.4. Immunohistochemistry and Western Blot were used to investigate the expression and distribution of ROKα, PKCδ, p-PKCδ, MARCKS, p-MARCKS in rats'cortex and hippocampus and changes after treatment. 5. Real-time quantitive PCR was used to investigate the expression of ROKα,PKCδ,MARCKS mRNA in rats'cortex and hippocampus and changes after treatment.6. The statistical software SPSS 10.0 was used to analysis the relevance between ROKα/PKCδ/MARCKS and neurological injury score.Results1. All the rat models had occurred neurological injury signs, and the neurological injury score were decreased significantly after treatment (P<0.01)2. The pathology of the model was found with obvious alterations of ischemia, and treatment with KuDieZi injection and/or Fasudil could inhibit the change.3. The expression of ROKα, PKCδ, p-PKCδ, MARCKS, p-MARCKS in cortex and hippocampus of acute ischemia brain were elevated comparing with the control group (P<0.05)4. The expression of ROKα, PKCδ, p-PKCδ, MARCKS, p-MARCKS were found decreased in ischemia brain tissues with treatment of KuDieZi and/or Fasudill(P< 0.05).5. The expression of ROKα,PKCδ,MARCKS mRNA were found decreased in ischemia brain tissues with treatment of KuDieZi and/or Fasudill(P<0.05).6. The expression of ROKα, PKCδ, p-PKCδ, MARCKS, p-MARCKS was positively correlated with neurological injury score (P<0.01 or P<0.05).Conclusion1. The neurological injury score and pathology were decreased by treatment with KuDieZi injection and/or Fasudil, suggesting that KuDieZi injection and/or Fasudil had function of cerebral protection.2. The expression of ROKa, PKC8, p-PKCδ, MARCKS, p-MARCKS and their mRNA were positively correlated with neurological injury score, suggesting that ROKa and PKCδ/MARCKS pathway was relevant with neurological injury.3. The expression and activation of ROKa and PKCδ/MARCKS were decreased by treatment with Fasudil, a novel and specific ROK inhibitor. We could speculate that ROKa located at upstream of PKCδ/MARCKS.4. The expression and activation of ROKαand PKCδ/MARCKS were decreased by treatment with KuDieZi injection with the function of Resolving Toxin and Dredging Collaterals. Re minding the relevance between the modern biology content of Injury of Brain Collaterals by Toxins and cell signal transduction.