Protective Effects And Mechanisms Of Diammonium Glycyrrhizinate On Cerebral Ischemia-reperfusion Injury In Mice

Cerebrovascular ischemia is a deadly disease that affects the health and life of many people. Traditional Chinese medicine considers that stroke is caused by reversed flow of Qi and blood which is made by disfunction of zang-fu viscera related to deficiency of Qi or Yin, fire of heart or liver, wind of outside or inside, sputum of wind or moist, qi stagnation or reversion, blood deficiency or stasis on the fundation of deficiency of Qi,the above factors interact with each other under certain conditions, resulting in dysfunctional organs, blood against the chaos and committing on the brain.The desease has been characterized with high incidence, high prevalence, high mortality, high morbidity, and high recurrence rates. Recent studies indicate that inflammatory reaction triggered by brain ischemia is an important factor leading to cerebral ischemic injury. The inflammatory reaction is a complex network process with multi-channel, multi-step, and multi-factor interactions. It is related to the infiltration of inflammatory cells, the expression of various cytokines, the activation of nuclear transcription factors (NF-κB) and other pathological links. The in vivo inflammatory metabolic network has a complicated dynamic regulatory mechanism. A variety of proinflammatory factors, cytokines, cyclooxygenase, nitric oxide synthase (NOS), NF-kappa B, and other pathways are involved in the regulation. Therefore, further studies on investigating the inflammatory pathway of cerebrovascular ischemia and understanding its action mechanism in the process of cerebral ischemic lesions have an important medical value.There is a growing interest in traditional medicines for treating ischemic stroke patients, partially due to the lack of effective and widely applicable pharmacological treatments. Radix Glycyrrhizae calm in nature and sweet in flavour is qi-supplementing drug with the function of invigorating spleen-stomach and replenishing qi, clearing heat-toxicity, moistening lung for arresting cough, relieving spasm and pain, harmonizing character of drug, its roots can be used as expectorant, diuretic, and clearing the lungs agent.Diammonium glycyrrhizinate (DG) is a salt form of glycyrrhizic acid(GA). GA is a triterpene glycoside extracted from licorice root (Glycyrrhiza glabra) which consists of one molecule of18b-glycyrrhetinic acid and two molecules of glucuronic acid. GA has been attributed to numerous pharmacologic effects like anti-inflammatory, antiviral, anti-tumor, and hepatoprotective activities. It has been shown tha GA inhibited the inflammation in mice model of liver injury. However, the protective role of D(or GA in cerebral ischemia reperfusion has not been clearly characterized.Objective:To investigate the neuroprotective effect and possible mechanism of DG on cerebra ischemia and provide a theoretical basis for treating ischemic brain injury.Methods:In the first part of this study, the mouse middle cerebral artery occlusion (MCAO model was employed. Diammonium glycyrrhizinate group was treated by intraperitonea injection of diammonium glycyrrhizinate. MCAO group was given the same amount of saline The sham operation group (sham group) served as controls. Neurological deficits, infarct size and brain water content (BWC) were measured after1,3, and7days of the MCAO. Th measurements were used to characterize neurologic impairment, brain water content and th volume of cerebral infarction. The results were used to evaluate protective effect or diammonium glycyrrhizinate in cerebral ischemia and reperfusion injury of rats. HE and Niss staining were used to evaluate the effect of diammonium glycyrrhizinate on pathological change of pathological tissue after cerebral ischemia reperfusion injury.In the second part of the study, RT-PCR was used to detect the expression level o inflammatory cytokines IL-1β, IL-6, TNF-amRNA, COX-2, and iNOS mRNA Immunohistochemical was used to detect the positive expression cell number of IL-6, TNF-alpha COX-2and GFAP. The inhibiting effect of diammonium glycyrrhizinate on the inflammatory reaction in cerebral ischemia reperfusion injury was evaluated.In the third part of study, the expression of NF-κB positive cell number was detected according to immunohistochemistry. Western blot was used to analyze protein activation of phosphorylated (p-IκB), NF-κB P50, and P65. The diammonium glycyrrhizinate's protective effect on mice brain ischemic reperfusion injury and NF-κB signaling pathway was evaluated.Results:The first part of the experiment showed that, compared with the MCAO group, diammonium glycyrrhizinate intervention after cerebral ischemia can significantly improve the degree of neurological deficits (P<0.05), mitigate cerebral edema (P<0.05), and infarct volume (P<0.05). The number of complete and legible nerve cells increased significantly (P<0.05) in HE staining. Degeneration and necrosis of tissue is relatively narrowed and lessened. Mice cerebral cortical neurons of Nissl bodies treated with the diammonium glycyrrhizinate was significantly increased (P<0.05).The second part of the experiment indicated that, compared with the MCAO group, the expression levels of IL-1β, TNF-alpha, COX-2, and iNOS mRNA in the mice brain cortex treated by diammonium glycyrrhizinate were significantly lower1day and3days after the reperfusion (P<0.05). The expression level of IL-1β, TNF-alpha, and COX-2mRNA were decreased significantly after7days (P<0.05). Yet, the expression level of iNOS mRNA was not significantly lower (P>0.05). The expression level for IL-6mRNA expression levels was significantly increased on day1and day3after the reperfusion (P<0.05) but there was no significant change after7days (P>0.05). Immunohistochemical results showed that the number of COX-2and TNF-a positive cell count in cerebral cortex treated with diammonium glycyrrhizinate was significantly lower after1day and3days of the reperfusion (P<0.05), and decreased significantly after7days (P<0.05). No significant change in COX-2was observed (P>0.05). The number of IL-6positive cells increased after1,3and7days of the reperfusion (P<0.05) and GFAP-positive cells was significantly reduced (P<0.05).The third part of the experiment showed that, compared with the MCAO group, the NF-KB-positive cells in the mice brain cortex treated with diammonium glycyrrhizinate were significantly lower on day1, day3, and day7after the reperfusion (P<0.05). The expression level of p-IκB, NF-κB P50, and P65decreased significantly (P<0.05).Conclusions:Diammonium glycyrrhizinate intervention may play the role of cerebral protection by reducing cerebral ischemia and reperfusion injury. The protection may occur through the down regulation of the NF-κB pathway and inhibition of ischemic inflammatory reaction within the local brain tissue.