| As a common high-risk disease,cerebral ischemia has been widely concerned by scholars.Judging from the current prevention and treatment of cerebral ischemic diseases,Tibetan medicine has its own unique advantages and distinct characteristics,whether it is the study of pathogenic theory or the means of prevention and treatment.The three Tibetan classics,namely Wishful-treasure Pills,25-Herb shanhu Pills and 20-Herb chenxiang Pills,have shown significant curative effects in long-term clinical studies of cerebral ischemic diseases.However,due to the complex composition of three Tibetan medicines,the mechanism research is not yet clear.Based on the above characteristics,in this study,the method of serum pharmacochemistry was used to trace and identify the active components of blood in three Tibetan medicines by HPLC,and at the same time by copy the right cerebral ischemiareperfusion(I/R)model in rats,to evaluate the efficacy of active monomer on cerebral ischemia.Methods:(1)Tibetan medicines were intragastrically administered to the rats according to different doses,and the serum was collected at 1 h after intragastric administration.Using serum pharmacochemistry method,the in vivo and in vitro biological samples of the three Tibetan medicines were analyzed by HPLC at 250 nm and 400 nm,and then compared with 25 standards to trace the active ingredients in the blood.(2)Healthy Sprague-Dawley rats were randomly divided into 6 groups: sham group,model group,positive group,monomer A low-dose group(10 mg/kg),middle-dose group(30 mg/kg)and high-dose group(50 mg/kg),18 rats in each group.The sham group and model group were given normal saline,the positive group was given nimodipine suspension(18.9 mg/kg).All the groups were administered once a day.Prophylactic administration for 3 days,the corresponding operation was performed in each group,and then treat for 5 days after operation.After the last dose,serum and brain tissue samples were collected for TTC staining,brain water content measurement,histopathological examination(HE staining,TUNEL staining,Nissl's staining),inflammatory factor detection(TNF-?,IL-6,IL-1?),oxidative stress level(SOD,MDA),coagulation factor XII detection and permeability of the blood-brain barrier.(3)Healthy Sprague-Dawley rats were randomly divided into 6 groups: sham group,model group,positive group,monomer B low-dose group(10 mg/kg),middle-dose group(30 mg/kg)and high-dose group(50 mg/kg),12 rats in each group.The model of right middle cerebral artery ischemia reperfusion were established by using middle cerebral artery occlusion(MCAO).The systemic efficacy of monomer B was evaluated in terms of cerebral infarction volume,brain water content,histopathology(HE staining,Nissl staining,TUNEL staining)and oxidative stress(MDA,SOD)in serum and brain tissue.Results:(1)Comparing the HPLC characteristic spectrum with standard of those three Tibetan medicines in vivo and vitro.The results showed that there are 14 components in total for the three Tibetan medicines in vitro,and 6 components in vivo.In other words,there are a total of 6 components prototype constituents absorbed into blood in the three Tibetan medicines.(2)Efficacy evaluation of monomer A in rats with ischemia-reperfusion injury: The high-dose group(50 mg/kg)can significantly reduce the cerebral infarct size and water content,improves Nissl's body damage,inhibits apoptosis in I/R rats.Significantly downregulates TNF-?,IL-6,IL-1? expression in rat serum and brain tissue,thereby inhibiting inflammatory responses.Significantly down-regulates the expression of AQP-4 and MMP-9 in brain tissue,up-regulation the expression of ZO-1,thereby improving brain edema and protecting and repairing the BBB.Significant upregulation the activity of SOD in serum and brain tissue,down-regulation the expression of MDA,thereby improving oxidative stress injury.In addition,there was no significant difference in coagulation factor XII between groups,indicating that oral administration of 10 mg/kg,30 mg/kg,and 50 mg/kg monomer A did not cause significant coagulation.(3)Efficacy evaluation of monomer B in rats with ischemia-reperfusion injury: Monomer B can improve cerebral ischemia reperfusion injury,and presented a dose-dependent relationship.Cerebral infarction area and water content of I/R rats were significantly reduced in the high-dose group(50 mg/kg).Nissl bodies increased significantly,and the number of TUNEL-positive cells was significant decreased,while SOD expression was up-regulated and MDA expression was significantly down-regulated.Conclusion: Monomer A and monomer B can effectively improve cerebral ischemic reperfusion injury in rats.The common mechanism is to reduce the infarct size,reduce brain water content,improve Nissl's body injury,and inhibit apoptosis,improve the damage of oxidative stress.At the same time,monomer A can effectively improve the body's inflammatory response,and play a certain role in the protection and repair of BBB. |
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