| Objective: Cerebral vascular disease is of great concern to human beings since it has a high incidence rate,mortality rate,reoccurrence rate and disability rate.Cerebral vascular disease also,therefore,puts significant pressure on the finances of publicly-funded medical services.Cerebral ischemia is a very common kind of cerebral vascular disease,but new born vessels could provide the damaged brain tissue with oxygen,nutrients and neurotrophic support.Accumulating evidence indicates that angiogenesis after cerebral ischemia could relieve the patients' symptomsand improve their conditions,and research intoangiogenesis mechanisms has become a new target for research into treatments for cerebral ishchemia.Many traditional Chinese medicines have been proved to be functional in treating cerebral ischemia via different mechanisms.Ginsenoside Rg1 is an ingredient of ginsenosides and mainly exists in ginseng medicines.Ginsenoside Rg1 could relieve fatigue quickly,improve learning and memory abilities,delay the aging process,stimulate the central nervous system and also inhibit the coagulation of blood platelets.It has been confirmed that Ginsenoside Rg1 could enhance angiogenesis in terms of myocardial infarction,limb infarction in diabetes,ischemia necrosis of derma and hypoxic-ischemic encephalopathy.It has also been proved by experiments that Ginsenoside Rg1 could enhance the regeneration of blood vessels and also enhance angiogenesis in endothelial progenitor cells.This research used the middle cerebral artery occlusion(MCAO)model in mice and Ginsenoside Rg1 was used as an intervention to evaluate the angiogenesis function of Ginsenoside Rg1 after cerebral ischemia,aiming to prove its ability to enhance angiogenesis and discuss possible mechanisms.Healthy,male and adult C57BL/6 mice were used.The right arteria carotis communis was ligated and the cortex branch of middle cerebral artery underwent galvanocautery in microscope to prepare middle cerebral artery occlusion(MCAO)model.The groups were as follows: sham group(sham-operation group),MCAO(middle cerebral artery occlusion group),GRg1-L(disposed group at low dose),GRg1-M(disposed group at middle dose),GRg1-H(disposed group at high dose).The mice of all the groups were administered with drugs right away after the successful conduction of the operation and the drug was administered by intraperitoneal injection.The administration dose were as follows: disposed group at low dose(12.5 mg/kg),disosed group at middle dose(25 mg/kg),disposed group at high dose(50 mg/kg)and the drug was administered for 14 days consecutively.Rotarod and m NSS neurological score was conducted at day 1,day 7,day 14,day 21 and day 28 respectively.HE staining was made to evaluate the infarction volume of the brain at day 28.Western blot was conducted at day 3,day 7,day 14 to measure protein expression of VEGF,HIF-1?,VEGFR-2.Laser speckles machine was used to detect the brain blood flow condition before,right after and at day 1,day 3,day 7 and day 14 after the operation was successfully done.Results:1 GRg1 would improve neurological function deficit after cerebral ischemia in mice.This experiment conducted Rotarod and m NSS neurological score at day 1,day 7,day 14,day 21 and day 28 after cerebral ischemia was induced to all the groups including sham group,MCAO group,GRg1-L group,GRg1-M group,GRg1-H group.Prism was used to do statistics.24 hours after the infarction was induced,that is,1 day after onset of cerebral ischemia,all the mice in all the groups excluding sham group showed hemiplegic paralysis of the left limb which was a neurological deficit symptom and the neurological score rose,but the difference among all the groups were not statistically significant(P>0.05).Compared with MCAO group,the neurological scores of GRg1-L group,GRg1-M group and GRg1-H group Methods: were a lot lower and the difference was statistically significant from day 7 after the operation.In terms of falling latencies in Rotarod test,there was a siginificant difference between MCAO(P<0.05)and GRg1-H from day 7 after the operation.There were intermittent occasions when GRg1-M and GRg1-L were significantly different from MCAO but there was a constant difference between the two groups and GRg1-H,indicating that GRg1 did improve neurological deficit symptoms after cerebral ischemia and the ideal dose was high dose(50 mg/kg).2 GRg1 could not significantly lower the infarction volume of mice after cerebral ischemiaThe brain tissues of all groups of mice were taken out at day 28 after operation and HE staining was conducted.It was found out that there was no infarction focus in mice of sham group and there were infarction focus in mice of all the other groups.Compared with the MCAO group,the infarction volume of brain itssue in all of the medicine-treated groups was reduced but the difference was not statistically significant(MCAO vs.GRg1-H vs.GRg1-M vs.GRg1-L: 11.76%±2.02% vs.9.98%±1.03% vs.10.03%±2.11% vs.10.99%±2.10%,P > 0.05).This result indicated that GRg1 could not significantly lower the infarction volume of brain tissues.3 GRg1 could upregulate the protein expression of VEGF,HIF-1?,VEGFR-2 thus enhancing angiogenesis.This research conducted western blot to test the protein relative expression of three proteins that are related to antiogenesis including VEGF,HIF-1?and VEGFR-2 after treatment with GRg1 in face of cerebral ischemia.3 days,7 days and 14 days after the MCAO operation(the medicine was administered immediately after the operation i.p.,once a day,14 days consecutively),the brain tissue of the mice were taken out and procedures of western blot were followed to test VEGF,HIF-1 ?,VEGFR-2 protein expression.Image J and Prism were used for statistical analysis and the results indicated that from day 14 after cerebral ischemia of the mice,VEGF,HIF-1?,VEGFR-2 protein relative expression upregulated significantly in all the medicine-treated group compared with MCAO group(P<0.05).However,there were no differences among the 3 medicated groups of protein relative expression(P>0.05).But it still can be concluded that the higher the dosage,the greater effects GRg1 has on enhancing angiogenesis by upregulating proteins that are related to angiogenesis according to the trend.4 GRg1 could improve brain blood flow after cerebral ischemiaLaser speckle instrument was used to detect the brain blood flow of the mice of all groups before,right after and at day 1,day 3,day 7 and day 14 after the operation was done.It was observed that the brain blood flow of all the other groups of mice were reduced after the operation excluding the sham group and the difference between after operation and before operation was statistically significant(P<0.05).From day 3,the brain blood flow of all the disposed groups were improved compared with that of MCAO group and the difference was statistically significant(P<0.05);from day 7,the brain blood flow of GRg1-H group was higher than that of GRg1-M group,GRg1-L group and MCAO group and the difference was statistically significant(P<0.05).This result showed that GRg1 would effectively improve the brain blood flow after cerebral ischemia and the high dose(50 mg/kg)of GRg1 worked the most effectively on improving brain blood flow.Conclusion:Intraperitoneal injection of GRg1 would improve the neurological function of mice after cerebral ischemia,and would work as neuroprotective drugs to reduce the infarction volume of brain tissue.GRg1 would also enhance angiogenesis after cerebral ischemia by upregulating VEGF,HIF-1?,VEGFR-2 and would restore focal cerebral blood flow after cerebral ischemia.It can be concluded that GRg1 protects brain tissue and improve cerebral ischemia outcome by enhancing angiogenesis. |
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