Ginkgolide K Promotes Angiogenesis After Ischemia Stroke Through JAK2/STAT3 Pathway

With the development of social economy and the improvement of people's living standards,stroke has become a more and more common disease,it has high mortality and morbidity.Studies have shown that about 80%of people after local cerebral ischemia will leave a different degree of sequelae,brought the community and the family a heavy burden.The main pathological features of Cerebral Ischemia are the brain stenosis and acute cerebral blood circulation disorders caused by occlusion.Modern theory suggests that energy depletion,calcium overload,oxidative stress,excitotoxicity,inflammation,autophagy and apoptosis all play important roles in the pathogenesis of cerebral ischemia.Stroke is refractory disease,there are still no effective therapeutic approaches on clinic,the current acute treatment is limited to r-tPA thrombolytic therapy,but there still exists some problems such as narrow treatment time window and side effects.The recovery period mainly focuses on neurogenesis nerve regeneration and angiogenesis.Angiogenesis after cerebral ischemia can improve the blood supply in the injured area,especially the ischemic penumbra,increase the transport of oxygen and nutrients,so that can reduce the infarct volume,regenerate the microenvironment around the blood vessels and facilitate neurogenesis and synaptic formation.At present,it has been gradually recognized that angiogenesis is conducive to improving the prognosis of patients with stroke,how to strengthen the endogenous angiogenesis,and promote neurological repair after cerebral ischemia,has become a hot topic in recent years.JAK2/STAT3 is an important signaling pathway of JAK/STAT family,which plays an important role in cell proliferation,differentiation,angiogenesis and other aspects.When ischemic stroke occurs,the expression of phosphor-STAT3 increased significantly,which is involved in neuroinflammation and angiogenesis.It was found that catalpol can increase the expression of VEGF and p-STAT3 in the cerebral ischemic brain,through which accelerating the angiogenesis and vascular remodeling.In addition,Danshen polyphenolic acid can regulate the level of VEGF,Ang-1 and Ang-2 through JAK2/STAT3 pathway to promote angiogenesis and improve neurological impairment after cerebral ischemia.Ginkgolide K(GK)is a new compound extracted from Ginkgo biloba leaves,which is very similar to the structure of Ginkgolide B and belongs to diterpene lactone compounds.At present,there are few researches about ginkgolide K,and the main research findings are the anti-oxidative stress and neuroprotective effect of ginkgolide K on ischemic stroke.The preliminary results of our laboratory indicated that ginkgolide K was superior to ginkgolide B and ginkgolide at the same dose in the acute phase of ischemic stroke,which suggested that GK may have potential pharmacological effects as a new Found ginkgo lactone drug,and it worth further study.Nowadays Ginkgolide has been used for clinical treatment of stroke in the recovery period.Ginkgolide K as one of ginkgolides,there still has no research on whether it exerts a protective effect on angiogenesis during ischemic stroke.The aim of this study was to investigate whether ginkgolide K has an effect on promoting angiogenesis and its mechanism after cerebral ischemia-reperfusion.In this study,C57BL/6 mice were used to do a transient middle cerebral artery occlusion(tMCAO)to subject cerebral ischemia/reperfusion model.After the reperfusion,the mice were treated with GK,and its protective effect and influence on angiogenesis were assessed 14 days after tMCAO.Meanwhile we used the oxygen-glucose deprivation/reperfusion(OGD/R)model on b End3 cells to research the effect of GK on HIF-1?,VEGF and JAK2/STAT3 in mice.The research clarifies that GK stimulates angiogenesis after cerebral ischemia-reperfusion injury,and its mechanism is related to the up-regulation of VEGF and the activation of JAK2/STAT3 pathway,which provides an academic basis for the clinical application of this drug.AIM:To investigate the protective effect and angiogenesis of GK on long-term repair of tMCAO model in mice,and the role of JAK2/STAT3 pathway and VEGF played in angiogenesis.METHODS:1)The transient middle cerebral artery occlusion model was subjected on 2-3 months old C57BL/6 mice.After 1 hour of ischemia and 1 hour of reperfusion,doses of GK(3.5,7.0,14.0 mg/kg,ip.Bid.,14 d)were given.The neurological deficits were assessed on day 1,4,7,14 after tMCAO.And rotarod test and MRI were performed on the 14th day.The IgG leakage and the morphological changes of collagen IV were observed by immunohistochemistry after perfusion.Through the above indicators to explore whether GK has a protective effect on the long-term repair after cerebral ischemia-reperfusion.2)On the 14th day,the blood flow was monitored by Doppler flow detector.And Glutl/Ki67 immunofluorescence co-lebelling was used to observe the effect of GK on angiogenesis after cerebral ischemia-reperfusion.3)2-3 month's old C57BL/6 mice were used to establish the tMCAO model.The dose of GK(3.5 mg/kg,ip,bid,14 d)was selected according to the preliminary results.And GK was administrated 1h after occlusion for 1h.Collect the brain tissue,RT-PCR and western blot were used to evaluate the expression of HIF-1? and VEGF.4)GK(0.1,1,10,50,100?M)were pre-treated to b End3 cells before OGD/R(3h),after culturing tor 24h,the viability of b End3 cells were detected by MTT and LDH to select the minimum effective concentration of GK.5)b End3 cells were cultured for OGD/R(3h),GK(10?M)was pre-treated to the cells before OGD/R,Western blot and RT-PCR were used to evaluate the expression of HIF-1? and VEGF.6)b End3 cells were cultured for OGD/R(3h),GK(10?M)was pre-treated to the cells before OGD/R,Western blot was used to detect the expression of JAK2?p-JAK2?STAT3?p-STAT3.7)b End3 cells were cultured for OGD/R(3h),GK(10?M)and AG490(10?M)were pre-treated to the cells before OGD/R,the morphological effects of GK on b End3 cells after OGD were observed by immunofluorescence of VEGF.8)b End3 cells were cultured for OGD/R(3h),GK(10?M)and AG490(10?M)were pre-treated to the cells before OGD/R,Western blot was used to detect the expression of JAK2,p-JAK2,STAT3,p-STAT3,HIF-1?,VEGF.RESULTS:1)GK(3.5,7.0,14.0 mg/kg)significantly promoted the recovery of neurological function after 14 days of cerebral ischemia-reperfusion,reduce the infarct volume and the leakage of the blood-brain barrier.2)GK promoted the recovery of injured ipsilateral cerebral blood flow after ischemia-reperfusion in mice.3)GK(3.5 mg/kg,ip,bid,14 d)significantly up-regulated the expression of HIF-la and VEGF in the mice brain of 14 days after tMCAO model.4)GK(10,50,100?M)significantly increased the viability of b End3 cells and decrease the LDH level.5)GK significantly up-regulated the expression of HIF-la and VEGF in b End3 cells after OGD for 3h.6)GK significantly up-regulated the protein expression of p-JAK2 and p-stat3 in b End3 cells after OGD.7)The cell immunofluorescence of VEGF showed that b End3 cells were arranged in a compact and tend to form a circular lumen,and after treatment with OGD,b End3 cells were swollen and the rounded lumen formed by cell was destroyed,while GK attenuated the morphological damage of b End3 cells,which can be partly abolished by AG490,an agonist of JAK2.8)Pretreatment of AG490 inhibited the up-regulation effect of GK on the expression of HIF-1?,VEGF,p-JAK2 and P-STAT3 to some extent.CONCLUSIONS:1)GK attenuated neurological impairment and promotes angiogenesis in long-term recovery after cerebral ischemia-reperfusion in mice.2)GK(3.5 mg/kg,ip,bid,14 d)promoted angiogenesis through activating JAK2/STAT3 pathway to upregulate the expression of HIF-la and VEGF.IN SUMMERY:1)GK exerted the protective effect in the long-term recovery of cerebral ischemia-reperfusion through promoting angiogenesis of the ischemic penumbra.GK promoted the angiogenesis of the ischemic penumbra in the tMCAO mice to improve cerebral blood flow,as well as reducing the volume of cerebral infarction and promoting the repair of nerve and motor function.2)GK promoted angiogenesis through increasing the expression of HIF-1?/VEGF by activating JAK2/STAT3 pathway.GK significantly increased the expression of HIF-la,VEGF in the ischemic mice brain.And GK significantly increased the expression of HIF-1?,VEGF,p-JAK2 and p-STAT3 in b End3 cells after OGD,while the protective effect of GK was partially abolished when the JAK2/STAT3 pathway was inhibited.