OBJECTIVE Progressive isch⁃emic stroke is characterized by aggravation of neurological dysfunction and poor prognosis.Neuroinflammation is involved in the pathological process of cerebral ischemia.Inflammasomes-activa...OBJECTIVE Progressive isch⁃emic stroke is characterized by aggravation of neurological dysfunction and poor prognosis.Neuroinflammation is involved in the pathological process of cerebral ischemia.Inflammasomes-activated caspase-1 has thus been considered a promising target for stroke therapy.However,it remains not fully understood how caspase-1 ag⁃gravates progressive functional impairment.We previously identified a novel caspase-1 inhibitor CZL-80,the present study is to explore whether CZL-80 protects against progressive ischemic stroke.METHODS Male C57/BL6 mice and cas⁃pase-1-/-mice were subjected to photothrombotic(PT)-induced cerebral ischemia.CZL-80 was in⁃traperitoneally injected daily during 1-7 d,1-4 d,4-7 d after PT.The grid-walking task and the cyl⁃inder task were used to determine the motor function.RESULTS Mice developed primary and the secondary neurological dysfunction at 1 d and 4-7 d after PT onset.The activation of cas⁃pase-1 peaked at 7 d after ischemic stroke and caspase-1 was mainly derived from activated microglia.Treatment with CZL-80(30 mg·kg-1)during 1-7 d significantly improved motor func⁃tion.Administration of CZL-80 during 1-4 d could not ameliorate motor function loss while administration during 4-7 d after PT onset signifi⁃cantly reduced foot faults and forelimb symme⁃try.Remarkably,treatment with CZL-80 during 4-7 d showed no significant difference in efficacy compared with the its administration during 1-7 d,which indicated a key therapeutic window.More⁃over,the neuroprotective effect of CZL-80 during 4-7 d was available at least until 43 d after isch⁃emic stroke,indicating CZL-80 can improve the long-term neurological function after cerebral ischemia.Furthermore,administration of CZL-80(30 mg·kg-1)during 4-7 d after PT onset in cas⁃pase-1-/-mice failed to improve the motor func⁃tion,which suggested that the neuroprotective effect of CZL-80 was caspase-1-dependent.The results showed that CZL-80 did not inhibit the expression of GSDMD and failed to reduce neu⁃ronal loss after ischemia.These results indicated the effect of CZL-80 was not attributable to inhib⁃it pyroptosis.We further found that CZL-80 signif⁃icantly reduced the number of activated microglia in the peri-infarct brain cortex after ischemic stroke,which might be involved in its neuropro⁃tective effect.CONCLUSION CZL-80,a novel caspase-1 inhibitor,improved motor function after progressive ischemic stroke in mice.The effective therapeutic window of CZL-80 would be 4-7 d after ischemia,when the secondary neuro⁃logical dysfunction occurred.Therefore,the inter⁃vention by targeting caspase-1 in this window phase provides a novel strategy for the function⁃al recovery of stroke survivors.展开更多
OBJECTIVE Only limited number of drugs are currently available for treating ischemic stroke.Therapeutic angiogenesis has recently emerged as one of the most promising therapies for cerebral ischemic injury.Isopropyl-...OBJECTIVE Only limited number of drugs are currently available for treating ischemic stroke.Therapeutic angiogenesis has recently emerged as one of the most promising therapies for cerebral ischemic injury.Isopropyl-β-(3,4-dihydroxyphenyl)-α-hydroxypropanoate(IDHP)is a metabolite derived from the botanical formulation for Dantonic®.Here,we investigated the angiogenic efficacy of IDHP in cerebral ischemia.METHODS The in vivo effects of IDHP were evaluated in the C57BL/6 mouse Matrigel plug and rat transient middle cerebral artery occlusion(tMCAO)models.Primary human umbilical vein endothelial cells(HUVEC)and human brain microvascular endothelial cells(HBMEC)were used to explore the effects of IDHP on stimulating proliferation,migration and tube formation in vitro.ELISA and Western blotting were used to quantitate the release and expression of relevant target molecules and signaling pathways.RESULTS IDHP reduced infarct volume and improved sensorimotor function in rats subjected to tMCAO by promoting angiogenesis,and promoted Matrigel neovascularization in mice.Moreover,IDHP produced a biphasic modulation on proliferation and migration both in HUVEC and HBMEC.It also induced tube formation in a 12-day HUVEC-HDF co-culture model and in Matrigel assays.IDHP-induced angiogenesis was accompanied by increased levels of p-AMPKα(Thr172)and p-eNOS(Ser1177)both in vitro and in vivo,and the decreased level of VEGF in rat brains on day 1 whereas enhanced level of VEGF on day 3 and 7 after tMCAO.Mechanistically,AMPK knockdown or pharmacologically inhibiting AMPK and its upstream kinases(CaMKKβ)inhibited the eNOS phosphorylation induced by IDHP in HUVEC.Furthermore,selective eNOS inhibitor(L-NIO),selective CaMKKβinhibitor(STO)and AMPKa inhibitor(Compound C)blocked the capillary-like tube formation in the co-culture model induced by IDHP(10 nmol·L^(-1)).CONCLUSION Collectively,these findings showed that IDHP protected rats from cerebral ischemia-reperfusion injury by promoting angiogenesis via activating CaMKKβ/AMPK(Thr172)/eNOS(Ser1177)signaling,and suggest it to be a promising new drug candidate for the prevention and/or treatment of cerebral ischemia and other vascular occlusive diseases.展开更多
文摘OBJECTIVE Progressive isch⁃emic stroke is characterized by aggravation of neurological dysfunction and poor prognosis.Neuroinflammation is involved in the pathological process of cerebral ischemia.Inflammasomes-activated caspase-1 has thus been considered a promising target for stroke therapy.However,it remains not fully understood how caspase-1 ag⁃gravates progressive functional impairment.We previously identified a novel caspase-1 inhibitor CZL-80,the present study is to explore whether CZL-80 protects against progressive ischemic stroke.METHODS Male C57/BL6 mice and cas⁃pase-1-/-mice were subjected to photothrombotic(PT)-induced cerebral ischemia.CZL-80 was in⁃traperitoneally injected daily during 1-7 d,1-4 d,4-7 d after PT.The grid-walking task and the cyl⁃inder task were used to determine the motor function.RESULTS Mice developed primary and the secondary neurological dysfunction at 1 d and 4-7 d after PT onset.The activation of cas⁃pase-1 peaked at 7 d after ischemic stroke and caspase-1 was mainly derived from activated microglia.Treatment with CZL-80(30 mg·kg-1)during 1-7 d significantly improved motor func⁃tion.Administration of CZL-80 during 1-4 d could not ameliorate motor function loss while administration during 4-7 d after PT onset signifi⁃cantly reduced foot faults and forelimb symme⁃try.Remarkably,treatment with CZL-80 during 4-7 d showed no significant difference in efficacy compared with the its administration during 1-7 d,which indicated a key therapeutic window.More⁃over,the neuroprotective effect of CZL-80 during 4-7 d was available at least until 43 d after isch⁃emic stroke,indicating CZL-80 can improve the long-term neurological function after cerebral ischemia.Furthermore,administration of CZL-80(30 mg·kg-1)during 4-7 d after PT onset in cas⁃pase-1-/-mice failed to improve the motor func⁃tion,which suggested that the neuroprotective effect of CZL-80 was caspase-1-dependent.The results showed that CZL-80 did not inhibit the expression of GSDMD and failed to reduce neu⁃ronal loss after ischemia.These results indicated the effect of CZL-80 was not attributable to inhib⁃it pyroptosis.We further found that CZL-80 signif⁃icantly reduced the number of activated microglia in the peri-infarct brain cortex after ischemic stroke,which might be involved in its neuropro⁃tective effect.CONCLUSION CZL-80,a novel caspase-1 inhibitor,improved motor function after progressive ischemic stroke in mice.The effective therapeutic window of CZL-80 would be 4-7 d after ischemia,when the secondary neuro⁃logical dysfunction occurred.Therefore,the inter⁃vention by targeting caspase-1 in this window phase provides a novel strategy for the function⁃al recovery of stroke survivors.
基金National Natural Science Foundation of China(31971143)and Primary R&D Plan of Shaanxi Province(2021KWZ-24)。
文摘OBJECTIVE Only limited number of drugs are currently available for treating ischemic stroke.Therapeutic angiogenesis has recently emerged as one of the most promising therapies for cerebral ischemic injury.Isopropyl-β-(3,4-dihydroxyphenyl)-α-hydroxypropanoate(IDHP)is a metabolite derived from the botanical formulation for Dantonic®.Here,we investigated the angiogenic efficacy of IDHP in cerebral ischemia.METHODS The in vivo effects of IDHP were evaluated in the C57BL/6 mouse Matrigel plug and rat transient middle cerebral artery occlusion(tMCAO)models.Primary human umbilical vein endothelial cells(HUVEC)and human brain microvascular endothelial cells(HBMEC)were used to explore the effects of IDHP on stimulating proliferation,migration and tube formation in vitro.ELISA and Western blotting were used to quantitate the release and expression of relevant target molecules and signaling pathways.RESULTS IDHP reduced infarct volume and improved sensorimotor function in rats subjected to tMCAO by promoting angiogenesis,and promoted Matrigel neovascularization in mice.Moreover,IDHP produced a biphasic modulation on proliferation and migration both in HUVEC and HBMEC.It also induced tube formation in a 12-day HUVEC-HDF co-culture model and in Matrigel assays.IDHP-induced angiogenesis was accompanied by increased levels of p-AMPKα(Thr172)and p-eNOS(Ser1177)both in vitro and in vivo,and the decreased level of VEGF in rat brains on day 1 whereas enhanced level of VEGF on day 3 and 7 after tMCAO.Mechanistically,AMPK knockdown or pharmacologically inhibiting AMPK and its upstream kinases(CaMKKβ)inhibited the eNOS phosphorylation induced by IDHP in HUVEC.Furthermore,selective eNOS inhibitor(L-NIO),selective CaMKKβinhibitor(STO)and AMPKa inhibitor(Compound C)blocked the capillary-like tube formation in the co-culture model induced by IDHP(10 nmol·L^(-1)).CONCLUSION Collectively,these findings showed that IDHP protected rats from cerebral ischemia-reperfusion injury by promoting angiogenesis via activating CaMKKβ/AMPK(Thr172)/eNOS(Ser1177)signaling,and suggest it to be a promising new drug candidate for the prevention and/or treatment of cerebral ischemia and other vascular occlusive diseases.
