Research Of Rock Signaling Pathway Involved In Rabbit Basilar Artery Smooth Muscle Proliferation Which Stimulated By TNF-α

BACKGROUND AND PURPOSE:Subarachnoid hemorrhage is a relatively common disease of hemorrhagic stroke. Inaddition to the high mortality of early pathogenesis, the incidence of cerebral vasospasmcaused by multiple factors which occured in the late stage of subarachnoid hemorrhage isalso high. It is ischemic brain damage caused by cerebral vasospasm that lead to death anddisability of patients in the late stage of subarachnoid hemorrhage. Therefore, preventionand treatment of cerebral vasospasm is an urgent problem in clinical.So far,the mechanism of cerebral vasospasm has been studied by many scholars.Studying the mechanism is to illustrate the pathogenesis of cerebral vasospasm in order toexplore an effective method of treatment.But the specific mechanisms have not been fullyelucidated so far. However, we have got a better understanding for the development ofcerebral vasospasm with the development of related research.Most of former researchersstudy the factors that inducing spasm after subarachnoid hemorrhage, and some scholarshave tried to apply the blockers of appropriate factor that inducing spasm to prevent andtreat cerebral vasospasm. However, these applied antispasmodic did not achieve the desiredresults. So the pathological changes of wall thickening and stenosis of related artery aftersubarachnoid hemorrhage attracted the attention of scholars and experts, and this become anew hot spot of studying cerebral vasospasm after subarachnoid hemorrhage currently. Theproliferation of cerebrovascular wall in cerebral vasospasm after subarachnoid hemorrhageis closely related to inflammatory cytokines.Rock signal is an important signaling pathway in vivo.Related studies have shown thatRock signal may be closely related to cerebral vasospasm by inducing vascular smoothmuscle contraction,and we use fasudil which is a Rock inhibitor to treat cerebral vasospasmin clinical. But whether Rock signal is involved in the abnormal proliferation of vascular smooth muscle cells which stimulated by TNF-α or other inflammatory cytokines and theincidence of cerebral vasospasm after subarachnoid hemorrhage are rarely reported. So weneed to further understand the specific role of the Rock signaling pathway in proliferation ofvascular smooth muscle cells induced by inflammatory factors,so that we can know thespecific mechanisms of cerebral vasospasm after subarachnoid hemorrhage. This may hasgreat significance for we can take appropriate measures to block the occurrence of cerebralvasospasm after subarachnoid hemorrhage in clinical.METHODS:1.Culturing and identifying vascular smooth muscle cells of rabbit basilar artery bytissue block method;2.The cells are divided into three groups first: negative control group, TNF-α-treatedgroup and interfered by Y-27632group.Then the proliferation and cell cycle of cells in eachgroup are detected;3. Using technology to detect the expression and distribution of PCNA protein ofvascular smooth muscle cells in each group.RESULTS:1.Rabbit basilar artery vascular smooth muscle cells are primary cultured successfullyby tissue block method and identified by immunofluorescence;2.The results of CCK-8assay show the proliferation of rabbit basilar artery vascularsmooth muscle cells can be promoted significantly(P <0.01),Y-27632can inhibit the effectof promoting cells proliferation significantly(P <0.01);3. The results of cell cycle detected by Flow Cytometer shows that comparing with thecontrol group the proportion of vascular smooth muscle cells which stimulated by TNF-α inG0/G1phase was significantly reduced and the proportion of cells in S phase+G2/M phasewas significantly rised (P <0.05), the proportion of cells pretreated by Y-27632in G0/G1phase was significantly decreased and S phase+G2/M phase cells was significantly raisedcompared with TNF-α treated group (P <0.05);4. The results of Immunocytochemistry and Western-blot shows that PCNA proteinexpress in the nucleus, TNF-α can increase the expression of PCNA protein in the nucleussignificantly(P <0.01) and Y-27632can significantly reverse the effect of TNF-α inpromoting the expression of PCNA protein(P <0.05). CONCLUSIONS:1.Rabbit basilar artery vascular smooth muscle cells can be successfully cultured bytissue block method;2.TNF-α can promote the proliferation of vascular smooth muscle cells by acceleratingthe process of cell cycle and Rock signaling pathway plays an important role in the effect ofpromoting proliferation.3. Y-27632arrest vascular smooth muscle cells in G0/G1phase by inhibiting theactivity of Rock signaling pathway, and thus reverse the effect of TNF-α that promotingproliferation and increasing PCNA protein expression.