| Background: Ischemic cerebrovascular disease is the common and multiple disease in central nervous system(CNS)diseases. It has high mutilation rates, mortality rate and relapse rate. It threatens people's life and health severely, gives heavy burden to the society and families. Scholars did lots of experiments and clinical studies about ischemic cerebrovascular disease and the recognition of its damage, prophylaxis, therapy and restoration were improved. With the development of therapeutic study on cerebral ischemia, the concept of whole brain protection postischemia was proposed gradually. All encephalic region, cell types and cell composition should be protected for the functional recovery. Because the glial were the major component of white matter, it is very necessary and important to investigate the mechanism and inhibition means of its ischemic damage.The glial reactive proliferate when subjected to inflammation, external injury et al. It includes cell number increasing, cell multiplication and hypertrophy, glia filament and protuberance increasing than normal, metabolic activity enhancement, expression of protein GFAP raise. The glial scar may be the focus of secondary epilepsy after brain ischemia. Inflammatory reaction after ischemic reperfusion enhance secondary brain damage, it is one cause of ischemic reperfusion damages. The reactive hyperplasia of glial has relationship to inflammation. So, paying close attention to the inflammation of glial after ischemic reperfusion and the anti-inflammation, methods of protecting nerves about the inflammation will have important and actual significance in the therapy and later stage convalescence of cerebral arterial thrombosis.Nuclear factor-кB is an important intranuclear transcription factor. It regulates the transcription and expression of a series of cell factor and erzymes. NF-кB is the central link in inflammation course and it is one of perspective therapeutic targets in interfering in ischemic reperfusion damages.3-n-butylphthalide(NBP)is a new drug which has the first national independence intellectual property rights in studies on cerebral vessels. It has three kinds of optical isomers: l-NBP, d-NBP, dl-NBP. l-NBP has the better pharmacodynamic action among them. l-NBP has curative effect obviously on therapy to the acute cerebral arterial thrombosis, it has better activity of anti-ischemic in brain. Therefore we studied on the inhibition of l-NBP to the inflammatory impair and reactive hyperplasia of glial was subjected to oxygen glucose deprivation/reoxygenation, studied the possible mechanism of it moreover. Study the protection of l-NBP to glial cell following oxygen glucose deprivation/reoxygenation. Presumed l-NBP may inhibit the form of glial scar. Predicted l-NBP may has therapy or adjunctive therapy effect on other inflammatory diseases in white matter(disseminated sclerosis, traumatic CNS injury, CNS infection).Objective: (1) Investigate the cultivate method of mouse brain glial cells ( GC ) , observed by morphology and identify them by immunocytochemistry.(2) Investigate GC cell population and activity in different time stages after oxygen glucose deprivation/ reoxygenation(OGD/R)by cell OGD/R model, study the dynamic rule of protein NF-κB p65 expression and activity in cell.(3) Observe the effect of l-NBP to GC cell population after OGD24h/R1d.(4) Observe the effect of l-NBP to expression of protein IκB-α, NF-κB p65 and IL-1βin GC cell after OGD24h/R1d.Methods: Took suckling Kunming mouse brain tissue, mechanical blowing to demesh,filtering by grit, centrifugalization etc. technique to obtain mouse brain glial cell and to cultivate. Observed the cultivated cell by inverted microscope and identify them by immunocytochemistry. Evaluated OGD model by LDH determination and immunocytochemistry, determined the OGD/R time point whose chang was obvious in GC population. Investigated expression of protein of IκB-α, NF-κB p65 and IL-1βwhich was NF-κB downstream inflammation factor by Western blot and ELISA in GC interfering with l-NBP after subjected to OGD/R.Results: (1) The cultivated cell was identified by morphology and immuocytochemistry(GFAP, MAC-1, NSE), it was mixed culture of astrocyte and microglia.(2) GC morphous changed obviously after OGD, LDH releasing increased and had significant difference from normal control group(p<0.01).(3) Expression of protein NF-κB p65 in GC nuclear increased after OGD by immuocytochemistry. It began to increase in OGD4h, got to the peak in OGD12h and began to descent, the amount in OGD24h was still more than normal control group. There was no significant difference between expression level in OGD12h and in OGD24h(p>0.05). OGD groups had significant difference from normal control(p<0.01). NF-κB p65 weak positive expression in endochylema before OGD and strong positive expression in nuclear after OGD, cell showed"solid center".(4) GC population was obviously less than normal control group and OGD24h/R groups after OGD24h by MTT(p<0.05). GC population gradually increased after OGD24h/R8h, OGD24h/R12h, OGD24h/R1d, OGD24h/R2d, OGD24h/R3d. The quantity of OGD24h/R1d, OGD24h/R 2d, OGD24h/R3d were significantly more than normal control group(p<0.05).(5) GC population of l-NBP groups ( 20μmol/L, 100μmol/L, 500μmol/L)had no significant difference from normal control group when normal cultured with l-NBP(p>0.05).GC population of l-NBP 500μmol/L group was significantly more than normal control group(p<0.01), but PDTC 100μmol/L group was significantly less than that(p<0.01). Population of GC in l-NBP group(s20μmol/L, 100μmol/L, 500μmol/L)and PDTC 100μmol/L group decreased after OGD24h/R1d while OGD24h/R1d group increased. PDTC 100μmol/L group had significant difference from other groups(p<0.05). l-NBP 500μmol/L group had significant difference from 20μmol/L and 100μmol/L group(p<0.05).(6) Straps in Western blot indicated expression of proteim NF-κB p65 in nuclear of OGD24h/R1d group was obviously more than normal control group(p<0.05). Expression of protein NF-κB p65 of l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)decreased, the higher density of l-NBP the less protein NF-κB p65 expression. l-NBP 100μmol/L group and l-NBP 500μmol/L group had significant difference from OGD24h/R1d group(p<0.01). NF-κB p65 protein amount in PDTC 100μmol/L group was also obviously less than OGD24h/R1d group(p<0.01). L-NBP 500μmol/L group had significant difference from 20μmol/L and 100μmol/L group(p<0.05). (7) Straps in Western blot indicated expression of proteim IκB-αin endochylema of OGD24h/R1d group was obviously less than normal control group(p<0.01). Expression of protein IκB-αof l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)increased, the higher density of l-NBP the more protein IκB-αexpression. l-NBP 100μmol/L group and l-NBP 500μmol/L group had significant difference from OGD24h/R1d group(p<0.01). IκB-αprotein amount in PDTC 100μmol/L group was also obviously more than OGD24h/R1d group(p<0.01).(8) IL-1βsecretory volume of OGD24h/R1d group obviously more than normal control group by ELISA(p<0.05). IL-1βsecretory volume of l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)decreased, the higher density of l-NBP the less IL-1βsecretory. l-NBP 100μmol/L group and l-NBP 500μmol/L group had no significant difference from normal control group(p>0.05). IL-1βsecretory amount in PDTC 100μmol/L group was also obviously less than other groups(p<0.01).Conclusions: (1) Done well in primary mixed culture of mouse brain astrocyte and microglia and in identifing them by immuocytochemistry.(2) l-NBP groups(20μmol/L,100μmol/L,500μmol/L)had no the promoted action to the growth of GC when normal cultured. L-NBP 1mmol/L group promoted the growth of GC and PDTC 100μmol/L group inhibited the growth of GC when normal cultured. It indicated that l-NBP has no toxic side effect to GC but PDTC may has . (3) Cell OGD/R model in vitro was convenient and utility, GC population decreased, cell activity attenuated and factor NF-κB was activated to get into the nulcear after OGD. OGD12h was the peak of expression of protein NF-κB.(4) GC reactive hyperplasia after OGD24h/R1d, l-NBP and PDTC inhibited the increase of cell, the inhibition of l-NBP 500μmol/L group was more stronger than other groups of lower concentration(20μmol/L, 100μmol/L).(5) The protein IκB-αdegradated and the activated protein NF-κB p65 entered the nucleus in GC after OGD24h/R1d. l-NBP prevented NF-κB p65 from entering the nucleus by reducing the degradation of IκB-α. The inhibition of l-NBP 500μmol/L group was more stronger than other groups of lower concentration(20μmol/L, 100μmol/L)and PDTC 100μmol/L group.(6) The volume secretion of factor IL-1βincreased after OGD24h/R1d, l-NBP and PDTC inhibited the secretion of factor IL-1β, the inhibition of l-NBP 100μmol/L and 500μmol/L group was more stronger than l-NBP 20μmol/L group.
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