Expression Of EphrinA3 And The Mechanism In The Lipopolysaccharide Induced Proliferation Of Astrocyte

BackgroundThe original function of central nervous system(CNS) usually can not recover completely after injury, the reason of which is mainly the limited regeneration of lesioned axons. The difficulty of axon regeneration may be elucidated by the lack of growth promoting molecules demanded for axson outgrowth and guidance on the one hand, together with the microenvironment of the injury site on the other hand. Resent research showed that the axon guidance molecules also involved into the axon regeneration. The Eph recepor tyrosine kinases(RTKs) and there ligands, the ephrins are important axon guidance molecules family, which take part in axon guidance during nervous development. In the audult CNS, Ephs and ephrins continue to be expressed, in spite of keeping lower levels, yet are upregulated while the injury to the CNS, such as the activation of neurons, astrocytes and oligodendrocytes. The upregulateion of expression may inhibit regrowth of regenerating axons directly. In addition, Ephs and ephrins could also be helpful for glial proliferation and glial scar formation, may have an impact on the neural stem cell proliferation, differentiation and migration. As a result, Eph/ephrin signaling system may affect the CNS regeneration and functional recovery through more than one mechanism and it is of vital importance to regulate the expression of Ephs and ephrins and the Eph/ephrin signal for the the CNS regeneration after injury, may also contribute to the treatment and rehabilitation of stroke and other CNS diseases. Following damage to the CNS and neurodegerative diseases, the inflammatory response is extraordinarily clear that astrocytes are significantly activated, resulting in a series of inflammatory cytokines, chemokines trophic factors and so on, which play an important role in the processes of the development and survival of neurons, axon regeneration and the differentiation of neural stem cells. Studies showed that the inflammatory response injury may change the expressions of Ephs and ephrins after injury, and the inflammatory fators may be associated with the expressions of Ephs and ephrins. EphA4 is widely distributed in the brain neurons, many studies have shown that it plays a negative role in the damage and repairment of the injury CNS. EphrinA3 is one of Eph receptor ligands, presenting in astrocyte, playing an important part in maintaining the activation of EphA4 and normal form of dendritic spines. This experiment based on the above mentioned studies established a inflammatory model induced by LPS, and the effects of LPS on the proliferation of the astrocytes and the changes of ephrinA3 expression after injury, and the relationship between the changes and the inflammation were observed and studied.Purpose1. Isolate, cultivate, and purify the astrocytes of the cerebral cortex in the rats.2. Explore the effects of lipopolysaccharide(LPS) on the proliferation of the astrocytes and the expression of ephrinA3 and the possible machanism in vitro.Method1. Astrocytes of the cerebral cortex were extracted from brain of newborn Wistar rats within 24 hours, cultured and purifed.2. The growth state and the morphologic changes of the astrocytes were observed under the inverted phase contrast microscope.3. The expressions of the specific marker glial fibrillary acidic protein(GFAP) and ephrinA3 on the astrocytes were identified with the method of immunohistochemical staining. Purity testing was used to measure the purity of the third generation astrocytes.4. The third generation astrocytes were used for the experiment and were divided into six groups at random:control group, exposure to LPS(10mg/L) for 30min, exposure to LPS(10mg/L) for 6h, exposure to LPS(10mg/L) for 24h, exposure to LPS(10mg/L) for 48h, exposure to LPS(10mg/L) for 72h.5. The survival rates of the astrocytes were determined using MTT method.6. The apoptotic rates of the astrocytes were determined by AO/EB fluorescence staining.7. The expressions of GFAP and ephrinA3 on the astrocytes were detected by fluorescein stain of CY3.8. The changes of the inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-six(IL-6) in the astrocytes culture soluton were measured by ELISA. 9. The Statistical Package for the Scocial Sciences(SPSS) 13.0 were used to analyze the datas obtaining. All the datas were demonstrated with mean±standard deviation(x±s). After the homogeneity test of variance, one way analysis of variance(ANOVA) was used to compare mutiple group means, and the least significant difference method(LSD) was used for the group comparison. The inspection level wasα= 0.05, and P<0.05 for diffference have statistical significance.Result1. The cell bodys greatened, the prominence elongated, the appearance became clear gradually of the astrocytes with the incubation time prolonging observed under the inverted phase contrast microscope. After purified, the shape of the astrocytes became simple, appearing fusiform or star, with apparente cell body and distinct realm. Comparing with the control group, the cell bodys were larger and the prominences were longer in the exposure to LPS groups.2. GFAP antibody was used to identify the purify of the astrocyte and the results showed that astrocytes more than 97% purity. EphrinA3 polyclonal antibody was used to identify the expression of the ephrinA3 on the astrocyte and the positive expression was found.3. MTT method using for determing the astrocyte survival rate showed that the survival rates were notably increased(P<0.05) in exposure to LPS(10mg/L) for 6h, 24h,48h,72h, and the exposure to LPS(10mg/L) for 30min group had no sinificant increase(P>0.05), comparing with the control group. Compared with the exposure to LPS(10mg/L) for 24h, the exposure to LPS(10mg/L) for 30min,48h, and 72h groups had notable difference(P<0.05).4. AO/EB fluorescence staining using for determing the astrocyte apoptotic rate displayed that the apoptotic rates were notably decreased(P<0.05) in exposure to LPS(10mg/L) for 6h,24h,48h, and 72h, and the exposure to LPS(10mg/L) for 30min group had no sinificant decrease(P>0.05), comparing with the control group. Compared with the exposure to LPS(10mg/L) for 24h, the exposure to LPS(10mg/L) for 30min,48h,72h groups had notable difference(P<0.05).5. The fluorescein stain of CY3 were used for detecting the expression of GFAP and ephrinA3. The cell body and the prominence of the astrocytes took on red fluorescein and the nucleus were colorless, presenting dark area. Comparing with the control group, the fluorescence intensity of the exposure to LPS(10mg/L) for 24h, 48h, and 72h group was significantly strong(P<0.05), and the exposure to LPS(10mg/L) for 30min and 6h groups had no significant changes(P>0.05). Compared with the exposure to LPS(10mg/L) for 24h, the exposure to LPS(10mg/L) for 30min,6h,48h, and 72h groups had notable difference(P<0.05)6. ELISA method using for anlyzing the changes of the inflammatory factors tumor necrosis factor-a(TNF-a) and interleukin-six(IL-6) in the astrocytes culture soluton showed that the content of TNF-αand IL-6 increaced sightly(P>0.05) within the exposure to PS(10mg/L) for 6h. Comparing with the control group, the content of TNF-a and IL-6 of the exposure to LPS(10mg/L) for 24h,48h, and 72h group was significantly increased(P<0.05), and compared with the exposure to LPS(10mg/L) for 24h, the exposure to LPS(10mg/L) for 30min,48h, and 72h groups had notable difference(P<0.05).Conclusion1. LPS(10mg/L) could induce and promote the proliferation of astrocyte, lasting 72h at least.2. The expression of ephrinA3 increased during the astrocyte induced by LPS in rats and its mechanism may be concerned with inflammatory cytokines.