Different Factors On The Proliferation And Migration Of NSCs From SVZ And Potential Mechanism

BackgroundThe neural stem cells(NSCs), residued in the subventricular zone(SVZ) hold the capacity of self-renewal, proliferation, migration and differentiation into the three main linages in the central nernous system(CNS). In the past decades, studies have revealed that NSCs in the SVZ could be in situ activated, and proliferate, migrate, differentiate into functional neurons and intigrate into the damaged neural network in the lesion after brain injury, such as: ischemia, intracerebral hemorrhage(ICH), traumatic brain injury(TBI) et al.. Although researches have confirmed these phenomena after injury, the capability of proliferation and migration of NSCs in the SVZ is still limited after brain injury; thus, the recovery outcome still remains unsatisfactory. Therefore, discovery of reagents and deciphering the underlying potential mechnisms to enhance proliferation and migration of NSCs in the SVZ appears to be significant and greatly contributes to basic and clinical research.Tissue p H plays an important role in the neural cells performing their functions among various microenvironmental parameters. With the accumulation of a large number of acidic substances under pathological conditions, such as cerebral ischemia, the tissue p H in the peri-infarct could be decreased to 6.5, even less than 6.0 in the infarct core. This microenvironmental alteration must have some effect on NSCs in the SVZ. Thus, we speculate that the microenvironmental acidosis may inhibit migration of NSCs in the SVZ. Meanwhile, artesunate(ART) has advantages of high efficiency, low toxicity, easy penetration through the blood brain barrier(BBB), which is widely used in clinical treatment of malaria. Furthermore, recent studies have suggested it’s curative effect on tumors. But there is no report about the relationship between ART and NSCs proliferation in the SVZ. We hypothesize that the suitable concentration of ART might promote proliferation of NSCs from SVZ. To verify the two thoughts above, this study is carried out to elaborate the phenomena and potential mechnisms about enhancement of ART on proliferation and inhibition of ASIC1 a on migration of NSCs in SVZ, combining in vitro and in vivo experiments with application of immunofluorescence, confocal laser scanning microscopy(CLSM), p Harmacology, biochemistry and molecular biology methods. The concrete research contents are as follows:Part I The effect of acid-sensing ion channel 1a on the migration of NSCs from SVZ and its potential mechanismObjectiveTo investigate the expression of acid-sensing ion channels 1a(ASIC1a) on the NSCs in SVZ and its potential mechanism on the migration with the usage of Pc TX1.MethodsNSCs were incubated with different groups of PH value for 24 h, as follows: control(p H 7.4), p H 6.0, p H 6.5, p H 6.8, p H 7.0, p H 7.2. And, the number and distance of migration cells were counted using p Hase contrast microscopy to obtain the most obvious impact on NSCs migration from SVZ. Furthmore, the CCK8 assay were carried out to evaluate the effect of each p H value on NSCs. Then, the immunofluence staining of ASIC1 a and nestin was performed to certify the expression of ASIC1 a in NSCs from SVZ. Followed by RT-PCR and Western Blotting to certify the expression of ASIC1 a in NSCs. The Pc TX1 was used to assess the effect of ASIC1 a on the migration of NSCs from SVZ with application of transwell and observation under p Hase contrast microscopy in differen groups, as follows: control(p H 7.4), p H 6.8 and p H 6.8+Pc TX1. WB was implemented to certify the expression of ASIC1 a at the same time. All the animals were divided into three groups as above. Brdu(50mg/Kg)was administered to all the animals after surgery. Then, the immunofluence staining was carried out to measure the role of ASIC1 a on the migration of Brdu+ and Nestin+ cell number with the application of Pc TX1 after 3 days. The role of ASIC1 a on the migration of NSCs from SVZ was certified with in vitro and in vivo experiments above.Results1. NSCs activity could be significantly affected at acidic environment of p H 6.5, compared with the control group(p<0.01).2. NSCs activity could not be affected at acidic environment of p H 6.8, but the cell number and distance were significantly decreased, compared with the control group(p<0.01).3. ASIC1a+ and nestin+ were co-labeled in the NSCs from SVZ; RT-PCR and WB were carried out to certify m RNA and protein expression, respectively; in other words, ASIC1 a expressed in NSCs from SVZ.4. The inhibitory effect of migration at p H 6.8 microenvironment could be attenuated with addition of Pc TX1.5. The number and distance of migration Nestin+ cells were greatly recuced at p H 6.8 group towards lesion, while this effect could be attenuated with the application of Pc TX1.ConclusionThe migration of NSCs from SVZ is inhibited at extracellular acidosis of p H 6.8. ASIC1 a, expressed in NSCs from SVZ, plays an important role in the migration related to microenvironmental acidification. Pc TX1 could attenuate the effect of inhibitory effect of NSCs in extracellular acidosis.Part II Artesunate promotes rat NSCs proliferation from SVZ via PI3K-AKT pathway in vitroObjectiveTo investigated the role of ART on the proliferation NSCs from SVZ and its potential mechnism using CCK8 assay, Ki-67 immunofluorescence, LY294002 and WB.MethodsNSCs were incubated with different concentrations of ART for 24 h, as follows: 400 nmol/L, 800 nmol/L, 1.6 μmol/L, 3.2 μmol/L. And CCK8 assay were carried out to evaluate the effect of ART on NSCs proliferation at wavelenth of 450 nm after 2.5h incubation. The PI3K-AKT signaling pathway inhibitor(10 μmol/L LY294002) was added in 800 nmol/L ART group to evaluate its inhibitory effect NSCs proliferation using CCK8 assay. The Ki-67 immuno?uorescence staining was used to confirm the results got from CCK8 assay. WB assays were used to valuate the expression of Nestin, p-AKT and AKT treated in different groups after 24 h, and groups are as follows: control, LY294002, 800 nmol/L ART and 800 nmol/L ART + LY294002.Results1. CCK8 assay indicated that that the absorbance values(450nm) were significantly higher in 800 nmol/L ART group than other groups(P<0.05), and decreased to the level of control group with addition of LY294002.2. The number of Ki-67+ was higher in 800 nmol/L ART group than others, and recuced to the level of control group with addition of LY294002.3. WB indicated that the expression of p-AKT and Nestin were significantly up-regulated in 800 nmol/L ART group, but reduced to the level of control with addition of LY294002(P <0.05).Conclusion800 nmol/L ART could enhance the proliferation of NSCs from SVZ in vitro, and PI3K-AKT signaling pathway might plan an important role in this process.