| Objective:Axonal injury after stroke has been considered to be the primary pathogenesis of the dysfunction of motor abilities. Our research simulated the pathological changes in axon after stroke by duplicating the model of Oxygen-glucose Deprivation (OGD) using organotypic cortical slices of neonatal Sprague Dawley rats (P7-12). To observe the axonal growth effect of Gua Lou Gui Zhi Decoction water extract (GLGZD) and explore whether GLGZD promoted axon regeneration through regulating the protein Nogo-A and Nogo-A mediated Rho-ROCK signaling pathways.Methods:The Stopinni, HW Dong and Foehring’s method of preparing organotypic cortical slices was modified from us. The OGD model was duplicated by using a three gas incubator. (1) Slices were divided into the control group, OGD 15 min group, OGD 30 min group, OGD 45 min group and OGD 60 min group. The release and accumulative release of lactate dehydrogenase (LDH) was then determined by using micro-enzyme-linked method to measure the best duration of OGD. (2) Slices were divided into control group, OGD group, GLGZD Low-dose group (GLGZD-L), GLGZD Medium-dose group (GLGZD-M), GLGZD High-dose group (GLGZD-H) and Fasudil group. The slices in control group received no treatment. The OGD process was then applied to the other groups following the established OGD duration. After the OGD process, the drug treatment groups were treated with respective doses of GLGZD or Fasudil, and culture for another 3 days. Brain tissues and culture medium were then collected. ①The release of LDH in the supernate of culture medium was detected by using micro-enzyme-linked method. ② The specific axonal protein NF68 was labeled using immunohistochemistry, the length of axons were measured using ImageJ software.③ Western blot was employed to detect Tau-1 and GAP43 expression.④ The mRNA expressions of Nogo-A, NgRl, RhoA, ROCK Ⅱ were detected using RT-qPCR. Western blot was applied to detect the level of protein expressions of Nogo-A, NgRl, RhoA, ROCK Ⅱ and phosphorylated CRMP2.Result:(1) The PI uptake in OGD 15 min and 30 min group showed no significant differences (P>0.05) comparing with control group while the PI uptake in OGD 45 min and 60 min increases significantly (P<0.01). And the release and accumulative release of LDH in OGD 15 min and 30 min group showed no significant (P>0.05) difference whereas the OGD 45 min and 60 min group displayed significant increases comparing with control group (P<0.01). The results above suggested that 15 or 30 min of OGD were not the proper duration in this experiment, and 45 min of OGD or above should be better OGD duration. Thus, we employed the duration of 45 min to duplicate OGD model.(2) The PI uptake in slices of OGD group significantly increased (P<0.01) after 3 days of administration; No significant PI uptake reduction was observed in GLGZD-L group (P>0.05); The degree of PI staining after OGD was significantly reduced (P<0.01) in the slices treated with GLGZD-M, GLGZD-H and Fasudil.(3) The LDH release in OGD group significantly increased (P<0.01) after 3 days of administration; However, no significant LDH release reduction was observed in GLGZD-L group (P>0.05); Additionally, LDH release in GLGZD-M, GLGZD-H and Fasudil group were significantly reduced. (P<0.01)(4) The results of immunohistochemistry showed that axons in control group had intact constructive and the length of axon fibers remained. However, the length of the axons in OGD group shortened in a relatively large degree (P<0.01). Simultaneously, the structure of axons collapsed that the segments of the axons were easily observed. In comparison to OGD group, the length of the axons in all GLGZD treatment groups and Fasudil group showed significant (P<0.01) increases with an obvious dose-dependent manner.(5) The detection of Tau-1 protein manifested that the expression of Tau-1 declined significantly (P<0.01) after OGD, and increased by different doses of GLGZD following a dose-dependent manner. The expression of GAP43 showed no significant increase after OGD process (P>0.05) in comparison to control group. However, the intervention of GLGZD could significantly increased the expression of GAP43 following a dose-dependent manner.(6) The detection of Nogo-A and NgR as well as relative index in Rho-ROCK signaling pathway illustrates that the mRNA and protein expression of Nogo-A, NgR1, RhoA, ROCK Ⅱ in cortex significantly upgrade after OGD 45min (P<0.01),the phosphorylation of CRMP2 rises (P<0.01) as well. After given GLGZD’s intervention, the mRNA and protein expression of Nogo-A, NgR1, RhoA, ROCKⅡ and phosphorylation of CRMP2 shows a visibly downgrade.Conclusion:Taken together, the results above demonstrated that GLGZD has the effect on promoting axon morphological remodeling and functional reconstruction after OGD injury. This effect may be related to its regulation on Nogo-A and its receptor (NgR1),the inhibition of the activation of Rho-ROCK signaling pathway, the reduction of the phosphorylation level of CRMP2 protein, maintain the stability of microtubules and the normal form of the growth cone. |
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