Effects Of Berberine On G0/G1Phase Of Neurons In Cerebral Ischemia Injury

The protective effect of berberine on cerebral ischemia is widely studied in recentyears. Neurological dysfunction and death of neurons will occur during the stroke. It hasbeen proved that once post-mitotic neurons enter cell cycle, apoptosis will be induced.Thus, it is noteworthy how to avoid neurons entering the cell cycle and then induceapoptosis during cerebral ischemia injury. We further investigated the effects ofberberine on cell cycle and antiapoptosis in cerebral ischemia process.After the apoptosis induced by cerebral ischemia was determined by MTT andAnnexin V-FITC/PI double staining, we found that cerebral ischemia also resulted inthe changes of cell cycle in vitro model and berberine could increase the percentage ofG0/G1phase. We used PC12cell line and primary neurons to verify the effects ofberberine on antiapoptotic and inducing cell cycle arrest. By western blot and real-timefluorescent quantitative PCR detection, we found that berberine could inhibit theactivation of Caspase3and the phosphorylation of Bad significantly; in addition,berberine could inhibit the G0/G1phase regulatory proteins of CyclinD1, p53, and thephosphorylation of Rb, but increase total Rb protein. The above effects of berberinewere verified in the mouse cerebral ischemia reperfusion model.In order to determine the importance of the cell cycle regulators on theneuroprotective effect of berberine, we introduced siRNA or construct the knockdownor overexpression stable cell lines using lentivirus. These results demonstrated that theprotective effect of berberine was achieved by CyclinD1, p53and Rb, and the inhibitionof phosphorylation of Rb was achieved by down-regulating CyclinD1. We also foundthat berberine could activate PI3K/AKT pathway during the reperfusion time. Duringthe neurons undergo oxygen-glucose deprivation and reperfusion stages, berberine acteda selective effect. That is: berberine could induce cell cycle arrest during the OGDphase, and activated the PI3K/AKT pathway during the reperfusion phase. The selectiveeffect of berberine was likened to “hibernate”-“recovery” effect.Based on the above work, we further investigate the mechanism of theup-regulated of Rb protein by berberine. By constructing the Rb promoter plasmid aswell as the plasmids with or without Rb polyadenylation tail signal, we have determined that the increased Rb protein by berberine was related to the increased Rb mRNA, andthe increased mRNA was achieved by a combination of berberine and poly(A) to inhibitRb mRNA degradation.This work provided new experimental evidence on the treatment of cerebralischemia-reperfusion of berberine. In addition, we provided a new insight for the studyof the mechanism of berberine.