Ghrelin Inhibits High Glucose-induced PC12Cells Apoptosis And Its Molecular Mechanisms

Objective:(1) To find out the effect of high glucose (HG) on PC12cells, obtain its IC50and observe its morphologies.(2) To clarify thechanges of Bcl-2and Caspase-3in this process.Methods: The density of PC12cells with logarithmic phase wasregulated to5×108/L. The effect of4.5—27mg/mL HG on the proliferationof PC12cells were examined by MTT assay at the time span of24—96h.Morphological alterations of PC12cells were observed under lightmicroscope and transmission electron microscope (TEM). And then, wedivided PC12cells into two groups in this study, which were (1) the controlgroup;(2) the HG group (13.5mg/mL high glucose DMEM). Theexpression of Bcl-2and Caspase-3were detected by RT-PCR and westernblot analysis.Results: HG inhibited PC12cells proliferation significantly in vitro， which exhibited a dose-dependent manner at the span of4.5—27mg/mLand a time-dependent manner at the span of24—96h. And the inhibitoryeffect reached IC50at72h with the concentration of13.5mg/mL HG.Compared with control group, apoptotic cells and morphologic changes canbe found under TEM in the HG group. The results of RT-PCR and westernblots demonstrated that the expression of Bcl-2was down-regulated by HG,while the expression of Caspase-3was up-regulated by HG.Conclusion: We discovered that HG can induce the apoptosis of PC12cells. The corresponding changes of Bcl-2and Caspase-3verified sucheffect of HG. Objective:(1) To study the effect of ghrelin on the PC12cells.(2) Toexplore the role of TLR4/NF-κB pathway in this process.Methods: We designed four experimental groups in this study, whichwere (1) the control group;(2) the HG group (13.5mg/mL high glucoseDMEM);(3) the HG (13.5mg/mL)+ghrelin (100ng/μL) group;(4) the HG (13.5mg/mL)+ghrelin (100ng/μL)+D-lys-3-GHRP-6(30g/L) group. Theeffects of the HG and ghrelin on the apoptosis of PC12cells weredetermined by annexin V analysis. The expression of TLR4，MyD88,TRAF6, and NF-κB p65were detected by RT-PCR and western blotanalysis.Results: By using annexin V analysis, the apoptosis rate of cells wassignificantly increased in the HG group (p<0.05versus the control group);the apoptosis rate of cells was significantly decreased in the HG+ghrelingroup; in the HG+ghrelin+D-lys-3-GHRP-6group, the effect of ghrelinwas offset (p<0.05versus the control group). The results of RT-PCR andwestern blots demonstrated that TLR4，MyD88, TRAF6, and NF-κB p65,the key proteins of TLR4/NF-κB pathway, were up-regulated by HG(p<0.05versus the control group) and corrected by ghrelin.Conclusion: We discovered that ghrelin can inhibit HG-induced PC12cells apoptosis. The mechanisms may be that ghrelin alleviates theinflammation of diabetic encephalopathy and diminished the expression ofNF-κB which is related with apoptosis by down-regulating theinflammation-related pathway——TLR4/NF-κB pathway. Objective:(1) To study the effects of high glucose and ghrelin onthe cell cycle of PC12cells.(2) To explore the role of Wnt/β-cateninpathway in this process.Methods: In this study, PC12cells were divided into four groups,which were (1) the control group;(2) the HG group (13.5mg/mL highglucose DMEM);(3) the HG (13.5mg/mL)+ghrelin (100ng/μL) group;(4)the HG (13.5mg/mL)+ghrelin (100ng/μL)+IWR-1(10μmol/L) group. Andthen, we tested the cell cycle distribution in each group by using flowcytometry. Finally, we investigated the changes of p-GSK3β, β-catenin, andCyclin D1by using immunofluorescence, immunocytochemistry, RT-PCR,and western blots.Results: The cell cycle of PC12stopped at G0/G1phase withoutgoing into S phase and G2/M phase in HG group. The abnormal cell cyclecan be regulated after the addition of ghrelin. The results ofimmunocytochemistry, immunofluorescence, RT-PCR, and western blotsdemonstrated that p-GSK3β, β-catenin, and cyclin D1, the key proteins ofWnt/β-catenin pathway, were down-regulated by HG and renovated byghrelin. Conclusion: Our findings revealed that HG can disturb the normalcell distribution, while ghrelin can inhibit HG-induced PC12cellsapoptosis by adjusting the abnormal cell cycle. The mechanisms may bethat ghrelin enhanced the expression of cyclin D1by up-regulatingWnt/β-catenin signaling pathway.