Based On The GRP78/CAMK? Signal Pathway, The Effect Of Liuwei Dihuang Pills On A?_1-42-induced Endoplasmic Reticulum Stress In SH-SY5Y Cells Was Investigated

Purpose: Based on the theoretical foundation of TCM "Shenchangjing,jingshengsui",Based on GRP78 / CAMK? signal pathway,To investigate the effect of Liuwei Dihuang Pill on endoplasmic reticulum stress in Alzheimer's disease(AD)cell model induced by A?1-42 induced SH-SY5 Y cells,and reveals the possible mechanism of action of Liuwei Dihuang Pill in preventing and treating Alzheimer's disease.Material and method:Preparation of Liuwei Dihuang Pills Containing Serum:30 rats were randomly divided into normal group and Liuwei Dihuang Pill group.Rats in Liuwei Dihuang Pill Group used crude drug with a concentration of 1.18 g.?kg-1 ? d-1 Liuwei Dihuang Pill(concentrated pill)aqueous solution was given by gavage,5 times a day,twice a day,and the blood was taken after the last 2h to extract the drug-containing serum of Liuwei Dihuang Pill,and the rats in the normal group were gavage with double distilled water under the same conditions.Extract serum from normal group;This experiment is divided into 3 groups: blank control group,model group and Liuwei Dihuang Pill drug-containing serum group,The blank control group and the model group were cultured with DMEM high glucose medium containing 10% normal group rat serum.Group and Liuwei Dihuang Pill medicated serum group added a final concentration of 20 umo L?L-1 A?1-42 oligomer,continue to be detected after 48 hours of culture;Lactate dehydrogenase kit to detect the activity of lactate dehydrogenase(LDH)in cell culture medium;transmission electron microscopy to detect the ultrastructural changes of endoplasmic reticulum of SH-SY5 Y cells;real-time fluorescence quantitative nucleic acid amplification detection system(Real-time PCR Quantitative Detecting System(QPCR)was used to detect the expression levels of GRP78,CAMK? m RNA in each group;Western Blot method was used to detect the expression levels of GRP78 ? CALM and CAMK?proteins in each group.ResuLts:1.Changes of LDH activity in cell culture supernatant of each group:Compared with the blank control group,the activity of lactate dehydrogenase in the cell supernatant of the model group was significantly increased(p<0.01);compared with the model group,Liuwei Dihuang Pill drug-containing serum group cells The content of lactate dehydrogenase in serum was significantly reduced(p<0.05).2.Observe the ultrastructural changes of cells in each group under transmission electron microscope : In the blank control group,the cell structure was clear,the double-layer nuclear membrane and cytoplasmic membrane were intact,and the endoplasmic reticulum was in normal shape with obvious structure;Compared with the blank control group,the endoplasmic reticulum was significantly expanded,showing vacuoles;compared with the model group,the cells in the Liuwei Dihuang Pill drug-containing serum group were close to normal cell morphology,and the endoplasmic reticulum expansion was significantly reduced.3.QPCR detection of m RNA expression of cell related genes in each group showed:compared with the blank control group,the expression level of CAMK? m RNA in the model group cells was significantly reduced(p<0.01),and the expression level of GRP78 m RNA was significantly increased(p<0.01);Compared with that,the expression level of CAMK? m RNA was significantly increased in Liuwei Dihuang Pill containing serum group(p<0.01),and the expression level of GRP78 m RNA was significantly decreased(p<0.05).4.Western Blot detection of cell-related protein expression in each group showed:compared with the blank control group,the expression level of CAMK? protein in model cells was significantly reduced(p<0.01),and the expression levels of GRP78 and CALM proteins were significantly increased(p<0.01);Compared with the model group,the expression level of CAMK? protein in the serum-containing serum group of Liuwei Dihuang Pill was significantly significant(p<0.01),and the expression levels of GRP78 and CALM protein were significantly reduced(p<0.01).Conclusion:1.Liuwei Dihuang Pill medicated serum can effectively improve the damage ofSH-SY5 Y cells due to endoplasmic reticulum stress.2.Liuwei Dihuang Pills-containing serum may play a role in improving endoplasmic reticulum stress and downstream calcium ion channels by regulating the gene expression of GRP78,CALM,and CAMK?.