Effects Of Liuwei Dihuang Pill-containing Serum On SH-SY5Y Cell Injury Induced By A?_1-42 Through Caspase-1/GSDMD-N Pathway

Purpose:Through the study of SH-SY5Y cell injury model induced by A?_1-42,the effect of Liuwei Dihuang pill containing serum on SH-SY5Y cell death was investigated by using Caspase-1/GSDMD-N as the breakthrough point.Materials and methods:1.Thirty healthy SD rats were randomly divided into two groups after 2-3 days of adaptive feeding,normal control group and Liuwei Dihuang Pill group.Each group had 15 rats,which were raised in separate cages.According to the effective dose conversion of human and rat body surface area,Liuwei Dihuang Pill group was given Liuwei Dihuang Pill powder water solution containing 1.18 g/kg crude drug of Liuwei Dihuang pill,while the normal control group was given double distilled water of the same volume.Twice a day for 5 days.Two hours after the last gavage,blood samples were collected from the abdominal aorta of the two groups at the same time,and the serum was separated as normal rat serum and drug containing rat serum for standby.2.SH-SY5Y cells were divided into three groups:normal control group,model group and Liuwei Dihuang pill containing serum group.The normal control group and model group were cultured in DMEM high glucose medium containing 10%normal rat serum.The Liuwei Dihuang pill containing serum group was cultured in DMEM high glucose medium containing 10%Liuwei Dihuang pill containing rat serum.The model group and Liuwei Dihuang pill containing serum group were cultured in DMEM high glucose medium containing 20 umol/L A?_1-42oligomer for 48 hours.3.Trypan blue was used to observe the cell morphology and calculate the living cell rate;Transmission electron microscope(TEM)was used to observe the morphology and structure changes of SH-SY5Y cells in each group;Enzyme linked immunosorbent assay(ELISA)was used to detect the contents of interleukin-1?(IL-1?)and interleukin-18(IL-18)in the supernatant of SH-SY5Y cells;Western blot(WB)was used to detect the protein expression of Caspase-1(Caspase-1)and Gasdermin-D-N(GSDMD-N)segments in cells.Results:1.Trypan blue staining method was used to observe the cell morphology and calculate the rate of living cells in each group:Under microscope,the cells in the normal control group had good morphology,clear structure,full cell body,synapses,good light transmittance,and became transparent;The cells in the model group had irregular shape,incomplete cell membrane,cell fragmentation,a large number of debris outflow,round cells,no synapses,more dead cells,and were dyed blue.In the serum group,the cell morphology was relatively good,the structure was relatively clear,the cell body was relatively full,there were only a few fragments,synapses,and a few were dyed blue.Compared with the normal control group,the cell survival rate of the model group was significantly lower(P<0.01),and compared with the model group,the cell survival rate of the drug containing serum group was significantly higher(P<0.01).2.The morphological structure of SH-SY5Y cells in each group was observed by transmission electron microscope:In the normal control group,the cell nuclear membrane was smooth,the shape was complete,regular,and the structure was clear;Compared with the normal control group,the cells in the model group appeared nuclear shrinkage,mitochondrial swelling,and chromatin edge gathering phenomenon;Compared with the model group,the cell nuclear membrane in the drug containing serum group was smooth,and the mitochondrial swelling was reduced.3.The results of comparison of the levels of IL-1?and IL-18 in the supernatant of each group were detected by ELISA:Compared with the normal control group,the levels of IL-1?and IL-18 in the supernatant of the model group were significantly increased(P<0.01);Compared with the model group,the levels of IL-1?and IL-18 in the supernatant of the drug containing serum group were significantly decreased(P<0.01).4.Protein expression of Caspase-1 and GSDMD-N in each group was detected by western blot:Compared with the normal control group,the protein expression of Caspase-1 and GSDMD-N in the model group was significantly increased(P<0.01);Compared with the model group,the protein expression of Caspase-1 and GSDMD-N in the drug containing serum group was significantly decreased(P<0.01).Conclusion:1.The serum containing Liuwei Dihuang pill can significantly improve the damage state of SH-SY5Y cells induced by A?_1-42,and has protective effect on the damaged SH-SY5Y cells.2.Liuwei Dihuang pill containing serum can inhibit cell death by regulating Caspase-1/GSDMD-N pathway,and then inhibit cellular immune inflammatory response,and play a role in the prevention and treatment of AD.