Study On The Protective Effect Of Active Components Of Gardenia Jasminoides On OGD/R-induced SH-SY5Y Cell Injury And Its Anti-pyroptosis Mechanis

Objective: This study predicted and observed the protective effect of chemical constituents of Gardenia on hypoxia and hypoxia/reoxygenation(OGD/R)SH-SY5 Y cells and their effects on NLRP3/Caspase-1/GSDMD The influence of the expression of cytokines related to the pyroptosis pathway,and to explore the possible mechanism of gardenia to protect SHSY5 Y cells by inhibiting pyroptosis and alleviating damage,and providing an experimental basis for its application in the treatment of cerebral ischemia-reperfusion injury.Methods: 1.Virtual screening: The molecular mechanism of Gardenia in anti-ischemic stroke was studied by using network pharmacology and molecular docking,and the potential active chemical components were screened out based on the literature.2.Research on the extraction,separation and purification process of the three major components of Gardenia:(1)Extraction: The crude extracts of total flavonoids,total iridoids and total pigments of Gardenia were obtained by single factor experiment.(2)Separation and purification: Use AB-8 macroporous resin to separate and purify total iridoids,YWD09A5 macroporous resin to separate and purify total pigments,and polyamide resin to separate and purify total flavonoids.(3)Determination of content: a.Drawing the standard curve of the three major components;b.Determination of the UV content of the three major components;c.Determination of the content of gardeniside in the total iridoid components.3.SH-SY5 Y cell culture and OGD/R modeling: SH-SY5 Y cells were cultured in DMEM complete medium containing 10% FBS and 1% double antibody,and inoculated into corresponding culture dishes according to the detected indicators and experimental purposes and orifice plate.After the cells adhered and grew logarithmically,the D-Hanks medium was replaced,and the cells were placed in a hypoxic chamber at a temperature of 37°C and cultured with 95% N2-5% CO2.The OGD time was 4 h,and then the original culture conditions were changed to restore the supply of sugar and oxygen for 12 h,and drug treatment was carried out according to the groups.4.Intervention of chemical components of Gardenia on SH-SY5 Y cells after OGD/R: 6groups in the administration group,1 group in the blank group,and 1 group in the model group.5.Detection of relevant data: The morphology of cells in each group was observed under an inverted microscope,the proliferation activity was detected by CCK8 method,the degree of cell damage was detected by flow cytometry,and the expression of pyroptosis pathway NLRP3/Caspase-1/GSDMD protein was detected by Western blot method.Results: 1.By constructing a "disease-component-target" network model for topology analysis,it was found that quercetin and kaempferol had the most targets,indicating that quercetin and kaempferol may be the potential active components of Gardenia in the treatment of CIS.At the same time,further analysis found that anti-CIS of gardenia mainly acts on NF-κB and other signaling pathways.In addition,the molecular docking results showed that the binding energies were all less than-5k J/mol.2.Research on the extraction,separation and purification process of the total iridoid components of Gardenia jasminoides: The content of iridoids in the purified iridoid extracts was detected by UV spectrophotometry,and the content of iridoids reached 94.65%.The content of geniposide was 43.2% by high performance liquid chromatography.3.Detection-related data: The maximum toxicity limits of quercetin,kaempferol,crocetin and crocetin detected by CCK8 are 25 μM,40 μM,100 μM,125 μg/m L,and the optimal doses are 4 μM,1 μM,50 μM,100 μg/m L,the optimal administration concentrations of geniposide and iridoids were 250 μg/m L and 578 μg/m L;compared with the model group,the degree of cell damage in the administration group was significantly decreased by flow cytometry;Western blot method Compared with the model group,the expressions of NLRP3,ASC,Caspase-1,GSDMD and other proteins in each administration group were decreased.Conclusion: Five chemical constituents(quercetin,kaempferol,crocus acid,crocetin,gardeniside)and gardenia iridoids in Gardenia have effects on glucose-oxygen deprivation/reperfusion-induced effects.Cell death has a protective effect,among which gardeniside,crocin and gardenia iridoids have a stronger effect.The mechanism is related to the NLRP3/Caspase-1/GSDMD pyroptosis signaling pathway.