Effect of Salvianolic Acid B Combined with Puerarin on Pyroptosis of SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate the effect and mechanism of salvianolic acid B combined with puerarin in protecting the SH-SY5Y cells from the damage by oxygen-glucose deprivation/reoxygenation （OGD/R） based on pyroptosis. MethodSH-SY5Y cells were used to establish the model of OGD/R， and cells were classified into the control， OGD/R， 10 μmol·L-1 salvianolic acid B， 100 μmol·L-1 puerarin， 10 μmol·L-1 salvianolic acid B + 100 μmol·L-1 puerarin， and 10 μmol·L-1 NOD-like receptor protein 3 （NLRP3） inhibitor MCC950 groups. Except the control group， other groups were rapidly reoxygenated for 12 h after 6 h OGD for modeling. The cell survival rate was determined by the methyl thiazolyl tetrazolium （MTT） assay. An optical microscope was used to observe the cell morphology. A spectrophotometer was used to determine the content of lactic dehydrogenase （LDH） in culture supernatant. Cell damage was measured by Hoechst/PI staining. The mRNA levels of NLRP3， cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， apoptosis-associated speck-like protein （ASC）， and interleukin-1β （IL-1β） were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein activation of Caspase-1 and NLRP3 was detected by immunofluorescence. Western blot was employed to determine the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1. ResultCompared with the control group， the OGD/R group showed decreased cell survival rate （P<0.01）， damaged cell morphology， increased leakage rate of LDH （P<0.01）， up-regulated mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.01）， and up-regulated protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.01）. Compared with the OGD/R group， salvianolic acid B， puerarin， and salvianolic acid B combined with puerarin improved cell survival rate （P<0.01）， and the combined treatment group outperformed salvianolic acid B and puerarin used alone （P<0.01）. Salvianolic acid B combined with puerarin and MCC950 both improved cell morphology， reduced the leakage of LDH （P<0.01）， alleviated cell damage， and down-regulated the mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.05， P<0.01） and also the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.05， P<0.01）. ConclusionThe results indicated that salvianolic acid B combined with puerarin can alleviate the OGD/R-induced damage of SH-SY5Y cells by inhibiting pyroptosis.