基于NLRP3/Caspase-1/GSDMD通路探讨蒌慈散结方诱导乳腺癌4T1细胞焦亡的机制OA

Objective To investigate the inhibitory effect of Louci Sanjie Formula(蒌慈散结方,LCSJ)on breast cancer 4T1 cells and its regulatory impact on the NLRP3/Caspase-1/GSDMD pathway.Methods A transplanted tumor model was established by subcutane-ously inoculating breast cancer 4T1 cells into BALB/c mice.After tumor formation,mice were randomly divided into a model group,low/medium/high-dose LCSJ groups(12,24,48 g/kg),and positive control group(epirubicin hydrochloride 8 mL/kg).Mouse body weight and subcutaneous tumor volume were measured,and tumor mass was weighed to calculate tumor inhibition rates.Hematoxylin-eosin(HE)staining was employed to observe morphological changes in tumor tissues.Quantitative Real-time PCR(qRT-PCR)was used to assess mRNA expression levels of NLRP3,Caspase-1,and GSDMD in tumor tissues.Cells were divided into 5%blank serum group,and 5%,10%,and 20%LCSJ-containing serum groups.Cell morphological changes were microscopically observed,while CCK-8 assay was employed to evaluate cell viability,plate clonogenicity assay was employed to assess clone-formation capacity,LDH release rate was determined via LDH assay kit,ELISA assay was employed to quantify IL-1β and IL-18 levels in culture supernatant,and West-ern blot(WB)was conducted to evaluate the expression levels of NLRP3,Caspase-1,and GSDMD.Results Compared with the model group,tumor volume and mass in the low/medium/high-dose LCSJ groups and the positive drug group were significantly decreased(P<0.05).With increasing doses of LCSJ,the tumor inhibition rate also increased.Tumor cells in the model group were tightly arranged with irregular morphology and rare necrosis;the positive drug group showed nuclear pyknosis and large necrotic areas,as well as cells in all LCSJ groups were loosely arranged with many ruptured cells in the necrotic areas.Compared with the model group,mRNA levels of NLRP3,caspase-1,and GSDMD were significantly increased in the low/medium/high-dose LCSJ groups(P<0.05),and NLRP3 mRNA level in the positive drug group was significantly higher than in the model group(P<0.05).CCK-8 assay revealed that,com-pared with the 5%blank serum group,all LCSJ-containing serum groups exhibited significantly reduced cell viability after 24,48,and 72h of intervention(P<0.05),and there was a trend of gradually decreasing cell viability with increasing concentration and intervention time(P<0.05).Thus 72-hour intervention was selected for subsequent experiments.Colony formation assays demonstrated the number of clones in the 5%,10%,and 15%LCSJ-containing serum groups was significantly reduced compared to the 5%blank serum group(P<0.001).Compared to the 5%blank serum group,LDH release rates,IL-1β,and IL-18 levels in the cell supernatant,as well as pro-tein expression levels of NLRP3,Caspase-1,and GSDMD were significantly increased in LCSJ-containing serum groups(P<0.05).4T1 cells in the various concentrations of LCSJ-containing serum groups showed varying degrees of swelling and round bubble-like pro-trusions,consistent with the morphological characteristics of pyroptosis.Conclusion LCSJ may inhibit the growth of breast cancer 4T1 cells,and its mechanism may be related to the activation of NLRP3/Caspase-1/GSDMD pathway-mediated pyroptosis.