Mechanism of action of Compound Zeqi Punch-containing serum on psoriasis cell model by regulating PI3K/Akt/NF-κB pathway and NLRP3 inflammasome
Objective To investigate the regulatory effect of Compound Zeqi Punch-containing serum on PI3K/Akt/NF-κB pathway and NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome in psoriasis cell model,so as to elucidate the intervention mechanism of Compound Zeqi Punch on psoriasis.Methods Preparation of Compound Zeqi Punch rat serum,NLRP3 gene silencing was performed on human immortalized keratinocyte cell line HaCaT cells,HaCaT cells were stimulated with 10 ng·mL-1 keratinocyte growth factor(KGF)in vitro to establish a psoriasis cell model.The cells were divided into control group,model group,Compound Zeqi Punch-containing serum at 5％,10％ and 20％ dose groups.CCK-8 method was used to detect the cell viability of HaCaT cells treated with different concentrations of Compound Zeqi Punch-containing serum at 0 h,24 h,48 h and 72 h,and the optimal concentration and action time of Compound Zeqi Punch-containing serum were determined.ELISA was used to detect the secretion levels of inflammatory factors IL-1β,IL-17α,IL-23,IL-8,chemokine CXCL1 and CXCL2 and the release of LDH before and after the intervention of Compound Zeqi Punch-containing serum.The mRNA expression levels of NLRP3,cysteinyl aspartate specific proteinase(Caspase-1),apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC),PI3K,Akt and NF-κB were analyzed by qRT-PCR.The protein expression levels of NLRP3,Caspase-1,ASC,p-PI3K,p-Akt,p-NF-κB,cleaved-Caspase-1 and gasdermin D(GSDMD were analyzed by Western blotting.The expression and distribution of p-PI3K,p-Akt,p-NF-κB,ASC and Caspase-1 were detected by immunofluorescence.Results The serum containing 10％Compound Zeqi Punch and 48 h were selected as the optimal concentration and time of drug-containing serum.In the psoriasis cell model of KGF-stimulated HaCaT cell,compared with the control group,the secretion levels of inflammatory factors IL-17α,IL-23,IL-1β,IL-8 and chemokines CXCL1 and CXCL2 were increased,and the release of lactate dehydrogenase(LDH)was significantly increased.Compared with the model group,the expression of inflammatory factors IL-17α,IL-23,IL-1β,IL-8 and CXCL1 and CXCL2 in the psoriasis cell model was significantly decreased after 48 h of intervention with 10％Compound Zeqi Punch-containing serum,and the release of LDH was significantly decreased.The mRNA expression levels of NLRP3,ASC,Caspase-1,PI3K,Akt and NF-κB were also decreased.After treatment with the optimal concentration of Compound Zeqi Punch-containing serum,the protein expression levels of NLRP3,Caspase-1,ASC,p-PI3K,p-Akt,p-NF-κB,cleaved-Caspase-1 and GSDMD decreased significantly,and the expression and distribution of p-PI3K,p-Akt,p-NF-κB,ASC and Caspase-1 also decreased.Conclusion Compound Zeqi Punch-containing serum exerts an intervention effect on the psoriasis cell model by affecting the PI3K/Akt/NF-κB signaling pathway and NLRP3 inflammasome.
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