Effect Of Berberine Hydrochloride On NLRP3 Inflammasome Stimulated By ?-glucan And Candida Albicans Cell Wall Integrity In Hyphal Phase

Objective:By detecting the effect of berberine hydrochloride on the TLR4/NF-?B signaling pathway and NLRP3 inflammasome in macrophages stimulated by Candida albicans-derived?-glucan,this study aimed to explore the regulatory mechanism of berberine hydrochloride on the innate immunity of Candida albicans infection.Furthermore,the effect of berberine hydrochloride on the cell wall integrity of Candida albicans was investigated.Methods:Human mononuclear leukemia cells(THP-1)were differentiated with 100 ng/ml propylene glycol methyl ether acetate(PMA).24 hours later,cell differentiation was identified by evaluating cell adhesion and spreading under an inverted microscope.The CD68 expression was analyzed by flow cytometry.CCK-8 assay was used to detect the cytotoxicity of berberine hydrochloride to PMA-differentiated macrophages.To determine the best dose of Candida albicans infection,the PMA-differentiated macrophages were treated with Candida albicans at different doses.The effects of Candida albicans on morphological changes of macrophages were investigated by evaluating cell adhesion and spreading under an inverted microscope.To investigate the regulatory mechanisms of berberine hydrochloride which inhibited Candida albicans infection,Candida albicans-infected macrophages were treated with berberine hydrochloride at a dose of 16?g/ml for 4 h.After this time,the cells were collected.ELISA detected IL-1?and IL-18 levels;western blot validated the expressions of TLR4,My D88,NF-?B,NLRP3,ASC,pro-caspase 1,and cleaved caspase 1.Detection of MIC by micro liquid-based dilution method;the effect of berberine hydrochloride on the colony formation of Candida albicans SC5314 was investigated by spot assay;the effect of berberine hydrochloride on the viability of Candida albicans SC5314 was checked by XTT assay and fluorescent staining assay.The effect of berberine hydrochloride on the morphology of Candida albicans SC5314 was examined by Scanning Electron Microscope.The changes in the cell wall of Candida albicans SC5314 after berberine hydrochloride treatment were checked by Transmission Electron Microscopy.The effect of berberine hydrochloride on?-glucan from Candida albicans SC5314 was detected by flow cytometry.The effect of berberine hydrochloride on mycelium-specific genes and?-glucan synthase genes in Candida albicans was examined by q RT-PCR.Results:In this study,we identified the compound PMA could effectively promote the differentiation of THP-1 cells to macrophage.After PMA treatment,the cells showed strong adhesion and spreading capacity.Flow cytometry analysis also suggested the expression of CD68,which is a specific marker of macrophage,increased significantly.The cytotoxicity of berberine hydrochloride to macrophages was determined.The concentrations ranged from 4?g/ml to16?g/ml.The optimal dose of Candida albicans infection in macrophages was 1.5×10~6 CFU per milliliter.Further analysis showed that Candida albicans infection can significantly induce the expression of related inflammatory factors IL-1?,IL-18,TLR4,NF-?B,NLRP3,and cleaved caspase 1 in macrophages,which are related to TLR4/NF-?B signaling pathway and NLRP3inflammasome.However,the expressions of these proteins decreased significantly after treatment with 16?g/ml berberine hydrochloride.Noticeably,the expressions of My D88,ASC,and Pro-caspase1 in Candida albicans-infected macrophages were not altered by the treatment of berberine hydrochloride.Berberine hydrochloride showed a strong inhibitory effect on both clinical and standard strains of Candida albicans,and the MIC was 64-128?g/ml,respectively.The spot assay,XTT assay and fluorescent staining assay showed that with the increase of berberine hydrochloride concentration,the viability of Candida albicans SC5314gradually decreased.The transmission electron microscopy scanning assay showed that this compound can cause cell wall damage of Candida albicans.The flow cytometry analysis showed that the fluorescence intensity of?-glucan gradually increased in a berberine hydrochloride dose-dependent way.The q RT-PCR further showed that berberine hydrochloride could significantly downregulate mycelium-specific genes and?-glucan synthase genes.Conclusion:Our results show that the compound Berberine hydrochloride can reduce the expression of NLRP3 inflammasome,which is related to the TLR4/NF-?B pathway.Also,this compound can inhibit Candida albicans?-glucan synthase-related genes and expose?-glucan,destroying the cell wall structure of Candida albicans and damaging the integrity of the wall.