Artesunate interacts with Vitamin D receptor to reverse mouse model of sepsis-induced immunosuppression via enhancing autophagy

Shenglan Shang; Jiaqi Wu; Xiaoli Li; Xin Liu; Pan Li; Chunli Zheng; Yonghua Wang; Songqing Liu; Jiang Zheng; Hong Zhou

. CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi. org/10.1101 org/10. /2020 Subsequently, immunostaining was used to determine whether AS affected their intracellular co-localization and distribution of VDR and NF-κB p65. The results revealed an obvious co-localization of VDR and NF-κB p65 in LPS tolerance cells, but AS significantly inhibited their co-localization (Figure 6b1, b2) . Moreover, nuclear translocation of NF-κB p65 markedly declined in LPS tolerance cells relative to LPS cells, which was reversed by AS treatment (Figure 61, b3) . These results indicated that high level of VDR intensively interacted with NF-κB p65, preventing the nuclear translocation of NF-κB p65. Noteworthily, AS inhibited the physical interaction between VDR and NF-κB p65, accelerating the nuclear translocation of NF-κB p65. To confirm it, the nuclear protein level of NF-κB p65 was investigated using ELISA and immunoblotting. The results showed NF-κB p65 level in nucleus obviously reduced in LPS tolerance cells relative to LPS cells, which was reversed by AS (Figure 6c1, c2) . In LPS tolerance cells, the nuclear NF-κB p65 level increased in VDR-KD group compared with negative control, leading to the loss of tolerance phenotype ( Figure 6d1 ). In VDR-OE cells, the NF-κB p65 nucleoprotein level remarkably suppressed compared to negative control, and AS treatment made no difference in LPS tolerance cells ( Figure 6d2 ). To determine whether AS-promoting pro-inflammatory cytokines releases in LPS tolerance cells is related to its influence on NF-κB p65 activity, NF-κB p65 expression in RAW264.7 was modified ( Figure S4a, b) . The results showed that knock-down of NF-κB p65 would lead to the decline in pro-inflammatory cytokines release, and loss of AS's effect ( Figure 6e1) . Simultaneously, overexpression of NF-κB p65 would lead to loss of LPS tolerance phenotype, and addition of AS making no difference (Figure 6e2 ). All the aforementioned findings indicated AS inhibited physically interacts between VDR and . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.02.26.966143 doi: bioRxiv preprint NF-κB p65 in LPS tolerance macrophages, and then promoted NF-κB p65 nuclear translocation as well as downstream pro-inflammatory cytokine release.