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

VDR is a nuclear receptor that mediates most biological functions of V D3 ; it can translocate into the nucleus to regulate the transcription of downstream target genes such as ATG16L1 via binding to their promotors , and then regulating the V D3 -mediated regulation of autophagy and antibacterial effects (Sun, 2016) . Therefore, nuclear translocation of VDR and its subsequent effect on target gene ATG16L1 was investigated firstly. The immunoblotting and immunostaining results showed that VDR protein level dramatically increased in the nucleus of LPS tolerance cells compared with LPS cells, which could be reversed by AS (Figure 4a, b) . Secondly, a ChIP assay results showed that AS induced binding of VDR to the promoter of ATG16L1 as analyzed by semiquantitative PCR and RT-PCR in LPS tolerance cells (Figure 4c, d) . Together, these data showed that AS's effect is closely related to the transcriptional regulation of VDR on ATG16L1. Thirdly, the immunoblotting results indicated that the protein level of ATG16L1 in LPS tolerance cells obviously decreased compared with LPS cells, but AS increased its . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 level (Figure 4e ), suggesting VDR negatively regulates the transcription of ATG16L1 in LPS tolerance cells, which could be prevented by AS. Additionally, our results demonstrated that the protein level of ATG16L1 in LPS tolerance cells markedly increased in VDR-KD group compared with Ctrl-KD group, and the effect of AS was diminished (Figure 4f ). However, AS-increasing ATG16L1 level in LPS tolerance cells was abrogated by overexpression of VDR ( Figure 4g ). All the aforementioned findings indicated AS inhibits the function of VDR via preventing the nuclear translocation of VDR, thus influences the transcription of its target genes ATG16L1.