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| NLRC3 alleviates hypoxia/reoxygenation induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6 |
| Zhong-Tang Li, Hang Liu, Wan-Qiu Zhang ∗ |
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
∗ Corresponding author
E-mail address: zhangwanqiu3916@163.com (W.-Q.Zhang). |
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Abstract Background: NOD-like receptor family CARD domain containing 3 (NLRC3) plays an important role in both innate and adaptive immunity. This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation (H/R)-induced inflammatory response in RAW264.7 cells.
Methods: Liver ischemia-reperfusion (I/R) model in mice and H/R model in RAW264.7 cells were con- structed. Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3, TRAF6, p-p65, p65, I κB-α, and the K63-linked ubiquitination level of TRAF6 in cells. The immunofluorescence assay was performed to evaluate the nuclear level of the NF-κB (p65). ELISA was conducted to measure the content of IL-1 βin serum and cell supernatant. The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay.
Results: The NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time ( P < 0.05). The expression of NLRC3 and I κB-αprotein in RAW264.7 was decreased gradually, while the expression of p-p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time ( P < 0.05). The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly. However, NLRC3 had no effect on the expression of TRAF6 protein. The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R + Lv-NLRC3 group was significantly lower than that in the H/R + negative control (NC) group ( P < 0.05). Moreover, the activation of NF-κB in H/R + Lv–NLRC3 group was inhibited compared with that in the H/R + NC group, and the level of the inflammatory factor IL-1 βin the cell culture supernatant was also decreased accordingly ( P < 0.05).
Conclusions: NLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.
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2020, Vol. 19 
