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/reoxy...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 constructed.Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3,TRAF6,p–p65,p65,IκB–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–1βin 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 IκB–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–1βin 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.展开更多
Background: Hepatic ischemia-reperfusion(I/R) injury(IRI) represents a crucial challenge in liver transplantation. Fisetin has anti-inflammatory, anti-aging and anti-oxidative properties. This study aimed to examine w...Background: Hepatic ischemia-reperfusion(I/R) injury(IRI) represents a crucial challenge in liver transplantation. Fisetin has anti-inflammatory, anti-aging and anti-oxidative properties. This study aimed to examine whether fisetin mitigates hepatic IRI and examine its underlying mechanisms. Methods: Sham or warm hepatic I/R operated mice were pretreated with fisetin(5, 10 or 20 mg/kg). Hepatic histological assessments, TUNEL assays and serum aminotransferase measurements were performed. An in vitro hypoxia/reoxygenation(H/R) model using RAW264.7 macrophages pretreated with fisetin(2.5, 5 or 10 μmol/L) was also used. Serum and cell supernatant concentrations of interleukin-1 β(IL-1 β), IL-18 and tumor necrosis factor-α(TNF-α) were determined by enzyme-linked immunosorbent assay(ELISA). Protein levels of p-GSK3 β, p-AMPK and NLR family pyrin domain-containing 3(NLRP3)-associated proteins were detected by Western blotting. Results: Compared with the I/R group, fisetin pretreatment reduced pathological liver damage, serum aminotransferase levels, serum concentrations of IL-1 β, IL-18 and TNF-α in the murine IRI model. Fisetin also reduced the expression of NLRP3 inflammasome-associated proteins(NLRP3, cleaved caspase-1, IL-1 β and IL-18) in I/R-operated liver. The experiments in vitro showed that fisetin decreased the release of IL-1 β, IL-18 and TNF-α, and reduced the expression of NLRP3 inflammasome-associated proteins in H/R-treated RAW264.7 cells. Moreover, fisetin increased the expressions of p-GSK3 β and p-AMPK in both models, indicating that its anti-inflammatory effects were dependent on GSK3 β/AMPK signaling. The antiinflammatory effects of fisetin were partially inhibited by the AMPK specific inhibitor compound C. Conclusions: Fisetin showed protective effects against hepatic IRI, countering inflammatory responses through mediating the GSK3 β/AMPK/NLRP3 inflammasome pathway.展开更多
基金This study was supported by grants from the National Natural Science Foundation of China(81873592)the graduate tutor team construction project of Chongqing Municipal Education Commission Foundation(dstd201801).
文摘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 constructed.Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3,TRAF6,p–p65,p65,IκB–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–1βin 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 IκB–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–1βin 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.
基金This study was supported by grants from the National Natural Science Foundation of China(81672959,81873592 and 81703063)the Science and Technology Commission Foundation of Chongqing,China(cstc2019jscx-gksb X0005)+1 种基金the Natural Science Foundation of Chongqing,China(cstc2018jscx-msyb X0133)the graduate tu-tor team construction project of Chongqing Municipal Education Commission Foundation,China(dstd201801).
文摘Background: Hepatic ischemia-reperfusion(I/R) injury(IRI) represents a crucial challenge in liver transplantation. Fisetin has anti-inflammatory, anti-aging and anti-oxidative properties. This study aimed to examine whether fisetin mitigates hepatic IRI and examine its underlying mechanisms. Methods: Sham or warm hepatic I/R operated mice were pretreated with fisetin(5, 10 or 20 mg/kg). Hepatic histological assessments, TUNEL assays and serum aminotransferase measurements were performed. An in vitro hypoxia/reoxygenation(H/R) model using RAW264.7 macrophages pretreated with fisetin(2.5, 5 or 10 μmol/L) was also used. Serum and cell supernatant concentrations of interleukin-1 β(IL-1 β), IL-18 and tumor necrosis factor-α(TNF-α) were determined by enzyme-linked immunosorbent assay(ELISA). Protein levels of p-GSK3 β, p-AMPK and NLR family pyrin domain-containing 3(NLRP3)-associated proteins were detected by Western blotting. Results: Compared with the I/R group, fisetin pretreatment reduced pathological liver damage, serum aminotransferase levels, serum concentrations of IL-1 β, IL-18 and TNF-α in the murine IRI model. Fisetin also reduced the expression of NLRP3 inflammasome-associated proteins(NLRP3, cleaved caspase-1, IL-1 β and IL-18) in I/R-operated liver. The experiments in vitro showed that fisetin decreased the release of IL-1 β, IL-18 and TNF-α, and reduced the expression of NLRP3 inflammasome-associated proteins in H/R-treated RAW264.7 cells. Moreover, fisetin increased the expressions of p-GSK3 β and p-AMPK in both models, indicating that its anti-inflammatory effects were dependent on GSK3 β/AMPK signaling. The antiinflammatory effects of fisetin were partially inhibited by the AMPK specific inhibitor compound C. Conclusions: Fisetin showed protective effects against hepatic IRI, countering inflammatory responses through mediating the GSK3 β/AMPK/NLRP3 inflammasome pathway.