Inhibiting ATP6V0D2 Aggravates Liver Ischemia-Reperfusion Injury by Promoting NLRP3 Activation via Impairing Autophagic Flux Independent of Notch1/Hes1

Abstract

At present, liver ischemia-reperfusion (IR) injury is still a great challenge for clinical liver partial resection and liver transplantation. The innate immunity regulated by liver macrophages orchestrates the cascade of IR inflammation and acts as a bridge. As a specific macrophage subunit of vacuolar ATPase, ATP6V0D2 (V-ATPase D2 subunit) has been shown to promote the formation of autophagolysosome in vitro. Our research fills a gap which has existed in the study of inflammatory stress about the V-ATPase subunit ATP6V0D2 in liver macrophages. We first found that the expression of specific ATP6V0D2 in liver macrophages was upregulated with the induction of inflammatory cascade after liver IR surgery, and knockdown of ATP6V0D2 resulted in increased secretion of proinflammatory factors and chemokines, which enhanced activation of NLRP3 and aggravation of liver injury. Further studies found that the exacerbated activation of NLRP3 was related to the autophagic flux regulated by ATP6V0D2. Knocking down ATP6V0D2 impaired the formation of autophagolysosome and aggravated liver IR injury through nonspecific V-ATPase activation independent of V-ATPase-Notchl-Hesl signal axis. In general, we illustrated that the expression of ATP6V0D2 in liver macrophages was upregulated after liver IR, and by gradually promoting the formation of autophagolysosomes to increase autophagy flux to limit the activation of liver inflammation, this regulation is independent of the Notch1-Hes1 signal axis.

Figure 1

The expression of ATP6V0D2 in liver macrophages is upregulated by IR. 90 min liver warm ischemia reperfusion model was constructed as described in the Methods and Materials. (a) Serum alanine aminotransferase (sALT) and aspartate aminotransferase (sAST) (n = 4‐6 mice/group) were used to evaluate the liver inflammatory injury at 1, 3, 6, 12, 24 h, and 72 h after IR, sham as control. (b) Protein level of liver ATP6V0D2 was detected by western blot at 3 h and 6 h after IR, sham as control. (c) The expression of ATP6V0D2 in liver macrophages at 6 h after IR was detected by immunofluorescence staining, sham as control (400x magnification; representative of three experiments). (d) The expression of ATP6V0D2 in liver Mφ at 3 h and 6 h after IR was measured by quantitative real-time-PCR (n = 3‐6 mice/group), sham as control. (e) Clodronate liposome was pretreated (i.v, 250 μl/mouse) to eliminate liver Mφ 24 h before IR surgery, then detect the expression of ATP6V0D2 in liver by qRT-PCR at 6 h after IR (n = 3‐6 mice/group), sham as control. ^∗P < 0.05. ^∗∗P < 0.01. ^∗∗∗P < 0.005.

Figure 2

Knocking down ATP6V0D2 aggravates IR inflammation and tissue damage. (a) H&E staining was used to evaluate the liver injury of siATP6V0D2 pretreated groups and siCtrl groups at 6 h after IR, sham as control. (b) sALT and sAST (n = 4‐6 mice/group) were used to evaluate the liver injury at 3 and 6 h after IR in siATP6V0D2 group, siCtrl as control. ^∗∗P < 0.01. (c) The expression of chemokines responsible for the inflammatory Mφs at 3 h and 6 h after IR in siATP6V0D2 and siCtrl groups was measured by quantitative real-time-PCR (n = 3‐6 mice/group), sham as control. (d) Suzuki scores were used to evaluate liver injury (n = 3‐6 mice/group). (e) Liver macrophages were transfected with siATP6V0D2 or siCtrl for 4 h and then treated with IR surgery. Serum IL-6 and IL-10 were measured by ELISA at 3 h or 6 h after IR (n = 4‐6 mice/group).

Figure 3

Knockdown of ATP6V0D2 increased the activation of NLRP3 in liver IR macrophages. (a) The protein levels of P62, NLRP3, cleaved-caspase1, pro-caspase1, ASC, cleaved-IL1β, pro-IL1β, and β-actin in siATP6V0D2 and siCtrl groups were harvested at IR 6 h, then detected by western blot (representative of 3-6 experiments). (b) The expressions of NLRP3 and IL18 in macrophages harvested from siATP6V0D2 and siCtrl IR6h groups were measured by quantitative real-time-PCR (n = 4‐6 mice/group). ^∗∗P < 0.01. (c) Isolated macrophages from different experimental groups were cultured for another 6 h in vitro. IL-1β and IL-18 levels were measured in the culture supernatant by ELISA (n = 4‐6 mice/group). (d) Liver macrophages were transfected with siATP6V0D2 or siCtrl for 24 h and then stimulated with LPS for 6 h in vitro; then, the expression of IL1β was measured in the culture supernatant by ELISA (n = 4‐6 mice/group).

Figure 4

Knockdown of ATP6V0D2 aggravates ROS-related mitochondrial damage after IR. (a) Mitochondria in liver macrophages were detected by transmission electron microscopy at IR6h; the areas marked by box were mitochondria (1000x magnification; scale bars, 1 μm; representative of three experiments). (b) NAC was pretreated (150 mg/kg, ip) 30 min before IR surgery, then detected the expression of NLRP3 and IL18 in macrophages harvested from siATP6V0D2 and siCtrl IR6h groups by quantitative real-time-PCR (n = 4‐6 mice/group). ^∗∗P < 0.01. (c) After pretreating NAC or vehicle, isolated macrophages from siATP6V0D2 and siCtrl groups were cultured for another 6 h in vitro. IL-1β and IL-18 levels were measured in the culture supernatant by ELISA (n = 4‐6 mice/group). (d) On the basis of pretreating NAC or vehicle, the levels represented for activation of NLRP3-related proteins were detected by western blot in siATP6V0D2 and siCtrl groups at IR6h (representative of three experiments). (e) BMDMs were transfected with siATP6V0D2 or siCtrl for 24 h and then stimulated with LPS for 0, 3, and 6 h in vitro. NAC (5 mM) or ctrl was treated 1 h before LPS stimulation. IL-1β in the culture supernatant was measured by ELISA (n = 4‐6 mice/group).

Figure 5

siATP6V0D2 induces NLRP3 activation by impairing IR autophagy flux. (a) The detection of autophagic microstructures in liver macrophages by transmission electron microscopy at IR 6 h; the areas marked by box were autophagolysosome, solid triangle arrows were lysosomes, and hollow triangle arrows were autophagosomes (1000x magnification; scale bars,1 μm; representative of three experiments). (b) Immunofluorescence staining of LC3B in macrophages from siATP6V0D2 and siCtrl groups was detected by confocal microscopy at IR 6 h, sham as control (100x magnification; representative of three experiments). (c) The protein levels of LC3B and P62 in Mφs from siATP6V0D2 and siCtrl groups were detected by western blot at 6 h after IR (representative of three experiments). (d) LC3II/I relative intensity and P62/GAPDH relative intensity were analyzed by Prism. (e) BMDMs were transfected with siATP6V0D2 or siCtrl for 24 h and then stimulated with LPS for 0 h and 3 h in vitro. The protein levels of LC3B and P62 in Mφs were detected by western blot (representative of three experiments). (f) LC3II/I relative intensity and P62/GAPDH relative intensity of macrophages stimulated with LPS were analyzed by Prism. (g, h) Macrophages at IR6h which pretreated with siATP6V0D2 or siCtrl for 4 h or additional rapamycin (5 mg/kg, ip) for 1 h were harvested in vitro. The protein levels of LC3B and P62 in Mφs were detected by western blot (representative of three experiments).

Figure 6

siATP6V0D2 impairs autophagolysosome formation after IR. (a) The average number of autophagosomes and autolysosomes at IR6h in each macrophage harvested from siATP6V0D2 and siCtrl groups. (b) Macrophages at 6 h after IR which pretreated with siATP6V0D2 or siCtrl were harvested and treated with CQ (20 nM) for another 4 h in vitro. The protein levels of P62 were detected by western blot (representative of three experiments). (c) The protein levels of LC3B and β-actin in siBECN1 or siBECN1 + ATP6V0D2 (200 μl, iv)or siCtrl groups were harvested at IR 6 h, then detected by western blot (representative of 3-6 experiments). (d) The protein levels of LC3B and β-actin in 3MA or 3MA + ATP6V0D2 (200 μl, iv) or siCtrl groups were harvested at IR 6 h, then detected by western blot (representative of 4-6 experiments).

Figure 7

The function of ATP6V0D2 is independent of the V-ATPase activity that induces Notch1 signaling. (a) Mannose-siATP6V0D2 conjugated vector was injected into the tail vein before IR operation, and then, the protein levels of NICD and Hes1 of macrophages were detected at 6 h after IR by western blot.(b) Injection of siATP6V0D2 (iv) or siNotch1 + siATP6V0D2 4 h before IR surgery, siCtrl as control. Then, detected the level of ALT and AST in serum at IR 6 h by ELISA. (c) Relative expression of IL1β, MCP-1, IL10, and Arg1 in liver macrophages quantitied by qPCR. (d) BMDMs were transfected with siATP6V0D2 or siCtrl for 24 h, then stimulated with LPS for 0 h or 4 h, harvested the cells to detect the protein levels of NICD and Hes1 by western blot. (e) Injection of siATP6V0D2 (iv) or siNotch1 + siATP6V0D2 4 h before IR surgery, siCtrl as control. Then, detected the mRNA levels of NLRP3 and IL18 at 6 h by RT-qPCR (representative of 4-6 experiments). (f) Macrophages at IR6h which pretreated with siATP6V0D2 or siATP6V0D2 + siNotch1 for 4 h or additional rapamycin (5 mg/kg, ip) for 1 h were harvested in vitro. The protein levels of NLRP3 and cle-IL1β in Mφs were detected by western blot (representative of three experiments).