Fig 1.
Transplantation-associated ischemia and warm reperfusion in vitro elevates mtDNA levels in both rat and human hepatocyte cell lines.
(A) Six hours of transplantation-associated ischemia and one hour of reperfusion significantly increased the mtDNA levels in the rat hepatocyte cell line, McA RH7777. (B) Six hours transplantation-associated ischemia and one hour reperfusion significantly increased the mtDNA levels in the human hepatocyte cell line, HepG2. Control: cells were cultured in a conventional cell culture incubator for seven hours; 6 h Isc: six hours incubation in cold and ischemic conditions; 6+1 h IR: six hours incubation in cold and ischemic conditions followed by one hour of warm reperfusion; CytB, cytochrome B; COXIII, cytochrome C oxidase subunit III; NADH, NADH dehydrogenase. *, P = 0.01, **, P = 0.001, ***, P = 0.0001.
Fig 2.
Warm ischemia and warm reperfusion in vitro elevates mtDNA levels in both rat and human hepatocyte cell lines.
(A) One hour of warm ischemia and two hours of warm IR significantly increased the mtDNA levels in the rat hepatocyte cell line, McA RH7777. (B) One hour warm ischemia and two hours warm IR significantly increased the mtDNA levels in the human hepatocyte cell line, HepG2. Control, cells were cultured in normal cell culture incubator for two hours; 1h Isc, one hour warm ischemia incubation; 1+1 IR, one hour warm ischemia incubation followed by one hour warm reperfusion treatment, CytB, cytochrome B; COXIII, cytochrome C oxidase subunit III; NADH, NADH dehydrogenase. *, P = 0.01, **, P = 0.001, ***, P = 0.0001.
Fig 3.
MTDs induce a similar pattern of cell death pattern compared to cells undergoing transplantation-associated ischemia and warm reperfusion in vitro.
Apoptosis assay in vitro in HepG2 hepatocytes undergoing IR treatment and MTD incubation. (A) Control: HepG2 hepatocytes were cultured in normal environment. (B) Ischemia group: HepG2 hepatocytes that were maintained in a hypoxic environment for six hours in vitro demonstrated increased apoptosis when compared to Control cells. (C) IR group: HepG2 hepatocytes that were incubated in a hypoxic environment for six hours and subsequently transferred to a normoxic incubator for 24 hours demonstrated an increased percentage of both apoptosis and necrosis. In addition, these conditions induced a sub-population of PI-positive cells (gated in circle). (D) MTDs incubation group: HepG2 hepatocytes that were co-cultured with 400ug/ml MTDs in a normal environment for 24 hours demonstrated an increased percentage of apoptosis and necrosis with an increased sub-population of PI-positive cells (gated in circle), similar to the IR group (C). The figures shown are representative of three experiments with similar results. The PI single positive population is the basal level cell death.
Fig 4.
MTDs cause cell death in vitro in a dose dependent manner.
(A) MTDs (Mitochondrial Damage-Associated Molecular Patterns) induce significant rat hepatocytes cell death at concentrations above 100 ug/ml, McA RH7777 co-cultured with different concentrations of MTDs (ug/mL) for 24 hours. (B) MTDs induce significant human hepatocytes cell death at concentrations above 100 ug/ml, HepG2 co-cultured with different concentrations of MTDs for 24 hours (ug/ml). The blank bar indicates Annexin V positive cells, the black bar indicates PI positive cells. *, P = 0.01, **, P = 0.001, ***, P = 0.0001 (n = 3/group).
Fig 5.
Hepatic IR increases mtDNA level in circulation in vivo.
(A) One hour of warm ischemia and one hour of warm reperfusion in vivo significantly increased the mtDNA levels during both ischemia and IR; OB, blood collection immediately following laparotomy; I, ischemia: the atraumatic clip was placed on the portal triad for 60 minutes; R, reperfusion; reperfusion was initiated by removal of the atraumatic clip and observed for another 60 minutes; I0, I30, I60: ischemia for 0/30/60 minutes; R0, R30, R60: after 60 minutes portal triad clamping and the clip was removed to achieve reperfusion for 0/30/60 minutes. (B) mtDNA levels were not altered after 2 hours following a sham operation. 0, 30, 60, 90,120 indicate minutes of sham operating time, laparotomy without clamping of the portal triad. *, P = 0.01, **, P = 0.001, ***, P = 0.0001 (n = 3/group).
Fig 6.
Histopathologic analyses of liver injury after IR.
Liver sections were obtained from IR and sham control rats at the time point of 120 minutes. (A) IR induce hepatic injury assessed by H&E staining from different liver lobes: median (M), left (L), right (R) and caudate (C) lobes of sham and IR group, black arrow and V points to the portal vein, yellow arrows indicate necrotic cells. (B) IR induce significant hepatic injury assessed by Suzuki injury score. ***, P = 0.0001(n = 3/group).
Fig 7.
Hepatocellular injury evaluated by AST and ALT.
IR induce significant hepatic injury as demonstrated by elevated AST and ALT levels. Serum samples were obtained from IR and sham rats at 120 minutes post reperfusion. A statistically significant difference was noted in mean serum ALT and AST levels following the induction of IR, compared with sham operated rats in vivo. ***, P = 0.0001(n = 3/group).
Fig 8.
Total hepatic IR causes apoptosis in vivo.
Liver sections were obtained from IR and sham control rats at 120 minutes post reperfusion. (A) and (B) the intensity of immunostaining of pro-caspase 3 significantly decreased after IRI compared with the sham group. (C) and (D) the intensity of immunostaining of cleaved-caspase 3 significantly increased after IRI compared with the sham group.
Fig 9.
Total hepatic IR cause systemic inflammation in in vivo.
Total hepatic IR induces significant IL-6 (A), TNF-α (B), and IL-10(C) cytokines release. Serum samples were obtained from IR and sham rats at 120 minutes post reperfusion. *, P = 0.01, ***, P = 0.0001 (n = 3/group).
Fig 10.
Fold changes in TLR2, TLR4, TLR9, MyD88 and NF-κB mRNA levels.
MTDs induce MyD88 and NFκB expression and up-regulate expression of TLR2, TLR4 and TLR9 similar to warm hepatic IR. (A)-(E), The expression level of TLR2, TLR4, TLR9, MyD88 and NF-κB significantly increased with MTDs co-culture, warm IR, and cold IR treatment compared to control; MTDs, co-culture MTDs with McA RH7777 hepatocyte cell line for 24 hours; W.IR, McA RH7777 hepatocyte cell line underwent warm ischemia for 1 hour followed by reperfusion for 24 hours in vitro; C.IR, McA RH7777 hepatocyte cell line underwent cold ischemia for 6 hours followed by reperfusion for 24 hours in vitro. *, P = 0.01, **, P = 0.001, ***, P = 0.0001 (n = 3/group).
Table 1.
TNFα was released by the hepatocytes in MTDs co-cultured and warm IR.
Fig 11.
Fold changes in TNFα mRNA levels.
MTDs-induced hepatocytes up-regulate expression of TNFα similar to warm IR treatment. MTDs, co-culture 400ug/ml MTDs with McA RH7777 hepatocyte cell line for 24 hours; W.IR, McA RH7777 hepatocyte cell line underwent warm ischemia for 1 hour and reperfusion for 24 hours in vitro. **, P = 0.001, ***, P = 0.0001 (n = 3/group).