Studies On Effects And Mechanism Of Resolvin E₁ Restrainingthe Kupffer Cells Activation In The Rats Following HepaticIschemia-Reperfusion Injury

Hepatic ischemia/reperfusion injury (HIRI) is a common pathologicalprocess of traumatic surgical diseases in the liver, such as severe liver trauma,extensive hepatic lobus excision, liver transplantation, shock and infection. Itresults in a series of injuries on metabolism, structure, and function in hepatictissues and cells, and even liver function failure. It is also one of the major factorsinfluencing the prognosis and survival of the patients. The mechanisms of hepaticischemia/reperfusion injury resemble those observed in other organs that aretransiently deprived of oxygen. These mechanisms involve Kupffer cell (KC)activation, cytokine release, and neutrophil activation, increased expression ofadhesion molecules, sinusoidal endothelial cell death, and hepatocyte injury.Among these, the activation and the release of the excessive cytokine in KCs playa major role.Resolvin E1 (RvE1) is an omega-3 eicosapentaenoicacid (EPA)–derived lipidmediator generated during resolution of inflammation and in human vasculaturevia leukocyte-endothelial cell interactions. RvE1 possesses anti-inflammatory andproresolving actions?Stops transepithelial and transendothelial migration? Stimulates Macrophage non-phlogistic phagocytosis of apoptotic neutrophils?Blocks IL-12 production of Dendritic cell?Upregulates CCR5 expression of Tcell?Regulates leukocyte expression of adhesion molecules?reduced leukocyterolling (40%)?selectively blocked both ADP-stimulated and thromboxanereceptor agonist U46619-stimulated platelet aggregation. An excessiveinflammatory response is clearly recognized as a key mechanism of HIRI. Wepostulate that RvE1 might have beneficial effects on HIRI through restraining theKCs activation. In order to test this hypothesis, we establish a model of hepaticischemia/reperfusion injury in rats at the normal temperature; isolate the KCs atdifferent times, to evaluate whether RvE1 attenuates the release of some cytokines(TNF-?, IL-1?, Chemerin and MIP-2) from KCs, which is probably an importantfactor in the protection mechanism against ischemia/reperfusion injury.ObjectiObjectiveTo study the protective effect of RvE1 on the rats suffering hepatic ischemiareperfusion injury and its influence to the expression of Chemerin and MIP-2 inthe KCs and determine the concentration of TNF-?and IL-1?in the supernatantsof primary cultures of rat KCs and explore the undering molecular mechanisms.Methods1. KCs were isolated from liver of SD rats by perfusion with collagenase anddensity gradient centrifugation with Percoll, and maintenance cultures of purifiedKCs were established according ro the attached time and cultured condition. KCswere identificated by Lysozyme immunohistochemical staining, trypan bluestaining, and ultrastructure, etc.2. The model of the partial warm hepatic ischemia/reperfusion was based onthe Nauta et al method. 0h, 1h, 6h, 12h and 24h after the reperfusion, KCs wereisolated and incubated immediately. The TNF-?and IL-1?in the supernatantswere measured by ELISA. Chemerin and MIP-2 in KCs was detected byimmunohistochemistry and RT-PCR.3. Seventy-two male Sprague Dawley rats were divided at random into three groups: the sham operation group (control group), the ischemia/reperfusion injurygroup (IRI group) and the RvE1 treatment group (RvE1 group). The model of thepartial warm hepatic ischemia/reperfusion was established. One hour, six hours,twelve hours and twenty-four hours after the reperfusion, blood was taken frominferior vena cava to test the hepatic enzyme level, including ALT and AST. KCswere isolated and incubated.The TNF-?and IL-1?in the supernatants of primarycultures of KCs were measured by TNF-?and IL-1?ELISA kits. The expressionsof Chemerin and MIP-2 in KCs were detected by immunohistochemistry and RTPCR.4. KCs isolated from the rats suffering hepatic ischemia reperfusion injurywere divided at random into five groups. The cells of group three, four and fivewere treated with different concentration of RvE1. The TNF-?and IL-1?in thesupernatants were measured by ELISA. Chemerin and MIP-2 mRNA in KCs wasdetected by RT-PCR.Results1. KCs were isolated sucessfully with high purity. the yield was7.34±1.54×106cells /liver. KCs showed particles of India ink in cytoplasm, andlysozyme positve by immunohistochemical staining.2. The concentration of TNF-?in supernatant of primary cultures of rat KCs inthe control group was 13.89±2.04ng/L and it increased at least ten times in 1hafter the reperfusion. Following reperfusion injury stimulate, the TNF-?level wasincreased with time, and reached the maximum (230.6±26.3ng/L) 6 h after thestimulation (P<0.01). Even 24 h after the reperfusion, the concentration of TNF-?(133.68±12.15ng/L) was still significantly high compared with that in the controlgroup(P<0.01). The concentration of IL-1?in the control group was64.65±4.63ng/L and significantly increased with time, reaching its maximum(189.8±13.13ng/L) 6 h after the reperfusion (P<0.01), and slightly declined(146.30±11.90ng/L) 24 h after the following reperfusion.The expressions ofChemerin and MIP-2 in KCs were detected by immunohistochemistry and RTPCR.No or few Chemerin and MIP-2 protein and mRNA were expressed in the KCs of control group. Its expression in the IRI group had a significant increaseafter the reperfusion (P<0.05) which was contrary to the control group. The activebehavior of the Chemerin and MIP-2 gene in KCs following liverischemia/reperfusion injury is assumed to be one of the major causes for thehepatic ischemia/reperfusion injury.3. The ALT and AST level of the RvE1 group was significantly lower than theischemia/reperfusion group(P?0.05). The concentrations of TNF-?, IL-1?in thesupernatants were significantly increased with time, reaching its maximum at 6 hfollowing reperfusion (P<0.01). Significant decreases were observed in the RvE1group when compared to the IRI group (P<0.01). Immunohistochemical studiesshowed Chemerin and MIP-2 protein was not or weakly expressed in the controlgroup. Its expressions in the IRI group had a significant increase at 6h,12h and24h interval (P<0.01), which were contrary to the control group, but there were nodifference at 1h interval (P>0.05). The levels of Chemerin and MIP-2 proteinlessened in the RvE1 group at 6h,12h and 24h intervals when compared with theIRI group(P<0.01), but there was no difference at 1h interval (P>0.05). RT-PCRanalysis showed that KCs from the control group had low but detectable levels ofChemerin and MIP-2 mRNA. The mRNA level was significantly increased withtime, reaching its maximum at 6 h following IR, and slightly declined 24 h afterIR, but was still significantly higher than that in the control group (P<0.01).However, in the RvE1 group, these changes were significantly inhibited by thetreatment of RvE1 (P<0.01).4. The mRNA expression of Chemerin and MIP-2 and the level of TNF-?andIL-1?in the KCs isolated from the rat following ischemia/reperfusion injurysignificantly increased compared to the control group (P<0.01). The expression ofthese factors were markedly decreased after RvE1 treatment compared with theIRI group (P<0.05).Conclusion1. The technique for isolation of KCs described here is simple and reliable?Itcan be used to study the biologic function of Kupffer cells. 2. The model of rat liver partial warm ischemia-reperfusion injury establishedin this study is similar to the clinical situation where the whole liver is renderedischemic during total vascular exclusion. At the early phase of theischemia/reperfusion injury, the activated KCs produce high concentrations ofTNF-?and IL-1?. The secretions of these toxic inflammatory cytokines appear tobe a key mediator of the inflammatory response to promote the release of othercytokines. The expressions of Chemerin and MIP-2 mRNA and protien in KCssignificant increase after the reperfusion.The active behavior of the Chemerin andMIP-2 gene in KCs is assumed to be one of the major causes for the hepaticischemia/reperfusion injury.3. RvE1 protect against rat liver ischemia/reperfusion injury partially byrestraining the KCs activation, reducing the excess release of TNF-?and IL-1?from KCs and decreasing the high expression of Chemerin and MIP-2 protein andmRNA.4. In vitro experiments, the mRNA expression of Chemerin and MIP-2 andthe level of TNF-?and IL-1?in KCs isolated from the rat followingischemia/reperfusion injury were inhibited by RvE1 treatment.