AIM: To study the protective effects of tumor necrosis factor α (TNF α ) antibody and ulinastatin on liver ischemic reperfusion in rats.

METHODS: One hundred and twenty male SD rats were randomly divided into four groups: Normal control group, ischemic group, TNFα antibody group and TNFα antibody + ulinastatin group. The animals were killed at 0, 3, 6, 9, 12 h after ischemia for 60 min and followed by reperfusion. Serum alanine aminotransferase (ALT), malondialdehyde (MDA) and liver histopathology were observed.

RESULTS: After ischemic reperfusion, the serum ALT and MDA were remarkably increased, and the hepatic congestion was obvious. Treatment of TNFα antibody and ulinastatin could significantly decrease serum ALT and MDA levels, and relieve hepatic congestion.

CONCLUSION: Ulinastatin and TNFα antibody can suppress the inflammatory reaction induced by hepatic ischemic reperfusion, and have protective effects on rat hepatic ischemic reperfusion injury.

Liver ischemic reperfusion injury is induced when liver gets the retrieval of its blood perfusion or oxygen supply, and hepatic injury would aggravate due to ischemia and hypoxia injury[1-4]. Hepatic insufficiency or primary liver graft non-function can be caused by liver ischemic reperfusion injury after portal blockage, hemorrhagic shock or liver transplantation. As liver ischemic reperfusion is hard to be avoided in hepatic surgical practice and the existing prevention and cure methods are not satisfactory, research of the mechanisms and therapy on liver ischemic reperfusion becomes one of the hotspots in hepatic surgery[5,6].

Cytokines are polypeptides with extensive biological activities, and play important roles in the immunoloregulation. They prevent body from diseases and accelerate tissue rehabilitation. But on the other hand, too many cytokines can also lead to or aggravate tissue damages[7,8]. Recent researches have demonstrated that TNF α plays an important role in ischemic reperfusion injury of liver[9-12]. At the same time, ulinastatin has been applied in the clinical treatment of pancreatitis, shock and extracorporeal circulation because of its significant inhibitory effect on inflammation[13-15]. In the present study, we attempted to relieve ischemic reperfusion injury of liver by using TNF α antibody and ulinastatin, so as to provide experimental and theoretic bases for prevention and treatment of liver ischemic reperfusion injury.

Liver ischemic reperfusion injury can be frequently seen in surgical practice, and plays impotatnt roles in severe infection, trauma, shock, cardiorespiratory malfunction, organ transplantation, etc.. Ischemic reperfusion can cause a series of injuries on metabolism, structure, and function in hepatic tissues and cells, and even liver function failure. So it is one of the major factors influencing the prognosis, operative success and survival of patients.

Cytokines, are the soluble polypeptides excreted by immunocytes, and play important roles in immunological activation and inflammatory reaction. The study of cytokine effects on ischemic reperfusion injury in liver has become highlights at present[17-22]. TNFα is the polypeptide excreted by activated macrophages, endothelial cells, neutrophilic granulocytes and B lymphocytes, and plays an important role in inflammatory reaction. Liver possesses tremendous Kupffer cells (KC) that have the great potency to produce TNFα in human body. At the time of liver ischemia, for the blockage of ATP production, calcium pump of liver is in dysfunction, which causes intracellular calcium overload. Aggravated calcium overload can activate Kupffer cells. On the other hand, Kupffer cells have complement receptors, intracellular protein was released during ischemic reperfusion, thus activating complements, which can also activate Kupffer cells. The activated Kupffer cells excrete TNFα. Liver has plenty of TNFα receptors, and also is the major target organ of TNFα [23-26]. Some mechanisms were used to explain the liver damage caused by TNFα [27-30]. Firstly, TNFα could directly injure hepatocytes. Secondly, TNFα could activate neutrophilic granulocytes and mononuclear macrophages to express IL-1 and IL-6, and phosphatidase A2 that can decompose arachidonic acids. Inflammatory media were produced such as platelet active factor, leukotriene, and thromboxane A2. So the inflammatory reaction was aggravated. Thirdly, the toxic effect of TNFα on endothelial cells could induce the circulatory disorder of hepatic sinusoid, and TNFα could activate complement system that aggravates tissue damage by cytotoxicity. Finally, the oxygen-derived free radidicals induced by TNFα could facilitate oxidation explosion of neutrophilic granulocytes, which could also result in liver damage. Therefore, the way to block the production of TNFα in liver can be used to prevent liver from injury by TNFα .

Ulinastatin is one kind of glycoproteins containing 143 amino acids with molecular weight of 67 ku. It is the typical urine protease inhibitor isolated and purified from human urine. Ulinastatin has two active function domains which have a wide restrained zymogram with no overlapped region each other. So ulinastatin can inhibit many hydrolytic enzymes in one time, including trypsin, phospholipase A₂, alidase and elastase. Moreover, the components of low molecular mass decomposed from ulinastatin can also inhibit hydrolytic enzymes strongly. On the other hand, ulinastatin could ameliorate the shock situation by blocking the production of myocardial depressant factors and stabilizing the membrane of lysosome[31-33]. Recent researches found that ulinastatin could block the release of inflammatory factors, prevent cascade reaction of cytokines, inhibit excessive activation of leukocytes and block vicious circles among cytokines, inflammatory factors and leukocytes. So when pancreatitis, shock or other severe infection occured, the action of multiple hydrolytic enzymes necessitated the use of ulinastatin, which can inhibit many hydrolytic enzymes at the same time and alleviate the vicious effects of multiple proteases on tissues and organs[34-36].

The present study was to verify the protective effect of TNFα antibody and ulinastatin on ischemic reperfusion injury of liver. The results showed that the levels of ALT and MDA in TNFα antibody and ulinastatin treatment group were remarkably decreased as compared with those in ischemic reperfusion injury group. Pathological changes of liver also demonstrated the significant protective effect of TNFα antibody and ulinastatin on ischemic reperfusion injury of liver. The present results suggest TNFα antibody and ulinastatin can effectively prevent ischemic reperfusion injury of liver. But further work is needed to make clear of their best concentrations, best ratio and their effect on immune system of the body.