The Effects Of Shenfu Injection On The Microcirculation Dysfunction During Hepatic Ischemia And Reperfusion In Rats

PrefaceHepatic ischemia - repeifusion injury is a common problem that often occurs in hepatic surgery, and an important reason that causes liver function failure after many liver operations. Though its exact mechanism is still well unknown , it is thought to be related to intracellular calcium overload, oxygen free radical, a series of cytokines, and microcirculation dysfunction. Presently, there are measures that may protect or attenuate ischemia - reperfusion injury, including ischemia preconditioning, inhibition of Kupffer's cell, many kinds of drugs protect, antibodies against many kinds of proinflammatory cytokines and recombinant products of anti - inflammatory cytokines. But few are able to used clinically.Recently researches have explored that shenfu injection attenuates hemor-rhagic shock - induced multiple organs injury and prevents spinal cord, brain, small intestine, and renal ischemia — reperfusion injury. It is known of its inhibiting xanthine oxidase and lipid hyperoxidastion, increasing nitrous oxide and carbon monoxide, enhancing ATP - ase, inhibiting cellular apoptosis, and reducing leukocyte aggregation. But it is unknown whether shenfu injection protects hepatic tissue from ischemia - reperfusion injury, inhibits Kupffer's cell activity , inhibits nuclear factor kappa B, and affects metabolization of arachidonic acid. The aim of study is to test the hypothesis that shenfu injection prevents hepatic sinusoidal endothelial cell injury and microcirculation dysfunction and influences the release of thromboxane A₂ and prostacyclin, two metabolites of arachidonic acid.Materials and methodsTwelve male wistar rats weighing 200 250g were divided into two groups randomly. SF group was treated with shenfu injection, 10ml/kg through intrap-eritoneal injection each day only once and continued 5 days. IR group was treated with 0.9% sodium chloride solution at the same dose and served as a control group. All rats were operated three hours reperfusion after fifteen minutes hepatic ischemia according to Pringles maneuver on the sixth day and were given the same dose drug once thirty minutes before operation. Arterial blood samples for liver function tests and venous ones for the measurement of TXB₂ and 6 - keto -PGF₁a (the stable metabolites of thromboxane A₂ and prostacyclin respectively) were collected three hour after reperfusion. An auto analyzer was used to determine serum ALT, AST and LDH level, while TXB₂ and 6 - keto - PGF₁a were measured with radioimmnoassay. Liver tissue samples were collected after three hour of reperfusion, and fixed in 2. 5% Glutaraldehyde solution and 10% formalin solution respectively for morphological studies. All values were expressed as x ± s. An independent - samples T test was performed with SPSS11. 0 software and P values of less than 0.05 was considered statistically significant.Resultsliver enzyme level. The levels of liver enzymes (ALT, AST and LDH) released into the serum were lower in SF group than in IR group. The AST concentration (262.3 ± 40.7 U/l vs 741.5 ±427.0 U/l, P <0.05) and the LDH concentration (1524.3 ± 614.6 U/l vs 2603. 8 ±866.4 U/l, P <0.05) 3 hour after reperfusion were significantly lower in SF group than in IR group.Plasma TXB₂ and 6 - keto - PGF₁a TXB₂( 118.7 ± 19.1 pg/ml vs 386.3±282.7pg/ml, P >0.05) 3 hour after reperfusion was lower in SF group thanin IR group, while 6 - keto - PGF₁a( 1081.7 ± 282.7 pg/ml vs 960.0 ± 209.9pg/ml, P >0.05) was higher in SF group than in IR group. The ratio of TXB₂and 6 - keto - PGF₁a(0.11 ± 0.03 vs 0.39 ± 0.24, P <0.05 ) was significant-ly lower in SF group than in IR group.Histopathology. A three - hour reperfusion after fifteen minutes ischemia caused important hepatic histologic alterations. Marked structural abnormalities were observed in IR group, such as massive hepatocyte and sinusoidal endotheli-al cell swelling, necrosis, vacuolar changes, nucleus chromatins coagulation, sinusoidal derangement and congestion, and leukocyte aggregation. In SF group, hepatic tissue injury was reduced significantly.DiscussionThe major efficacious ingredients of shenfu injection are ginsenoside and a-conitine. Shenfu injection is used to cure clinically many kinds of shock, heart failure and so on. Recently researches have explored that shenfu injection protects tissue from ischemia - reperfusion injury, such as spinal cord, brain, small intestine. This study demonstrated that shenfu injection which was given before operation attenuated liver injury. Shenfu injection reduced serum ALT, AST and LDH release, suppressed TXB₂ release and increase 6 - keto - PGF₁a, thus shenfu injection reduced significantly the ratio of TXB₂ and 6 - keto - PGF₁a. Secondly, shenfu injection reduced sinusoidal endothelial cell injury, alleviated leukocyte aggregation in microvascular.Previously studies indicated that microcirculation dysfunction induced by ischemia and reperfusion induced hepatic tissue injury. After ischemia and reperfusion, sinusoidal endothelial cell injury, leukocyte aggregation, hepatocyte swelling and so on, caused sinusoidal block and microcirculation dysfunction. Many vasoconstrictors, such as TXA₂, endothelin, caused hepatic microvascular contraction, which induced sinusoidal no - flow and aggravated tissue hypohe-mia. 20 minutes of normothermic hepatic ischemia and reperfusion leads to a 25% decrease in the sinusoidal diameter and a 20% decrease in the postsinusoi-dal venular diameter. Prostacyclin and thromboxane A₂ are vasoactive cytokines. Thromboxane A₂ increases significantly early during postischemic reperfusion and decreases in the late phase of ischemia - reperfusion. The level of prostacylin also increases, but its increasing extent is lower than that of thromboxane A₂. Theratio of TXA₂/PGI₂ increases significantly early during postischemic reperfusion and approachs to be normal after twenty four hours reperfusion. Thromboxane A₂ is a potent vasoconstrictor and chemoattractant, enhances microvascular permeability and induces platelet aggregation. Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation. Endogenous prostacyclin activates calcium - activated potassium channels (K_Ca channels) and K_ATP channels. The activation of these channels enhances the hyperpoarization of cellular membrane and reduces intracellular calcium overload. Thus, it is a major mechanism which prevents ischemia - reperfusion injury that the ratio of TXA₂/PGI₂ is improved. This study suggests that shenfu injection might not only protect sinusoidal endothelial cell from ischemia - reperfusion injury, attenuate leukocyte aggregation and alleviate hepatic sinusoidal block, but also decrease thromboxane A₂ release and increase prostacyclin release, consequently improve hepatic microcirculation perfusion, thus it prevents hepatic ischemia -reperfusion injury. In addition, aconitine, one of active ingredients of shenfu injection, might dilate vessel and reduce vascular resistance, which improves organ perfusion.The release of thromboxane A₂ and prostacyclin, two metabolites of arachi-donic acid, is regulated by key enzyme such as phospholipase A₂, cyclooxygen-ase. Previous studies have explored that nuclear factor kappa B modulates the transcription and expression of phospholipase A₂ and cyclooxygenase, and the inhibition of nuclear factor kappa B reduces the synthesis of prostanoids. These studies indicate that the modulation of nuclear factor kappa B activity is a mechanism of shenfu injection, which regulates the release of thromboxane A₂ and prostacyclin. The precise mechanisms, however, remain to be elucidated.ConclusionsIn summary, the present study shows that shenfu injection protects hepatic tissue from ischemia - reperfusion injury, and its protective effects are due to defend sinusoidal endothelial cell, alleviate leukocyte aggregation, and inflammatory cell response within hepatic sinusoid, and reduce the ratio of thromboxane A₂ and prostacyclin, consequently improve hepatic microvascular perfusion.