A Study Of Positive Effect Of SFN On Endotoxin-Induced Acute Hepatic Injury

Background:Endotoxin, named lipopolysaccharide (LPS), was the element of all the gram negative bacilli, and the main factor that induced Pyemia, systemic inflammatory response syndrome. Endotoximia occured in the process of the occurrence and development of serious insult, infection, hepatopathy and numerous serious diseases. Meanwhile, Pyemia could induce multiple organ failure. Liver was one of the most vulnerable organs. Therefore, Pyemia and liver interacted as both cause and effect, and influenced each other. Once Pyemia occured in liver, vicious circle might come. Thus obstruction some chains of the circle and prevention endotoxin's damaget to liver was a critical problem. Up to now, there was not unique and effective mean to cure hepatic injury induced by endotoxin in clinical practice.At present, pharmaceutical therapy was common in clinical practice. Medicines most in use were as follows:1. Dexamethasone: it inhibited the overproduction of nitrogen monoxidum (NO) and lipid peroxidation, and increased antioxidative activity;2. Platelet activating factor and receptor antagonist: it antagonized degradation function of membrane phospholipid and inhibited the production of oxygen free radical;3. Glycine;glycocine: it inhibited Kupffer's cells'secretory function and decreased the secretion of tumor necrosis factor (TNF-α), inhibited the entero-mast cells degranulation, and decreased histamine release, and decreased the enterogenic endotoxin level and endotoxin level in the blood plasma;4. LPS antagonist:it antagonized LPS's biological activity, accelerated its eradication rate of passing red blood cell and heterophil granulocyte's CRI receptor. Some experiments had proved that the protection of anti-endotoxin monoclonal antibody on animals was effected by decreasing the sensitivity of vascular endothelial cell to TNF-αand other cell factors such as IL-6, instead of decreasing TNF-αdirectly.5. Traditional Chinese medicine: such as powder preparation for infusion Jiedu Hugan Granule,herba Artemisiae Scopariae, but the mechanism was not known yet. However, the present therapy had obvious shortages, for example, dexamethasone had obvious side effects, especially in the decreasing of body immune function and the function of eradicating bacteria, and causing diffusion of the infection and increasing in the possibility of Pyemia exacerbation; LPS antagonist was expensive, could not prevent the development of hepatopathy furtherly;the present therapies were relatively unitary, and neglected that the increasing of bilirubin would induce secondary liver injury, and had not involved self -immunoregulation. Therefore, their comprehensive therapeutic effectiveness were weak. The combination of different kinds of medicines would overload the liver certainly, and increased patients'economic burden..Sulforaphane (SFN), a kind of isothiocyanate contained in Cruciferae plant, had been a focus of study either at home or abroad since being found to perform the function of chemical protection. Researches showed that SFN could resist oxidative damage by inhibiting phaseⅠenzyme and inducing phaseⅡenzyme. It could restrain tumors also by inducing the division cycle of tumor cells to be stagnant , making them apoptotic and inhibiting the epithelialis of tumor blood vessels. In addition, SFN could perform the function of immunoregulation by improving the vitality of natural killer cells, reinforcing the phagocytic power of phagocytic cells and lowering the level of TNF-αreleased by macrophage stimulated by endotoxin. Moreover, SFN could resist bacteria by inhibiting the growth of helicobacter pylori (HP). There was not studies about SFN both abroad and at home. Therefore, this study attempt to make clear whether SFN can protect endotoxin-induced acute hepatic injury and to provide theoretical evidence.Objective:To establish the rat models of endotoxin-induced acute hepatic injury, and even to make clear the physiopathologic characteristics and further illuminate the rules and mechanisms of endotoxin-induced acute hepatic injury. Then, SFN was used to intervene in rats with endotoxin- induced acute hepatic injury. Tests of various indicatrixs were detected to identify the positive effect of SFN on endotoxin-induced acute hepatic injury to provide some experiment basements for clinical use of SFN. Materials and Methods:(1) The rat models of endotoxin-induced acute hepatic injury by LPS and D-GalN of sublethal dose were constructed. Fourty eight healthy Wistar rats were randomly divided into 3 groups with 16 rats in each group. Every group was then randomly divided into control group and experimental group. The experimental group was received peritoneal injection of LPS(50μg/kg)+D-GalN(300mg/kg) of sublethal dose in 1ml stroke-physiological saline solution(SPSS),while the rats in the control group was treated SPSS only. Blood sample was collected from abdominal artery at 6,24,48h and serum was taken after centrifugalization,then the rats were sacrificed respectively to observe morphological changes in hight microscope, got the serum and examined the levels of ALT, AST and TBIL with automatic biochemistry analyzer, counts of apoptotic cells of hepatic tissue were observed in hight microscope. The level of IL-1βwas tested by ELISA. TNF-αand the expression of UDP-glucuronyltransferase(UGT1A1) mRNA were examined by RT-PCR.(2) The positive effect of SFN on endotoxin-induced acute hepatic injury was examined. Twinty four healthy Wistar rats were randomly divided into 3 groups: the control group, the DEX intervention group and the SFN intervention group with 8 in each group. Three groups were injected with LPS(50μg/kg)+D-GalN(300mg/kg) into peritoneal respectively. The DEX intervention group was injected with 1ml of DEX (5mg/kg) into peritoneal. The SFN intervention group was injected with 1ml of SFN (0.5mg/kg) into peritoneal. The control group was only injected with 1ml of stroke-physiological saline solution. After 24 hours, rats were killed and observed morphological changes in hight microscope, the levels of ALT, AST and TBIL were tested with automatic biochemistry analyzer. Counts of apoptotic cells of hepatic tissue were observed in light microscope. The level of IL-1βwas tested by ELISA. TNF-αand the expression of UDP-glucuronyltransferase (UGT1A1) mRNA were examined by RT-PCR.Results:Experiment of the animal model constructing:1. The characteristics of Histopathology:(1) Control group: The structure organization of lobuli hepatis was intact without any degeneration and necrosis.(2) 6 h treated group: The hepatic cells is swelling light, but without necrosis.(3) 24 h treated group: The hepatic cells were swelled generally, with a small amount of hepatocyte punctiform necrosis, inflammation cell invaded lobuli hepatis(4) 48 h treated group: The hepatic cells were swelled generally, with a great amount of hepatocyte punctiform necrosis, inflammation cell invaded lobuli hepatis2. The Serum Levels of ALT, AST and TBIL:Six hours after injecting LPS/D-GalN, the levels of ALT, AST and BIL of the experiment group rised significantly compared with the control group. With the time going, the levels of ALT, AST and TBIL rised gradually and reached the peak (3204.875+791.400,3662.500+725.661, 65.100 +23.150) respectively in 24 hours. Then ALT, AST and TBIL remained at high levels. There was no significant difference (P>0.05) between the 24h group and the 48h group.3. The results of TUNEL analysis of the withered cell of liver tissue:The apoptotic index (AI) of liver tissue cell increased in 6 hours and had significant difference compared with the treated group (P<0.01), and changed with time. The number of apoptotic cell increased, and reached the peak(29.225+4.315) in 24 hours, 24 h group and 6 hours of group had significant difference (P <0.05).4. The level of IL- 1βand TNF-αmRNA, UGT1A1 mRNA:The level IL- 1βrised obviously in 6h, and was significant difference compared with the control group(P<0.01), d ecreased in 24h and in 48h obviously,but it stillwas higer than the control group(P<0.01). The expression of TNF-αmRNA rised in 6 h, and was significant difference with control group (P<0.01), high level was still maintained in 24 h, but decreased obviously in 48h, and there was not significant differance compared with control group (P>0.05). The expression of UGT1A1mRNA rised in 6h, and was significant difference with control group(P<0.01), higher level was still maintained in 24h, but decreased obviously in 48h , and there was significant differance compared with 24h Group(P<0.01)and notsignificant differance compared with control group(P>0.05).Experimental research of positive effect of SFN on Endotoxin-Induced Acute Hepatic Injury:5. Histopathologic Characteristics: (1) The control group: structure of hepatic lobule was complete, with a small amount of hepatocyte punctiform necrosis, inflammation cell invaded lobuli hepatis.(2) The DEX intervention group: mucoid degeneration by side of hepatic,vascellum, with a small amount of conjugate nuclei hepatic cells and hepatocyte punctiform necrosis.(3) The SFN intervention group: structure of hepatic lobule was complete, with a few swelling and necrotic hepatic cells and the infiltration of inflammation.6. The Serum Levels of ALT, AST and TBIL:The Serum Levels of ALT, AST and TBIL decreased by SFN intervention group and DEX intervention group and and there was not significant difference between SFN and DEX intervention group (P>0.05), but were remarkably different compared with the control group (P<0.01).7. The apoptotic results of hepatic tissues based on TUNELApoptotic cells reduced significantly in both SFN intervention group and DEX intervention group compared with the control group (P<0.01), but there was not significant difference between SFN and DEX intervention group (P>0.05).8. The results of IL-1β, TNF-αand UGT1A1 mRNAOnly SFN intervention group could reduce the IL-1βproduction,there was not significant difference compared with the control group (P>0.05); both SFN intervention group and DEX intervention group could decrease the TNF-αmRNA expression, and there was significant difference compared with the control group (P<0.01). The reducing effect of SFN intervention group was more remarkable than DEX intervention group (P<0.01); both SFN intervention group and DEX intervention group increased the UGT1A1 mRNA expression, and there was significant difference compared with the control group (P<0.01), there was not remarkable difference between SFN and DEX intervention group (P>0.05).Conclusion:1. Sublethal dose of LPS can cause the endotoxin-induced acute hepatic injury of rats which sensitivity to D-GalN. This model is suitable for further research on endotoxin-induced acute hepatic injury.2. Apoptosis of cells is the important morphology change,attention should be paid to it.3. The levels of IL-1β,TNF-αrise significantly at early stages (6h). Therefore early intervention can reduce the second strike of inflammation mediators on the liver.4. The bilirubin metabolism ability of rats with endotoxin-induced acute hepatic injury improved at the early stage (6h and 24h) and weakened later (48h). It is very important to protect hepatic cells for lowering hyperbilirubinemia.5. Both SFN and DEX can decrease the levels of ALT and AST, the number of apoptotic cells and the expression of TNF-αmRNA, reinforce the expression of UGT1A1mRNA. They can not only reduce the second strike of TNF-αmRNA to liver, but also decrease the level of jaundice. The results show that SFN and DEX have positive effect on endotoxin-induced acute hepatic injury.6. SFN can decrease the level of IL-1βand TBIL, it is more effective in decreasing the expression of TNF-аmRNA, these results shows that SFN are more effective than DEX to protect the liver from endotoxin-induced acute hepatic injury. Thus, SFN is valuable in practice.