| Sepsis is a systemic inflammatory response syndrome caused by infection,which is a common complication of severe trauma,burn,or major surgery,which often lead to septic shock and even multiple organ dysfunction syndrome.Liver is not only one of the most easily damaged target organs in sepsis,also plays an important role in the occurrence and development of sepsis.During sepsis,Kupffer cells,the liver resident macrophages,were activated by endotoxin in blood and released a large number of inflammatory factors,directly leading to liver inflammation and causing systemic inflammatory response;Kupffer cells and other inflammatory cells produced large amounts of oxygen free radicals and lipid peroxidation products such as malondialdehyde,causing liver cell membrane damage and mitochondrial function damage which induced liver transaminase and bilirubin releasing into the blood;at the same time,the synthesis of liver coagulation factor,anticoagulant and fibrinolysis inhibitor were reduced and scavenging activity were also decreased in the liver,leading to coagulation dysfunction,which aggravated the systemic inflammatory response and organ damage.Nowadays the hepatic dysfunction is considered to be an independent predictive factors for the early phase of death in patients with sepsis.If early treatment is effective and the liver function is well improved,it may help to improve the prognosis of patients with sepsis.Nobiletin(5,6,7,8,3,4'-hexame-thoxy flavones),also called Sichuan Pavilion or flavonoids in tangerine,is an multi methoxy flavone compounds extracted from orange peel of citrus reticulaia Blanco which belong to Rutaceae citrus genus.In recent years,studies have shown that nobiletin has anti-inflammatory,antioxidant,anti-tumor and nerve protection and so on many kinds of biological activity,its role may be related to the suppression of NF-kB and activator protein-1(AP-1)transcription factors,and no obvious toxic side effects were reported.Therefore,nobiletin can be used for the clinical treatment of various inflammatory diseases and tumors and has great development prospects.However so far,nobiletin pharmacological effect and mechanism of anti-inflammatory effect has not been clarified,whether it can be used as the treatment of sepsis induced liver injury also needs further evaluation.In this study the sepsis induced liver injury animal model were established and the mortality change in septic mice were surveyed,the alanine transaminase in serum(ALT)and aspartate aminotransferase(AST),total bilirubin(TBil)level,serum and liver tissue TNF-alpha,IL-1beta and IL-6 were measured.The inflammatory mediators such as i NOS and COX-2 were measured in LPS induced Kupffer cells,the NF-kappa B,MAPK(ERK,JNK and p38MAPK)and Nrf2-HO-1 signal transduction pathway were also detected,to clarify the molecular mechanism of nobiletin in treatment of liver injury and to provide a new research approach for the prevention of sepsis induced liver injury.The first part: The protective effect of nobiletin on liver injury in septic miceObjective:To observe the protective effect of nobiletin on liver injury in septic mice.Methods:LPS induced liver injury mice model were established by intraperitoneal injection of endotoxin.75 adult C57BL/6 mice were randomized into five groups: normal control group(15 mices,phosphate buffer),LPS group(15 mices,LPS 10mg/kg)and nobiletin low dose group(15 mices,LPS 10mg/kg and nobiletin 50mg/kg),nobiletin in dose group(15 mices,LPS 10mg/kg and nobiletin 100mg/kg)and nobiletin high dose group(15 mices,LPS 10mg/kg and nobiletin 200mg/kg).After intraperitoneal injection of drugs at different time points(6h,12 h,24h),mices were killed and the blood and liver tissues were collected.The pathological changes of liver tissue were observed by HE staining,the level of serum alanine aminotransferase(ALT),aspartic acid transaminase(AST),total bilirubin(TBIL),TNF alpha and IL-1beta and IL-6 were measured.The other 50 mice were selected and the survival rate of mice within 72 hours were observed.Result:Compared with the normal control group,model group and drug intervention group were changed significantly,the results showed that the model was constructed successfully.(1)2h after LPS injection,the model group and drug intervention group mice were depressed,shortness of breath,hypothermia,not drink or eat,while the mice in NOB intervention group were relatively good;(2)24h after LPS injection,the liver pathology of mice significantly changed.Tissue swelling,necrosis,hyperemia and infiltration of inflammatory cells were observed in the liver tissue,but tissue injury were significantly alleviated in the NOB intervention group;(3)Serum ALT,AST and TBil levels in the model group increased significantly compared with normal control,while these levels decreased significantly in the NOB intervention group(P<0.05 or P< 0.01);(4)The inflammatory factor TNF-alpha and IL-1beta and IL-6 increased significantly in the model group,while these levels decreased significantly in the NOB intervention group(P<0.05 or P<0.01);(5)The 72 h survival rate was 100% in normal control group,the 72 h survival rate was 0% in the model group.Nob intervention can effectively improve the survival rate: the 72 h survival rate was 40% in the dose of 50 mg/kg group,the 72 h survival rate was 50% in the dose of 100 mg/kg group,the 72 h survival rate was 80% in the dose of 200 mg/kg group;Conclusion:Nobiletin plays a protective role in LPS induced liver injury mice by inhibiting systemic inflammation and hepatic inflammatory factors release,relieving liver cell edema,necrosis and inhibit the infiltration of inflammatory cells,and reducing the release of liver enzymes.The second part: The effect of nobiletin on IkB/NF-kB signal pathway in LPS activated mouse Kupffer cellsObjective:To observe the effect of nobiletin on IkB/NF-kB signal pathway in LPS activated mouse Kupffer cells,to explains the molecular mechanisms of nobiletin on the liver injury in septic mice.Method:Kupffer cells were separated by collagenase digestion,density gradient centrifugation combined with selective separation method.Then Kupffer cells were inoculated in 6-well plates and randomly divided into five groups: normal control group(phosphate buffer),LPS group(LPS 10ug/ml),nobiletin low dose group(LPS 10ug/ml and nobiletin 10 m M),nobiletin medium dose group(LPS 10ug/ml and nobiletin 20 m M)and nobiletin high dose group(LPS 10ug/ml and nobiletin 40 m M).Cell and cell culture supernatant were collected and determined as follows: the cytokine TNF-?,IL-1? and IL-6 levels in 12 h cell culture supernatant was detected by ELISA;the expression of i NOS and COX-2 protein were measured by Western blotting;p-IkB,IkB,p-p65 and p65 were detected by Western blotting;nuclear NF-kB p65 DNA binding activity was detected by EMSA.Result:12h after LPS stimulation on cultured Kupffer cells,the TNF-?,IL-1? and IL-6 levels in cell culture supernatant increased significantly,while after different doses of nobiletin treatment these cytokines significantly decreased(P<0.05 or P<0.01);1h after LPS stimulation,the proportion of p-IkB/IkB and p-p65/p65 in Kupffer cells increased significantly(P<0.05 or P<0.01),nucleus NF-kappa B p65 DNA binding activity also significantly increased(P<0.05 or P<0.01),while after different doses of nobiletin treatment it significantly decreased(P<0.05 or P<0.01)Conclusion:Nobiletin plays a protective role in LPS induced liver injury mice by inhibiting IkB/NF-kB signaling pathway and thus reducing the release of inflammatory cytokines.The third part: The effect of nobiletin on MAPK and Nrf2-HO-1 signal pathway in LPS activated mouse Kupffer cellsObjective:To observe the effect of nobiletin on MAPK(ERK,JNK and p38MAPK)and Nrf2-HO-1 signal pathway in LPS activated mouse Kupffer cells,to explains the molecular mechanisms of nobiletin on the liver injury in septic mice.Method:Kupffer cells were separated by collagenase digestion,density gradient centrifugation combined with selective separation method.Then Kupffer cells were inoculated in 6-well plates and randomly divided into five groups: normal control group(phosphate buffer),LPS group(LPS 10 u g/ml),nobiletin low dose group(LPS 10ug/ml and nobiletin 10 m M),nobiletin medium dose group(LPS 10ug/ml and nobiletin 20 m M)and nobiletin high dose group(LPS 10ug/ml and nobiletin 40 m M).1h,6h and 12 h after drug stimulation,the expression of p-ERK,p-JNK,p-p38 MAPK,Nrf2,HO-1,i NOS and COX-2 protein were measured by Western blotting.Result:1h after LPS stimulation on cultured Kupffer cells,p-ERK,p-JNK,P-P38 MAPK expression increased significantly,while after different doses of nobiletin treatment these expression significantly decreased(P<0.05 or P<0.01);6h after LPS stimulation on cultured Kupffer cells,the cytoplasm HO-1 and nuclear Nrf2 expression increased slightly,while after different doses of nobiletin treatment these expression significantly increased(P<0.05 or P<0.01);12h after LPS stimulation on cultured Kupffer cells,i NOS and COX-2 expression increased significantly,while after different doses of nobiletin treatment these expression significantly increased(P<0.05 or P<0.01).Conclusion:Nobiletin plays a protective role in LPS induced liver injury mice by inhibiting mitogen activated protein kinase(ERK,JNK and p38 MAPK)and increase Nrf2 signaling pathway and thus reducing the expression of i NOS and COX-2. |
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