Experimental Study On The Molecular Mechanism Of The Effect Of Astilbin On Liver Warm Ischemia-reperfusion Injury

Experimental Study on the molecular mechanism of the effect of astilbin on liver warm ischemia-reperfusion injuryLiver transplantation has become the method of choice for treatment of the majority of acute and chronic end-stage liver disease.However,ischemia-reperfusion injury(IRI) existing in every case of liver transplantation is still the main reason of primary graft dysfunction or non-function.The shortage of donor leads to the growing use of the marginal donor in most transplant centres,including the aging donor,non-heart-beating donors,small size and fatty liver donor,while the basic problem of the marginal donor is still IRI.So,reducing IRI maximatily would not only increase the appropriate donors,but also allow more patients recovery from liver transplant surgery.And to achieve this desire, firstly,we should understand the mechanisms of I/R injury more fully.Liver is one of the most sensitive organs to ischemia-reperfusion injury.The activation of sinusoidal endothelial cells,Kupfer cells and neutrophil and the formation of oxygen free radicals plays an important role in the pathological process of ischemia-reperfusion injury.Oxygen free radicals and inflammatory cytokines,adhesion molecules further promote neutrophil activation and aggregation,mediate lipid peroxidation injury process, which eventually lead to liver damage.The Toll-like receptors(TLRs) found in 1997 play an important role in the disease of infection and inflammation.The TLR-4 signaling pathway was considered to be dominated in the pathophysiological process of heart,kidney and liver IRI.TLR-4 molecule exists in all of hepatic parenchyma,non-parenchymal cells,each group of liver cells has a complete TLR-4 signaling pathway.Activated hepatic non-parenchymal cells(especially Kupffer cells) play an important role in the liver warm ischemia-reperfusion injury through TLR-4 signaling pathway.Activated TLR-4 leads to the activation of NF-κB,which further triggers great increase of inflammatory cytokines such as TNF-a,IL-1,IL-6,et al.Because of the important role of TLR-4 signaling pathway in liver ischemia-reperfusion injury,the purpose of reducing liver IRI by inhibiting this signaling pathway is of enticing prospects. HO-1 is a heat shock protein 32 members.Liver HO-1 was first produced by Kupffer cells. As one of the TLR-4 ligands,it has strong antioxidant,anti-inflammatory function thus protecting liver IRI by inhibiting TLR-4 signaling pathway.On the other hand,SOCS1 is a negative regulator molecule,which inhibit ILs,IFN-γ, GH,and other cytokine signal transduction pathway through negative regulation of JAK-STAT signaling pathway.SOCS1 also involves in the regulation of other signaling pathways,such as TNF-a,LPS.The over-expression of SOCS1 in vitro can significantly reduce the hepatocytes apoptosis induced by TNF-a,while TNF-a is considered TLR-4 activation marker,implying that SOCS1 may be one of the endogenous negative regulators of TLR-4 signal transduction systems.Studies show that IL-10 can promote the formation of SOCS protein,in particular the SOCS1 and SOCS3.The role of inhibiting inflammation and protecting organizations of IL-10 can be observed in many models of ischemia -reperfusion injury,including liver.So if drugs can promote IL-10 expression in vivo,it could play a protective role in the liver IRI through regulating SOCS1 negative signal pathway.Astilbin is one of the three flavanonols isolated from the ethanol extract of rhizoma. Flavonoids have strong function of antioxidant and can be used as delivery of hydrogen to scavenging oxygen free radicals,resulting in anti-inflammatory and reducing ischemia-reperfusion injury.Researches have confirmed its role of diuretic,analgesic, anti-inflammatory and reduce myocardial ischemia-reperfusion injury.As reported that astilbin could significantly reduce the liver damage induced by carbon tetrachloride(CC14) through the intervention of oxygen free radicals and lipid peroxide formation;In concanavalin A(Con A)-induced hepatitis,astilbin could significantly reduce TNF-a production and the proliferation of liver Kupffer cells.And in a contact dermatitis model, astilbin could significantly promote the expression of IL-10 and SOCS1 and SOCS3, reducing inflammatory response.Based on these research backgrounds,further study will be involved in this topic: 1)The protective effects of astilbin on liver warm ischemia-reperfusion injury,including liver function,liver pathology,SOD,MDA,the MPO detection;2) The effects of astilbin on the expression of TLR-4,HO-1,NF-κB,TNF-a protein and mRNA in the liver ischemia-reperfusion injury model,to investigate the molecular mechanisms of inhibiting inflammation signal pathway;3) The effects of astilbin on the expression of IL-10,SOCS1 protein and mRNA,to explore the molecular mechanism of promoting anti-inflammation signaling pathway.Based on these researches,to prepare for the further study on the mechanisms of protective and immunosuppressive effects of astilbin on liver ischemia and reperfusion injury,in the liver transplantation model of allogeneic mice.PARTⅠ.Effect of astilbin on liver warm ischemia-reperfusion injuryObjective:To investigate the protective effect of astilbin on liver function and hepatic tissues in the liver warm ischemia-reperfusion injury.Methods:C57BL/6 mice were randomly divided into four groups(n=8):sham-operated group(Sham),model control group(I/R),low dosage of astilbin treatment group(10mg/kg) and large dosage of astilbin(40mg/kg) treatment group.24 hours and one hour before Ischemia,treatment group mice were intraperitoneally injected 10 or 40 mg/kg astilbin.Then the hepatic ischemia-reperfusion model of 70 percent of liver,including the left and middle hepatic lobe,were established.The I/R model control group and the sham operation group were administered with the same volume of normal saline.After 90 min ischemia and 6h reperfusion of the partial hepatic lobe,blood and liver tissue samples were collected from the experimental groups.Serum ALT activity was detected as an indicator of liver function damage,and the content of MDA,MPO,SOD in liver tissues were detected by ELISA. Histopathology detection was performed.Results:Compared with the I/R model control group,serum ALT in both treatment group of the low and large dosage astilbin were significantly decreased(P＜0.01),even lower in the large dosage group than in the low dosage group(P＜0.01).Hepatic pathology performance improved significantly in the large dosage group;The content of MDA,MPO in liver tissues were significantly decreased in both treatment groups when compared with the I/R model control group(P＜0.01),also lower in the large dosage group than in the low dosage group(P＜0.01).And SOD levels significantly increased in treatment groups(P＜0.01),higher in the large dosage group than in the low dosage group(P＜0.05).Couclusiou:Treatment with astilbin can effectively improve the mouse liver function and liver pathology damage induced by liver warm ischemia-reperfusion injury;Intervention with astilbin can reduce the aggregation, infiltration and activation of neutrophil in the mouse liver induced by warm ischemia-reperfusion injury,thereby reducing inflammatory response;Intervention with astilbin can inhibit the formation of oxygen free radicals,and reduce the damage of lipid peroxidation induced by liver ischemia-reperfusion injury. PARTⅡ.Inhibiting Effect of Astilbin on TLR-4 Signaling Pathway in Liver warm ischemia-reperfusion injuryObjective:To study the molecular mechanism of the effect of astilbin on inhibiting the inflammation signaling pathway in liver warm ischemia-reperfusion injury.Methods: C57BL/6 mice were randomly divided into four groups(n=5):sham-operated group (Sham),model control group(I/R),low dosage of astilbin treatment group(10mg/kg) and large dosage of astilbin(40mg/kg) treatment group.24 hours and one hour before Ischemia, treatment group mice were intraperitoneally injected 10 or 40 mg/kg astilbin.Then the hepatic ischemia-reperfusion model of 70 percent of liver,including the left and middle hepatic lobe,were established.The I/R model control group and the sham operation group were administered with the same volume of normal saline.After 90 min ischemia and 6h reperfusion of the partial hepatic lobe,blood and liver tissue samples were collected from the experimental groups.Serum TNF-a activity was detected,and the content of TNF-a,TLR-4,NF-κB and HO-1 in liver tissues were detected by western blot.mRNA expression of these proteins was detected by semiquantitative RT-PCR.Results:Compared with the I/R model control group,serum TNF-a in both treatment group of the low and large dosage astilbin were significantly decreased(P＜0.01),even lower in the large dosage group than in the low dosage group(P＜0.01).The protein content of TNF-a,TLR-4,NF-κB in liver tissues were gradually decreased in both treatment groups when compared with the I/R model control group,also lower in the large dosage group than in the low dosage group. Same trends were observed in the mRNA expression of these proteins showed by semiquantitative RT-PCR(low dosage group P＜0.05;large dosage group P＜0.01).HO-1 levels gradually increased in treatment groups,higher in the large dosage group than in the low dosage group,the result of mRNA showed a same trend(low dosage group P＜0.05; large dosage group P＜0.01).Conclusion:Intervention with astilbin can reduce the high levels of serum TNF-a caused by ischemia-reperfusion injury,as well as increased expression of TNF-a protein and mRNA in the liver tissues,thereby reducing TNF-a-mediated further injury;Intervention with astilbin can reduce the high expression of TLR-4 protein and mRNA,and NF-κB protein in IRI liver tissues,demonstrating its effect of inhibiting TLR-4 inflammation signaling pathway.Treatment with astilbin can promote the expression of HO-1 protein and mRNA in IRI liver,demonstrating its effect of TLR-4 pathway inhibition and promotion of the expression of HO-1 is closely related.PARTⅢ.Effect of Astilbin on Expression of SOCS1,IL-10 in Liver warm ischemia-reperfusion injuryObjective:To study the molecular mechanism of the effect of astilbin on the anti-inflammation signaling pathway in liver warm ischemia-repeffusion injury.Methods: C57BL/6 mice were randomly divided into four groups(n=5):sham-operated group (Sham),model control group(I/R),low dosage of astilbin treatment group(10mg/kg) and large dosage of astilbin(40mg/kg) treatment group.24 hours and one hour before Ischemia, treatment group mice were intraperitoneally injected 10 or 40 mg/kg astilbin.Then the hepatic ischemia-reperfusion model of 70 percent of liver,including the left and middle hepatic lobe,were established.The I/R model control group and the sham operation group were administered with the same volume of normal saline.After 90 min ischemia and 6h reperfusion of the partial hepatic lobe,liver tissue samples were collected from the experimental groups.The content of SOCS-1 and IL-10 in liver tissues were detected by western blot.mRNA expression of the same proteins was detected by semiquantitative RT-PCR.Results:Compared with the I/R model control group,SOCS-1 and IL-10 protein levels gradually increased in treatment groups,higher in the large dosage group than in the low dosage group,the result of mRNA showed a same trend(low dosage group P＜0.05; large dosage group P＜0.01).Conclusion:Intervention with astilbin can promote the expression of IL-10 protein and mRNA in IRI liver,thus inhibiting the inflammation; Intervention with astilbin can promote the expression of SOCS-1 protein and mRNA in IRI liver,showing its role of negative regulation by upregulating SOCS1 pathway.