Acanthus Ilicifolius Alkaloid Aameliorates Acute Liver Injury By Inhibiting TLR4/NF-κB And MAPK Signaling Pathways In Mice

Objective:According to the previous research results of our group,Acanthus ilicifolius alkaloid A(4-hydroxybenzo[d]oxazol-2(3H)-one,HBO)has good liver protection function.However,the effects of HBO on LPS/D-Gal N-induced acute liver injury have not been investigated,and its potential mechanisms remain unclear so far.Thus,the present study aimed to explore whether HBO could alleviate LPS/D-Gal N-induced acute liver injury.Additionally,the study investigated whether HBO exerted the hepatoprotective effects by regulating the TLR4/NF-κB and MAPK pathways.Method:Sixty male C57BL/6J mice were divided into six groups randomly: normal control group(n=10 per group),model group,positive control group,and HBO-treated groups(high-,medium-and low-dosages).The normal control group and model group were pre-treated intragastrically with 0.5% CMC-Na.In addition,the HBO and Bifendate were prepared with 0.5% CMC-Na,and the positive control group was pre-treated intragastrically with Bifendate(150mg/kg/d),and the HBO-treated groups were pre-treated intragastrically with HBO(200,100 and 50 mg/kg/d).After 10 days,the normal control group was given one intraperitoneal injection of normal saline.The LPS and D-Gal N were dissolved in normal saline,and all mice except the normal control group were given one intraperitoneal injection of LPS(10 μl/kg)and D-Gal N(700 mg/kg).About 6 h later,all mice were sacrificed;moreover,the serum was collected and liver samples were separated immediately for further analysis.The levels of ALT,AST and TBIL in serum samples were determined.The liver sample sections were stained by hematoxylin and eosin(H&E)and examined under a light microscope.Moreover,the Terminal d UTP nick end labeling(TUNEL)method was used to detect hepatocyte apoptosis.And the protein expression of c-jun in livers was detected by immunohistochemical analysis method.The levels of cytokines in tissue homogenates such as TNF-αand IL-1β were examined using ELISA kits in accordance with the kit instructions.Moreover,the activities of NO,i NOS,MDA,GSH-PX,GSH,SOD and CAT in liver homogenates were measured by assay kits.Additionally,the m RNA levels of TNF-α,IL-1β,TLR4,NF-κB p65,NF-κB p50,ERK,p38,JNK and c-jun were tested by RT-PCR.The m RNA levels of TNF-α,IL-1β,TLR4,NF-κB p65,NF-κB p50,ERK,p38,JNK and c-jun were tested by RT-PCR.In addition,the related proteins expression of TLR4 / NF-κB signaling pathway,MAPK signaling pathway and apoptosis in liver tissue were detected by WB method.Results:1 HBO exerted a protective effect on LPS/D-Gal N-induced acute liver injuryH&E staining revealed that the livers in the normal control group exhibited the normal structure with hepatic cells in ordered arrangement and no pathological lesion area.But LPS/D-Gal N-treated group was found to have the architecture disruption,characterized by massive necrosis,haemorrhage and inflammatory infiltration.It should be noted that the damaged tissues induced by LPS/D-Gal N were alleviated both in Bifendate and HBO treatment groups.In addition,serum AST,ALT and TBIL levels were strongly elevated by LPS/D-Gal N treatment,however,their activities were decreased by the pre-treatment of Bifendate and HBO.The levels of GSH,GSH-PX,SOD and CAT were significantly decreased in LPS/D-Gal N-treated group as compared to the normal control group,and the content of MDA,NO and i NOS was significantly increased,interestingly,these alterations induced by LPS/D-Gal N were reversed by HBO and Bifendate treatment.These data clearly indicated that HBO could alleviate the severity of LPS/D-Gal N-induced acute liver injury.2 HBO alleviated inflammatory response.The content of TNF-α and IL-1β was strikingly increased after LPS/D-Gal N treatment,whereas HBO and Bifendate pre-treatment significantly reduced the over-expressions and the m RNA levels of these inflammatory cytokines.These data suggested that HBO was able to ameliorate LPS/D-Gal N-induced acute liver injury by alleviating inflammatory response.3 HBO mitigated LPS/D-Gal N-induced hepatocyte apoptosisThe TUNEL result was shown that LPS/Gal N induced massive hepatocyte apoptosis,whereas HBO treatment markedly decreased these effects.Moreover,the expression levels of apoptosis-related proteins were examined by Western blotting,LPS/D-Gal N treatment significantly up-regulated the expression of pro-apoptotic protein BAX and BAK,which were decreased when treated with HBO;furthermore,the expression of anti-apoptotic protein Bcl-2 and Bcl-xl were contrary to the pro-apoptotic protein.In the present work,the ratio of mitochondrial to cytosolic Cyt C in normal group was markedly higher than the model group,however,HBO pre-treatment effectively restored this ratio.These results indicated that HBO had the strong inhibitory effect on apoptosis.4 HBO inhibited the activation of the TLR4/NF-κB pathwayThe results showed that the protein expression levels of NF-κBp50,TLR4 and My D88,and the phosphorylation of IKK-α/β,IκB and NF-κBp65 in the LPS/D-Gal N-treated mice liver were distinctly elevated compared with the normal control mice.After treatment with HBO,the expressions of NF-κBp50,TLR4 and My D88,and the phosphorylation of IKK-α/β,IκB and NF-κBp65aforementioned were significantly decreased.Moreover,compared to normal group,the m RNA expressions of TLR4,NF-κBp65 and NF-κBp50 were remarkably enhanced by LPS/D-Gal N,however,the m RNA expression levels of these genes were partially inhibited by HBO and treatment.The above evidence demonstrated that HBO could suppress the activation of TLR4/NF-κB pathway induced by LPS/D-Gal N.5 HBO inhibited the activation of the MAPK signaling pathwayThe WB result showed that liver tissues levels of MEK,ERK1/2,JNK and p38 phosphorylations were significantly enhanced in LPS/D-Gal N treatment mice compared to the normal group.Strikingly,the phosphorylation of these molecules was remarkably suppressed by HBO treatment.In our Immunohistochemical staining experiment,liver section from the normal group revealed that c-jun was only faintly expressed;after challenged by LPS/D-Gal N,the expression of c-jun was significantly observed.However,the expression of c-jun was markedly inhibited by treatment with HBO compared to the model group.As expected,with the treatments of LPS/D-Gal N,ERK1/2,JNK,p38 and c-jun m RNA expressions were evidently enhanced.Nevertheless,the m RNA levels of ERK1/2,JNK,p38 and c-jun were remarkably reduced by treatment with HBO and.These data indicated that HBO might alleviate LPS/D-Gal N-induced acute liver injury by restraining the activation of the MAPK pathway.Conclusion:Taken together,our study demonstrates that HBO can ameliorate LPS/D-Gal N-induced acute liver injury in mice.HBO could improve the damage of liver structure,reverse the abnormal levels of ALT,AST and TBIL,inhibit the inflammatory reaction,and HBO has good antioxidant capacity.The hepatoprotective mechanisms of HBO may be associated with the inhibition of the TLR4/NF-κB and MAPK signaling pathways.