| Objective:Liver,acting important roles in metabolism and detoxification in human body,is one of the most sensitive organs to a series of stimuli such as viral infection,drugs,poisons,ischemia,metabolic disorders,autoimmune disease and various chemical toxins.Excessive stimulation often leads to acute liver injury,which may result in life-threatening clinical problems such as serious blood coagulation disorders and eventually develop into acute hepatic failure.Although the rates of hepatic failure survival have been improved substantially on recent years because of critical care management and emergency liver transplantation,a great of challenges is still exist to find a new available treatment for acute liver injury and interdict the progression of acute liver injury to acute hepatic failure.Nowadays,the research on preventive and therapeutic methods of acute liver inj ury depends largely on various ALI animal models similar to the pathogenesis of acute liver injury in humans.Therefore,we choose different types of ALI animal models to find a common agent which can effectively protect various types of ALI,in order to facilitate further development and application in the future.Garlic(Allium sativum L.),which is cultivated globally and can be found in everywhere or every country's daily diet as a spice or condiment in various raw or cooked dishes,possess various beneficial effects and protect against several diseases,such as microbial infections,hyperlipidemia,heart diseases and acute or chronic liver injury.The various pharmacological activities exhibited by garlic are highly related to the various organosulfur compounds produced by metabolic transformation.Diallyl sulfide(DAS)is a colorless,water-insoluble a bioactive organosulfur compound extracted from garlic and the degradation product of allicin.Allicin is unstable and readily decomposed into oil-soluble allyl sulfur compounds with similar chemical structure,including DAS,diallyl disulfide(DADS)and diallyl trisulfide(DATS),there are no reports comparing the preventive and therapeutic effects of DAS,DADS and DATS on different types of ALI.Therefore,in the current study,we first used lipopolysaccharide(LPS)combined with D-Galactosamine(D-GalN),acetaminophen(APAP)and carbon tetrachloride(CCl4)to build acute immune,drug and chemical liver injury,respectively,in mice.Next,in order to screen out the best agents for further research,we compared the preventive and therapeutic effects of DAS,DADS and DATS on different types of ALI,and then investigate the different mechanisms of DAS on immune,drug and chemical ALI in mice.Methods:1.The effects of DAS,DADS and DATS on different types of acute liver injury in mice.Chemical-induced ALI in mice was established by intraperitoneal injection of 0.25%CCl4(dissolved in corn oil).To observe the preventive and therapeutic effects,a range of doses of DAS,DADS,DATS(100,200,400 ?mol/kg)and 60 mg/kg silibinin were orally administered before and 1 hour after CC1U intraperitoneal inj ection,respectively.And the liver and serum were immediately collected24 hours after CCl4 inj ection.Drug-induced ALI in mice was established by intraperitoneal injection of 300 mg/kg acetaminophen(APAP).To observe the preventive and therapeutic effects,a range of doses of DAS,DADS,DATS(100,200,400 ?mol/kg)and 200 mg/kg NAC were orally administered before and 1 hour after APAP intraperitoneal injection,respectively.And the liver and serum were immediately collected 24 hours after APAP injection.Immunological ALI in mice was established by intraperitoneal injection of lipopolysaccharide(LPS,10 ug/kg)and D-galactosamine(D-GalN,500 mg/kg).To observe the prevention and treatment effect 100,400 ?mol/kg doses of DAS,DADS,DATS and 100 ?mol/kg DAS,DADS,DATS were orally given before and 1 hour after LPS/D-GalN injection,respectively.And the liver and serum were immediately collected 8 hours after LPS/D-GalN injection.Serum biochemical test was performed using Automatic Biochemical Analyzer,and the pathological changes of liver were observed by H&E staining.2.DAS protects against different types of ALI by inhibiting oxidative stress and inflammation in mice.50,100 and 200 ?mol/kg DAS were orally administered one hour before LPS/D-GalN intraperitoneal injection,and the liver and serum were immediately collected 8 hours after LPS/D-GalN injection.In addition,100,200 and 400 ?mol/kg DAS were orally administered one hour before APAP(300 mg/kg)or CCl4(0.25%)intraperitoneal injection,and the liver and serum were immediately collected 24 hours after APAP or CCl4 injection.The levels of ALT,AST,TBIL and TNF-?,IL-1? and MCP-1 in serum were measured using Automatic Biochemical Analyzer or ELISA,the pathological changes of liver were observed by HE staining,the levels of MDA,GSH,SDO,CAT and TNF-?,IL-1? and MCP-1 in liver tissue were measured using commercial kits or ELISA.the protein expression of IKB-?,p-IKB-? and NF-?B p65 in liver tissue were determined by Western blotting.3.DAS protects against different types of ALI by inhibiting hepatocytes apoptosis in mice.The liver samples collected in the second part experiment were used.TUNEL staining was used to observe hepatocyte apoptosis.Western blotting was used to detect the protein expression of cleaved caspase 3,Bcl-2,Bax,PI3K,p-PI3K p85,Akt and p-Akt(Ser473)in liver tissue.MK-2206 was used to inhibit the phosphorylation of Akt to observe whether DAS could inhibit LPS/D-GalN-induced hepatocyte apoptosis by regulating PI3K-Akt signaling pathway.4.DAS protects against different types of ALI by inhibiting the activation of macrophage in mice.The experiment was also divided into three parts.LPS/D-GalN,APAP and CCl4 were used to induce different types of ALI.The experimental groups were divided into:control group,DAS control group,ALI model group,ALI+DAS group,ALI+GdCl3 group,ALI+GdCl3+DAS group.The Kupffer cells(KCs)were blocked by tail intravenous injection of GdCl3 48 and 24 hours before DAS administration.DAS was orally administered with 400 ?mol/kg dose.LPS/D-GalN,APAP and CCl4 were injected at 1 hour after DAS administration,and serum and liver tissue were collected 8 hours after LPS/D-GalN inj ection or 24 hours after APAP,CCl4 injection.The levels of ALT,AST and TNF-?,IL-1?,IL-10 and MCP-1 were detected in serum.Liver tissues were stained with HE to observe pathological changes.The levels of NO and TNF-?,IL-1?,IL-10 and MCP-1 in liver tissues were detected by commercial kits or ELISA.The mRNA expressions of TNF-?,IL-1?,MCP-1,IL-6,IL-10,iNOS and Arginase-1 in liver tissues were detected by RT-qPCR.Results:1.The effects of DAS,DADS and DATS on different types of acute liver injury in mice.DAS,DADS,DATS and Silibinin significantly reduced the increase of serum ALT,AST activity induced by CCl4(p<0.05),DAS and DADS in medium and high dose groups could significantly reduce the increase of serum TBIL concentration caused by CCl4(p<0.05).In addition,high dose of DAS treatment after CCl4 injection could significantly reduce the serum transaminase activity(p<0.05),but failed to decrease the TBIL in serum,meanwhile,DADS?DATS and silibinin did not show significant therapeutic protective effect on ALI.Pretreatment or treatment with DAS and NAC significantly decreased the elevation of ALT,AST activity in serum induced by APAP and showed significant preventive effect on APAP-induced ALI(p<0.05),DADS and DATS failed to decrease the elevated ALT,AST activity in serum whenever given before of after APAP injection.Besides,pretreatment or treatment with DAS,DADS and DATS in each dose group could significantly reduce the increase of serum ALT,AST activity induced by LPS/D-GalN and showed significant preventive and therapeutic effects(p<0.05).2.DAS protects against different types of ALI by inhibiting oxidative stress and inflammation in mice.LPS/D-GalN,APAP and CCI4 exposure significantly increased the activity of ALT,AST and TBIL in serum(p<0.05),and pretreatment with different doses of DAS reduced the increase of AST,ALT activity and TBIL concentration in serum caused by LPS/D-GalN,APAP and CCl4 injection(p<0.05).H&E staining results shows that control group and DAS control group exhibited normal liver cell morphology,well-preserved cytoplasm and prominent nucleus,without inflammation or necrosis.In contrast,LPS/D-GalN injection induced severe diffuse necrosis in liver,showed hemorrhage,swelling,necrosis,inflammatory cell infiltration and apoptotic bodies,while APAP and CCl4 cause central necrosis of hepatic lobules,showed obviously pathological changes such as extensive hemorrhage,necrosis,infiltration of inflammatory cells and edema of hepatocytes.However,these pathological changes induced by LPS/D-GalN,APAP and CCl4 were significantly ameliorated or even vanished under DAS pretreatment.In addition,LPS/D-GalN,APAP,CCl4 exposure significantly increased MDA content in liver(p<0.05),and different doses of DAS could effectively reverse the increase of MDA content in liver tissue induced by LPS/D-GalN,APAP and CCl4(p<0.05).Beyond that,compared with control group,LPS/D?GalN injection markedly decreased the content of GSH and the activities of SOD and CAT in liver(p<0.05),and different dosages of DAS pretreatment could significantly up-regulate the level of GSH,SOD and CAT in liver tissue compared with LPS/D-GalN group(p<0.05).The activities of SOD and CAT in liver were significantly decreased alter APAP or CCl4 exposure compared with control group(p<0.05),while DAS pretreatment significantly boosted the activities of SOD and CAT compared with APAP or CCl4 group(p<0.05).Beyond that,APAP or CCl4 exposure led to the significant decreased concentration of GSH and the increased concentration of GSSG in the liver,and the ratio of GSH/GSSG was correspondingly significantly reduced compared with the control group(p<0.05).However,DAS pretreatment significantly restored the GSH concentration and increased the ratio of GSH/GSSH in liver compared with APAP or CCl4 group(p<0.05).The serum and liver levels of TNF-?,IL-1? and MCP-1 were significantly increased after LPS/D-GalN treatment compared with control group(p<0.05).In contrast,compared with the LPS/D-GalN model group,pretreatment with different dosages of DAS significantly decreased the serum and liver tissue levels of TNF-?,IL-1? and MCP-1 in a dose-dependent manner(p<0.05).APAP or CCl4 exposure significantly increased the level of TNF-a in serum(p<0.05),and pretreatment with DAS(100,200,400 ?mol/kg)significantly decreased the level of TNF-? in a dose-dependent manner(p<0.05).Meanwhile,the expression of inhibitor of kappa B alpha(I?B?)in cytoplasm was markedly decreased after APAP or CCl4 exposure and different doses of DAS pretreatment prevented this degradation in a dose-dependent manner(p<0.05).Inversely,the phosphorylation of I?B? and the ratio of p-I?Ba/I?B? was dramatically increased after APAP or CCl4 exposure,which was inhibited by DAS in a dose-dependent manner(p<0.05).APAP or CCl4 exposure markedly increased the amounts of NF-?B p65 subunits in the cytoplasm and nucleus(p<0.05),however,pretreatment with DAS normalized the level of NF-?B p65 in the cytoplasm and nucleus.3.DAS protects against different types of ALI by inhibiting hepatocytes apoptosis in mice.LPS/D-GalN,APAP,CCl4 injection induced excessive hepatocyte apoptosis,which showed the significantly increased number of TUNEL-positive stained cells in liver tissue sections(p<0.05),among which the hepatocyte apoptosis induced by LPS/D-GalN was the most obvious,the number of TUNEL-positive cells was significantly decreased by DAS pretreatment,which indicated that the pretreatment of DAS could significantly inhibit LPS/D-GalN,APAP or CCl4-induced hepatocyte apoptosis(p<0.05).LPS/D-GalN injection significantly increased the protein expression of cleaved caspase3 and Bax by 148%,78.3%and decreased the protein expression of Bcl-2 by 77.1%in the liver(p<0.05).However,pretreatment with different dosages of DAS significant reversed the rise in cleaved caspase 3 and Bax expression and reversed the decrease in Bcl-2 expression caused by LPS/D-GalN treatment(p<0.05).APAP exposure significantly increased the protein expression of cleaved caspase3 and Bax by 82.3%,61.2%and decreased the protein expression of Bcl-2 by 40.5%in liver(p<0.05).DAS pretreatment significantly decreased cleaved caspase 3 expression and increased Bcl-2 expression compared with APAP group(p<0.05).Beyond that,CCl4 exposure significantly increased the protein expression of cleaved caspase3 and Bax by 137.7%,88.9%and decreased the protein expression of Bcl-2 by 45.6%in liver(p<0.05).However,pretreatment with DAS significant reversed the increased expression of cleaved caspase 3 and Bax and the decreased expression of Bcl-2 caused CCl4 injection(p<0.05).In addition,LPS/D-GalN,APAP and CCl4 did not significantly change the expression of PI3K and Akt in liver tissue,but decreased their phosphorylation.Compared with the control group,LPS/D-GalN reduced p-PI3k p85/PI3K and p-Akt(Ser473)/Akt by 63.3%and 62.3%(p<0.05),respectively.APAP reduced p-PI3k p85/PI3K and p-Akt(Ser473)/Akt by 22.3%and 80.8%(p<0.05),respectively.P-PI3k p85/PI3K and p-Akt(Ser473)/Akt in tissues decreased by 30.0%and 37.0%respectively(p<0.05).After the pretreatment of DAS,the phosphorylation of PI3K and Akt was up-regulated,although the expression of PI3K and Akt in liver tissue remained unchanged.Compared with LPS/D-GalN,APAP and CCl4 model groups,the ratio of p-PI3k p85/PI3K and p-Akt(473)/Akt in tissues increased after DAS pretreatment(p<0.05).Furthermore,compared with control group,the expression of p-Akt(Ser473)was significantly decreased after intraperitoneal inj ection of MK-2206(66 mg/kg)for two times(p<0.05).MK-2206 treatment was able to weaken the effects of DAS on the reduction of serum transaminase activity in LP S/D-GalN-induced ALI.In addition,a large number of TUNEL-positive hepatocytes were observed in LPS/D-GalN treated liver sections with or without MK-2206 treatment,and the number of TUNEL-positive hepatocytes was significantly increased when DAS was combined with MK-2206 pretreatment compared with the liver sections without MK-2206 treatment,indicating that MK2206 could suppresses the inhibitory effect of DAS on hepatocyte apoptosis induced by LPS/D-GalN.4.DAS protects against different types of ALI by inhibiting the activation of macrophage in mice.After blocking KCs with GdCl3,the increase of serum transaminase activity induced by LPS/D-GalN,APAP and CCl4 was alleviated,but H&E staining showed that there still exist pathological manifestations such as hemorrhage,swelling,inflammatory cell infiltration and cell necrosis in the liver after GdCl3 pretreatment.Meanwhile,after GdCl3 blocked KCs,DAS pretreatment still significantly reduced the increase of serum transaminase activity induced by LPS/D-GalN,APAP and CCl4(p<0.05)and significantly alleviate various pathological changes in liver tissue.On the other side,compared with the control group,the number of F4/80 positive cells in liver tissue was significantly increased after LPS/D-GalN,APAP and CCl4 injection(p<0.05),and the LPS/D-GalN model group was the most obvious and scattered,while the activated macrophages caused by APAP and CCl4 inj ection mainly revolved around the hepatic band necrosis area,DAS pretreatment could significantly inhibit the activation of F4/80 labeled macrophages,which significantly reduced the F4/80 positive staining area compared with the model group(p<0.05).After blocking KCs with GdCl3,F4/80-labeled macrophages were observed to be scattered or distributed around the necrotic area,and the combination of DAS and GdCl3 significantly reduced the F4/80 positive staining area in the sections(p<0.05).Moreover,LPS/D-GalN,APAP and CCl4 injection increased the levels of iNOS expression and NO production in liver tissues(p<0.05),and compared with LPS/D-GalN model group,DAS pretreatment reduced NO content and iNOS expression by 61.3%and 96.2%respectively(p<0.05).Compared with APAP model group,DAS pretreatment reduced NO content and iNOS expression by 48.29%and 97.42%,respectively(p<0.05).Compared with CCl4 model group,DAS pretreatment significantly reduced NO content and iNOS expression by 49.53%and 88.60%,respectively(p<0.05).DAS pretreatment significantly reduced the excessive production of TNF-?,IL-1? and MCP-1 caused by LPS/D-GalN,APAP,CCl4 injection,both in serum and liver(p<0.05).Under the combined pretreatment of DAS and GdCl3,the levels of TNF-?,IL-1?and MCP-1 in liver and serum were not significantly different from those in DAS alone pretreatment(p>0.05).Compared with the control group,LPS/D-GalN,APAP and CCl4 injection significantly increased the level of anti-inflammatory factor IL-10 in liver and serum(p<0.05),DAS pretreatment did not increase,but decreased the level of IL-10 in serum,compared with the model group(p<0.05).In addition,compared with the control group,LPS/D-GalN,APAP,CCl4 injection significantly increased the mRNA expression of TNF-?,IL-1?,IL-6 and MCP-1 in liver tissues(p<0.05),while the excessive increased mRNA expression of these pro-inflammatory factors was inhibited by DAS or GdCl3 pretreatment(p<0.05).LPS/D-GalN increased the expression of IL-10 mRNA in liver tissue,while the expression of IL-10 mRNA in liver tissue was decreased by APAP or CCl4 exposure(p<0.05).Compared with each model group,DAS pretreatment did not effectively increase IL-10 mRNA expression in liver tissue(p<0.05).Conclusion:1.Compared with DADS and DATS,DAS showed more efficient preventive and therapeutic effects on different type of ALI caused by LPS/D-GalN,APAP and CCl4.And the preventive effect of DAS on ALI was better than therapeutic effect.2.DAS pretreatment exert the hepatoprotective effects on ALI induced by LPS/D-GalN,APAP and CCl4 by inhibiting oxidative stress and inflammation in mice.3.DAS pretreatment inhibited LPS/D-GalN,APAP or CCl4 induced hepatocyte apoptosis by activating PI3K/Akt signaling pathway,and then play a protective role on ALI in mice.4.DAS pretreatment reduced the production of pro-inflammatory TNF-?,IL-1? and MCP-1 by inhibiting the activation of macrophage in liver,and showing better effects than blocking KCs with GdCl3. |
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