| BackgroundLiver injury is a common clinical disease.Although we have made substantial progress in understanding the mechanism of liver injury in the past few decades,there is no specific drugs that can deal with liver disease effectively apart from keeping away from primarily pathogenic factors.In recent years,some extracts from herbal medicine show liver protective effects,but the mechanisms and targets of these extracts remain unclear.The metabolism of drugs itself also increase the liver burden,leading to worsen damage sometimes.A largy body of literatures have demonstrated aryl hydrocarbon receptor(AHR)plays a vital role in mediating toxicity and control of disease tolerance.Ketoconazole is an AHR agonist with dual effects——liver protection and liver toxicity;puerarin also has the dual effects toward liver injury,but thereare few reports on the effect of AHR.Lipopolysaccharide(LPS)and carbon tetrachloride(CCl4)are classical hepatotoxicants for studing the acute liver damage.In the previous preliminary experiments,we found pretreatment with puerarin aggravated acute liver injury induced by lipopolysaccharide,but no effects on carbon tetrachloride-induced acute liver injury;while pretreatment with ketoconazole protected against acute liver injury induced by LPS or CCl4.Therefore,we explored the effects of puerarin and ketoconazole on acute liver injury,and the effect of puerarin on AHR.Objective1.To study the effects of puerarin and ketoconazole pretreatment on acute liver injury induced by LPS.2.To study the effects of puerarin on liver AHR.3.To study the protective effect of ketoconazole pretreated on CCl4-induced acute liver injury and its mechanism.Method1.Effects of Puerarin and Ketoconazole on Lipopolysaccharide-induced liver injury in Rats.Sixty SD adult rats were randomly divided into five groups:(Ⅰ)control group;(Ⅱ)model group:rats were injected 5 mg/kg LPS via tail vein;(Ⅲ)puerarin+model group:rats were administered 200 mg/kg PUE daily for three consecutive days,and injected with 5 mg/kg LPS through tail vein 8 hours after the last administration;(Ⅳ)ketoconazole+model group:rats were administered 100 mg/kg KCZ daily for three consecutive days,and injected with 5 mg/kg LPS through tail vein 8 hours after the last administration;(V)Puerarin group:rats were given PUE of 200 mg/kg daily for three days.12 hours after LPS injection,rats were anaesthetized with 0.3%pentobarbital(1.6mL/kg).Blood was collected from abdominal aorta and some liver tissues were cut to be cryopreservation or fixed with 10%neutral formalin.The serum was separated,and the enzymes related to liver injury as AST,ALT and LDH were detected immediately by an automatic biochemical analyzer;the serum nitrite content was detected by a nitric oxide detection kit;The concentration of reactive oxygen species(ROS)was detected by enzyme-linked immunosorbent assay(ELISA).Histological examination were stained with HE and examined under a light microscope;the expression of inflammatory factors TNF α and IL-6 in liver tissue was detected by ELISA;2.Effects of Puerarin on liver tissue AHR and related proteins or genesSamples were from the control group and puerarin group in the first experiment.AHR protein in liver was tested with immunohistochemistry,genes expression of AHR,CYP1A1,UGT1A1,TIPARP in the liver tissue were detected by real-time quantitative PCR(qPCR).Western blot(WB)was used to detect the expression of ERα in liver tissue.Serum estradiol concentrations were testd by radioimmunoassay.3.Effect of Ketoconazole on Carbon Tetrachloride-induced liver injury in rats.Forty-eight SD rats were randomly divided into four groups;(Ⅰ)control;(Ⅱ)model:rats were injected with a single intraperitoneal(i.p.)dose of CC14-paraffin oil mixture(50%CCl4,v/v,2ml/kg body weight);(Ⅲ)ketoconazole+model:rats were treated with ketoconazole(100mg/kg body weight,p.o.)once a day for 3 days and injected a dose of CCl4-paraffin oil mixture(50%CCl4,v/v,2ml/kg body weight)4 h after the last gavage;and(IV)ketoconazole group:rats were administered with ketoconazole(100mg/kg body weight,p.0.)once a day for 3 days.24 hours after the injection of CCl4-liquid paraffin mixture,the rat abdominal cavity was opened under 0.3%pentobarbital(1.6mL/kg)anesthesia,2 mL of blood was collected through the abdominal aorta;liver tissues were cut to be cryopreservation or fixed with 10%neutral formalin.Serum was separated,and the activity of transaminase AST and ALT were detected immediately by automatic biochemical analyzer;pathological changes were detected by HE staining;GSH/GSSG kit was used to detect the contentsof GSH,GSSG and GSH/GSSG ratio;concentrations of TNF-α,IL-1β,and IL-6,were determined using enzyme-linked immunosorbent assay(ELISA)kits.The gene expressions of AHR,AHRR,CYP1A1,GSR,GSTα and NQ01 were detected by qPCR.The total protein of HO-1,CYP2E1 and Nrf2,NF-κB/p65 in the nucleus were detected by WB.Result1.Puerarin aggravates liver injury and ketoconazole alleviates liver injury.(1)Mortality:Within 12 hours of model establishment,one rat died in the model group,and two rats died in the PUE+MOD group,but no rats died in the other groups;the difference of mortality among the groups was not statistically significant.(2)Pathological result:Puerarin aggravated liver damage and ketoconazole relieved liver damage.After LPS stimulation,the liver tissue showed flaky,bridge-like necrosis,nucleus pyknosis and even dissolution,and inflammatory cell infiltration in the portal area.In the PUE+MOD group,the liver damage was more serious.In the KCZ+MOD group,the liver tissue also had flaky necrosis and inflammatory cell infiltration,but the damage degree was lighter than the model group.(3)Enzymes:The activities of AST,ALT and LDH in the model group were significantly higher than those in the control group(P<0.01).Compared with the model group,the serum enzyme level from the PUE+MOD group was higher than that of the model group(P<0.05),while the activity of AST,ALT and LDH in the KCZ+MOD group was lower than that in the model group(P<0.05 or P<0.01);compared with the normal control group,puerarin alone did not cause an increase in liver enzyme-related enzymology.(4)Inflammatory-related factors:The nitrite concentration test showed that the model group was significantly higher than the normal control group(P<0.01),and the PUE+MOD group showed higher concentration of nitrite than the model group(P<0.05),while the nitrite concentration in the KCZ+MOD group was lower than that in the model group(P<0.05).Serum ROS levels in the model group were significantly higher than those in the normal control group(P<0.01).Compared with the model group,serum ROS levels in the PUE+MOD group were higher than those in the model group(P<0.05),The level of ROS in the KCZ+MOD group was lower than that in the model group(P<0.05).(5)Inflammatory factors:The levels of TNFa and IL-6 in the liver tissue of the model group were significantly higher than those in the normal control group(P<0.01).Compared with the model group,the TNF a and IL-6 in the liver tissue from the PUE+MOD group were higher than that of the model group(P<0.05);the levels of TNF a and IL-6 in the KCZ+MOD group were significantly lower than those in the model group(P<0.05).2.The effect of puerarin on AHR and its related proteins and genes expression.After 3 days of puerarin pretreatment,immunohistochemistry showed that the expression of AHR in liver tissue sections were also significantly inhibited after puerarin pretrentment for 3 days.The result of immunological score analysis showed that the puerarin group was significantly lower than the control group(P<0.05).The relative expression of AHR mRNA in the liver was significantly inhibited compared with the control group(P<0.05).In contrast,puerarin significantly up-regulated the expression of TiPARP mRNA in liver tissue(P<0.05).The content of estradiol in puerarin group has no significant difference over the control group,but puerarin significantly up-regulated the relative expression of ERα in liver tissue(P<0.05).3.Ketoconazole pretreatment protects carbon tetrachloride-induced liver damage(1)Pathological section:After CCl4 stimulation,large areas of liver tissue damage could be seen at low magnification.Severe destruction of liver structure,vacuolization of balloon-like vacuoles,and significant hepatocyte expansion could be found at high magnification;for rats that were pretreated with ketoconazole,the characters of liver tissue damage were obviously milder than the model group.(2)Transaminase:The levels of AST and ALT in the model group were significantly increased compared to the control group(P<0.01),the activity of transaminase in the KCZ+MOD group was lower than that in the model group(P<0.01);the ALT level in the ketoconazole group was slightly elevated,but there was no significant difference between the two groups(P>0.05).Ketoconazole had no effect on AST in rats.(3)Inflammatory cytokines and NF-κB/p65:The levels of inflammatory cytokines in the liver tissue of the model group were significantly higher than that in the control group(p<0.01),while the release of inflammatory cytokines in the KCZ+MOD group were lower than that in the model group(p<0.05).The relative expression of NF-κB/p65 in the nucleus of the model group was significantly higher than that of the normal control group(p<0.05),and the relative expression of NF-κB/p65 in the KCZ+MOD rats was lower thanthat of the model group(p<0.05).(4)Antioxidant:The content of GSSG in the model group was significantly higher than that in the normal control group(p<0.01),and the GSSG content in the KCZ+MOD group was significantly lower than that in the model group(p<0.05).The GSH/GSSG ratio in the model group was significantly lower than that in the control group(p<0.01),and the GSH/GSSG ratio in the KCZ+MOD group was significantly higher than that in the model group(p<0.05).The expression of GSR mRNA in the model group was not different compared to the control group.The expression of GSR mRNA in the KCZ+MOD group was higher than that in the model group(p<0.05),and the expression of GSR mRNA in the ketoconazole group was higher than that in the control group(p<0.05).The expression of NQO1 mRNA in the model group was not different over the control group.The expression of NQ01 mRNA in the KCZ+MOD group was significantly higher than that in the model group(p<0.05).The expression of NQ01 mRNA in the ketoconazole group was significantly higher than that in the control group(p<0.01).The expression of GST a mRNA in the model group was significantly lower than that in the control group(p<0.01).The expression of GST α mRNA in the KCZ+MOD group was significantly higher than that in the model group(p<0.01).The expression of GST a mRNA in the ketoconazole group showed no difference compared with the control group.The expression of HO-1 protein in the model group was not different over the control group.The expression of HO-1 protein in the KCZ+MOD group was significantly higher than that in the model group(p<0.05),and the expression of HO-1 protein in the ketoconazole group showed no difference over the control group.The expression of Nrf2 in the model group was not different from that in the control group.But the expression of Nrf2 in the KCZ+MOD group was significantly higher than that in the model group(p<0.05).The expression of Nrf2 in the ketoconazole group was significantly higher than that in the control group(p<0.05).(5)AHR-related genes:The expression of AHR mRNA in the liver tissue of the model group was significantly lower than that of the normal control group(p<0.05),and the expression of AHR mRNA in the KCZ+MOD group was significantly higher than that of the model group(p<0.05).The expression of AHR mRNA in the ketoconazole group was significantly higher than that in the control group(p<0.01).The expression of AHRR mRNA in the model group was not different from that in the control group.The expression of AHRR mRNA in the KCZ+MOD group was significantly higher than that in the model group(p<0.05).The expression of AHRR mRNA in the ketoconazole group was significantly higher than that in the control group(p<0.05).The expression of CYP1A1 mRNA in the model group was not different from that in the control group.The expression of CYP1A1 mRNA in the KCZ+MOD group was significantly higher than that in the model group(p<0.01).The expression of CYP1A1 mRNA in the ketoconazole group was significantly higher than that in the control group(p<0.01).(6)CYP2E1:Compared with the control group,the expression of CYP2E1 protein in the model group was inhibited obviously(p<0.05),while the ketoconazole+model group showed higher expression of CYP2E1 protein than the model group(p<0.05).The expression of CYP2E1 protein in ketoconazole group was not different from that in control group.Conclusion1.Puerarin increased liver damage,promoted the release of ROS and NO,and increased the concentration of TNF a and IL-6 in the liver for the acute liver injury induced by LPS;which indicating that puerarin has a potential risk of aggravating liver injury.Ketoconazole attenuates liver damage;indicating that mild activation of AHR enhances liver tolerance to LPS challenge.2.Puerarin pretreatment inhibited the expression of AHR gene and protein in the liver tissue,while up-regulated the expression of TIPARP mRNA,and promoted the expression of ERα in liver tissue.Puerarin may be an inhibitor of liver AHR and an ERα agonist.Puerarin pretreatment increased the liver sensitivity to LPS may involve AHR inhibition.3.Ketoconazole preconditioning attenuates the hepatotoxicity of CCl4.This protective mechanism may be in that ketoconazole activated the AHR/Nrf2 signaling pathway in the liver,thereby enhancing the anti-inflammatory and anti-oxidative capacity in the liver;which indicate appropriate activation of AHR could enhance liver tolerance to CCl4.4.Mild activation of AHR enhenced the liver tolerance to hepatotoxic agent.Inhibition of AHR increases liver sensitivity to LPS. |
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