Preventive Effect Of Silymarin And Selenium-enriched Cordyceps Militaris On Triptolide-induced Acute Hepatotoxicity In Rats

ObjectiveThis study aimed to assess the preventive effect of silymarin and comparatively investigated that in selenium-enriched Cordyceps militaris(CS)and ordinary Cordyceps militaris(CM)against triptolide(TP)induced acute hepatotoxicity.Methods1.60 male Wistar rats were randomly divided into five experimental groups as follows(n = 12):Normal control(NC)group and triptolide(TP)group received normal saline by intragastric administration(lml/100g).Silymarin treated groups were orally administrated with silymarin(50,100,200 mg/kg,respectively).After treatment for 7 consecutive days,all groups except NC group were given TP(2.0 mg/kg)by intraperitoneal injection to establish the liver injury animal model.At the end of the experimental period(the 8th day),all the rats were fasted for 12 h.On the following day,the animals were intraperitoneally injected with 10%chloral hydrate to be anesthetized.The blood samples were collected from abdominal aortic for determining the level of serum aminotransferase and total cholesterol.After that all rats were sacrificed and collected liver tissue from the left lobe.One portion was prepared to make 1:9(w/v)homogenates with cold saline or phosphate buffered saline for measurement of antioxidant enzymatic activities,content of malonaldehyde,inflammatory cytokines and cytochrome C.One portion was fixed in 10%formalin to make H&E,immunohistochemical and TUNEL staining sections.One portion was used to extract total protein and determined expressions of phosphorylated p38,and phosphorylated JNK,cleaved caspase3 and Nrf2.2.84 male Wistar rats were randomly divided into five experimental groups as follows(n=12):Normal control(NC)group and triptolide(TP)group received normal saline by intragastric administration(1ml/100g).CS control group received 1.5g/kg CS,silymarin treated group received 200mg/kg silymarin,CS high dose and low dose treated groups received 1.5g/kg and 0.75g/kg CS respectively and CM high dose treated group received 1.5g/kg CM.After treatment for 7 consecutive days,all groups except NC and CS control group were given TP(2.0mg/kg)by intraperitoneal injection to establish the liver injury animal model.At the end of the experimental period(the 8th day),all the rats were fasted for 12h.On the following day,the animals were intraperitoneally injected with 10%chloral hydrate to be anesthetized.The blood samples were collected from abdominal aortic for determining the level of serum aminotransferase and total cholesterol.After that all rats were sacrificed and collected liver tissue from the left lobe.One portion was prepared to make 1:9(w/v)homogenates with cold saline or phosphate buffered saline for measurement of antioxidant enzymatic activities,content of malonaldehyde and cytochrome C.One portion was fixed in 10%formalin to make H&E,immunohistochemical and TUNEL staining sections.One portion was used to extract total protein and determined expressions of cleaved caspase3 and Nrf2.One portion was used to extract total RNA and determined expressions of GCLC,HO-1 and NQ01 mRNA.3.Graphite furnace atomic absorption spectrometry(GF-AAS)was used to determin selenium content and high performance liquid chromatography-electronic spray ion-mass spectrometry(HPLC-ESI-MS)was used to determin cordycepin and selenoaminoacids in CS and CM.Results:1.TP-increased serum parameters(ALT,AST,ALP,GGT)and total cholesterol.Histopathological alterations and hepatocytes apoptosis were prevented by silymarin pretreatment in a dose-dependent manner,TP-induced depletions in antioxidant enzymes(SOD,CAT,GSH,GSH-Px,GST)were also significantly recovered by silymarin.Additionally,silymarin dose-dependently exhibited inhibitory effects on MDA content,and production of pro-inflammatory cytokines such as TNF-α,IL-6,IL-10 and IL-1β.To illustrate the mechanism of silymarin,expressions of phosphorylated p38 and JNK in TNF-α induced inflammatory response and apoptotic pathway were assessed.Silymarin significantly blocked phosphorylated p38 and JNK activation and expression of pro-apoptotic proteins(cytochrome C,cleaved caspase-3 and Bax).2.CS significantly prevented the elevation in serum parameters(ALT,AST,ALP,GGT)and total cholesterol as well as pathological damage.CS pretreated groups showed activated SOD,CAT,GSH,GSH-Px,GST activities and suppressed malondialdehyde.Triptolide induced cell apoptosis in liver was also prevented by CS through up-regulating Bcl-2/Bax ratio and inhibiting expression of cytochrome C and cleaved caspase-3.CS also triggered Nrf2 translocation and promoted the genes expression of GCLC,HO-1 and-NQO1 mRNA to enhance endogenous antioxidant defense.3.Selenium,cordycepin and selenocystine content in CS was much higher than those in CM.Conclusion:Silymarin and selenium-enriched Cordyceps militaris both could dose-dependently strengthen endogenous antioxidative defense,relieve oxidative stress and inhibit lipid peroxidation to prevent triptolide induced acute liver injury.High dose selenium-enriched Cordyceps militaris showed parallel effect with high dose silymarin and was significantly superior to high dose ordinary Cordyceps militaris.The mechanism of silymarin might associated with mitigating over-expression of pro-inflammatory cytokines,inhibiting inflammatory signaling and ameliorating cell apoptosis while that in selenium-enriched Cordyceps militaris might elevating antioxidant enzymes activities via activating Nrf2 pathway.The two medicine both exerted satisfactory effects on preventing TP-induced acute hepatotoxicity.