| ObjectiveThe study aims to investigate the effect and uncover the potential mechanisms of total diterpenoids extracts from Isodon serra(Maxim.)Hara on hepatic mitochondria protection and antioxidation using acute alcoholic liver injury rats and concanavalin A-induced immunological liver injury mice.The model of ethanol-induced damage on L-02 cells was established and effect of total diterpenoids on damaged cells was detected as well.Methods1.Measure the growth curves of L-02 cells in different densities.Then,treated normal cells with ethanol in different concentrations for four hour and filtrated the suitable one for modeling along with MTT measurement.Besides,treated with normal cells in different concentrations for twelve hour,the IC50 value of total diterpenoids was calculated.Moreover,after damaging normal cells in 3%(v/v)ethanol for four hour,treated the damaged cells with total diterpenoids in 5.00 ?g/mL,10.00 ?g/mL,20.00 ?g/mL respectively,and measured the ALT and AST levels in culture medium.2.The SD rats were randomized into normal control group,model group,silymarin group(46.7 mg/kg/d)and total diterpenoids at high,medium,low dosages(100.0,50.0,25.0 mg/kg/d)group.Except from normal group,each group was intragastric administration with 55%(v/v)ethanol once in the morning,and orally treated with investigational samples once in the afternoon after a-four-hour fasting.After a-ten-day period,blood samples were collected from aorta abdominalis after anesthesia to separate the serum for ALT and AST detection;the livers,spleens and thymuses were excised.Using HE staining,the pathological changes were observed in hepatic tissues.The mitochondrial membrane potential(MMP),mitochondrial permeability transition pore(MPTP)opening pretreated with Ca2+,manganese superoxide dismutase(Mn-SOD)activities,8'-Hydroxy-2'-deoxyguanosine(8-OHdG)levels,activities of mitochondrial respiration chain complex ? and ? were detected separately in purified hepatic mitochondria.The MDA levels and GSH,GSH-Px activities were checked in liver homogenate.Nrf2,Bachl and HO-1 total mRNA expression were examined with RT-PCR technology.Protein expression levels ofNrf2,Bachl in nucleus and total protein expression level of HO-1 were analyzed with western blotting.3.The KM mice were apart into normal control group,model group,silymarin group(46.7 mg/kg/d)and total diterpenoids at high,medium,low dosages(100.0,50.0,25.0 mg/kg/d)group randomly.All mice pre-treated with investigational samples once in oral dose for seven days.Twelve hours after last administration of investigational samples,all mice were i.v.concanavalin A(Con A)at a 25 mg/kg dosage except normal control.Four hours after injection,serum was separated from blood samples to determine the levels of ALT and AST;the livers,spleens and thymuses were also excised.Using HE staining to observe pathological changes in hepatic tissues.The mitochondrial membrane potential(MMP),manganese superoxide dismutase(Mn-SOD)activities,8'-Hydroxy-2'-deoxyguanosine(8-OHdG)levels were detected using purified hepatic mitochondria.Prepare liver homogenate for examining levels of MDA,GSH and GSH-Px activities.4.In order to find out the potential targets of hepatoprotective effect treated with total diterpenoids,the Pearson correlation coefficient analysis was use to evaluate relationship of indexes between hepatic mitochondria and 'Nrf2/Bachl-ARE-antioxidant enzymes”pathway in alcoholic liver injury rats.Results1.Treated with ethanol in a range between 2%and 15%(v/v)could lead normal L-02 cells to a downfall of cell viability rate(P<0.01).Besides,a concentration range between 7.81 and 62.50?g/mL of total diterpenoids had no significant effect on inhibiting cells'multiplication,the IC50 value of total diterpenoids is 138.10?g/mL.L-02 cells were damaged with 3%ethanol,the increase of ALT and AST levels in culture medium were observed.Using 5.00,10.00 and 20.00?g/mL of total diterpenoids to treat damaged cells,and a non-significant decrease of ALT and AST levels was found compared to model group.2.With intragastric administration of 55%alcohol for ten days,the acute alcoholic liver injury was induced.Compared with normal control,the ALT and AST levels in serum of model group were at noticed higher levels(P<0.01 or P<0.05),the edema of hepatic cells was observed through pathology.The MMP was significantly dropped(P<0.05)as well as the increase of MPTP opening(P<0.05).The activities of Mn-SOD in mitochondrial matrix were decreased obviously(P<0.01),with the high levels of 8-OHdG(P<0.01).The activities of complex ? and complex ? were remarkably dropped too(P<0.01).After ten days' treatment,the total diterpenoids had remarkable effects on lowered the levels of ALT and AST(P<0.01 or P<0.05),improved microstructure of liver cells,recovered the MMP(P<0.05),inhibited MPTP opening(P<0.05),increased Mn-SOD,complex ? and complex ? activities,decreased 8-OHdG levels.The correlation analysis showed that the activities of Mn-SOD could become a sensitive parameter to reflect the liver function status and detection of AST levels in serum was beneficial to understand the status and damage of liver mitochondria in acute alcoholic liver damage rats.3.With a tail intravenous injection of Con A at a 25 mg/kg dosage,immunological liver injury was successfully reestablished.Compared with normal control,serious edema of hepatic cells considerable observed in model group,with the ALT and AST activities in serum were at a significantly higher level(P<0.01 or P<0.05).The MMP was noticed decreased(P<0.05),as well as the Mn-SOD activities in matrix(P<0.01).In contrast,the 8-OHdG level within mitochondrial matrix was significantly increased(P<0.01).Pre-treated with total diterpenoids,the edema of hepatic cells was prevented,along with the remarkable decrease of ALT levels,AST levels(P<0.01 or P<0.05),the slightly drop of 8-OHdG levels,and a bit up-regulation of MMP and Mn-SOD activities.Interestingly,the correlation analysis also indicated that the activities of Mn-SOD could become a sensitive parameter to reflect the liver function status and detection of AST levels in serum was beneficial to understand the status and damage of liver mitochondria in this particular model too.4.As for the rats,the MDA levels were obviously increased(P<0.01)in model group,while the GSH and GSH-Px activities were decreased(P<0.01 or P<0.05).The mRNA expression of Nrf2,Bachl and HO-1 were all up-regulated remarkably(P<0.01 or P<0.05).Moreover,the protein expression of total HO-1 were significantly increased(P<0.05).With total diterpenoids' treatment,the MDA levels were lower significantly in all dosage groups(P<0.01),with the recovery of GSH activities in high dosage(P<0.01)and GSH-Px activities in all dosage groups(P<0.01 or P<0.05).Compared with model group,both the mRNA and total protein expression of HO-1 expression in high and medium dosage grops were increased and remarkable margins were found between model group and these two group separately(P<0.01 or P<0.05).5.In immunological liver injury mice,compared to normal control,the MDA levels were obviously increased(P<0.01)in liver homogenate,and the GSH and GSH-Px activities were decreased(P<0.01)in model group.With prophylactic administrations,the total diterpenoids had positive effect on lowering MDA level in all dosage grops(P<0.01),restoring GSH activities in high,medium dosage groups(P<0.01 or P<0.05)and GSH-Px activities in high,low dosage groups(P<0.05).6.Pearson correlation analysis indicated that,when the liver injury occurred,the Nrf2 mRNA and complex I acitivities had significant positive correlation(r=0.997,P<0.05).After treatment of total diterpenoids,the HO-1 mRNA expression level and MMP had highly negative correlation(r=-0.878,P<0.01),the total HO-1 protein expression level and MPTP ??540 value had highly negative correlation(r=-0.806,P<0.01).Conclusions1.Total diterpenoids extracts from Isodon serra(Maxim.)Hara has a considerable hepatoprotective effect which can resist the leakage of ALT and AST from ethanol-damaged L-02 cells,decrease the aminotransferase activities in serum of different hepatic injury animal models.and ease edema of liver cells.2.The hepatoprotection of total diterpenoids is link to hepatic mitochondria.This effect might related to protection of mitochondrial membrane integrity,improvement of oxidation resistance,repair the mtDNA and recovery of OXPHOS system.3.The total diterpenoids also showed its hepatoprotective effect by adjusting endogenous antioxidative system This effect is relying on LPO inhibition,improvement of oxidation resistance and antioxidant pathway activation.Particularly on lower MDA levels,increase acitivities of antioxidant enzymes,up-regulating HO-1 mRNA and total protein expression.4.Along with the total diterpenoids' treatment,there is a certern correlation between HO-1 mRNA and total protein expression up-regulation and hepatic mitochondria protection. |
PDF Full Text Request