| Objective:Human hepatocytes(L-02)oxidative damage model were prepared by using glucose oxidase(GO).Testing the liver protective function and studying the effect on the oxidative damage model development process(Oh、4h、22h after model was prepared)of traditional Tibetan medicine-Jia Ga Song Tong.Clarifying the liver protective function mechanism of Jia Ga Song Tong by using three chemical interventions(t-BHQ、ATRA、Genistein)of transcription factors-Nrf2 and Bachl.Methods:1.Jia Ga Song Tang Drug-containing Serum were prepared by feeding with different Jia Ga Song Tong dosages(0.66g crude drugs/kg body weight、0.33g crude drugs/kg body weight)for SD rats.The male and female rats were half.Normal human hepatocytes(L-02)were fed respectively with different volumes(20 percent、10 percent、5 percent)of Jia Ga Song Tong containing medicineserum and different concentrations(60μmol/L、30μmol/L、15μmol/L)of curcumin for different hours(6h、12h、24h).The vitality of L02 cells was measured by MTT assay.2.Human hepatocytes(L-02)oxidative damage model was prepared successfully by feeding with 100U/L GO and acting 2 hours after normal human hepatocytes(L-02)were fed with 20 percent of Jia Ga Song Tong containing medicineserum for 12 hours.The vitality of L02 cells was measured by MTT assay;the AST,ALT of cell culture supernatant were tested by Reitman-Frankel;the MDA content in the cell culture supernatant were detected by Thibabituric Acid method;the enzyme activity of GSH-Px in the cell culture supernatant were measured by spectrophotometer.3.Setting three representative points in time(Ohour、4hour、22hour)of the damage development phase in the model induced by GO,and using transcription factors chemical agent interventions(t-BHQ、ATRA、Genistein),to studying the function mechanism of Jia Ga Song Tang:3.1 Using GO at the dosage of 100U/L to injure the L-02 cells after feeding with Jia Ga Song Tang Drug-containing Serum for 12 hours.Then taking samples after giving normal culture medium for different times(Ohour、4hour、22hour).3.2 Using GO at the dosage of 100U/L to injure the L-02 cells after feeding with Jia Ga Song Tang Drug-containing Serum for 12 hours based on feesing preferentially with three transcription factors chemical agent interventions(t-BHQ、ATRA、Genistein)for 12 hours.Then taking samples after giving normal culture medium for different times(Ohour、4hour、22hour).Tseting the samples at Ohour、4hour、22hour:the vitality of L02 cells、the activity of ALT and AST、the contents of MDA and the enzyme activity of GSH-Px.Preparing the slides of cells and using immunofluorescence to survey the position change of Nrf2 and Bachl in cells.Scraping cells and using Western blot to test the protein expression of Nrf2 and Bachl in nucleus.Using linear correlation analysis to analyse GSH-Px enzyme activity、MDA contents、Nrf2 proteins、Bach proteins in the damage development phase in the model of Jia Ga Song Tang.Results:1.Jia Ga Song Tang Drug-containing Serum were prepared by lavaging rats with 0.66g crude drugs/kg body weight.The cell activity of normal human hepatocytes(L-02)which were fed with 20 percent Jia Ga Song Tang Drug-containing Serum and 30μ mol/L curcumin 12 hours were significantly increased(P<0.01).2.Compared with blank serum control groups,L-02 in GO group,cell viability and GSH-Px activity both decreased significantly(P<0.01);AST、ALT activity and MDA contents both increased remarkably(P<0.01).Compared with blank serum model groups,Jia Ga Song Tang Drug-containing Serum were respectively able to increase significantly the cell viability and GSH-Px activity(P<0.01);and can decreased remarkably the AST、ALT activity and MDA content(P<0.01).3.Feeding primarily without transcription factors chemical agent interventions of Nrf2 and Bachl,to study the law of function in the three representative points in time(Ohour、4hour、22hour)of the damage development phase in the L-02 oxidative stress injury model induced by GO:3.1 At the three representative points in time of the damage development phase in the model(Ohour、4hour、22hour),compared with solvent control groups,solvent model groups not only can decrease significantly cell viability and GSH-Px activity(P<0.01),4hour was significantly lower than Ohour and 22hour(P<0.01);but also increase remarkably AST、ALT activity and MDA content(P<0.01),4hour was significantly higher than Ohour and 22hour(P<0.01).The results of immunofluorescence and Western blot showed that:at Ohour、4hour、22hour,the Nrf2 proteins in nucleus in 4hour is lowest;the Bachl protein expression quantity is decreasing with time.3.2 Compared with solvent model groups,Jia Ga Song Tang Drug-containing Serum and curcumin were able to increase significantly the cell viability and GSH-Px activity(P<0.01),4hour is lower than Ohour and 22hour;and can decreased remarkably the AST、ALT activity and MDA content(P<0.01),4hour is higher than Ohour and 22hour.The results of immunofluorescence and Western blot showed that the Nrf2 protein expression were significantly increased at Ohour、4hour、22hour by Jia Ga Song Tang(P<0.01),but the expression quantity in 4hour is lower than Ohour and 22hour;at the same time,the Bachl protein expression were significantly decreased in Ohour、4hour、22hour of Jia Ga Song Tang(P<0.01),but the Bachl protein expression quantity is decreasing with time.3.3 By linear correlation analysis,we found that there was positive correlation between Nrf2 expressed in nucleus and GSH-Px activity(r=0.999,P =0.024).4.Feeding primarily with transcription factors chemical agent interventions of Nrf2 and Bachl,to study the law of function in the three representative points in time(Ohour、4hour、22hour)of the damage development phase in the L-02 oxidative stress injury model induced by GO:4.1 Under the t-BHQ intervention,promote Nrf2 into nucleus:Compared with blank serum model groups,"t-BHQ+blank serum model”group can increase significantly the cell viability and GSH-Px activity(P<0.01),4hour is lower than Ohour and 22hour;and can decreased remarkably the AST、ALT activity and MDA content(P<0.01),4hour is higher than Ohour and 22hour.The results of immunofluorescence and Western blot showed that:compared with blank serum model groups,"t-BHQ+blank serum model" may increase significantly the Nrf2 protein expression quantity at 4h and 22h(P<0.01);and can increase significantly the Bachl protein expression quantity at three time points(P<0.01).Compared with"t-BHQ+blank serum model "group,"t-BHQ+Jia Ga Song Tang"group was able to increase significantly the cell viability and GSH-Px activity(P<0.01)and can decreased remarkably the AST、ALT activity and MDA content(P<0.01)at three points-in-time(Ohour、4hour、22hour).The results of immunofluorescence and Western blot showed that:compared with"t-BHQ+blank serum model" group,the Nrf2 protein expression were significantly increased at Ohour、4hour、22hour by "t-BHQ+Jia Ga Song Tang"(P<0.01),but the expression quantity in 4hour is lower than Ohour and 22hour;at the same time,the Bachl protein expression were significantly decreased in Ohour、4hour、22hour by "t-BHQ+Jia Ga Song Tang",(P<0.01),but the expression quantity in 22hour is higher than Ohour and 4hour.4.2 Under the ATRA intervention,restrain Nrf2 into nucleus:Compared with blank serum model groups,"ATRA+blank serum model"group can decrease significantly the cell viability at 22hour(P<0.01);have no effect on the AST、ALT activity at three time points(P>0,05);can increase MDA contents at Ohour and 22 hour(P<0.01);can increase GSH-Px activitof at three time points(P>0.05).The results of immunofluorescence and Western blot showed that:compared with blank serum model groups,the Nrf2 protein expression quantity were decreased significantly at Ohour and were increased markedly at 4hour and 22hourby"ATRA+blank serum model"(P<0.01);at the same time,the Bachl protein expression quantity were increased markedly at three time points by“ATRA+blank serum model"(P<0.01).Compared with "ATRA+blank serum model" group,"ATRA+Jia Ga Song Tang"group was able to increase significantly the cell viability and GSH-Px activity(P<0.01)and can decreased remarkably the AST、ALT activity and MDA content(P<0.01)at three points-in-time(Ohour、4hour、22hour).The results of immunofluorescence and Western blot showed that:compared with“ATRA+blank serum model”group,the Nrf2 protein expression were significantly increased at Ohour、4hour、22hour by "ATRA+Jia Ga Song Tang"(P<0.01),but the expression quantity in 4hour is highest;at the same time,the Bachl protein expression were significantly decreased in Ohour、4hour、22hour by "ATRA+Jia Ga Song Tang"(P<0.01),but the Bachl protein expression quantity is increasing with time.4.3 Under the Genistein intervention,restrain Bachl shift out nucleus:Compared with blank serum model groups,"Genistein+blank serum model "group can decrease significantly the cell viability at Ohour and 22hour(P<0.01);can decrease significantly the ALT activity at three time points(P<0.01)and increase the AST activity(P>0.05);can increase MDA contents at Ohour and 22 hour(P<0.01);can increase GSH-Px activitof at three time points(P>0.05).The results of immunofluorescence and Western blot showed that:compared with blank serum model groups,"Genistein+blank serum model" may increase significantly the Nrf2 protein expression quantity at three time points(P<0.01),and it is highest at 4hour;at the same time,the Bachl protein expression quantity were increased markedly at three time points by "Genistein+blank serum model"(P<0.01),and it increased with time.Compared with "Genistein+blank serummodel" group,"Genistein+Jia Ga Song Tang" group was able to increase significantly the cell viability and GSH-Px activity(P<0.01)and can decreased remarkably the AST、ALT activity and MDA content(P<0.01)at three points-in-time(Ohour、4hour、22hour).The results of immunofluorescence and Western blot showed that:compared with“Genistein+blank serum model”group,the Nrf2 protein expression were significantly decreased at Ohour、4hour、22hour by“Genistein +Jia Ga Song Tang"(P<0.01),but the expression quantity in 4hour is highest;at the same time,the Bachl protein expression were significantly decreased in Ohour、4hour、22hour by "Genistein+Jia Ga Song Tang"(P<0.01),but the expression quantity in 4hour is highest.Conclusion:1.Jia Ga Song Tang is good for normal human hepatocytes(L-02).20%Jia Ga Song Tang can obviously improve the oxidative stress injury,to come true the effect of antioxidants protective in liver.2.Human hepatocytes(L-02)were injured by GO for 2 hours,the rule of damage changing with time is that:from Ohour to 4hour after injury,damage degree increased;from 4hour to 22hour after injury,extent of damage gradually weakened.Damage degree is most serious at 4hour after injury.3.Jia Ga Song Tang can promote Nrf2 into nucleus and make Bachl shift out nucleus at Ohour、4hour、22hour.So it can increase the activity of GSH-Px.The protective effect of it at 4hour is weaker than ohour and 22hour.4.Our studies proved that t-BHQ promotes Nrf2 into nucleus、ATRA restrains Nrf2 into nucleus、Genistein prevents Bachl shift out nucleus.Our studies discovered that t-BHQ can promotes Bachl in nucleus at the the three representative points in time(Ohour、4hour、22hour)of the damage development phase in the L-02 oxidative stress injury model induced by GO;discovered that ATRA can increase Nrf2 protein expression in nucleus at 4hour and 22hour,and at the same time increase Bachl protein expression in nucleus;discovered that Genistein can both increase the Nrf2 and Bachl protein expression and decrease the GSH-Px activity.5.Using intervention agents,we fund that Jia Ga Song Tang cooperate with t-BHQ in increasing the Nrf2 protein expression.Jia Ga Song Tang can restrain ATRA to increase the Nrf2 protein expression.Jia Ga Song Tang can restrain Genistein to promote Bachl shift out nucleus and decrease the Nrf2 protein expression which were increased by Genistein,to balance the Nrf2 and Bachl in nucleus.In conclusion,Jia Ga Song Tang can regulate Nrf2 and Bachl protein expression in nucleus,to balance the Nrf2 proteins and Bachl proteins in nucleus,and then to regulate and control GSH-Px activity,so to come true the antioxidant protective of liver. |
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