Inhibitory Effect Of Iridoid Glucosides From Boschniakia Rossica On Liver Cancer And Its Mechanism

Objective:1.To study the inhibitory effect of Iridoid glucosides from Boschniakia Rossica(IGBR)containing serum on the growth on SMMC-7721 and HepG2 and SK-Hepl human hepatoma cell lines.2.To ’study the apoptosis-inducing effect of IGBR containing serum on SMMC-7721 human hepatoma cells and investigate the corresponding action mechanism.3.To observe the pathological changes of liver function and liver tissues in diethyl nitrosamine(DEN)induced rats with hepatic carcinoma and study the antioxidant activity,apoptosis-inducing effect and the corresponding action mechanism of IGBR.Methods:1.In vitro experiment:(1)Preparation of containing serum:thirty male Wistar rats were randomly divided into control group,5-fluorouracil(5-FU)group,low,medium and high IGBR dose groups with six rats in each group.Each below group rats were given by gavage once a day for one month consecutively:2 mL saline for control group,75 mg/kg(b.w.)5-FU for 5-FU group,125 mg/kg,250 mg/kg and 500 mg/kg(b.w.)IGBR for low,medium and high IGBR dose groups respectively.After that,all rats were executed to collect heart blood.The blood was centrifuged for 20 min at 3000 rpm and then put into 56 ℃ constant temperature water bath for 30 min to inactivate complement and other active ingredients in the serum.All samples were stocked at-20 ℃ for later use.(2)HepG2,SK-Hep1,SMMC-7721 human hepatoma cell lines were cultured in vitro.With standard chemotherapy drug 5-FU as the positive control,methyl thiazolyl tetrazolium(MTT)method was used to detect proliferation activity of heap-toma cells.Flow cytometry was applied to measure apoptosis ratio of hepatoma cells,fluorescence microscopy were used to observe morpholo-gical changes of hepatoma cells.Protein imprinting method(Western Blot)was employed to detect the expression of apoptosis related proteins.2.In vivo experiment:(1)Establishment of DEN induced rat model with Primary liver cancer:male Wistar rats were randomly divided into control group,hepatoma model group,5-FU group and IGBR group with thirty three rats in each group.On the first modeling day,control group rats were given 0.1 mL/100 g(b.w.)normal saline once by intraperitoneal injection.The other three groups were administrated 200 mg/kg(b.w.)DEN once by intraperitoneal injection and then they were given 0.05%(w/v)DEN aqueous solution as freely drinking water.From the next day,5-FU group rats were dosed 25 mg/kg(b.w.)5-FU by intraperitoneal injection three times a week.IGBR group rats were dosed 500 mg/kg(b.w.)IGBR by gavage once a day.Six rats in each group were executed at the end of 12 th week and 20 th week while twenty rats in each group were killed at the end of 28 th week.Heart blood was collected and livers were fixed in formalin.(2)The liver function of rats was tested by fully automatic biochemical analyzer.The content of GSH-S-transferase(GST)and y-glutamyl transpeptidase(γ-GT)was measured according to kit protocols.Antioxidant enzyme activity of liver tissue was detected though colorimetric method.Pathological changes of liver tissue and apoptosis of liver cell rate were acquired by HE staining and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)method respectively.The expression of apoptosis-associated proteins was presented by Western Blot method.Results:1.In vitro experiment:(1)5-FU containing serum and IGBR containing serum inhibited the proliferation of SMMC-7721,HepG2,SK-Hepl human hepatoma cells in concentration and time-dependent manner.(2)Morphologically,the typical apoptotic changes such as cell shrinkage,turning round,and nuclear retraction were observed in 5-FU containing serum group and IGBR containing serum group.The apoptosis rate of the two groups on SMMC-7721 human hepatoma cells was statistically higher than that in the control group(P<0.05)and there was no significant difference between the High dose IGBR group and 5-FU group(P>0.05).(3)Compared with control group,expression quantity of theCysteinyl aspartate specific protease-3(Caspase-3),Bcl-2 related X protein(Bax),phosphorylated c-jun amino terminal kinase(p-JNK)and phosphorylated p38MAPK kinase(p-p338)in SMMC-7721 human hepatoma cells increased after 5-FU containing serum and IGBR containing serum treatment.However,expression quantity of the B cell lymphoma/leukemia-2(Bcl-2),phosphorylated extracellular signal regulating kinase(p-ERK),phosphorylated protein kinase B(p-Akt)expression reduced.The difference in the above protein expression quantity between two groups and control group was all statistically significant(P<0.05),and there was no significant difference between the High dose IGBR group and 5-FU group(P>0.05).However,the expression quantity of ERK,JNK,p38 and Akt protein showed no obvious change.Through the use of MAPK and PI3K/Akt inhibitors,it was further confirmed that IGBR-containing serum induced apoptosis and inhibited growth of hepatoma cells by participating in the regulation of the above signal trans-duction pathways.2.In vivo experiment:(1)The rat model of Primary liver cancer was successfully induced after intragastric administration DEN for twenty eight weeks.Compared with the control group,the rats in hepatoma group exhibited dull and rough hair,low spirits,decreased liver weight.Furthermore,the livers in hepatoma group appeared dark red,hard texture,and the hepatic nodules were fused to form a lump.Compared with the hepatoma group,the above changes in 5-FU group and IGBR group were relatively mild,but there was no significant change in liver weight.(2)Compared with the control group,the content of ALT,AST,GST,y-GT and the antioxidant enzyme levels of liver tissues all increased in hepatoma group,and the difference was statistically significant(P<0.05).Compared with hepatoma group,the all above measured indicators all reduced in IGBR group and 5-FU group.The difference was statistically significant(P<0.05)and there was no obvious difference between the IGBR group and 5-FU group(P>0.05).(3)HE staining showed that pathological changes of liver tissues in hepatoma group mainly presented three stages,namely liver inflammation,cirrhosis and hepatoma.Compared with the control group,liver cells in rats of hepatoma group exhibited deep-dyed big nucleolus,cancerous cells with atypical hyperplasia.Compared with the hepatoma group,hepatic lobule structure basically existed,and there appeared visible dotted,focal necrosis and larger nuclear atypia in rats of 5-FU group and IGBR group.Moreover,the abnormal pathological changes in the three stages of induced hepatic carcinoma obviously improved.There was no obvious difference between the IGBR group and 5-FU group.(4)TUNEL results suggested that compared with control group,apoptosis increased significantly in the hepatoma group with the tan cytoplasm,cell shrinkage,nuclear pyknosis and absent nucleoli.Compared with hepatoma group,apoptosis in 5-FU group and IGBR group significantly increased.The difference was statistically significant(P<0.05),but there was no significant difference between the IGBR group and 5-FU group(P>0.05).(5)Immunohistochemical analysis results showed that cytoplasmic p53,Bcl-2,p21 and c-myc positive cells mainly distributed in the atypical hyperplasia and neoplastci foci.Compared with control group,the expression of p53,Bcl-2,p21 and c-myc in hepatoma group increased remarkably.The difference was statistically significant(P<0.05).Compared with hepatoma group,p53 and p21 expression in 5-FU group and IGBR group increased,while the Bcl-2 and c-myc expression decreased.The difference was statistically significant(P<0.05),but there was no significant difference between the IGBR group and 5-FU group(P>0.05).(6)Western Blot results suggested that compared with control group,p-ERK,p-Akt expression decreased whereas p-JNK and p-p38 expression increased in hepatoma group,which was statistically different(P<0.05).However,ERK,p38,JNK,Akt expression has no obvious change between hepatoma group and control group.Compared with hepatoma group,p-ERK,p-Akt protein expression decreased and p-JNK,p-p38 protein expression increased in 5-FU group and IGBR group.The difference was statistically significant(P<0.05),but there was no significant difference between the IGBR group and 5-FU group(P>0.05).Conclusion:1.IGBR containing serum can inhibit the proliferation of human hepatoma SMMC-7721 cells2.IGBR containing serum induced apoptosis of human hepatoma SMMC-7721 cells in a concentration manner.3.MAPK and PI3K/Akt signaling pathways participate in the regulation of apoptosis induced by IGBR containing serum in human hepatoma SMMC-7721 cells.4.IGBR could protect rat liver tissue of DEN-induced hepatic carcinoma through increasing the antioxidant activity of liver tissue.5.IGBR could induce apoptosis and suppress cell growth of DEN-induced hepatic carcinoma of rats by regulating the expression of apoptosis related proteins and proteins of hepatoma related signal transduction pathways.