The Hepatotoxic Response Of Triptolide To Tumor-bearing Mice Was Evaluated Based On The Theory Of “definitely Innocent”

ObjectivesAccording to 'You-Gu-Wu-Yun',the theory of Traditional Chinese Medicine(TCM),CT26 cells and CT26 tumor-bearing mice were adopted as experimental meterials for observing antitumor efficacy of Triptolide.Furthermore,hepatotoxicity of triptolide was compared on normal mice and tumor-bearing mice.The presented studies could provide more experimental data for clinical application and deep develpment of Tripterygium wilfordii.Material and MethodsIn vitro experiment:CT26 cells was incubated with Triptolide(50 nM)for a period of 12 hours or 24 hours.Cell viability was measured by MTS assay,and the proliferation inhibition rate was calculated;Then Flow cytometry was used to detect the apoptosis of CT26 cells induced by Triptolide(25nM and 50nM).In vivo experiment:In this study,male BALB/C mice were used as the research object.Two million CT26 cells were injected into the subcutaneous part of the right forelimb of mice to establish the tumor-bearing mice model.The mice were randomly divided into sham operated group,tumor model group,triptolide treatment group(0.4mg/kg)and there are 10 rats in each group.After intraperitoneal injection for 21 days,all mice were sacrificed to observe the effect of triptolide on CT26 colon cancer after.The observation indexes included the weight of mice,the growth of the tumor,the behavioral changes of mice and the length and width of tumor were measured to calculate the tumor volume.The anti-tumor effect of triptolide was observed by mice weight,tumor monitoring,tumor tissue frozen sections stained with TUNEL,and immunohistochemical detection of tumor tissue sections in the expression of Caspase-3.Then the normal mice and tumor-bearing mice were exposed to low dose of triptolide(O.lmg/kg),medium dose of triptolide(0.2mg/kg)and high dose of triptolide(0.4mg/kg)under the same conditions to observe the hepatotoxicity of different doses of triptolide on normal physiological and pathological conditions of tumor-bearing mice after intraperitoneal injection for 25 days by the general observation,serum biochemical detection(ALT,AST),liver homogenate assay(MDA,SOD,GSH-PX),histological observation,the expression of iNOS and Nrf 2 protein in liver,etc.Results(1)Triptolide could significantly inhibit the growth and proliferation of CT26 and induce its apoptosis.(2)0.4mg/kg triptolide intraperitoneal injection could significantly inhibit the tumor growth in tumor-bearing mice,the mice behavior and body weight had significant change and significant apoptosis appeared in tumor tissue.(3)For alanine aminotransferase(ALT),with the increase of triptolide dose,the content of ALT of the normal group and tumor group were increased.In high dose,the normal group's ALT was higher than that of the tumor group.For aspartate aminotransferase(AST),with the increase of triptolide dose,the change of AST of the normal group and tumor group was not obvious,the content of AST of the tumor group was significantly higher than that of the normal group.(4)The detection results of liver homogenate:For the malondialdehyde(MDA),with the increase of triptolide dose,the content of malondialdehyde were increased in the normal group and tumor group.In low dose and medium dose,the normal group's malondialdehyde was higher than that of the tumor group.For the superoxide dismutase(SOD),with the increase of triptolide dose,the superoxide dismutase activity in normal group and tumor group were reduced,but there was no significant difference between normal group and tumor group.For the glutathione peroxidase(GSH-PX),with the increase of triptolide dose,the glutathione peroxidase activity in normal group and tumor group were reduced.In low dose and high dose,the tumor groups' glutathione peroxidase activity is higher than that of the normal group.(5)HE staining of liver tissue showed that with the increase of triptolide dose,the pathological changes of liver tissue were more obvious and the damage degree of liver tissue in normal group was higher than that in tumor group.(6)The results of iNOS immunohistochemical staining showed that with the increase of triptolide dose,the expression of iNOS in liver tissues were increased.In low dose and medium dose,there is no significant difference between the two groups.But in high dose,the expression of iNOS in the normal group were higher than that of the tumor group.(7)The results of Nrf 2 immunohistochemical staining and the western blot results show that with the increase of triptolide dose,the expression of Nrf 2 protein in liver tissues were increased.In low dose,there is no significant difference between the two groups.But in the middle dose and high dose,the expression of Nrf 2 in the normal group were higher than that of the tumor group.Conclusion1.Triptolide has significant anti-tumor effect,and its mechanism is related to the induction of apoptosis;2.The hepatotoxicity induced by triptolide in pathological state was lower than that in normal physiological state;3.The mechanism of hepatotoxicity induced by triptolide is related to the induction of oxidative stress;4.High dose of triptolide can produce significant hepatotoxicity both in the normal group and tumor group,suggesting that if the dosage of triptolide is beyond a certain range,whether the body is in physiological or pathological state,the degree of hepatotoxicity is very serious.