Study On The Toxicity Of Ciji Hua’ai Baosheng Ⅱ Formula And Its Effects On Cell Apoptosis Of Tumor After Receiving Chemotherapy

Part Ⅰ Study on tocixity1 Study on the absorbed components of Ciji Hua’ai Baosheng Ⅱ FormulaObject:Ciji Hua’ai Baosheng Ⅱ formula(CHB-Ⅱ-F)is composed of eight medicinals:Fulin,Dangsheng,Muli,Suanzaoren,Chenpi,(dry-fried)Maiya and Danshen,which has been applied in clinic for many years,and its curative effects are good,but the material basis of effects is unknown.The present study focused on the absorbed bioactive components of CHB-Ⅱ-F to clarify its material basis.Method:(1)Eight medicinals of CHB-Ⅱ-F are soaked and boiled,concentrated and made into freeze-dried powder.We accurately weigh the freeze-dried powder.Ultra-high performance liquid chromatography(UHPLC)and a high resolution electrospray ionization mass(HR-ESⅠ-MS)were used to analyze the bioactive components in the water solution of CHB-Ⅱ-F.(2)A total of 20 specific pathogen-free(SPF)Kunming(KM)mice were subcutaneously injected with suspension of 2×107/mL H22 hepatocellular carcinoma cells(HCC)into the right anterior armpit.All mice were received intraperitoneal injection with 5-fluorine pyrimidine(5-FU)(200 mg/kg)once to establish the chemotherapeutic model of HCC.Mice were randomly divided into two groups.One was treated with physiologic saline(untreated),one was treated with CHB-Ⅱ-F(32.5 g/kg).Mice freely access to food and water.Two groups were treated by intragastric administration once a day for 7 days.The 7th day after treatment 1 hour,peripheral blood was collected through removing mice’s eyeballs.Result:(1)CHB-Ⅱ-F was isolated with UHPLC system and its chromatographic fingerprinting established.Comparing the retention time,UV and MS slpectra with reference sample,the following 10 major bioactive components were identified:3,4-Dihydroxybenzaldehyde,Caffeic acid,Spinosin,Baicalin,Salvianolic acid C,Hesperidin,Rosmarinic acid,Salvianolic acid B,Lithospermic acid and Nobiletin.(2)The serum containing CHB-Ⅱ-F was collected.And four constituents(P-Sitosterol,Salvianolic acid,Ⅰsobavachalcone and Bakuchiol)were identified.Conclusion:Both CHB-Ⅱ-F solution and CHB-Ⅱ-F-contained serum of tumor bearing mice had absorbed bioactive components.The material basis of its clinical application may be related to the components detected.2 Acute and chronic toxicity evaluation for CHB-Ⅱ-FObject:According to the requirements of Chinese medicine efficacy and toxicity test,we evaluated whether acute and/or chronic toxicological reactions were showed from mice and rats after taking CHB-Ⅱ-F orally.Method:Ⅰn acute toxicity test,CHB-Ⅱ-F was administered to mice with 117 g/kg.No treatment-related death or toxic signs were observed.The maximum tolerated dose to mouse was more than 351 g/kg,equivalent to 108 times of clinical dose.Ⅰn the chronic toxicity test,after 4 weeks of continuous gastric perfusion with CHB-Ⅱ-F,the biochemical index,cholesterol total(TCHO)of male rats treated with 97.5 g/kg significantly increased,and the indices of heart and spleen increased relatively.The body weights of female rats were significantly increased as well as mean corpuscular hemoglobin contentration in hematological tests and y-glutamyl transpeptidase(TGG)of male rats treated with 48.75 g/kg.The liver index of male rats treated with 24,375 g/kg was increased as well as the spleen index.Hematological parameters,biochemical parameters,urinalysis,stool routine analysis,food intake and histopathological examination had no abnormalities after 4 weeks of continuous gastric perfusion and 2 weeks after convalescence.Conclusion:The maximum tolerated dose of mice is more than 351 g/kg.The rats were given 97.5 g/kg,48.75 g/kg,24.375 g/kg for 4 weeks,which was equal to 30,15 and 7.5 times of the clinical dose.No obvious adverse reactions were observed in the rats.CHB-Ⅱ-F is considered safe either with a daily dose of less than 351 g/kg or with a daily dose of 24.375 g/kg for 4 weeks.Part Ⅱ Effect and mechanism studyEffects and mechanisms of CHB-Ⅱ-F on cell apoptosis of tumor bearing mice with transplanted H22 hepatocellular carcinoma after receiving chemotherapyObjective:CHB-Ⅱ-F is a traditional Chinese medical formula,which can relieve the uncomfortable symptoms and improve the quality of life of cancer patients after chemotherapy for a long time.To study the mechanisms of CHB-Ⅱ-F on tumor cell apoptosis of mice with transplanted H22 hepatocellular carcinoma after receiving chemotherapy.Method:Seventy-two KM mice were subcutaneously injected with suspension of H22 hepatocellular carcinoma cells(2×107/mL)(HCC)into the right anterior armpit.All mice were received intraperitoneal injection with 5-fluorine pyrimidine(5-FU)(200 mg/kg)once to establish the chemotherapeutic murine model of HCC.Mice were randomized into six groups:physiologic saline(untreated)group,5-FU(20 mg/kg)group,three CHB-Ⅱ-F(16.25 g/kg,32.5 g/kg,65 g/kg)[CHB-Ⅱ-F(L),CHB-Ⅱ-F(M),CHB-Ⅱ-F(H)]groups and Yangzheng Xiaoji Capsule(YZXJC)(0.78 g/kg)(positive control)group.Mice were allowed free access to food and water.The CHB-Ⅱ-F(L),CHB-Ⅱ-F(M)and CHB-Ⅱ-F(H)treated groups received treatments by intragastric administration of CHB-Ⅱ-F aqueous solution once a day.5-Fu group received intraperitoneal injection of 5-FU every 2 days.YZXJC group was administrated once a day.The untreated group was administrated with 0.2 mL/lOg sodium chloride physiological Solution every day.After 14 days of administration,tumors were peeled,weighed,tumor coefficient was measured,cell cycle and apoptosis were detected by flow cytometry,morphological changes of tumor tissues were stained by HE and observed under the light microscope and electron microscope,respectively.The protein expressions of Caspase-3,Caspase-8,Caspase-9,Bcl-2 and Bax in mouse tumor tissues were determined by Western blot.Results:CHB-Ⅱ-F could ameliorate the general survival conditions of mice H22 transplanted tumor after chemotherapy.Compared with the model group,the weight gain of three CHB-Ⅱ-F groups was significantly different,the tumor volume in three CHB-Ⅱ-F groups was significantly smaller on the 7th day.And on the 14th day,the tumor volume in the CHB-Ⅱ-F(H)group decreased significantly,the difference was statistically significant.Compared with the model group,the pathological changes of tumor tissue were alleviated in different degree,and apoptosis was observed by ultramicro electron microscope.The percentage of tumor cells in the G0-G1 and S phase in CHB-Ⅱ-F(M)and CHB-Ⅱ-F(H)groups were significantly different compared with model group.The apoptosis rate of tumor.cells in CHB-Ⅱ-F(M)and CHB-Ⅱ-F(H)groups were higher than that in the model group.Western blot(WB)analysis showed that the protein expressions of cleaved Caspase-3 in the CHB-Ⅱ-F(L),CHB-Ⅱ-F(M),CHB-Ⅱ-F(H)was higher than that in the model group,and the protein expressions of cleaved Caspase-8 in the CHB-Ⅱ-F(M)and YZXJC groups were significantly increased.The level of Caspase-9 in the CHB-Ⅱ-F(L),CHB-Ⅱ-F(M)was higher than that in the model group.The protein expressions of cleaved Caspase-9 in CHB-Ⅱ-F(M)group were significantly higher than that in the model group.The expressions of pro-apoptotic protein Bax in CHB-Ⅱ-F(L)and CHB-Ⅱ-F(M)groups were significantly higher than that in model group.The results showed that the ratio of Bax/Bcl-2 increased in CHB-Ⅱ-F(L)and CHB-Ⅱ-F(M)groups.Conclusion:CHB-Ⅱ-F could improve the general stations of mice with transplanted H22 after receiving chemotherapy,such as inhibiting hair loss and increasing diet,reducing pathological changes of tumor tissue and inhibiting proliferation of tumor cells.The mechanisms of CHB-Ⅱ-F may be related to regulating the expression of apoptosis-related proteins,thus promoting the apoptosis of tumor cells.