| The previous research of this programme initially clarified that the extract of Centipedes(ECP) has some therapeutic effect on Hepatocellular carcinoma(HCC), and made an initial probe into the mechanism of ECP for the treatment of hepatocellular carcinoma. On the basis of the previous research, we plan to further approach inhibitive effect of extract from different parts of Centipedes(ECPs) on Bel-7404hepatic carcinoma xenografts and make a systematic toxicological study on ECPs-through observing the different toxicological reactions caused by ECPs try to determine the latter's toxicity and the target organs of the toxicity, and to probe into the mechanism of the toxicity on the target organs, so that an experimental basis can be provided for reasonable choice of clinical medication.Chapter One:Inhibitive Effect of ECPs on Bel-7404hepatic carcinoma xenograftsObjective:To investigate the inhibitive effect of ECPs on Bel-7404hepatic carcinoma xenografts.Method:To set up model of Bel-7404hepatic carcinoma xenografts in nude mouse,and observe its tumor growth and metastasis by intragastric administration of ECPs.Result:①The achievement ratio for Bel-7404hepatic carcinoma xenografts in nude mouse is100%.②ECPs had distinctive inhibitive effect on Bel-7404hepatic carcinoma xenografts in nude mouse. At the30st day postgraft of nude mouse,the average cancer volume of the contrast group was1000.5±87.4mm3, and that of the treatment groups are460.3±61.7,526.1±75.7,530.7±81.3mmj respectively; the average grafting carcinoma weight of the contrast group was1120.01±101.94mg, but that of the treatment groups were593.73±37.01,659.57±62.88,669.42±45.00mg respectively; all these differences are all distinctive (P<0.05). The inhibitive ratio of the head&foot group, the holo-centipede group and the truck group reach46.990%,41.11%,40.23%respectively. The average weight of the grafting carcinoma in the holo-centipede group and the truck group are of no significant difference; but that of the head&foot gorup is less than the other two groups, the difference is significant (P<0.05).Conclusion:①The model of Bel-7404hepatic carcinoma xenografts in nude mouse is set up successfully;②ECPs(10g/kg) can inhibit Bel-7404hepatic carcinoma xenografts in nude mouse; Extract from the head&foot of centipede has the most significant inhibitive effect. Chapter Two:Toxicity study on extract from different parts of centipedesObjective:To evaluate the general toxicity of the ECPs through acute toxicity test and long-term toxicity test. To evaluate the genetic toxicity of the ECPs through Micronucleus test, Sperm Malformation Test and Ames Test.Method:(1)Acute toxicity test:The KM mice were lavaged with the different dose of ECPs, and their activities, reactions and death were observed.(2)Long-term toxicity test:The SD rats were lavaged with the different dose of ECPs in successive4weeks and13weeks, and their reactions and weight changes were observed. The blood routine test and biochemical test were detected. Then pathological anatomy was done on the rats, and the organ coefficient was calculated.(3)Genetic toxicity test:①Micronucleus test:The different dose of ECPs were lavaged to the rats, Then1000polychromatic erythrocytes (PCE) per rat were counted on each smear and get the micronucleus rates.②Sperm Malformation Test: The different dose of ECPs were the same to the micronucleus test.1000sperm cells per rat were observed through the light microscope and the aberration rate was got.③Ames test:Standard plate method was used. The numbers of the strains in each culture dish were scored.Result:(1)Acute toxicity test:Through the acute toxicity test, the LD50of the head&food group was39.18g/kg, with the average95%confidence interval (CI)32.89～45.01g/kg; the LD50of the holo-centipede group was51.20g/kg, with the average95%CI41.81～59.97g/kg; and the LD50of the trunk group was53.85g/kg, with the average95%CI45.09～62.12g/kg.(2)Long-term toxicity test:①General physiological indices:Early in the experiment, ECPs caused some animals vomit and changes of stool trait, then the symptom could be relieved. Compared with the contrast group of the same period, no influence on the SD rats'food intake was found in any group during the experiment.But the group of rats lavaged with high dose of extract from the head&food of centipede were found conspicuous hold-back in their weight growth.②Hematological indices: During the period of administration (in the4th and the13th week), compared with the contrast group of the same period, it was found that, ECPs caused a drop in RBC and Hb, a raise in ALT, AST, BUN, and CREA, and extended PT,TT,APTT, and was found significant difference (P<0.01, P<0.05). When the convalescence is over (in the15th week), all the other indicators mentioned above, except ALT and AST, basically recovered to their normal level. Comparison among the groups of the same period with the same treatment but different dose level:significant differences were found between the high dose group and the low dose group.Comparison among the groups of the same period with the same dose level but different treatment:In the level of low dose, no differences was found having statistical sense; In the level of high dose, Significant differences were found in ALT,AST,BUN,CREA,PT,APTT between the head&foot group and the holo-centipede group, and between the head&foot group and the trunk group(P<0.01, P<0.05);but no difference was found between the holo-centipede group and the trunk group(P>0.05).③Pathological examination:After the experiment, the rats were killed and pathological examination was made on them. The results show that the groups of any dose level have no significant influence on organ coefficient. During the administration, high dose of head&foot extract, holo-centipede extract, and trunk extract caused swelling in the liver cells, especially the high dose of head&foot extract even caused liver necrosis, and mild cellular edema in kidney. When the convalescence was over, the damage on liver and kidney was reduced, the kidney basically recovered, hepatic necrosis remained. None group made significant damage on the rats' heart, brain, lung or spleen.(3)Genetic toxicity test:①Micronucleus test:No significant deviation was found in micronucleus rate between all the groups lavaged with ECPs and their negative contrast groups (P>0.05); while high significant deviation was found between cyclophosphamide positive contrast group and negative contrast group (P<0.01). This shows that the ECPs have no effect on proliferation of mice polychromatic erythrocytes.②Sperm Malformation Test:No manifest effect by the ECPs was found on the sperm malformation of the mice; no significant deviation was found in any group (at different dose level) compared with their negative contrast group (P>0.05); and significant deviation was found in the positive control group compared with the negative contrast group (P<0.01). This shows that the ECPs have no effect on sperm malformation.③Ames test: The number of colony with back mutation in the positive contrast group exceeded the twice number of that in the negative contrast group; the number of colony with back mutation in varied strains of Salmonella typhimurium at different doses did not exceed the twice number of those with spontaneous mutation. The result of the test was negative.Conclusion:①Extract from head&foot of centipedes had low toxicity, while the toxicity of extract from holo-centipede is similar to that of the trunk.②When at the same dose level, toxicity of ECPs is:head&foot extract>holo-centipede extract>trunk extract.③ECPs can cause dysfunction in liver, kidney, and coagulopathy; the main target organs may be liver, kidney, etc., while more damage will be caused on liver, and less on kidney and hematological system.④No genetic toxicity of ECPs was found under our experiment condition.⑤It is advisable to use holo-centipede, and to increase the dose appropriately in the clinical treatment of HCC. Chapter Three:Experimental study on mechanisms of hepatotoxical damage in rats caused by ECPsObjective:To probe into the toxicological mechanism of ECPs on liver damage through detecting the level of MDA, SOD, CytP450, TNF-α, and IL-6in the livers of SD rats.Method:The MDA contents in livers were measured by the method of thiobarbituric acid reaction (TBA); the SOD activity of the livers was measured by Xanthine oxidase; the CytP450contents in livers were measured by Omura; TNF-a and IL-6in livers were measured by ELISA.Result:Compared with the contrast group of the same period, it was found that, during the period of administration, ECPs caused a drop in SOD and CytP450, a raise in MDA, IL-6, TNF-α (P<0.01, P<0.05).When the convalescence is over, all the other indicators mentioned above, except IL-6and TNF-α, basically recovered to their normal level. Comparison among the groups of the same period with the same treatment but different dose level:significant differences were found between the high dose group and the low dose group.Comparison among the groups of the same period with the same dose level but different treatment:Significant differences in MDA. SOD. CytP450,TNF-α,IL-6were found between the head&foot group and the holo-centipede group, and between the head&foot group and the trunk group(P<0.01, P<0.05),but no significant difference was found between the holo-centipede group and the trunk group (P>0.05).Conclusion:①ECPs can cause a raise in MDA contents and a drop in SOD activity, and may cause a chemical injury in liver cells through lipid peroxidation.②ECPs can cause a raise in TNF,IL-6contents and immunological injury in liver cells through inducing cytokine and inflammatory factors (such as TNF, IL-6,etc.).③ECPs can cause a drop in CytP450contents in liver, and the damage may be caused through affecting Histamine-HP450in the animals'body. |
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